Pharma Focus Asia - Issue 07

Strategy

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Expert Talk

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Research & Development |

Clinical Trials

Issue 7

2008

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Manufacturing

ÂŁ12 â‚Ź18 $25 Rs.300

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Research Insights

Nanoencapsulation | Nanobiomechanics | Nanofabrication w w w. p h a r m a f o c u s a s i a . c o m

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Foreword

Research & Development

Nanotechnology Promise “Nanotech presents many opportunities to pharmaceutical giants, ranging from better delivery of existing drugs to entirely new therapies based on nanomaterials.”

Matthew Nordan Vice President, Research, Lux Research

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nvestments in nanotechnology by governments and industries have been accelerating at a good pace globally. However, the pharmaceutical industry has been slow in investing and adopting the technology.

With the failure of the blockbuster model, dwindling revenues, pressure from generics, patent losses, declining product pipelines and ever rising R&D costs, the pharmaceutical industry has been forced to look at other avenues for growth. Personalised medicine, demand for safe and effective drugs, and target-based drug delivery systems are expected to drive future growth.

Nanotechnology is already playing a major role here, helping companies engineer drugs that can cross the blood-brain barrier, have greater solubility, stability and bioavailability and target-based drug delivery systems.

According to Freedonia Group, nanotechnology-based pharmaceuticals are expected to fetch revenues of US$ 16.6 billion by 2014.

The cover story features articles on research carried out by some of the premier institutions in applying nanotechnology to improve the properties of existing drugs and identify new drug candidates. The article “Sonication-Assisted Nanoencapsulation” presents the novel approach to build capsule walls with a

thickness of few tens of nanometres to adjust drug release rate and attach an antibody at the outer shell layer for targeted delivery.

“Nanobiomechanics and Human Diseases” discusses the role of biomechanics to better understand the pathophysiology of human diseases. This helps in developing new and improved high-throughput assays and diagnostic devices which are sensitive and accurate in detecting diseases during their early stages. “Unconventional Micro and Nanofabrication” dwells on some unconventional nanofabrication techniques that allow microengineering of the environment surrounding a cell. These techniques facilitate better understanding of the response of a cell to its environment in a predictive manner leading to more reproducible and meaningful assays.

Though its use is limited to a few areas, nanotechnology holds great promise and could play an important role in deciding the fortunes of the industry.

Aala Santhosh Reddy Editor

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Sonication-Assisted Nanoencapsulation

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Yuri Lvov, Chemistry Professor, Tolbert Pipes Eminent Endowed Chair on Micro and Nanosystems, Institute for Micromanufacturing Anshul Agarwal, Doctoral Candidate in Biomedical Engineering Department of Biomedical Engineering, Louisiana Tech University, USA Vladimir Torchilin, Distinguished Professor of Pharmaceutical Sciences Chair, Department of Pharmaceutical Sciences and Director, Center for Pharmaceutical Biotechnology and Nanomedicine Rishikesh Sawant, Doctoral Candidate in Pharmaceutics and Drug Delivery Systems, Department of Pharmaceutical Sciences Northeastern University, USA

Nanobiomechanics and Human Diseases

Insights into the pathophysiology

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Chwee Teck Lim, Deputy Director, NUS Life Sciences Institute, Associate Professor, Division of Bioengineering & Department of Mechanical Engineering, National University of Singapore, Singapore

Unconventional Micro and Nanofabrication

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George M Whitesides, Woodford L and Ann A Flowers University Professor Michael D Dickey, Post Doctoral Fellow, Whitesides Group Department of Chemistry, Harvard University, USA

Strategy Empowering Patients in Asia

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Chris Lee, Regional Head, Bayer Schering Pharma, Asia Pacific

Breaking Down Borders Asian biotechnology industry

10 Ying Luo Chief Executive Officer, GNI Ltd., Japan and Shanghai Genomics, Inc., China

Open Source Drug Discovery A feasible business model?

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Jagadeesh Napa Assistant Editor, Pharma Focus Asia

RegionFocus

The Arab Drug Industry Challenges and future potential Abdel Aziz Saleh Ex-Special Adviser to the Regional Director for Medicines, WHO / EMRO, Egypt

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Effective Interactions Institute - Industry

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R S Gaud Dean, School of Pharmacy & Technology Management, SVKM’s Nursee Monji Institute of Management Studies University, India

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Contents Research & Development Current Advances in Proteomics

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Afif Abdel Nour, Research Scientist in Molecular Biotechnology

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Automating PAT for process control Frank E Sistare, Principal PAT Scientist, CAMO Technologies Inc., USA

Thierry Aussenac, Scientific Director Institut Polytechnique LaSalle Beauvais, France

Getting to the Bone

Eliminating Guesswork

Using Chemometrics in PAT Investigating data from the process

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Petter Mörée, Director, Online Products, Umetrics AB, Sweden

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Fighting skeletal cancer metastases with specific T-lymphocytes

PLM solution in packaging and labelling

Dominik Rüttinger, Surgical Oncologist, Laboratory of Clinical and Experimental Tumor Immunology, Department of Surgery, Grosshadern Medical Center, Ludwig-Maximilians-University, Germany

Marc Sluijs EMEA Business Development Director Life Sciences, Oracle Healthcare & Life Sciences, Europe, Middle East & Africa

Bernard A Fox, NIH-funded Researcher Hong-Ming Hu, NIH-funded Immunologist Robert W Franz Cancer Research Center, Earle A Chiles Research Institute, Providence Cancer Center, Oregon Health and Science University, USA

Target Deconvolution in the Post-genomic Era

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Georg C Terstappen, Chief Scientific Officer, Siena Biotech S.p.A., Italy

CaseStudy

Improving Pharmaceutical R&D Using Lean Sigma

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Clinical Trials Retention of Subjects The question of informed consent J Findlay Walker, Vice President, Administration, Daiichi Sankyo Development Ltd., UK

Keith Russell, Director, Enhancing Product Delivery, AstraZeneca, USA

Manufacturing

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Cell-based Potency Assays

Adhering to GMP standards

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Clinical Trials in China Mark Engel Chairman, Excel PharmaStudies, China

Daniel N Galbraith, Head, Operations Andrew Upsall, Head, In vitro Services BioOutsource Ltd., UK

Single-use Bioprocess Containers Economics of usage in Asia

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Swapnil Ballal, Head, Biopharmaceutical Bulk Manufacturing, Intas Biopharmaceuticals Ltd., India

Large-scale Biochromatography

What lies ahead?

Uwe Gottschalk, Vice-President, Purification Technologies, Sartorius-Stedim Biotech, Germany

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Leticia Cano IRTA Postdoctoral Fellow, Laboratory of Applied Mass Spectrometry, National Heart, Lung and Blood Institute, National Institutes of Health, USA

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Issue 7

2008

Editor

: Aala Santhosh Reddy

Assistant Editor

: Jagadeesh Napa

Editorial Team : Bhamoti Basu T P Venu Language Editor

: G Srinivas Reddy

Consulting Editor

: P Sudhir

Editorial Advisor : Sasikant Misra Deputy Director,

Confederation of Indian Industry

Art Director

: M A Hannan

Visualiser

: Sk Mastan Sharief

Graphic Designers : K Ravi Kanth Ayodhya Pendem Copy Editor

: Jagadeesh Napa

Production

: Suresh Giriraj

Head - Sales

: Rajeev Kumar

Manager - Intl. Sales

: Naveed Iqbal

Sales Manager

: Sunita John

Sales Associates :

Compliance

Vinod P Sirwani Sylas Makam Veena Raj Sowjanya

: P Bhavani Prasad

CRM :

Rajkiran Boda Yahiya Sultan Savitha Devi Murali Manohar G Vijay

IT Team :

Shadaan Osmani Ifthakhar Mohammed Azeemuddin Mohammed Sankar Kodali Thirupathi Botla N Saritha

Chief Executive Officer : Vijay Chintamaneni Managing Director : Ashok Nair Pharma Focus Asia is published by

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Empowering Patients in Asia

Persistent changes in the healthcare landscape in Asia are driving the demand for increased information to patients to empower them as consumers. Better access to information, more patient responsibility and a greater focus on prevention of disease should be the basis for modern healthcare in Asia.

Chris Lee, Regional Head, Bayer Schering Pharma, Asia Pacific

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discussion of patient as a consumer should take into account the economic status of country to which he / she belongs Economic status to a great extent affects the power of patients as consumers in those markets. This is because the economic maturity of a country in many cases also defines its healthcare system.

There is no homogenous model of healthcare across the Asia Pacific region.

Australia’s medical care system resembles that of the United Kingdom, with free medical care and reimbursement for a broad range of medicines. Governmentsubsidised private health insurance has stemmed the tide of people leaving health funds, now covering around 30 per cent of Australians. Both Korea and Taiwan have introduced national health insurance systems that cover nearly all of their population, but cracks are beginning to show with the increasing financial burden due to the prevalence of the diseases that ageing population and changing lifestyles bring about.

Malaysia and China, on the other hand, have health systems that feature a mix of a private and public healthcare catering to different segments of the society.

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In developed countries, national health insurances are struggling with a hefty financial burden. In developing countries, where a significant proportion of the population is unable to pay for their own healthcare, the governments are making efforts to reduce costs to provide healthcare access for the poorer segments of their society. Many of the measures implemented are short-term and are only aimed at alleviating the symptoms. Here, the patient information that is geared towards disease prevention and disease awareness can enable early detection and contribute to containing the rising costs of healthcare.

Indicators show that the debate on patient empowerment in diagnosis and treatment is increasing in Asia. An increasing number of Asian consumers, instead of relying only on healthcare providers, are accessing other sources such as the media and Internet for healthcare information.

Small but persistent changes in the healthcare landscape in Asia are driving this demand. Improved primary care in rural areas has led to an increase in access to generic as well as innovative drugs.

Better diagnosis options and increased disease awareness coupled with increased

health insurance coverage, further prompt the Asian population to be more involved in their health matters and to seek and demand better healthcare information.

With these trends and changing patient habits and behaviour, it is very much evident that patient empowerment is the basis for modern healthcare in Asia.

What is patient empowerment? It simply implies better access to information, more patient responsibility and a greater focus on prevention of disease.

Patient empowerment

In many Asia Pacific countries the doctorpatient relationship is strong with the doctor acting as the ultimate source of medical information for the patient and his / her family. Such a paternalistic model demands heavy intervention from the state (government) and the medical professionals as this system works on the assumption that the patient has no knowledge on medical and healthcare matters. However, modern healthcare systems should take the patients seriously as a dialogue partner in the management of disease. One can only benefit from a healthcare system that is patient centred—a healthcare system which is designed and delivered to address each individual patient’s needs and preferences. This model allows more choice, personalised care and effective patient involvement.

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While it is acknowledged that this may not yet be possible in all Asian communities due to a number of factors, such as limited information channels and education levels, an increased interest is being observed from the patients to obtain information on how to manage health issues beyond the hospital or general practitioner’s office in many developing countries. For example, many young Asian working women in urban centres seek information on medical solutions that allow them to plan their families and space their pregnancies.

It is clear that patients in the future will no longer accept being passive recipients of healthcare and advice. Instead, they will like to become proactive consumers, with better access to quality information about their diseases and treatments and more control on decisions affecting their health.

This is particularly relevant when it comes to chronic disease prevention and control, where the actions of the patient outside the ambit of clinic or hospital, in terms of diet and lifestyle modification, have a major impact on optimum management of the disease.

Unfortunately, unlike in most other industries, the consumer does not have a say in what he or she consumes.

Therefore, any move towards more patient empowerment will require at a minimum, a process of education and better patient information.

Information to patients

In some parts of Asia, government regulations mean that medicine or treatment information to patients by the industry is restricted or not allowed. Yet better access to health and drug information would empower citizens to take a more active role in managing their health, leading to healthier behaviours, better compliance and more efficient use of healthcare resources.

Sharing of quality health and medicine information from multiple sources including the pharmaceutical industry should be allowed. This is particularly

Bayer’s initiatives in increasing patient empowerment Bayer is making serious efforts to empower patients by cooperating with governments, patient groups and medical associations to provide information. The emphasis is on providing training to experts, sharing patient information, improving awareness and prevention of diseases. One such initiative is supported by the Chinese Ministry of Health Disease Control Bureau, called Bayer Cup – Diabetes TV contest. Ten thousand diabetics from more than 100 municipal hospitals in Beijing, Shanghai, Guangzhou and Qingdao take part in the national quiz on diabetes in China. With the broadcast of the final nationwide round attracting more than 100 million TV viewers.

Another programme is to train 10,000 healthcare professionals in Western China in partnership with China’s Ministry of Health. A second programme with the Ministry of Health aimed at healthcare professionals was launched in April 2008. Bayer Healthcare’s HOME campaign aims at improving the capability of community healthcare practitioners in China and build strong chronic disease management system in the community through a three year training programme. In Thailand, Bayer supports Multiple Sclerosis Society to provide a platform to communicate, exchange experiences and identify treatment options.

important in countries where general disease awareness on even the most common health problems is low. Being able to recognise symptoms and seeking medical help early are crucial steps in keeping the population healthy.

Pharmaceutical companies are a key source of information that patients can access. In particular, they have a part to play in raising disease awareness and encouraging patients to take a more

active role in managing their conditions.

However, the information from pharmaceutical companies has come under increasing scrutiny. Thus, the challenge they face as an industry is to provide unbiased and useful information in an ethical manner. This requires a significant amount of self-regulation and urge to accept greater responsibility towards the society and patients in particular.

Care should be taken to ensure that

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information is evidence-based, of high quality and reliable to gain public trust.

Regulatory authorities may restrict the manner in which pharmaceutical companies provide information, but ultimately, health information to the consumer should be acceptable and be judged depending on its quality, rather than the source which provides it.

About 17 million people per year die due to chronic, non-infectious diseases, such as heart disease, stroke, cancer, chronic respiratory diseases and diabetes.

region to non-communicable, lifestyle diseases.

Long deemed to be a scourge of the West alone, obesity is an emerging problem in the Asian region. Obesity is considered to be a major risk factor in the development of diabetes and cardiovascular heart diseases. The changes in lifestyles such as decreased physical activity and an unhealthy diet have tripled obesity rates in the past 10 years in developing countries.

If the number of deaths from the so called “lifestyle” diseases—such as cardiovascular and respiratory diseases, diabetes and obesity—is to be reduced, public authorities and healthcare stakeholders, including the industry, will have to adopt a comprehensive approach to disease prevention and control including all segments of the society.

Way forward

Focus on prevention and better disease management

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In the future all stakeholders of healthcare must focus more on prevention of disease. The main cause of death in the world today—about 17 million people per year—is the result of chronic, noninfectious diseases, such as heart disease, stroke, cancer, chronic respiratory diseases and diabetes. They are most often caused by known and preventable risk factors like unhealthy diets, physical inactivity and alcohol and tobacco consumption.

While the disease burden in Asia Pacific in the past decades has mainly been driven by communicable diseases associated with poverty and poor standards of living, rising affluence and urbanisation have shifted the disease burden in the

Sharing patient information will ultimately lead to better disease awareness, disease prevention and early detection.

Effective treatment can then be provided to the patient immediately upon being diagnosed at the outset of a disease reducing long-term financial burden and suffering.

Knowledgeable and responsible patients make responsible consumers of healthcare. To achieve this, governments, doctors and the industry need to work constructively together to empower patients and encourage them to take responsibility to look after their health. These efforts will contribute greatly to addressing some of the issues that ail current healthcare systems and will ultimately make healthcare affordable.

Chris Lee is the Asia Pacific Region Head of Bayer Schering Pharma, based in Singapore. Prior to this, he was the Head of Bayer HealthCare based in China. Lee obtained his Bachelor of Arts degree from University of Arizona USA in 1988 and his Masters degree (MBA) from Thunderbird, USA in 1990.

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BOOK Shelf

Raman and SERS Investigations of Pharmaceuticals (1st Edition) Authors: M Baia, S Astilean and T Iliescu Year of Publication: 2008 Pages: 224 Published by: Springer ISBN-10: 3540782826 ISBN-13: 978-3540782827 Description: Over the last several years it has become apparent to most researchers that interdisciplinary research is the key to success in the sciences future. The present book exemplifies such interdisciplinary work. Thus, some new derivatives have been prepared by chemists and consecutively analysed by physicists in order to better understand their physicalchemical properties for future tests to be performed by pharmacists. The book consists of an introductory section and eight chapters. First, the fundamentals of infrared, Raman and surface-enhanced Raman spectroscopy and those of the theoretical methods employed for the vibrational prediction modes are highlighted.

The SERS investigations illustrated in the following chapters are focussed on different kinds of drugs: tranquilisers and sedatives, anti-inflammatory drugs, vitamins, drugs with anti-bacterial properties, etc. Since there is an increased interest in designing highly effective and controllable SERSactive substrates, a few newly developed substrates that could contribute to a deeper understanding and knowledge of the adsorption behaviour of various types of molecules of pharmaceutical and medical interest are also presented.

For more books, visit Knowledge Bank section of www.pharmafocusasia.com

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S trategy

Breaking Down Borders Asian biotechnology industry

Ying Luo Chief Executive Officer, GNI Ltd., Japan and Shanghai Genomics, Inc., China

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f you had run into Mark Engel, CEO and founder of Excel PharmaStudies, at the recent 2008 China Pharmaceutical R&D Summit at Shanghai, you would realise that even the young biotech industry in Asia is not immune to the rapid globalisation of drug discovery and development. Excel, a leading CRO, was started by a western businessman in China and serves customers all over the world. Moreover, some of the multinational pharmaceutical companies sent more than 20 delegates to the conference. Investment banks, law firms, accounting companies and government agencies also showed up in large numbers to scout for new industry trends. Meanwhile, delegates from many Chinese biotech companies were also there reaching out to other geographies by offering contract research services or trying to attract international investors. With such an international mix of people at the conference, it is not hard to imagine that in a few years

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Cross-border collaboration in biotech industry has been emphasised by both the public and private sectors of many Asian countries. Although there are still many obstacles to overcome, such efforts have started to bear fruits. it may be difficult to find a mid-size Asian biotech company whose CEO, shareholders, employees, scientific advisors and customers are all from a single country.

Opportunity in Asia

About 60 per cent of the world’s 6.5 billion people live in Asia. Yet, because of differences in genetics, environment and food, the prevalence of major diseases in Asia is different from those in the West. Bacterial infection is still a major healthcare problem in most parts of Asia, leaving enough scope for large-scale production of antibiotics in China and India. Stomach cancer and lung cancer are more common in Japan and China than in Western countries. Liver disease, the ‘National Disease’ of China, is much less of a threat in Europe than in Asia.

Fortunately, multinational pharmaceutical companies have started to take a closer look at Asian opportunities, especially after clinical trial results demonstrated

that Iressa, the anti-cancer drug, was more efficacious in Asian populations.

Even when the US is still recovering from subprime crisis, the GDPs of China and India, the two most populated countries on earth, are growing between 8 and 10 per cent per year. Economic growth is driving the Chinese healthcare market to grow at double-digit rates. With Japan as the second largest healthcare market in the world, developing drugs for diseases prevalent in Asia has now become a priority for Asian biotech companies. Currently, most Asian countries lack sufficient economic power, established business models, or scientific talents to develop strong domestic pharma industries. This situation demonstrates the urgent need for Asian biotech companies to pool their resources across borders.

Government initiatives

From late 1990s to the early 2000s Asian government policies encouraged the growth of local biotech companies

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by offering tax incentives, cheaper land, incubators and direct investment. Since 2000, the Chinese government has attracted Roche, AstraZeneca, Novartis and Pfizer to set up R&D centres in Shanghai’s Zhangjiang Hi-Tech Park, hoping to eventually build up close ties between the Chinese biotech industry and multinational pharma companies. Singapore’s government has also been consistently investing in biotech infrastructure. Since the late 1980s, Singapore National University has recruited talent from all over the world to fill important faculty positions in the biological sciences. For example, Dr Xin Yuan Fu is one such person, who grew up in China, received his PhD in the United States, and has now moved to Cancer Research Centers of Excellence in Singapore.

Struggling with the high cost and slow speed of clinical trials, which are the major bottlenecks of drug development, Western pharma companies are looking at India and China as the solution. At the same time, many Asian governments have also realised that their countries’ limited financial resources, human talent and market size do not justify the development of drugs limited to their own country. To facilitate cross-border clinical

Struggling with the high cost and slow speed of clinical trials, which are the major bottlenecks of drug development, Western pharma companies are looking at India and China as the solution.

research among Asian countries, Japan’s Pharmaceuticals and Medical Devices Agency (PMDA), the Chinese State Food and Drug Administration (SFDA) and the Korea Food & Drug Administration (KFDA) have recently held the East Asia Pharmaceutical Regulatory Symposium in Tokyo. With increasing numbers of Japanese pharma companies, such as Eisai and Sankyo, moving their clinical trials to China and India, it is natural for the regulatory bodies of those countries to start thinking about harmonising their different standards.

Collaborative research and outsourcing

Today, it would be surprising if a life science department in an American university does not have many Chinese

and / or Indian graduate students. Since the late 1990s, waves of ‘returnees’ have come back to Asia. In fact, most of the 200 biotech companies in Shanghai’s Zhangjiang Hi-Tech Park were founded by returnees, who bring back not only international investment, but also their previous academic and industry networks.

For example, one of the biotech companies in Shenzhen, China has its company registration in the Caribbean, its operation in China, scientific advisors in the United States, and board members in Hong Kong and Singapore. Likewise, the target markets of these companies’ products are also not limited to China alone. Even at the clinical development stage, their products are already licensed out to pharma companies from the United States and Europe.

A mixture of collaborative research, contract research, outsourcing, and co-development is also sprouting all over Asia. Following the success of their Indian colleagues, Chinese entrepreneurs are playing a quick catch-up game in the chemistry outsourcing field, represented by Wuxi Pharmatech and Shanghai ChemExplorer. After successfully outsourcing chemistry on a large scale, the next growth opportunity is testing those compounds in various

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International investment

The growth potential of Asian biotech companies is by no means off the radar screen of international investors. Venture capitalists, private equity groups and brokerage houses are combing China and India for new opportunities. Recently, Lead Therapeutics completed a seed financing round of US$ 17 million in the US. The company’s business model is to have experienced chemists in California generate novel and differentiated small molecule pre-IND candidates against clinically validated therapeutic targets, and then synthesise and test those compounds in China. This approach is one way to reduce the burn rates of young biotech companies in the West. Other leading biotech companies in China, such as MicroPort Medical, Medicilon, Bridge and Ambrosia all have major shareholders from Japan, the United States, or Singapore.

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Merger is probably the most direct way to create cross-border advantages.

In 2005, Tokyo-based GNI merged with Shanghai-based Shanghai Genomics. The combine has more than 100 people and two drugs in clinical trials in China.

GNI was later listed on the Tokyo Stock Exchange. Recently, Wuxi Pharmatech of Shanghai acquired AppTec, and Beijing-based Venturepharm Laboratories acquired 39 per cent of Commonwealth Biotechnologies. Acquiring companies from United States enabled these Chinese companies to be proactive in reaching out to a broad range of

Merger is probably the most direct way to create cross-border advantages.

customers in the West. After M&A, it is plausible that business development, financing, and some research may remain with high-cost countries, while development, testing production, and support may be moved to low-cost regions.

Challenges

Recently, GNI / Shanghai Genomics published two target discovery-related articles in the Proceedings of the National Academy of Sciences, USA, demonstrating that the quality gap in discovery research between the West and the East is closing. However, lack of original discovery research is still the major weakness of Asian biotech companies.

Closer collaboration with universities all over the world may be a feasible way

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biological assays locally. However, biological discovery outsourcing is growing at a relatively slow rate due to the complexity of discovery projects. Because quality control and training of teams require more effort, Western pharma companies are yet to feel confident about outsourcing their discovery research projects to Asia. Recently however, GNI / Shanghai Genomics have extended and expanded its FTE-based research programme with Organon (part of Schering-Plough), an achievement that represents a breakthrough in this area.

Although outsourcing or contract research does not generate intellectual property for Asian biotech companies, it provides stable revenue streams and important platforms for their future opportunities. In 2007, Lilly signed drug development agreements with Nicholas Piramal of India and Hutchison MediPharma of China. Such agreements demonstrate that Asian biotech companies are moving up the value chain from pay-for-service to risk-sharing with pharma companies.

for Asian biotech companies to access cutting-edge knowledge and new discoveries.

Cooperation among government regulatory agencies is also an urgent issue.

Not every Asian country has the size of market or human resources to develop full-fledged pharmaceutical companies.

American and European markets are still much larger than that of any Asian country, except Japan. If regulation of clinical studies can be standardised and the clinical results obtained in one Asian country can be accepted by the others, it will create a Pan-Asian pharmaceutical market large enough to encourage all the biotech companies to find their niches, and thus provide a major competitive advantage for the Asian biotech industry. As of today, the borders have not yet broken down, though there are positive signs that it will occur sooner than people think.

The next generation

When a bird carries a flu virus all over the world, it does not recognise borders.

If the dissemination of drugs was not restricted by borders, drugs developed in one country in Asia could also benefit its neighbours as well as patients all over the world. Although the borders are breaking down, the collaboration among Asian companies is less compared to that between Asian biotech and Western pharma companies. However, it may be difficult, in the next decade, to label an Asian biotech company as a Chinese biotech company, an Indian biotech company, or a Singaporean biotech company. Most likely, by then the “pure� Asian biotech company will be an extinct species!

Ying Luo received his PhD degree in 1991 and did his postdoctoral research on HIV gene regulation at the University of California at San Francisco. He is currently CEO of Tokyo based GNI and Shanghai Genomics. His articles have been published in more than 30 scientific publications. In 2007, he received the Magnolia Award from Shanghai Municipal Government for his contribution to the economical development of Shanghai.

RegionFocus

The Arab Drug Industry Challenges and future potential

Abdel Aziz Saleh Ex-Special Adviser to the Regional Director for Medicines, WHO / EMRO, Egypt

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n response to the 21st century challenges to the health sector, WHO established an important global commission on Macroeconomics and Health. One of the important conclusions of the commission report indicated that by the year 2015, the world could save up to 10.5 million lives every year by scaling up access to existing health interventions to prevent or treat infectious diseases, maternal and perinatal conditions, childhood diseases, and non-communicable

Though Arab drug industry is increasingly contributing to the production of biotechnology products, particularly vaccines and herbal medicines, the main challenge is to achieve the regional objective of self-sufficiency in the production of essential drugs and vaccines.

diseases. Most of these interventions depend on the universal access to essential medicines and vaccines.

To achieve this objective, national and regional production of essential medicines and vaccines are important strategies that have been endorsed by ministries of health in the Arab countries.

The Arab world has 22 countries in the Middle East of which 19 are members of WHO Eastern Mediterranean Region. They have many

important commonalities—they speak one language (Arabic), they are predominantly Muslim and share a common history. However, they differ greatly in socio-economic and demographic indicators. The population ranged from 0.725 to 72 million in 2005, and per capita GDP ranged from US$ 192 to 32,193 in 2004. Total health expenditure in Arab countries ranges from US$ 6 to 862 per capita, while medicines expenditure ranges from US$ 1 to 80 per capita.

w w w . p h a r m a f o c u s a s i a . c o m 13

Salient Features

Arab Pharma Industry To improve universal access to the essential medicines and reduce costs, Arab countries have been encouraging local production of medicines since four decades.

Year

Local production (million US$)

Drug consumption by local production (%)

Coverage of consumption by local production (%)

Drug consumption per capita (US$)

1975

345

785

44

05.5

1980

700

1800

39

11.0

1987

815

2400

34

13.0

1993

1300

3200

41

14.0

1995

1540

3873

40

16.8

1996

1696

4144

41

17.1

Source: ACDIMA

Arab drug market

Most Arab pharma companies limited themselves to the manufacture of patent generic or branded generic medicinal chemicals and pharmaceutical preparations under licence.

Saudi Arabia and Egypt are the two major markets in the region.

Research and developmental activities in Arab pharmaceutical industry, except in a few large firms, are still at a primary stage, mostly focussing on manufacturing process or improving product quality.

Although Arab drug market grew significantly in last two decades, the local drugs constitute only

40-50

per cent of total consumption.

14 P h a r m a F o c u s A si A

Drug consumption and production in the Arab region, 1975–96

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Data on the consumption and local production of medicines in the Arab countries are based on estimation from various studies. The total drug market rose from US$ 785 million in 1975 to US$ 4,144 million in 1996. However, the proportion of locally produced drugs in total consumption remained at just over 40 per cent (Table 1).

Data from the Union of Arab Manufacturers indicates that the Arab pharmaceutical market was worth US$ 6.20 billion accounting for 1.25 per cent of the global pharmaceutical market in the year 2003. In Arab countries, the local drug production constitutes various percentages of national drug market ranging from 0 per cent up to more than 90 per cent. In average, the local Arab drug industry covers 50 per cent of the total Arab drug market. It is estimated that the Arab drug market was worth US$ 10 billion in 2007. Saudi Arabia and Egypt are the two major markets in the region with a market size of more than US$ 1.2 billion each.

The Egyptian market in some studies has been estimated to be around US$ 1.7 billion. Most of the locally produced products are formulations. In some countries, about 40 per cent of drugs are produced under license or in subsidiary plants of multinational companies. More than 90 per cent of the raw

Table 1

materials needed for local production are imported.

Arab drug industry

According to the World Health Organization report on World Medicines Situation, trends from 1985 to 1999, the value of total medicine produced has grown four times more than the world’s income. Data in this report has also shown that only five industrialised countries produce almost two-third of the world medicines production.

It is important to note that the cost-effective local production of essential medicines can contribute to the universal access of essential medicines.

Arab countries have, therefore, encouraged local production of medicines during the last four decades.

Arab pharmaceutical industry, which started in Egypt in 1930s, had passed through several phases that are characterised by state domination in the 1960s, followed by liberalisation in the 1970s and 1980s which attracted more private investment. The pharmaceutical industries nowadays are attracting more investments from the private sector in all the Arab countries.

The pharmaceutical industry in Middle East region recorded a combined annual growth rate of 10.6 per cent between 1998 and 2002. There are more than 245 pharmaceutical plants in the Arab states, and the number is

RegionFocus Estimated vaccine requirements for EPI programmes in the Eastern Mediterranean region, 2003–2010 DPTHepB-Hib

Year

BCG

DTP

MCV

MMR

OPV

Hib

HepB

TT

DPT-HepB

DPT-Hib

2008

20,579,406

50,838,649

18,379,929

10,981,612

85,097,896

10,792,167

24,335,478

36,585,610

7,462,634

10,095,863

988,826

2009

21,051,488

51,993,752

18,797,135

11,224,619

87,002,632

11,030,218

24,907,866

37,424,868

7,611,887

10,336,793

1,015,582

2010

21,535,231

53,177,709

19,224,505

11,473,758

88,953,970

11,274,130

25,495,784

38,284,855

7,764,125

10,584,109

1,043,063 Table 2

increasing every year. They belong to the Reproductive Pharmaceutical Industries (RPI) and range from small-scale family enterprises to medium-scale public and private shareholding companies with diverse strategies.

Production is typically limited to the manufacture of patent-expired generic or branded generic medicinal chemicals and pharmaceutical preparations under license. Other main characteristics of the Arab drug industry include: • Production mainly caters to local markets with the exception of Jordan • Some therapeutic groups and specialised dosage forms, like certain hormones, antineoplastics and immunosuppressive agents and advanced drug delivery systems are yet to be produced • Pharmaceutical raw materials are produced in Egypt, and to a limited extent in Oman and Algeria. Local production of these materials is still in the early stage of development, and output does not cover more than 10 per cent of demand at national and regional levels.

Vaccine production

In all national immunisation programmes for prevention of serious communicable diseases particularly in children, the availability of good quality drug affordable vaccines represents one of the most efficient interventions. The sustainability

of the continuous supply of the required vaccine represents a serious challenge to the national immunisation programmes in developing countries. New global changes and challenges include increased competition, difficulties in accessing cutting-edge technology, reduction in the number of vaccine suppliers, limited market and profit margins and decreased interest in vaccine production by the industrialised countries. They are putting additional strain on ensuring universal access and affordability of essential vaccines.

Data from 18 countries of the WHO / EMR region including 15 Arab countries show that in 2003, a total of 590 million doses of vaccines were purchased in the region, of which 12.5 per cent were from local producers.

Table 2 shows the estimated needs of essential vaccines in the WHO / EMR countries. In Arab countries, only Egypt and Tunisia are vaccine producing countries. Table 3 shows production capabilities in Egypt and Tunisia.

innovative research activities based on local medicinal plants.

Recently efforts are being made in Egypt, Saudi Arabia, Jordan and the UAE to start the production of biotechnology products, particularly vaccines and other biological products. The major areas that need to be addressed in order to develop research and development include lack of funding, lack of experienced human resources in certain specialised areas and lack of suitable environment to promote research and development. To address these challenges, the following strategies are suggested: • Provision of incentives to companies to increase their research and development budget • Establishment of companies for research, financed by national or regional pharmaceutical industries • Pooling of regional resources to serve large-scale research and development projects.

Research and development

WHO / EMRO initiative on the production of essential medicines and vaccines

Research and development activities in the Arab pharmaceutical industries focus on the manufacturing process and are often aimed at improving product quality. Small firms may not be involved in research and development activities.

Recently, few companies particularly in Jordan and Egypt initiated some

WHO / EMRO in collaboration with its member states including the 19 Arab countries developed the regional initiative on regional self-sufficiency in the production of essential vaccines and medicines.

The guiding principles for the development of this initiative are:

w w w . p h a r m a f o c u s a s i a . c o m 15

S trategy

Vaccine production in Egypt and Tunisia

Recently, new facilities for the production of new vaccines were established in Saudi Arabia.

Country

Vaccine 1

Current production capacity Future production capacity 2010

Egypt

DPT

30 million doses

50 million doses

Measles

30 million doses

60 million doses

OPV

300 million (vial) 400 million doses (plastic tubes)

Tetanus toxoid

30 million doses (vial) 20 million doses (uniject)

30 million doses (vial) 20 million doses (uniject)

Hep B

150 million doses (vial) 6 million doses (uniject)

150 million doses (vial) 6 million doses (uniject)

DT

20 million doses

6 million doses

Meningitis

27 million doses

60 million doses

Tunisia

BCG

2 million doses

10 million doses Table 3

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Conclusion

In conclusion, the challenges that Arab countries face in medicine production are: • Promoting innovations and development of new medicines and ensure universal access and affordability worldwide • Complying to national and global codes of ethics for medicine promotion • Complying to WTO and bilateral free trade agreements and safeguarding the interest of public health • Combating the serious problem of counterfeit medicines nationally and globally • Utilising traditional medicines properly.

Therefore, the following important strategies should be considered: • Strengthen systematic data collection on consumption patterns, needs, local markets and local production of medicines and vaccines with special emphasis on coverage of essential medicine needs • Develop policies for promoting the production of essential medicines

A uthor

• Local production is an important component of national drug and vaccines policies and is one of the strategies for achieving equity in the access to essential drugs and vaccines • Quality is the leading parameter in relation to decision-making • Advancing technology and changing global trade arrangements created a new reality which countries and manufacturers have to consider • Governments are responsible for creating a conducive environment that strengthens local pharmaceutical and vaccine production industries.

The regional vaccine self-sufficiency was discussed in 2004. It was agreed among the experts from the region that the regional vaccine self-sufficiency is a broad and inclusive concept. It means sustained and sufficient supply, of existing and new quality vaccines to meet present and future needs for priority diseases in the member states. The initiative also contributes to the establishment and strengthening of national vaccine production facilities and effective National Regulatory. Research, development and local production of vaccines, where feasible, needs to be strengthened to first meet the national requirements and then as a priority, the needs of other member states in the region. Effective and feasible regional and other cooperative mechanisms need to be established to foster such vision.

and vaccines of acceptable quality and support the quality assurance (GMP and ISO 9000) and environment management (ISO 14000) systems • Establish holding companies at the national or regional levels charged with three main responsibilities, coordination in drug production, strengthening quality assurance and promoting research and development • Improve the capacity of the national regulatory authority to enforce GMP requirements, and meet WHO prequalification requirements • Develop and implement new strategies (generic medicine policies, hospital formularies etc.) to maintain the continuous supply of essential medicines and vaccines of good quality at reasonable prices • Encourage academic and research institutions to orient research activities towards the development of new medicines for diseases of national priority.

Full references are available on www.pharmafocusasia.com/magazine/

Abdel Aziz Saleh completed his PhD in Pharmaceutical Science from School of Pharmacy, University of Alexandria, in 1968. He has also worked as Visiting Phrofessor in schools of Pharmacy in Nigeria, Sudan and Libya. In 1989 Saleh joined WHO / EMR as the Regional Adviser for Pharmaceuticals. In 2001, he held the position of Deputy Regional Director until retirement in July 2002.

S trategy

Open Source Drug Discovery

A feasible business model?

The concept of open source model which is a huge success in the software industry holds great promise for the pharma industry as it strives to lower the cost of drug discovery and improve the bottom lines.

Jagadeesh Napa Assistant Editor, Pharma Focus Asia

O

pen source model in the pharma industry is currently used by non-profit organisations and governments to discover drugs for diseases neglected by industry. It is hoped that the open source model can be suitably adapted by the pharma industry and used as a sustainable business model to discover costefficient drugs, and to offer other tangible benefits that will improve bottom lines.

The pharmaceutical industry is yet to find a cure for certain life-threatening diseases prevalent in the tropical regions of Asia and Africa. These include Kala-azar, African Sleeping Sickness, Malaria, Tuberculosis, Chagas, Schistosomiasis, Lymphatic Filariasis etc., diseases commonly found in tropical regions. Such diseases, sometimes highly infectious, continue to claim thousands of lives in these parts of the world.

While vaccinations and other preventive

w w w . p h a r m a f o c u s a s i a . c o m 17

S trategy

The pioneers The idea of open source was first used in pharma and biotech industries in bioinformatics to produce tools to understand the mechanics of proteins. This was followed by the Human Genome Project and other initiatives where open source model made further inroads into the pharma and biotech domains. For instance, the Human Genome Project used the services of hundreds of biologists, microbiologists and other researchers worldwide to understand and sequence the human genes. Other major open source initiatives include Tropical Disease Initiative (TDI), Collaborative Drug Discovery (CDD), Innocentive etc. TDI, as the name implies, focusses on tropical diseases like Malaria, Tuberculosis, Schistosomiasis etc. and works in partnership with The Synaptic Leap, a virtual platform that provides the technology support to carry out research while TDI provides intellectual and scientific support.

of their time to work on drug development. Traditional drug discovery requires a closed-door environment. For security reasons, only the researchers working for the company have access to the research facilities. On the contrary, an open source model encourages inputs and insights from as many individual researchers as possible. It allows hundreds of scientists from all corners of the world to share their experience and expertise, and infuse speed and flexibility into the discovery process.

The Indian initiative

methods have been used to keep these diseases at bay, little or no research was undertaken to develop drugs and therapies to treat them.

The apathy of the major pharma companies to fund such research is the inability of the patients in these regions to afford the high prices for such drugs.

Taking the drug discovery process to the open source table promises great potential in finding cures for such hitherto neglected diseases.

IT industry – An early adopter

Open source, though new to the pharma industry, has been tried and tested, notably in the software industry where it has worked wonders. The robust yet cost-effective operating system, Linux, is a testimony to that. Supporters of cost-effective drug discovery have taken a cue from the open source model that led to the development of Linux, and portend that its adoption would augur well for pharma research.

A virtual platform

Open source drug discovery essentially brings researchers and scientists, from industry and academia, together onto a single virtual platform with all the necessary tools and resources at their disposal.

A list of medical problems (for which drug candidates are to be developed) are identified and posted on a specially developed website along with necessary tools and resources like compound

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libraries, biological information, screening facilities etc. Anyone can use these resources, propose insights and put forward their inputs. The inputs can range from identifying targets to lead optimisation. In this way, collaborative drug discovery process is accelerated through the open source model.

Pre-clinical and clinical trials are usually outsourced by the sponsors of the research to competent players from low-cost areas.

Drugs so developed can be used to treat patients in the third world countries where a majority of the patients live below poverty line. The issues of IP and patents are dealt by different players in different ways. In any case, set procedures to tackle the issue of intellectual property in an open source drug discovery model are yet to take shape.

Can it lower costs?

Cost reduction is the prime benefit in an open source model. R&D costs can be reduced considerably with the voluntary participation of researchers.

Hence, the drugs so developed will be comparatively cheaper. Further, these drugs can be licensed to generic drug makers and contract manufacturers who compete for market share by trimming costs and keeping drug prices low.

The discovery-to-market time of the drug would also be reduced as more researchers and institutions give much

The benefits of the open source model have prompted the Indian government to use this model to develop cost-effective and novel drugs for neglected diseases.

The Centre for Scientific and Industrial Research (CSIR), a state-funded apex body responsible for research & development activities, has taken an initiative in this direction. S K Brahmachari, Director General of CSIR, has been instrumental in piloting this initiative. A grant of US$ 34 million has been sanctioned for the project that uses open source model to discover drugs for neglected diseases.

CSIR’s initiative, named Open Source Drug Discovery (OSDD), targets neglected diseases that are widespread in India. Though similar to Tropical Diseases Initiative and The Synaptic Leap, OSDD differs in certain aspects. For instance, under TDI, IP rights of the drugs discovered would rest with Virtual Pharma, a group of non-profit venture capital firms. In contrast, under OSDD, the drugs developed would immediately become generics and hence the question of IP and patents does not arise.

Another difference lies in providing incentives to participants. Though the participation is voluntary in both the initiatives, CSIR is a step ahead as it encourages participation through its incentive scheme.

With research on tuberculosis as its maiden project, OSDD has plans to extend the open source model to discover

S trategy

drugs for AIDS. A first of its kind, OSDD has been developed through Public-Private Partnership (PPP) that includes the likes of Sun Microsystems and TCG Lifesciences contributing to the initiative. If OSDD is successful, it would attract a lot of sponsorships for its future projects, making it self-sustaining in the long run.

A relook at business models

In the light of these developments, is there a need for the pharma industry to review its business models? The answer is an emphatic yes. The traditional RiskInnovation-Reward model followed by the pharma industry has stopped fetching timely rewards, at least for now. Further to this, the concept of personalised medicine is slowly evolving in the pharma industry and is catching up fast in the healthcare industry. Decision-makers in the healthcare industry are seriously evaluating patient-centric strategies and in the not too distant future, these strategies will be implemented industry-wide. As a consequence, developing drugs customised to target populations and genetic traits will soon become the norm.

In such a scenario, the current “onedrug-fits-all” concept would be outdated and the traditional innovation-based business model will no longer hold good.

Therefore, in enlightened self-interest, pharma majors will need to give serious thought to the open source model, and tweak it to suit their requirements.

Open source drug discovery brings with it many advantages to pharma companies and patients alike. The biggest advantage of this model being huge cost reductions, pharma companies can leverage the open source model to outsource the drug discovery process and save a fortune in the process. Some costs thus saved could be passed on to produce and market the final drug at much lower prices. Thus, open source proves to be the most viable model for discovering life-saving drugs. Further, the model can also be used to discover lifestyle drugs

OSDD – A public private partnership The Open Source Drug Discovery (OSDD) initiative is the brain child of Dr S K Brahmachari, Director General, CSIR. The initiative took off as public private partnership with the objective of creating a Team India Consortium. The public private partnership includes TCG Lifesciences, Sun Microsystems, Institute of Genomics & Integrative Biology, Sky Quest Labs and others. OSDD aims to create a Team

India Consortium with global partners from public and private sectors lending their hands in cause of developing cost effective drugs for the third world countries. This initiative encompasses all the activities in the drug discovery process from identification of druggable nontoxic targets, in vitro and in vivo validation, in-silico screening of small molecules, lead optimisation, pre-clinical toxicity and clinical trials.

that pharma companies develop and market endearingly.

appropriate metrics and compensated through attractive rewards.

The issue of IP

A feasible model!

Till now, the issue of patents has been a major hurdle that has kept the pharma industry away from this model. Bernard Munos Advisor, Corporate Strategy at Eli Lilly had proposed in 2006 that open source models can also generate intellectual property. He pointed out that most open source activities happen at the pre-commercial R&D stage and these activities are similar to exchange of ideas among scientists. The scientist who makes a breakthrough publishes the findings and files for a patent.

The pharma company that wishes to discover a drug can throw open the research in an open source forum or launch its own website with the necessary tools to support the research. It can also announce payments / rewards for every credible milestone result that can carry forward the research. Thus, the pharma company gets the research running and at the same time remunerates the efforts of achievers. In this way it can own the intellectual property rights for the drug so developed and can file for patent. As an alternative, the pharma company can also share the patent with the researcher(s) crucial to development of the drug. Sharing of the patent can reduce the difference of opinions between the researchers and the sponsor, if any.

Open source research, therefore, brings down R&D costs drastically and allows pharma companies to maintain same profit levels despite the low premiums the drugs would command. The resulting market volumes would offset the premiums while the huge untapped market potential would fuel the future growth.

While Munos’ model of open source drug discovery is inclined towards PublicPrivate Partnerships (PPP), this model addresses the concerns of commercial pharmaceutical companies that wish to

Rewarding the researcher

This theory of Munos can be extended further with cue from the publishing industry. One can compare the drug discovery activity using open source model with that of the publishing industry—pharma companies can be compared with publishers and the researchers with those of the authors publishing their content. The authors from different fields get their articles published in various publications, but the copyrights of the published articles mostly rest with the publisher. Similarly, the pharma company that sponsors the research can retain the IP while rewarding the researcher(s) handsomely. All the researchers whose contributions are crucial to the drug discovery can be identified through

w w w . p h a r m a f o c u s a s i a . c o m 19

S trategy

Drug discovery ­– Open source models a

Open source model followed by non-profit organisations.

Open source web portal sponsored by non-profit organisations

b

BOOK Shelf

A probable open source model for the pharma industry.

Open source web portal sponsored by pharma company

Innovation and the Pharmaceutical Industry Milestone discovery

Milestone discovery (Reward the researcher(s))

Milestone discovery

Milestone discovery (Reward the researcher(s))

New Drug Candidate

New Drug Candidate

(Becomes a generic)

(To be patented by the sponsor)

(Ready for trials)

(Ready for trials)

Inputs by individual researches and institutions

reduce their drug discovery costs while benefiting from a global talent pool.

Open source activities in drug discovery process can happen only until a drug candidate is confirmed (i.e. until lead optimisation). Once confirmed, the subsequent stages—pre-clinical and clinical trials—can be outsourced to lowcost centres in India, China and other Asian countries where cGCP-compliant CROs will help move the drug from the lab to the market.

The way ahead

The pharma industry is gradually realising the potential of open source

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model. As a result, traditional mindsets have begun to change. The success of the present initiatives like TDI and OSDD will only attract more players onto this turf. Adopting open source model can help pharma companies to develop cost-effective drugs for both neglected as well as lifestyle diseases.

Further, this model can help in bringing the concept of personalised medicine closer to the masses. But the rate of adoption by the pharma companies depends on how quickly they change their mindsets and tread towards realising the benefits of this model.

Critical Reflections on the Virtues of Profit

Editor: H Tristram Engelhardt Year of Publication: 2008 Pages: 250 Published by: M & Scrivener Press ISBN-10: 0980209447 ISBN-13: 978-0980209440 Description: This book examines the central role of profit in the development of pharmaceuticals, medical devices, and healthcare generally.

Recent efforts to understand this role have often underestimated and even dismissed its importance, arguing for its replacement by other means and mechanisms. However, as the essays in this volume attest, it would be impossible to account adequately for the range of pharmaceuticals and medical devices that have become part of everyday medicine without recognising that the depth and scope of innovations are tied not simply to altruism, a concern for the common good, or the pursuit of knowledge for its own sake, but crucially to the pursuit of private good and of individual profit.

Balancing a concern for theory and practice, the analyses and evaluations provided in these essays touch directly on many of the most heated and important debates in pharmaceutical ethics, such as profit margins, corporate social responsibility, drug advertising, litigation, patents, and parallel trade. Reflecting critically on the problems and prospects of medical innovation, they invite a rethinking of the foundations of the bioethics and business ethics of the pharmaceutical and medical device industries.

For more books, visit Knowledge Bank section of www.pharmafocusasia.com

w w w . p h a r m a f o c u s a s i a . c o m 21

Effective Interactions Institute - Industry

R S Gaud

Dean, School of Pharmacy & Technology Management, SVKM’s Nursee Monji Institute of Management Studies University, India

How different are the industry-academia relations in India compared with the US and European countries?

In India, industry-academia interrelations are not very strong. Both industry and academia are not taking necessary initiatives to strengthen the relations as they do not consider this as a priority issue.

On the contrary, in the US and European countries there is a strong bond between industry and academia with industry sponsoring many projects in universities and reputed institutions. Many universities have undertaken pioneering research projects and also established research companies on a commercial basis. They have identified faculty exclusively to carry out such research activities with few academic commitments.

In the Indian scenario, teachers are overburdened and are involved in too many non-academic activities resulting in poor research output. There are only a handful of universities and institutions

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with a serious focus on research. Moreover, the industries here are very conservative and do not encourage funding for research activities carried out in institutions.

Indian pharma companies are shifting from generic-based business model to that of an innovation-based business model. How do you see the industry-academia interactions in light of these developments?

In post-GATT era with product patents in place, the Indian pharma companies have to come up with their own drug molecules and formulations which need innovative research. Academic institutions and universities can carry out basic research to achieve these targets. Industry with the institutional involvement will be able to accelerate their New Chemical Entities projects and other research activities using younger scientists and faculty from the institutions. They can as well use the infrastructure and human resources provided by these institutions in

other priority areas. Academia has more vision and expertise in developing newer technology which is essential for innovative research. Academia also has enough database for such research approaches due to continuous education programmes.

Many educational centres have not commercialised their research activities and limited research to academic purposes. With industry involvement, these ideas can be brought to reality for the benefit of the society. Thus, industry can also benefit provided they encourage tie-ups with academia.

E x pert talk

critical evaluation and suggestions made by the industry to improve their curriculum.

All the changes made to the curriculum must be acknowledged by the industry experts. Industry experts should voluntarily interact with university academia for making curriculum more relevant to the end-user i.e. the industry as it is meant for their own survival. The changes thus made to curriculum should reflect the respective changes in the industry.

Unfortunately, industry experts take it as a burden and quite often they remain absent on such occasions. I have experienced this many times in my 30 years of academic experience. While suggesting change in the syllabus, industry should also provide a framework for “train the trainer” approaches. The industry should consider it as a long-term investment.

Can you suggest ways to overcome these hurdles?

We must encourage younger faculty to engage themselves in research activities by providing more grants and opportunities to interact with pharma companies and by arranging awareness workshops on Intellectual Property Rights (IPR).

Incentives should be given to faculty engaged in research activities.

All postgraduate and doctoral research should be focussed on industrial needs and should not be only of academic value. Workshops on newer research approaches must be conducted regularly.

Faculty / student exchange programmes at national / international levels must be encouraged.

Government funding procedures must be time-bound and simple in operation.

Faculty with research inclination should be identified and encouraged to carry out research activities.

What is the role of government in promoting the relationship between industry and the academia?

The Government of India through its various agencies provides huge funds for such interactions. However, there is lack of awareness about such facilities and lack of commitment amongst the faculty. Also, institutions are not encouraging such activities as faculty members are already overburdened. They have limited time for academic delivery and most of the managements are casual in their approach to strengthen industryinstitute interaction. All India Council for Technical Education, Department of Science and Technology, Universal Grants Commission, Indian Council of Medical Research are a few bodies that look towards participating actively in such interactions. But response from majority of the academia is far from satisfactory.

Also, these grants are given mainly to the limited government / government-aided

Recruiting right talent is very crucial for the success of the industry. In what ways can the academia help the industry in this direction?

Academia should interact with companies of repute to provide training to their students. They should also look into the

P rofile

Academia in the US holds more patents than its counterparts elsewhere in the world. What are the factors that hinder the Indian academia to actively participate in the drug R&D? In India, research is not a priority and wherever research is carried out is not focussed and is of purely academic interest.

Academia and industry lack the patent awareness and are mostly dependant on research that is done abroad.

Secondly, there is lack of infrastructure and motivation for innovative research.

Even if research facilities are available, more often they are not accessible to young scientists. The availability and operation of research grants for academia from government bodies involves a lot of formalities due to which the academia stay away from utilising these facilities.

institutions. Unfortunately, these grants are not given to the capable investigators from private sector which constitutes a major chunk of the education system.

What are the benefits that the industry and academia can derive from each other in the long run?

The interaction would result in developing faculty and providing necessary human resources to the industry. Faculty members have to update their knowledge bank and industry must get workforce of the quality they look for. This is possible only through this interaction. The industry, by using the innovative approaches of the younger generation, can face the global competition effectively.

The benefits that industry can derive from the interaction with academia are many. Firstly, the industry will save many precious man hours in training their employees at its own cost. They can get the human resources of acceptable quality right at the entry point. They can also develop their Intellectual Property and improve their research potential. They will also have an opportunity to improve and economise their products and activities.

There will be a fresh flow of information to increase their data bank. Such efforts may lead the industry to new discoveries.

Academic institutions can redesign their courses, teaching, learning methodologies and research activities as per the needs of the industry. This will facilitate students’ placement and will improve the quality of the curriculum. Institutions can develop themselves as focussed and need-based research centres by utilising opportunities from both government agencies and the industry.

Interview conducted by Jagadeesh Napa, Assistant Editor, Pharma Focus Asia

R S Gaud is a senior academician in Pharma Sciences since the last 30 years and is responsible for shaping the Pharmacy division at NMIMS University. He also has worked as an advisor at All India Council Technical Education for five years. He is also a member of CII National Committee of Drugs & Pharmaceuticals. He has presented and published more than 150 research papers. He has authored nine pharmacy books and has two patents under process.

w w w . p h a r m a f o c u s a s i a . c o m 23

R esearch & D evel o pment

Current Advances in Proteomics Every single protein in the human body is a potential target for diagnosis and treatment of diseases. This article presents a brief description of biotechnology methods applied in proteomics that may help discovering new drugs.

Afif Abdel Nour, Research Scientist in Molecular Biotechnology Thierry Aussenac, Scientific Director Institut Polytechnique LaSalle Beauvais, France

T

he fact that more than 80 per cent of pharmaceutical drugs act through proteins reflects the importance of proteins as targets for diagnosis and treatment of diseases. “-omics� disciplines were developed to discover new biomarkers for health and medicine. Among these disciplines proteomics is directly involved in drug development because disease processes and treatments often manifest at the protein level.

Studying the functions of proteins seems to be the next important phase after the Human Genome Project.

The expected outcomes from the human proteomics are firstly to identify new targets, which will act as the basis for the development of new drugs, by deciphering the intracellular signalling pathways leading to the initiation and progression of pathologies and secondly, to discover new biomarkers for early diagnosis and profiling of pathologies. But technical challenges abound in proteomic research and they arise because of the large number of proteins (100,000 in human cells) present in the human body and possible modifications (acetylation, methylation, phophorylation etc.) to each of these proteins. The comprehensive analysis of protein complexes and signal transduction pathways can be achieved using new proteomic tools (Figure 1).

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Two-dimension gel electrophoresis and mass spectrometry

Almost all proteomics analytical techniques are based on protein separation. In the late 1970s, Leigh Anderson observed the potential of Two-Dimension (2D) gel electrophoresis as a mechanism to conduct proteomic studies of blood proteins and leukocytes. The first step is to separate the protein mixture of the samples. The proteins in a sample are applied to a rectangular piece of synthetic

gel (mostly polyacrylamid gel). First, the proteins are separated according to their charge and their size. The proteins are then rendered visible, resulting in a pattern of spots in which each spot contains a specific protein. The bigger the spot, the more would be the number of the concerned proteins present in the sample. Next, the spot is excised and characterised by Mass Spectrometry (MS). For many scientists, the latter invention enables the analysis of fragile biomolecules at a global level.

In the past few years, there has been an exponential increase in publications using the two methods (2D gels and MS) in the pharmaceutical field. Together these methods form the most powerful technology for the discovery of drug targets. Biomarkers

Various proteomics techniques

Mass Spectrometry 2-D gel

FFF

Proteomics Techniques Protein arrays

Proximity ligation Lab-on-a-chip

Figure 1

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Field-flow fractionation

With the development of analytical methods, the need for a simple separation technique is increasing. The Field-Flow Fractionation (FFF) is a flow-assisted separation technique for separating molecules in a 105 molar mass range, from macromolecules such as proteins to micron-sized particles such as whole cells.

The first application of FFF to proteins was reported in 1972. This technique allows the study of protein conformation, self-dissociation and dissociation, which are strongly related to the biological activity and to the interaction of proteins with other proteins, protein receptors, cell metabolites, and last but not least, drugs. This technique finds application in fields like biotechnology, pharmacology and genetic engineering.

Real-time Polymerase Chain Reaction and protein detection

It is surprising that even after 25 years of its invention, the Polymerase Chain Reaction (PCR), which is the most commonly used technique in genomics and transcriptomics, is still the most trusted technique in proteomics. In fact, sensitive methods are needed for biomarker discovery and validation. One of these methods is the Proximity Ligation PCR Analysis, a PCR-based method.

It consists of antibodies that can bind a target protein and is equipped with DNA strands capable of being joined by ligation when pairs of antibodies bind the same target protein molecule. The process can amplify and identify proteins with considerable sensitivity. Recently multiplex PLA was used to identify

and to validate sets of putative disease biomarkers relevant to cancer.

Developed at Harvard University in 2000, microarray proteins are also called as protein arrays or protein chips modelled after DNA microarrays. The types of protein chips available include antibody arrays, antigen arrays and tissue arrays.

This technique allows the detection and the comparison of large number of different proteins simultaneously. Antibody chips have been applied for protein profiling of intestinal, bladder, lung, pancreatic and prostate cancer; the list of applications can be very long. Hence, this tool has huge potential to alter the ways in which complex biological processes are analysed and interpreted. This would consequently open doors for personalised medicine in the clinics.

separation, detection and subsequent data analysis, essential to protein analysis. The Lab-On-Chip protein assay accomplishes these tasks in less than three hours, while the traditional SDS-PAGE consumes three to six hours for electrophoretic separation and detection. In fact, the automation of the SDS-PAGE process allows scientists to spend their valuable time on experimentation and research, rather than on processing slab gels. This method was developed and successfully used at the LaSalle Beauvais laboratory to characterise and compare proteins from different wheat cultivars. According to Prof. T Aussenac, this technique might be the backbone of the phenomics database development. It is worth mentioning that recently, researchers were showing lot of interest in phenomics, a field of study concerned with the interaction of the genome with the environment.

Capillary electrophoresis

Conclusion

Microfluidics-based techniques like LabOn-Chip and Capillary Electrophoresis were developed by many companies and academia. These techniques are automated, sensitive, reproducible, and give high-resolution separation proteins, differentiating almost all genotypes. They do not need toxic reagents or a long analysis time. Based on a microfluidic version of SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE), Lab-on-Chip protein assay chip is intended to perform automated protein sizing and quantitation. The system samples directly using 96-well plates and integrates all manual operations including staining, de-staining,

Technological advances of proteomics are still in their infancy as compared to genomic research. Standardised highthroughput procedures are necessary to fully leverage the potential of proteomics. Omics techniques are developing in India, the Far East and Gulf countries.

In a recent paper published in Nature, a genetics scientist identified the top 10 biotechnologies for improving health in developing countries the first place being for proteomics, second and third places were for techniques related to drug and vaccines development.

Microarray protein

A uthors

of disease can also be discovered by these techniques. The increase of the Glial fibrillary acidic protein in Alzheimer’s disease is an example of biomarkers discovered by the 2D gels and the MS. Though advancements in mass spectrometry have brought many a new drug targets to market, it is developments in chemistry, biology and bioinformatics that will lead to discovery of new drugs in the future.

Full references are available on www.pharmafocusasia.com/magazine/

Afif Abdel Nour has a PhD in Nutrigenomics and specialises in applied techniques in Molecular Biotechnology. He did his Agricultural Engineering and Masters in Molecular Microbiology from Pasteur Institut Lille. Currently, he is working as a Biotechnology teacher at the LaSalle Beauvais Institute, France and as a Molecular Biotechnology consultant in several countries in the Middle East.

Thierry Aussenac has a PhD in plant physiology and biotechnology from National polytechnic institution in Toulouse-France. He is employed by LaSalle Beauvais as a research director, with focus on plant biochemistry especially Wheat and Linum. He is a member of the American Association of Cereal Chemist and the French association of plant biology.

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Getting to the Bone Fighting skeletal cancer metastases with specific T-lymphocytest Breast, prostate and lung cancer most frequently metastasise to the skeleton.

Adoptive immunotherapy with antigen-specific T-lymphocytes may represent an alternative to standard treatment modalities such as chemotherapy and radiation, which can only provide palliation.

Dominik RĂźttinger, Surgical Oncologist, Laboratory of Clinical and Experimental Tumor Immunology, Department of Surgery, Grosshadern Medical Center, Ludwig-Maximilians-University, Germany Bernard A Fox, NIH-funded Researcher Hong-Ming Hu, NIH-funded Immunologist Robert W Franz Cancer Research Center, Earle A Chiles Research Institute, Providence Cancer Center, Oregon Health and Science University, USA

T

he skeleton is one of the most common target organs of metastases in human cancer.

Depending on the site of origin of the primary cancer, bone metastases are diagnosed in 23 to 84 per cent of the patients. Although breast, prostate and lung cancers most frequently metastasise to the bone or bone marrow, up to 45 per cent of melanoma patients also develop skeletal metastases. Bone

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metastases often result in complications such as severe pain, hypercalcemia, pathologic fractures and spinal cord or nerve root compression. Since there is no curative treatment for metastases currently, only palliative treatments such as bisphosphonates, chemotherapy, local radiation, surgery and analgesics are given.

Adoptive Immunotherapy (AIT) may represent an innovative treatment

strategy for bone metastases but, thus far, has not been assessed in this setting.

The adoptive transfer of in vitro activated and expanded T-lymphocytes has, however, demonstrated significant anti-tumour activity in various animal models using different murine cancer cell lines. Recently, Dudley et al. though not studying bone metastases, also reported promising findings in the clinical setting using adoptive transfer of selected tumour-reactive T-cells in patients with metastatic melanoma following a nonmyeloablative chemotherapy regimen. In the authors’ laboratory, a pre-clinical model of bone metastases was evaluated for the study of AIT. This model was modified from Arguello et al. and leads to a high incidence of bone metastases following injection of tumour cells into left cardiac ventricle.

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Several properties of the bone contribute to its uniqueness as a metastatic target. Bones constitute a wellvascularised and physically defined microcompartment. Due to the lack of a basement membrane and the presence of fenestrae between the endothelial cells, bone metastases form most frequently within the red bone marrow. In addition, spreading cancer cells might also profit from a rich supply of bone-derived and hematopoietic growth factors. At the same time, the bone marrow is also an important part of systemic immune responses including antigen presentation and various reports describe it as a rich source of protective immune cells in preclinical models investigating activespecific immunotherapy.

In summary, bone and bone marrow with their unique microenvironment support the establishment of tumour micrometastases, but, paradoxically, are also a yielding source of protective immune cells. In patients, the detection of bone marrow micrometastases has been described as an independent predictor for subsequent clinical relapse in distant organs. Several other clinical observations suggest that the bone marrow may be an important site of persistence of dormant tumour cells (or even so-called cancer stem cells) in patients treated with curative intent.

Immunologic control of these dormant tumour cells may fail at some point and new metastases may arise. Potential explanations for this phenomenon include impaired immunosurveillance or the development of tolerance.

The data set available on trafficking and function of adoptively transferred T-cells in the bone microenvironment is currently very limited. Several authors have, however, reported on adoptively transferred Tumour Infiltrating Lymphocytes (TIL) and their potential to migrate to metastases throughout the body including the skeleton.

Experimental model of bone metastases

In order to closely mimic the clinical setting, an experimental model of bone metastases was used where tumour cells (breast cancer, lung carcinoma and melanoma) are inoculated into the left cardiac ventricle of immunocompetent laboratory animals under general anaesthesia (Systemic Intra-arterial Administration, SIA). Skeletal metastases develop over time (Figures 1). Tumour colonies of pigmented melanoma cells to the bone can readily be seen on various parts of the skeleton but particularly on well-vascularised portions of long bones, such as the femur (Figure 2b). In a different set of experiments,

the authors used melanoma cells that had been transduced with a retrovirus containing the cDNA encoding Green Fluorescent Protein (GFP). This allows for assessment of tumour burden in various regions of the body either by Fluorescence Activated Cell Sorting (FACS) or even in vivo by direct visualisation through fluorescence microscopy (Figure 3).

Generation of effector T-lymphocytes and adoptive immunotherapy

To generate specific effector T-lymphocytes (TE), melanoma cells, that secrete granulocyte-macrophagecolony-stimulating factor (GM-CSF), are injected subcutaneously into the flanks of B6 mice. Eight days following vaccination, the draining superficial axillary and inguinal lymph nodes are harvested. These Tumour Vaccine-Draining Lymph Node cells (TVDLN) are resuspended and activated with an anti-CD3 antibody. After two days of activation the T-cells are harvested and expanded in medium containing IL-2 to generate TE. TE are then harvested, washed and injected intravenously or via SIA into mice with established bone metastases from SIA of D5 melanoma cells.

Femur bones (long bones) before and after the skeletal metastase development a

Figure 1

b

a

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Figure 2

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Figure 3a

Figure 3b

Figure 4a

Figure 4b

Regression of bone metastases requires high numbers of adoptively transferred effector T-cells

The adoptive transfer (intravenous infusion) of in vitro activated and expanded T-cells mediated tumour regression in the pre-clinical model of melanoma skeletal metastases used in this study (Figure 2) in a dose-dependent manner. Whereas 10 × 106 and 50 × 106 effector T-cells were not sufficient to induce a significant anti-tumour effect in the long bones of mice bearing bone metastases, the adoptive transfer of 100 × 106 TE cells resulted in macroscopic and microscopic tumour-free distal femurs and proximal tibiae of examined mice (Figure 2a/3/4b).

It required a large number of TE cells to induce an anti-tumour effect. It takes half as many TE cells to obtain significant tumour regression in a standard model of pulmonary metastases from the same melanoma cell line as used in the present study.

Furthermore, to observe reproducible metastatic colonisation of the skeleton,

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injection of 20 times fewer tumour cells into the left heart ventricle is sufficient, compared to intravenous application of 2 × 105 cells, that is required in the pulmonary metastases model. Low-dose interleukin-2, a cytokine co-administered to support persistence of transferred T-cells, did not influence T-cell mediated regression of bony metastases in the authors’ model.

In vivo depletion of CD8+ T-cells abrogates the therapeutic efficacy of AIT for bone metastases

CD8+ T-cells are generally considered to be the main effector population within a specific cellular immune response. In a second set of experiments, CD4+ and CD8+ subsets of T-lymphocytes, were depleted in vivo. Anti-CD4 and antiCD8 mAB were administered intraperitoneally to deplete the respective T-cell population. Because the depleting antibodies were given after the adoptive transfer, CD4+ and CD8+ subsets of both the transferred and endogenous population of cells were depleted by at

least 97 per cent as measured by flow cytometry. Bone metastases were detectable in only one animal in the IgG antibody control group and one animal in the group receiving the antibody for the depletion of CD4+ cells. In contrast, all animals receiving in vivo depletion of CD8+ cells exhibited pigmented tumour colonies on long bones. The histological examination of long bones, which was performed 17 days following SIA of tumour cells showed the bone marrow cavity to be almost entirely occupied by tumour cells (Figure 4b). Without CD8+ depletion, tumour cells were undetectable and the bone marrow appeared normal (Figure 4a), clearly demonstrating the key role of CD8+ cells in the immunemediated regression of bone metastases in this setting.

Efficiency depends on the route of administration

The authors hypothesised that intravenous injection might limit trafficking of TE to bone or bone marrow due to early entrapment in the lungs. To circumvent this problem, TE cells were also administered directly into the arterial systemic circulation via the left cardiac ventricle bypassing the pulmonary microvasculature.

50 × 106 TE cells failed to mediate regression of bone metastases when injected intravenously. In contrast, transfer of the same number of TE cells into the arterial circulation through the left cardiac ventricle completely cleared bones of visible tumour colonies. But using even lower number of TE cells (25 × 106) did not lead to a significant reduction in animals with bone metastases.

TE traffic to bone or bone marrow following IV injection and division of transferred cells is detectable

Since AIT had proven to be an effective treatment protocol for experimental metastases to the skeleton in this model, it was hypothesised that this effect was mediated by the transferred TE and that these cells were able to migrate to the bone marrow of animals

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bearing 3-day bone metastases. To address this question, TE cells were generated from B6. PL-Thy1a/Cy mice, which carry the T-lymphocyte-specific Thy1a (Thy1.1) (CD90.1) allele. Thus, donor T-cells could be easily distinguished from recipient T-cells using flow cytometric analysis.

To assess cell division of transferred cells, TE cells were labelled with CFSE before adoptive transfer. Not surprisingly, in tumour-bearing animals, adoptively transferred cells were present in the spleen after 24 hours following IV injection.

Sixteen per cent of all CD8+ cells originated from Thy1.1 donor mice at this point of time. This number declined to 8 per cent by day 3 and 3 per cent by day 6 following AIT. A sequential reduction of CFSE fluorescence was detected in the spleen at all time points indicating that transferred cells had also divided.

Thy1.1+ / CD8+ donor cells were also present in the bone marrow at each of the three times of measurement with 18 per cent of CD8+ cells in the bone marrow deriving from the transferred population after 24 hours. This number was reduced to and remained at 5 per cent on day 3 and 6. At two of the three time points, CFSE fluorescence indicated that Thy1.1+ had undergone cell division in the bone marrow. Since divided donor cells were also detectable in peripheral blood of animals on days 3 and 6, it remained uncertain if cell division had occurred within the bone or bone marrow or if donor cells had migrated to this location after dividing elsewhere.

demonstrated for the first time. Further investigation seems obvious considering the fact that the skeleton is among the most common sites of metastatic disease and that skeletal metastases represent a devastating complication for patients.

The sheer presence of single tumour cells in the bone marrow has been identified as a (poor) prognostic indicator for the development of distant metastases following curative treatment of the primary tumour. In general, the rationale for adjuvant therapy is based on the assumption that clinically undetectable, viable tumour cells are present at distant sites, e.g. the bone marrow, at the time of curative treatment. One key feature of micrometastatic tumour cells in the bone marrow is, however, their relative resistance to chemotherapeutic agents. In breast cancer patients this was attributed to overexpression of the erbB-2 proto-oncogene possibly inducing tumour cell resistance. In addition, most of the tumour cells isolated from the bone marrow failed to express typical proliferation markers such as Ki-67 and p120 indicating that these cells might be resting in the G0 phase of the cell cycle and therefore be less susceptible to cytotoxic agents.

The immunotherapeutic approach used in this study might represent a cell-cycle independent treatment modality which can recognise and eradicate tumour cells in the bone or bone marrow independent of their state of proliferation.

However, as one can imagine, there are major obstacles to translating these strategies into a clinical application.

In conclusion, adoptively transferred TE cells showed a remarkable ability to migrate to bone or bone marrow and mediate tumour regression in the authors’ pre-clinical model of skeletal metastatic disease. Therefore, further studies of adoptive T-cell immunotherapy for macrometastatic and micrometastatic disease to the skeleton seem warranted with the long-term objective to implement this cell-cycle independent treatment regimen as a valuable addition to established treatment modalities.

Acknowledgements: This work was supported by the Chiles Foundation, Portland, Oregon, USA, NIH grants CA80964, CA92254, CA107243, CA119123 and the Walter-Schulz-Foundation, Munich, Germany.

Full references are available on www.pharmafocusasia.com/magazine/

Dominik RĂźttinger is a board-certified surgical oncologist. He spent his postdoctoral fellowship in tumour immunology at the Earle A Chiles Research Institute in Portland, USA. He is now an associate professor at the University of Munich (Germany) and serves as Medical Director/Clinical Development for Micromet Inc. He is an advisor to GTAC UK, the Gene Therapy Advisory Committee.

Thus far, the adoptive transfer of TE to animals with systemic metastases has demonstrated therapeutic efficacy in disease models as different as melanoma, colorectal and brain cancer. However, to date, no attempt has been made to define premises for the investigation of adoptive T-cell transfer as immunotherapeutic approach to bone metastases. In this study, the therapeutic value of adoptive immunotherapy in a model of metastatic disease to the skeleton has been

A uthors

Summary and perspective Bernard A Fox is an NIH-funded Researcher in pre-clinical and translational cancer immunotherapy, on four editorial boards, consults for Government and Biotechnology sector, Presidentelect of International Society for Biological Therapy of Cancer (www.iSBTc.org), co-founder of UBIVAC and on Board of NeoPharm Inc. Hong-Ming Hu is an NIH-funded immunologist focussed on understanding immune recognition of cancer and development of new cancer vaccines and immunotherapies. He is Chief of Cancer Immunobiology, on the faculty at Oregon Health and Science University and co-founder of UBIVAC, a cancer vaccine biotechnology company.

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Target Deconvolution in the Post-genomic Era In times of reduced productivity coupled with increasing costs within the pharmaceutical industry, “reductionistic” target-based drug discovery has come under pressure and phenotype-based discovery has gained momentum. Target deconvolution is an important aspect for efficient progression of compounds towards development.

Georg C Terstappen, Chief Scientific Officer, Siena Biotech S.p.A., Italy

W

hile the growing demand for innovative medicines is creating increasing opportunities for the pharmaceutical industry, it is exactly this industry that suffers from an expensive disease itself, known by the name “innovation deficit”. In fact, while the FDA approved 53 New Molecular Entities (NMEs) and biologics in 1996, a nearly steady decline since then has led to a disappointing 22 NME approvals in 2006, which is even more disappointing owing to the fact that twice as much money was spent. The cause of this malaise is of multiple nature and many different legal, political and financial factors have increased the complexity of current pharmaceutical R&D. Most compounds—especially those with a new mechanism of action—fail due to a lack of efficacy observed during clinical development. This is mainly a

consequence of insufficient understanding of disease pathophysiology in conjunction with a “reductionistic” target-based approach for drug discovery. In the 1990s, this approach largely replaced the more holistic approach of screening compounds for phenotypic changes in complex systems such as living animals and isolated organs (Figure 1).

The target-based approach was thought to be more rational and efficient, largely corroborated by the sequencing of the human genome which was in progress at that time and promised the advent of a new era with all potential drug targets known.

Renaissance of phenotype-based drug discovery

The perceived “failure” of target-based drug discovery in current post-genomic times has led to a renaissance of the more holistic approach which allows identifica-

tion of compounds with multiple cellular targets (“polypharmacologic compounds”) since phenotypic effects are measured as an integral of the response of an entire system, thus better matching the polygenic and / or multifactorial nature of most diseases. Although many model systems such as worm, fly, zebrafish and mouse can be employed for smallscale drug screening, at present mainly mammalian cells in culture are compatible with High Throughput Screening (HTS) with a phenotypic read-out.

Permanent cell lines are easy to handle, can be grown in large quantities and many are commercially available as well, and therefore are used predominantly.

Furthermore, through recombinant gene technology aspects of disease biology can be engineered into these cells.

In the research laboratories of Siena Biotech S.p.A, dedicated to the study of Huntington’s disease—a progressive neurodegenerative disorder—a stable human HEK293 cell line was developed which expresses the mutated huntingtin gene under the control of an inducible promoter. The recombinant cell system

Phenotype-based drug discovery Assay development

Screening

Target-based drug discovery Hits & Leads

Target deconvolution

TARGET

Target validation

Assay development

Screening

Hits & Leads

Figure 1

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Metho ds DNA m based on icr and pr oarrays oteom ics

Target deconvolution strategies

mR

NA

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isplay

Whatever the disease model system that might have been used, the retrospective identification of the molecular targets underlying the observed phenotypic responses, termed target deconvolution, is important. From a basic research perspective, knowledge of the molecular targets will help to understand the (patho) mechanism(s) of the disease. But this knowledge will also aid rational drug design and allow efficient Structure-Activity Relationship (SAR) studies in a chemical optimisation programme, thereby developing target-specific assays. In addition, the aspects related to targetspecific toxicity and side effects can be addressed, reducing later-stage “attrition” early on.

A wide range of different technologies can be employed for target deconvolution (Figure 2). However, no “best practice” or “standard operating procedure” is available and the method of choice will have to be decided on a case-bycase basis often mainly governed by the properties of the “drug” molecule.

Methods leading to direct identification of targets typically exploit the affinity between the “drug” molecule and its target protein assuming that physical interaction is a prerequisite for functional effects.

c

d Phase

Target deconvolution

Thr ee sys hybrid tem s

y nity Affi graph ato m hro

ted fec ns ys tra arra rse icro ve Re cell m

also contains a luciferase reporter gene under control of a cAMP-Responsive Element (CRE) which allows measurement of transcriptional activation. If the mutated huntingtin, which causes the disease in humans, is switched on, cells start to degenerate leading to transcriptional dysregulation and cell death. While the first is measured with the reporter gene, the latter phenotypic effect is simply determined by using a toxicity assay. A HTS with this recombinant cellular disease system has led to the identification of drug-like organic molecules that rescue the phenotypic effects caused by expression of the mutated huntingtin.

Protein microarrays Figure 2

Direct target deconvolution methods

“Classical” affinity chromatographybased methods have been successful for target deconvolution. An important advantage in these methods is that target proteins maintain their 3-D structure including post-translational modifications. Protein microarray-based strategies represent an alternative to affinity chromatography and identification of proteins which are expressed at low levels in vivo is not a priori compromised since large numbers of individually purified proteins are arrayed in equal amounts.

Clearly, only those proteins can be identified that have been immobilised on the microarray. A method referred to as “reverse-transfected” microarrays employs microarrays of cells expressing defined cDNAs thus circumventing the need to use large numbers of individually purified proteins.

Technologies based on expression cloning (e.g. three hybrid systems, phage display and mRNA display) also include an affinity step. Since proteins are expressed from fusion constructs of

cDNA libraries, the properties of these recombinant proteins might be different from their native counterparts, especially in terms of post-translational modifications. In addition, sophisticated molecular biology skills are needed, which is not the case for affinity chromatography.

Common to all these direct methods is that the “drug” molecule has to be modified chemically for labelling and / or immobilisation purposes. This means that access to synthetic chemistry might be necessary and often a limited structure-activity relationship study will also have to be carried out in order to identify a position where the molecule can be modified under retention of its biological activity, which has to be tested.

Methods supporting target deconvolution

Target deconvolution can be supported by technologies based on comprehensive DNA microarray and proteomics analyses. In a more indirect way, application of these technologies, which do not need modification of the chemical molecules, can eventually also lead to the

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identification of the molecular targets.

For instance, investigation of global gene expression changes of an organism induced by the “drug” molecule and determined by DNA microarray analysis can help to understand the MoA of the molecule and in the form of “connectivity maps” can be used to connect chemical molecules, genes and disease in a systematic manner. Global changes of the protein constitution of an organism in response to exposure to a “drug” molecule, studied by proteomics technologies, allow the generation of a “proteomic compound signature”, again aiding to understand the MoA of the chemical molecule.

Confirmation of ‘deconvoluted’ targets

A uthor

The ultimate goal of all strategies and methods for target deconvolution is the identification of the biological molecules that directly interact with the “drug” molecule. In addition, it is also important to confirm that modulation of the identified biological target is associated with the functional effects detectable in the phenotypic assay system that was used for identification of the chemical molecule.

If 3-D protein structures are available, a structural analysis of the proteincompound interaction can be used to evaluate the interaction profile further.

Since these in silico analyses generate predictions, direct proof of “drug”protein interaction and its functional consequences is necessary. A variety of methods can be used to confirm physical interaction. For instance, surface plasmon resonance, and associated phenotypic changes can be confirmed by functional studies employing genetic methods

such as RNA interference, leading to a knock-down of the target gene or gene overexpression. In vivo, transgenic and knock-out animals can be generated for detailed studies of phenotype.

BOOK Shelf

Conclusion

Around the world, target-based drug discovery, carried out by major pharmaceutical companies and the biotechnology industry, has so far resulted in disappointing results in terms of delivering efficacious new medicines for therapy of diseases. This perceived “failure”, which is associated with tremendous costs, has recently led to the renaissance of phenotype-based drug discovery. For this approach, typically cell-based assay systems are used that mimic aspects of disease biology or host disease-relevant pathways. Employing these “unbiased” systems, small organic molecules are screened for relevant phenotypic effects which might be mediated by an active compound’s interaction with one or more cellular targets. Identification of these targets is important in order to progress active compounds efficiently through the drug discovery process. Although a broad panel of experimental target deconvolution strategies exist, none of these methods could universally be applied and hence a case-by-case evaluation is necessary. Apart from the choice of the method another important decision will be when to start deconvolution activities during the progression of active chemical molecules since timelines might be long and outcomes unpredictable. In this context, it might be comforting to know that for some of the approved drugs on the market, as of today the molecular target(s) are still not known.

Georg C Terstappen is the Chief Scientific Officer of Siena Biotech.

He is the coordinator of ADIT, a research project in Alzheimer’s disease and an adjunct Professor of Biotechnology at the University of Siena. He has 16 years of experience in drug discovery and has worked at Bayer AG, GlaxoWellcome and GlaxoSmithKline as Director of Systems Research. He received his PhD for work conducted at the Research Centre Juelich, and was a postdoctoral research scientist at the Max-Planck-Institute in Cologne.

Pharmacogenomics and Personalized Medicine Editor: Nadine Cohen Year of Publication: 2008 Pages: 528 Published by: Humana Press ISBN-10: 1934115045 ISBN-13: 978-1934115046 Description: With all of the multitude of challenges facing the pharmaceutical research and development process, the industry is actively exploring the relationships between human genetics and drug responsiveness, susceptibility to disease and disease severity. In this book, leading experts from the pharmaceutical industry, the scientific community and the government provide guidance for conducting pharmacogenomic research from discovery to the market, while also presenting a realistic perspective on the challenges, practicalities and obstacles in its application. Focussing on DNA data and associated analytical methodologies, with a consideration for complementary RNA-based studies, this volume includes a wide array of vital, cutting-edge research.

Comprehensive and timely, this book will assist novice and experienced investigators alike in the understanding of the current scientific challenges in applying pharmacogenomics to drug discovery and clinical development.

For more books, visit Knowledge Bank section of www.pharmafocusasia.com

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CoverStory

Nanoencapsulation Nanobiomechanics Nanofabrication

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anotechnology in pharmaceutical R&D, though in its infancy, is attracting lot of attention from the industry. Nanotechnology will have a bigger role to play in the coming days for the pharma industry, more so with the problems that the industry is facing, especially with the diminishing returns on investment in research and development.

The technology has been instrumental in understanding the human pathology at nano level and is helping researchers immensely in understanding the nature of the human cells and the associated diseases to design and produce better drugs.

Nanotechnology has made it possible to engineer drugs that are more stable, soluble and allow for targeted delivery thereby increasing the efficacy and reducing the side effects. The technology has proved to be a boon to the industry as it helps in developing efficient drug delivery systems and also in improving diagnostic processes and tools. With so many benefits to leverage, pharma companies have started adopting nanotechnology and nano tools in their most crucial function—R&D.

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Sonication-Assisted

Nanoencapsulation Nanoencapsulation of low soluble cancer drugs has been elaborated through powerful ultrasonication of the drug powder and simultaneous sequential polyelectrolyte deposition.

This is a novel approach that allows change in capsule wall thickness to adjust drug release rate, and to attach an antibody at the outer shell layer for targeted delivery.

Yuri Lvov, Chemistry Professor, Tolbert Pipes Eminent Endowed Chair on Micro and Nanosystems, Institute for Micromanufacturing, Department of Biomedical Engineering, Louisiana Tech University, USA Vladimir Torchilin, Distinguished Professor of Pharmaceutical Sciences Chair, Department of Pharmaceutical Sciences and Director, Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, USA Anshul Agarwal, Doctoral Candidate in Biomedical Engineering, Department of Biomedical Engineering, Louisiana Tech University, USA Rishikesh Sawant, Doctoral Candidate in Pharmaceutics and Drug Delivery Systems, Department of Pharmaceutical Sciences, Northeastern University, USA

T

he desired features of pharmaceutical drug delivery for intravenous administration (their small size, biodegradability, high content of a drug in a final preparation, prolonged circulation in the blood, and the ability to target required areas) are reasonably well met by liposomes, microcapsules and nanoparticles for water-soluble drugs. The development of nanoparticulate drugs displaying all of these properties for poorly soluble pharmaceuticals still represents a challenge. Low solubility in water, however, tends to be an intrinsic property of many drugs, including some powerful anti-cancer agents. Intravenous administration of relatively large aggregates of an insoluble drug may result in embolisation of these particles into small blood capillaries and may cause unwanted

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effects like tissue ischemia. Hence, it does not allow for achieving therapeutically significant concentrations.

Many promising drug candidates never enter further development processes because of solubility problems. On multiple occasions, micelles can serve as drug delivery systems for poorly soluble pharmaceuticals. However, there are problems, which include low loading efficacy of the drug into the micelles; problems with controlling the release rate of the drug, and with micelle stability.

Optimal drug delivery and reduction of systemic adverse effects have been age-old problems in chemotherapeutics of human cancer. With the advent of the use of water insoluble cancer drugs like paclitaxel and tamoxifen, it has been realised that better formulations are still needed for more specific and

controlled delivery of these agents. In a novel approach to form stable nanocolloids of these drugs with high content of the active drug and controllable release rate, using a sonicated Layer-by-Layer (LbL) polyelectrolyte coating technology is suggested. This is development of traditional LbL microencapsulation based on alternate adsorption of oppositely charged components (linear polyelectrolytes, proteins, and nanoparticles). To achieve nanosize cores, aqueous suspensions of poorly soluble drugs are subjected to the powerful ultrasonic treatment. To keep the nanoparticles formed under the sonication from fast re-aggregation, they are stabilised in a solution by sequential addition of polycations and polyanions and by assembling ultra-thin polyelectrolyte shells on them (Figure 1). LbL assembly allows preparation of multilayer shells with thickness of 5 to 50 nm and necessary composition. In this process, a nano-architectural approach for designing shells of different components, including ones serving as diffusion barrier and outermost layers containing targeting agents, was realised.

The procedure using synthetic polyelectrolyte was first established and then was transferred to biocompatible polymers such as cationic (polylysine, protamine sulphate) and anionic (dextran sulfate, and bovine serum albumin). After depositing the first polycation layer on

high-concentration drug colloids were kept in a low volume of saturated solution to prevent drug release. To study the release rate of drugs from the colloidal particles, the samples were placed in horizontal diffusion chambers made of cellulose acetate membrane and stirred in large volume of PBS, pH 7.2, to mimic sink conditions expected in vivo.

Scheme of LbL nanocolloidal particles formation from insoluble drugs I. Formation of colloidal nanoparticles Ultrasonicator Probe

Coolent Insoluble drug

Attachment of ligand moieties to the LbL nanocolloids of poorly soluble drugs

II. Alternate coating of nanoparticles with biopolymer layers

Subsequent drug release from the final LbL nanoparticle

Formation of multibilayered nanoshell

Formation of the second layer by the oppositely charged of polyelectrolyte (assembly of the first layer)

Drug nanoparticles stabilised by first layer of biocompatible polyelectrolyte once sonication is removed Figure 1

the surface of a drug nanoparticle, an oppositely charged polyanion is added.

This results in the formation of a stable inter-polyelectrolyte complex shell around each drug nanoparticle. By varying the charge density of each polymer or the number of coating cycles, particles with a different surface charge and different composition can be prepared. The use of a polymer containing reactive groups (such as amino groups) for the last “outer” surface layer allows for the attachment of specific ligands and other moieties of interest to drug nanoparticles. This article presents a proprietary method (Nemucore Medical Innovations Inc., Boston) of sonicated LbL assembly for the preparation of stable nanocolloids of paclitaxel and tamoxifen with very high drug content.

Materials and methods

Poorly soluble and potent anti-cancer drugs paclitaxel and tamoxifen have been used. Polyelectrolyte that was used included positively charged polylysine, poly(dimethyldiallyl ammonium

chloride) (PDDA), protamine sulphate (PS); and negatively charged sodium poly(styrene sulphonate) (PSS) and bovine serum albumin (BSA). Ultra Sonicator 3000 (Misonix Inc) was used for drug crystals disintegration at the power of 18W. To prevent the sample from overheating during the sonication and to keep the temperature in the range of 20-25°C, liquid nitrogen was used to cool the sample tubes.

For disintegration, all drug samples were ultrasonicated at reduced temperatures for 10 minutes before polyelectrolyte was added. Once this was added, the samples were sonicated for another 20 minutes. Polycations were used to form the first surface layer, since drug nanoparticles were found to bear the intrinsic negative charge. Drug samples were then centrifuged, washed and re-suspended in PBS buffer to get rid of the excessive polycation before further zeta potential readings were taken. Then, the coating process was repeated with polyanion, followed by polycations, and so on. The resultant

To prepare nanocolloids with the “reactive” surface suitable for the covalent attachment of various ligands to their surface, polycation containing free amino groups was used to form the outer layer on drug nanoparticles. To conjugate the monoclonal nucleosome-specific 2C5 antibody (mAb 2C5) recognising a broad variety of cancer cells, the reaction was carried out in two steps. In the first step, the carboxylate groups on the mAb 2C5 were activated using 1-ethyl-3carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) to make it amine-reactive. In the second step, the activated antibody was added to LbL paclitaxel nanoparticles coated with polyamino-containing polymer.

To verify the preservation of mAb 2C5 specific activity after the conjugation with LbL-paclitaxel nanoparticles, a standard ELISA was performed. The cytotoxicity of various concentrations of LbL-paclitaxel nanoparticles against and MCF-7 and BT-20 cells was studied using a MTT test.

Results and discussion

After 30 minutes of LbL-assisted sonication, paclitaxel particle sizes of about 50 × 50 × 120 nm were obtained.

Further increase in the sonication time did not result in significant decrease in drug particle size (Figure 2). The charged bare drug nanoparticles, which were negative initially became positive and formed stable colloids after the sonication with the addition of cationic polylysine or protamine sulphate.

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Ultra sonication of the drug in polyelectrolyte solution

R esearch & D evel o pment

SEM images (Hitachi-2006) of LbL encapsulated drug nanoparticles (Paclitaxel) a

b

20 min sonication + LbL coating

c

Smaller concentration sample with 30 min sonication + LbL coating with protamine sulfate / albumin, size 50 × 50 × 120 nm

120 nm diameter tamoxifen particles prepared via sonication in the presence of polylysine Figure 2

Further, assembly with the corresponding changes in zeta potential values allowed formation of the two bilayer shell with biocompatible protamine sulfate and albumin: (PS/BSA) 2. Figure 3 gives values of the surface zeta potential during the process of sequential four-step protamine sulfate / albumin adsorption on paclitaxel nanocores. Thickness of

such shell estimated with Quartz Crystal Microbalance was ca. 6 nm in dry state and 12 nm in water. Similar Zeta potential alternation was observed during shell assembly on tamoxifen nanoparticles.

Nanoparticle imaging and drug release

The particle size of all the drug samples

Changes on paclitaxel particle zeta-potential Changes on paclitaxel particle zeta-potential in the process of the LbL coating with two bilayers of protamine sulphate (PS) and bovine serum albumin (BSA) (ZetaPlus, Brookhaven Instruments).

50

Paclitaxel-(PS-BSA-PS)

40

Paclitaxel-(PS)

Zeta Potential (mV)

30 20 10 0 -10

1

2

3

4

5

6

-20 -30

Paclitaxel (1 mg)

-40

Paclitaxel-(PS-BSA) Paclitaxel-(PS-BSA)2

-50 Adsorption cycle

Figure 3

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formulated by the LbL technology into nanocolloidal state was confirmed by scanning electron microscopy and confocal fluorescent microscopy (Figure 2). At 20 min sonication, submicron particles were obtained, and further sonication with polycations and liquid nitrogen cooling resulted in nanosized particles.

LbL-coated paclitaxel has the elongated rod-like shape of approximately 50 × 50 × 120 nm. Particles of tamoxifen demonstrate mainly a spherical shape and have a diameter of 120 ± 30 nm.

Tamoxifen’s spherical shape probably is due to melting and then solidifying of paclitaxel nanoparticles during powerful sonication (instantaneous local temperature during collapsing cavitations’ microbubbles reaches hundreds of degrees).

This was confirmed by the amorphous form of the resultant tamoxifen nanoparticles. The SEM images were obtained after drying the samples, and during this process the nanoparticles become partially aggregated.

This aggregation does not proceed in aqueous suspension, as it was demonstrated with fluorescence confocal images of the samples.

Taking into account that the thickness of a single polymeric layer is approximately 3 nm in dry state, we can calculate

Release of tamoxifen nanocapsules at sink condition Top to bottom: bare uncoated nanoparticles, nanoparticles coated with (PDDA/PSS)3, nanoparticles coated with PDdA and bare tamoxifen without any sonication.

Tamoxifen (PDDA-PSS)3 Bilayers

Tamoxifen (30 min)

No sonication

Tamoxifen (30 min sonication) Figure 4

curves for 2 mg/mL tamoxifen in PBS buffer at pH 7.2 through a 0.2 micrometer pore nitrocellulose membrane. Two upper curves represent bare uncoated micronised drug, and two lower curves show release from 125-nm diameter nanoparticles coated with one bilayer and PDDA monolayer and thicker shell of three bilayers of (PDD/PSS)3. Similar results were obtained for paclitaxel also.

These are initial release experiments done with synthetic polymers. Experiments with biocompatible shells of (protomine sulfate / albumin)3 gave similar release rate.

Surface modification of LbL-coated drug nanoparticles

To confirm that LbL-coated drug nanoparticles can be easily derivatised on the surface in order to impart them various additional properties including targeted

delivery, tumour-specific mAb 2C5 have been attached to the paclitaxel nanoparticles via free amino groups belonging to the surface layer of PAH. ELISA with the nucleosome monolayer (specific antigen for mAb 2C5), the results of which clearly confirms that 2C5-modified LbL-coated paclitaxel nanoparticles acquire the ability to specifically recognise the target antigen, i.e. become targeted.

Increased cytotoxicity of tumour cell-targeted LbL drug nanocolloids in vitro

In vitro experiments with MCF-7 and BT20 cancer cell lines clearly confirmed that targeted drug nanoparticles demonstrate higher cytotoxicity than the non-targeted counterparts. At paclitaxel concentration of 100 ng/mL in case of MCF-7 cells and 30 ng/mL in case of BT-20 cells, when virtually no cytotoxic effect can be

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that the drug content in stable nanocolloidal drug particles is from 90 per cent wt (in case of paclitaxel particles with a double layer coating) to 85 per cent wt (in case of tamoxifen particles with a triple bilayer coating) which is dramatically higher compared to other solubilisation methods. Colloidal suspensions of both drugs were stable during a month of observation.

LbL technology allows control of drug release rate from polymer-stabilised colloidal nanoparticles by simple changes in coating thickness or composition. As one could expect, a slower release rate is observed as the number of polyelectrolyte layers in the shell increases. At sink conditions, non-coated tamoxifen particles (after sonication) dissolve within approximately two hours, while LbL coating allows to extend this time to 10 hours. Figure 4 gives the release

R esearch & D evel o pment

observed with non-targeted LbL-coated paclitaxel nanoparticles (around 95 per cent of cancer cells remain alive after the incubation for 48 or 72 hours), 2C5-targeted LbL-coated paclitaxel particles kill at least 30 per cent of cancer cells. This result confirms that LbL technology allows for decorating the surface of stable colloidal drug particles with very high drug content with various additional functions as needed.

Results

In conclusion, the following formulation technique was demonstrated: 1. Stable nanocolloids of insoluble drugs with very high drug content can be

prepared through the application of the sonicated LbL technology, i.e. combination of ultrasonication and alternate adsorption of oppositely charged polyelectrolytes, resulting in coated nanoparticles with the content of the drug far exceeding other known systems; 2. Drug release rate from such nanoparticles can be controlled by assembling organised multilayer shells with required wall composition, density and thickness; 3. Various additional functions, such as specific targeted ligands, can be attached to the surface on nanocolloidal particles of poorly soluble drugs by using a polymer with free

reactive groups for the “outer” coating and preserve their specific properties upon the attachment; 4. Since drugs are not modified in any way in the process of solubilisation and release as free drug molecules, there is no concern regarding any possible change in drug activity in vivo, however, to deliver a desired dose of a poorly soluble drug in the body, a very small quantity of polymeric carrier is required compared to any other protocol currently used for administration of poorly soluble pharmaceuticals.

Full references are available on www.pharmafocusasia.com/magazine/

A uthors

Yuri M Lvov is a Professor of Chemistry, and T Pipes Endowed Chair on Micro and Nanosystems at the Institute for Micromanufacturing, Louisiana Tech University. He was among pioneers of the polyelectrolyte layer-by-layer assembly. In 2007, Lvov got Best of Small Tech National Innovator Award in recognition of his achievements in nanotechnology.

Vladimir P Torchilin is a Distinguished Professor and Chair of the Department of Pharmaceutical Sciences and Director, Center for Pharmaceutical Biotechnology and Nanomedicine. His research interests include biomedical polymers, polymeric drugs, among others. He received the 2005 Research Achievements in Pharmaceutics and Drug Delivery Award from the AAPS. A founding member of American Academy for Nanomedicine, he is on the Board of Directors of the International Liposome Society.

Anshul Agarwal is a medical graduate from University of Bombay, India. He did MS in biomedical engineering from LA Tech University, with research on recombinant human haemoglobin. He has worked in Fiest Wieller Cancer Center in Shreveport, on FDA-approved head and neck cancer clinical trials. He is carrying out research on nano formulations/delivery of cancer drugs with Dr Lvov for PhD in Biomedical Engineering from LA Tech University, USA.

Rishikesh M Sawant is a PhD student in Pharmaceutics and Drug Delivery Systems at Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University. His thesis dissertation title is “Polyethylene glycol as the key component of long circulating delivery systems for therapy and imaging”. He received “The John L Neumeyer Research Fellowship” award in 2007. One of his articles submitted to Bioconjugate Chemistry was cited as one of the most accessed articles and “hot” paper in January 2008.

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Nanobiomechanics and Human Diseases Insights into the pathophysiology

Nanobiomechanics research carried out on human diseases provides new and important insights into their pathophysiology and may suggest new methods for early detection and diagnosis.

H

uman diseases not only impair bodily structures and functions but they also threaten the health and well-being of humans.

Current research on diseases focusses mainly on the microbiological, immunological and pathological aspects rather than on the biomechanics of the diseases, which may have direct link to their pathophysiological outcomes. In fact, some studies have suggested that the cause and origin of some of these diseases may have resulted from the changes in the structures and mechanical properties of cells and biomolecules.

Examples of such diseases include malaria, Alzheimer’s disease, sickle cell anaemia and cancer.

Connection to human diseases

Nanobiomechanics, an area which involves the study of the mechanics of living cells and biomolecules, is now increasingly being used to study human diseases.

One reason for this is the availability of nanotechnological tools that can now mechanically probe into cells and biomolecules in their physiological states and at forces and displacements of piconewtons and nanometres, respectively. Such tools

and equipment, include the Atomic Force Microscopy (AFM), Molecular Force Spectroscopy, Nanoindenter, Magnetic Twisting Cytometry, Microfluidics, Magnetic Tweezers, Optical Tweezers and Optical Stretcher. Using biomechanics to study human diseases can lead to better understanding of the pathophysiology and pathogenesis of some of the human diseases as changes occurring at the molecular and cellular levels may affect or correlate to changes occurring at the macroscopic level. Here, some of the nanobiomechanics research to study two human diseases (malaria and cancer) that manifest structural and mechanical property changes are discussed.

Mechanistic insights into the pathophysiology of human diseases Malaria

The healthy Red Blood Cells (RBCs) deliver oxygen by deforming their way through small capillaries to various parts of the body. When the RBCs are infected with malaria, they experience two important pathophysiological outcomes: increased rigidity and cytoadherence (or cell stickiness). They not only cause serious disruption in blood flow, but can also cause severe anaemia, coma or even death.

The study of how an infected RBC can undergo extensive molecular and structural changes will be important and help us to understand malarial pathophysiology.

In the past, biomechanical tools used to study malaria included the viscometer, rheoscope and the laminar shear flow system and these investigations involved the study of a population of cells.

However, there is an increasing trend now towards studying the mechanical behaviour of a single infected cell using the micropipette aspiration, which applies a suction pressure to aspirate the membrane of the cell via a micropipette or the optical tweezers which use laser trap system to stretch or deform a cell. These tools can allow accurate probing of the infected cells at a single cell level. In fact, optical tweezers have now been used to determine the mechanical property differences between healthy and infected RBCs at the different stages of infection and results

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Chwee Teck Lim, Deputy Director, NUS Life Sciences Institute, Associate Professor, Division of Bioengineering & Department of Mechanical Engineering, National University of Singapore, Singapore

R esearch & D evel o pment

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ation on the effectiveness of drugs being developed to prevent or inhibit stiffening and cytoadherence of infected RBCs. Cancer

The uncontrolled division and growth of abnormal cells is fast in a cancer patient.

Cancer can spread quickly to various parts of a human body and destroy the healthy body tissues. A distinctive outcome of cancer is that the affected cells can become more deformable than healthy cells. This difference in cell deformability can thus be exploited to detect and identify cancer cells. As of now, there is no breakthrough on how changes in biomechanical properties of cancer cells can contribute to cancer metastasis.

To determine the elasticity of normal and cancerous cells, researchers have used AFM to indent a single cell using a sharp tip with tip radius ranging from a few nanometers to micrometres. For example, some research has shown that the human bladder epithelial cancer cells are significantly softer than that of the healthy ones.

Another nanotechnological tool, the optical stretcher has also been recently developed to study the deformation of single suspended cells. This technique uses two counter-propagating lasers to deform a single cell in a microfluidic channel and is employed to investigate cancerous mouse fibroblast cells and malignant human breast epithelial cells. Cancer cells are found to be more deformable compared to the normal cells and metastatic cancer cells are stretched more in comparison to non-metastatic ones.

Metastasis is the process where cancer cells proliferate from their primary location to other parts of the body. During

A uthor

have shown that infected RBCs become more rigid as the disease progresses. The quantitative measure of rigidity is done through determining the changes in the shear elastic modulus of the membrane of infected RBCs. Recently, microfluidics has also been used to observe the flow of malaria-infected RBCs by squeezing them through narrow channels with sizes typical of the narrow capillaries found in the human body. This study can demonstrate how the late-stage infected cells may cause blockage in these narrow microchannels, thus mimicking what happens when the more rigid infected cells try to squeeze through the narrow capillaries.

For the pathophysiological outcome of cytoadherence, relatively less research has been done to quantify the physical adhesion involved between the infected RBCs and the endothelial blood vessel wall.

Studies have shown that certain knob-like structures are manifested on the infected cell membranes. They act as focal adherent points in which the infected RBC sticks to the endothelial cells lining the blood vessel and capillary. These knob-like structures are actually parasite proteins secreted by the malarial parasite which get exported to the membrane. Almost all cytoadherence is mediated by the parasite protein with the various endothelial receptor proteins.

Here, single molecular force spectroscopy using the atomic force microscope can be employed to quantitatively probe the single parasite protein-endothelial protein interaction that results in cytoadherence.

For example, an AFM tip functionalised with the endothelial receptor proteins can be made to probe the adhesion of these proteins with the knob which contains the parasite protein found on the infected cell membrane.

These studies show the importance of using mechanics in helping us understand malaria. With this better understanding from a biomechanics perspective, one can hope to provide useful information to clinicians on how they can better reduce parasite virulence. Also, these tests can possibly be developed into testing strategies that can provide quantitative evalu-

this process, Circulating Tumour Cells (CTCs) are made to traverse through the blood vessels and squeeze through narrow capillaries. These CTCs in peripheral blood have clinical significance of both highlighting disease status and even predicting survival for cancer patients. In isolating CTCs from blood, the challenge lies in the rarity of these cells. Researchers are now developing microsystems that can isolate CTCs by utilising the distinctive difference in the biorheological properties of CTCs and the accompanying blood constituents.

Future directions

Currently, there is still little research done on using biomechanics approaches to study human diseases. This article demonstrates how biomechanics can play an important role in helping to better understand the pathophysiology of human diseases.

Although there are several state-of-theart tools to probe human diseases at the cellular and molecular levels, they are tedious and difficult to use. There is now a need to probe individual and populations of cells more rapidly, and with enhanced sensitivity and accuracy. It is hoped that knowledge gained from biomechanics research may eventually assist in developing new and improved high-throughput assays and diagnostic devices that can be rapid, sensitive and accurate enough to detect diseases at the earliest stage possible even when symptoms of the diseases are still not discernable. This is especially needed for diseases where early diagnosis and detection are extremely crucial for their prevention and control.

Full references are available on www.pharmafocusasia.com/magazine/

Chwee Teck Lim is Associate Professor at both the Departments of Bioengineering and Mechanical Engineering at the National University of Singapore.

He is also the Deputy Director of the University’s Life Sciences Institute. His research on cell and molecular mechanics of human diseases was cited

by MIT Technology Review as one of the ten emerging technologies of 2006.

Unconventional

Micro and Nanofabrication Biological tools and assays developed through unconventional nanofabrication techniques are proving to be inexpensive.

Michael D Dickey, Post Doctoral Fellow, Whitesides Group Department of Chemistry, Harvard University, USA

D

iscovering and developing a new drug is a costly and timeconsuming process. Recent reports suggest that it costs around US$ 800 million (pre-tax) to bring a new drug to market. One approach to improve the efficiency (and lower the cost) of this process is to use appropriate models and assays early in the discovery phase to identify candidates that are likely to be safe and effective in human beings. Organismic assays are better and more predictive for testing putative therapeutics than molecular assays, but it is difficult and expensive to work with animals, and the relevance of these assays’ results in humans is often low. Human cell-based assays—which are less complicated than many organismic assays—are an attractive option for drug screening because cells are human-like (i.e. they contain the human genome, enzymes, proteins, etc.). The ability to microengineer the environment surrounding a cell allows a better understanding of the response of a cell to its environment in a predictive manner. This understanding leads to more reproducible and meaningful assays.

Our research group has developed simple tools to study the behaviour of

cells under controlled conditions. These tools allow fundamental mechanistic studies of cells to be performed that can lead to new assays for drug delivery and efficacy. The ultimate goal of this work is to develop highly parallel, inexpensive assays (cell-based, organismic, and molecular assays). Discussed here are some of the biological tools and assays developed in our laboratory and the methods by which they are fabricated. These devices often contain small, functional components that are fabricated using “unconventional” methods. These methods provide some benefits (e.g. a reduction in cost) over conventional methods.

Why “small”?

Here, new methods are sought to fabricate small structures in a simple, inexpensive, and reproducible manner because: 1. It is advantageous to use systems that require small volumes of sample.

2. Tools used to probe and study cells are typically on a similar or smaller size scale than a cell (~10-50 µm). Small length scales allow control over those aspects of the environment that influence the behaviour of the cell. Small tools can also exhibit useful, non-intuitive behaviours. For example, fluid flow through

Conventional fabrication

Although there is no formal definition of “conventional” fabrication, it is generally accepted that it includes photolithography—a state-of-the-art process in the field of industrial-scale micro and nanofabrication. Photolithography is a patterning process in which a focussed pattern of light is transferred to a substrate covered with a photosensitive material. Photolithography is the cornerstone technology used to pattern the components of computer chips and is largely responsible for the growth of the semiconductor industry over the past 50 years.

Although photolithography is a powerful tool, it is not a universally relevant fabrication technique because of its inherent limitations: it is expensive, limited in resolution by the diffraction of light, restricted to planar substrates and incompatible with many organic and biological materials. “Unconventional” methods to fabricate micro and nanostructures are developed to overcome one or more of these limitations.

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George M Whitesides, Woodford L and Ann A Flowers University Professor

a channel with a small cross-section is laminar (i.e. non-turbulent). Laminar flow provides controlled delivery of drugs to cells in microchannels.

3. Assays developed on a small length scale can have high-throughput and are low-cost (on a per assay basis) because many assays can be processed in parallel. These attributes are well-suited for patient-specific therapy and population screening.

R esearch & D evel o pment

A schematic depiction of the formation of a PDMS stamp using a silicon (Si) master

Master

Si

PDMS

Pour prepolymer and cure

Si

PDMS

Microfluidics

PDMS

PDMS

metal layer

Si

alkanethiol

Fluidic channels

Si Figure 1

A demonstration of microcontact printing to pattern cells on a surface Cells adhere selectively to a protein (fibronectin) that preferentially adsorbs onto regions of “bioselective” molecules patterned by microcontact printing. The cells adopt the shape of the patterns on the substrate and their ability to proliferate is dependent on the area of contact.

Soluble growth factors Fibronectin

30 m

G1 arrest

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Fibronectin

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50 m

Proliferation

“Unconventional fabrication” is broadly defined as any fabrication method that provides some advantage over conventional patterning. This article discusses three unconventional fabrication techniques developed in our laboratory: soft lithography, paper patterning, and nanoskiving.

Soft lithography

Remove stamp

Microcontact Printing

Unconventional fabrication

Figure 2

Soft lithography refers to a set of methods for fabricating or replicating structures using elastomeric stamps or moulds. It is called “soft” because it uses elastomeric materials, such as polydimethylsiloxane (PDMS). Figure 1 shows the general procedure for soft lithography. Casting and curing a prepolymer (e.g. PDMS) against a master generates an inverse replica of the master. Peeling the PDMS from the master forms the “stamp” or “mould”. The master is a topographically patterned substrate and can either be purchased, or fabricated using photolithography. Multiple stamps can be formed from a single master, thereby reducing the reliance on conventional fabrication techniques. The soft nature of the stamp provides added processing capabilities (e.g. patterning on curved surfaces) that are not possible with conventional techniques. PDMS stamps are optically transparent and permeable to oxygen and carbon dioxide, properties that are useful for studying cells.

Two examples of techniques that utilise PDMS stamps are highlighted at the bottom of Figure 1. Microcontact printing uses the relief structures on a PDMS stamp to transfer patterns of inks (i.e. molecules, such as alkanethiols) onto the surface of a substrate through conformal contact. Microfluidics is a method to manipulate small volumes of fluid in microchannels formed by sealing a PDMS stamp against a substrate. Both of these techniques are useful tools to study the behaviour of cells in a controlled environment. The following examples highlight the capabilities of these techniques.

Partial treatment of a cell (bovine capillary endothelial cell) with two different fluorescent dyes using a microfluidic channel (c-e). Fluorescent microscope images show that the mitochondria on the left half of the cell are stained red and those on the right half are stained green through the use of laminar flow (the nucleus is stained blue). The dotted line denotes the interface between the two adjacent streams in the microchannel (f). After two and half hours, the subpopulations of dyed mitochondria are intermixed.

Inlets Outlet

Coverglass

C

D

E

F

PDMS A

Cell B Objective

50 m

Figure 3

Adapted from: Takayama, S.; Ostuni, E.; LeDuc, P.; Naruse, K.; Ingber, D. E.; Whitesides, G. M. Selective Chemical Treatment of Cellular Microdomains Using Multiple Laminar Streams. Chem. Biol. 2003, 10, 123-130, and Takayama, S.; Ostuni, E.; LeDuc, P.; Whitesides, G. M. Laminar Flows: Subcellular Positioning of Small Molecules.

Nature 2001, 411, 1018.

Microcontact printing

Microcontact printing can pattern a surface chemically with “bioselective” molecules amongst a background of “bioinert” molecules. Certain proteins (e.g. fibronectin) selectively bind to the bioselective regions and cells, in turn, bind to the proteins. Microcontact printing, therefore, is a method to pattern cells into arbitrary shapes, sizes and distributions on a surface. Figure 2 shows an example that demonstrates the utility of this technique.

Microcontact printing generates arrays of fibronectin-coated squares of different sizes and spacing on a surface. This study showed that cells ceased to proliferate when their total projected area of attachment was below a threshold value. The ability to pattern cells allows for a dense array of cells to be organised on a surface for assays and has implications in the study of cell signalling and cytoskeletal behaviour.

Recently another use of microcontact printing has been demonstrated by

patterning parallel lines of protein to seed the alignment of cardiac myocytes.

When placed on a thin film of PDMS, the synchronised and spontaneous contraction of the myocytes causes the PDMS to curl with a synchronised rhythm (the contractions could also be paced externally). This cell-PDMS composite system is biomimetic and thus has implications as a model system to study cardiovascular diseases and therapeutics.

A small voltage pulse applied to the surface provides a method to release cells patterned on a surface by microcontact printing. The pulse, which does not harm the cells, releases the molecules from the surface, thereby freeing the cells.

These methods provide the basis for cell motility assays.

Microfluidics

Microfluidics is an effective method to control the microenvironment surrounding a cell. Fluids injected into a microchannel placed directly over a cell can

deliver nutrients and stimulants to the cell in a controlled manner. Because the flow in a microchannel is laminar (i.e. non-turbulent), two adjacent streams in the channel will mix only by diffusion slowly. This behaviour can be harnessed to expose a cell to two distinct fluids at the same time, as shown in Figure 4. This technique is useful for studying cell dynamics, chemotaxis, cell polarity, spatially regulated signalling, drug screening, and other subcellular processes and possibly also to expose part of a cell to a drug and monitor the reaction of the sub-cellular components.

We have recently shown that microfluidic channels can be used to immobilise live worms (C. Elegans).

Worms are a model system for organismic studies because of their simplicity.

Immobilising worms for morphological analysis, fluorescence imaging, and laser microsurgery requires drugging the worm or gluing it to a surface. We have shown that live worms can be immobilised in a minimally invasive, reversible manner by flowing them into narrowing microfluidic channels. The worms become pinned in the channels, at which point they can be studied, and then reversibly released using mild pressure. This approach is well-suited for the testing of drugs and the incorporation of other forms of external stimuli such as heat, mechanical stress, electrical stimulation and oxygen depletion.

Microfluidic channels are also useful for forming droplets with controlled size and shape. Droplets form in channels when two immiscible fluids flow simultaneously through a constriction in a channel. The ability to form droplets with controlled composition and geometry is well-suited for drug formulation and delivery.

Patterned paper

We have begun efforts to develop exceedingly inexpensive diagnostic devices made out of paper. This work involves fabricating the microfluidic channels in paper to distribute a single sample of fluid

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Partial treatment of a bovine capillary endothelial cell

R esearch & D evel o pment

Paper-based assays a

Hydrophobic polymer patterns are fabricated on a piece of paper using lithographic techniques.

chromatography paper soak in photoresist

i. pre-bake ii. align under a mask mask i. expose to UV light ii. post-bake

(by capillary action) into separate regions of paper for running multiple bioassays simultaneously (Figure 4). These paper-based microfluidic devices can serve as delivery systems in assays to detect metabolites, small molecule pollutants, proteins, antibodies and possibly DNA, and should be useful for detecting disease in humans, livestock and plants.

Paper has been chosen as a substrate because it is inexpensive, lightweight, disposable and easy to use / carry. Paper based assays require no external equipment, reagents or power sources. These types of assays are believed to be useful in less-industrialised countries, but may also find use in domestic healthcare or emergency response settings.

The channels in the paper (Figure 4) are defined by soaking the paper in a photosensitive polymer. Regions of the polymer exposed to light become insoluble and the unexposed regions are dissolved away.

The paper serves as a wicking medium and the hydrophobic polymer defines the edges of the channels. As a proof of principle, the detection of glucose and proteins in artificial urine has been demonstrated using colorimetric assays (Figure 4).

Nanoskiving

Nanoskiving is a nanofabrication technique that utilises an ultramicrotome (a tool featuring a knife capable of sectioning slabs as thin as 30 nm) to form nanostructures by sectioning thin-films embedded in polymer. Figure 5 is a schematic depiction of the nanoskiving process. A thin metallic film is embedded in a polymer matrix and sectioned with a microtome. In principle, the technique is only limited to materials that can be sectioned and deposited as a thin film (there are numerous techniques to deposit thin films, such as spin-coating and physical vapour deposition).

Nanoskiving is appealing because it is: i) simple, ii) inexpensive (it does not require the use of expensive conventional fabrication equipment or clean-rooms), iii) versatile (it can fabricate structures of various composition and geometry, including arrays of nanostructures), and iv) high-fidelity (it reproduces the same features with each section).

The nanostructures formed by nanoskiving are embedded in a thin slab of polymer which can be macroscopically positioned on various substrates.

Although this technique is still being developed, it is envisioned that the nanostructures formed by nanoskiving will be useful as sub-cellular sensors for probing cells. One desirable characteristic of nanoskiving is that it enables fine control over the dimensions of the nanostructures. The way in which certain nanostructures absorb light is dependent on both their geometry and their surrounding environment. This characteristic offers a route by which nanowires may act as sensors. We have begun to characterise the optical properties of the nanostructures formed by nanoskiving and have shown that they can change the response of the nanostructures to light by simply changing their geometry.

i. develop ii. wash with 2-propanol

i. plasma oxidize ii. cut out pattern

i. spot reagents ii. dry control protein assay glucose 0.5 b

A photograph of a device showing colorimetric read-outs for a drop of artificial urine.

Glucose

Protein

Outlook Hydrophobic

Hydrophilic

Adapted from: Martinez, A. W.; Phillips, S. T.; Butte, M. J.; Whitesides, G. M. Patterned Paper as a Platform for Inexpensive, Low-Volume, Portable Bioassays. Angew. Chem. 2007, 46, 1318-1320.

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Figure 4

Soft lithography is a mature technology that continues to be used as a tool to study cell behaviour under controlled conditions. Patterned paper is relatively a new area that has great potential to be used for healthcare assays in less industrialised countries. Nanoskiving is a new method for

Process of Nanoskiving a

A schematic diagram of the process of nanoskiving.

b

A scanning electron micrograph of gold nanorings on a glass (silicon dioxide) substrate.

Epoxy Deposit metal 70 nm

Au i. Embed in epoxy ii. Cure thermally Section direction

1 m

z

SiO2

Section with a microtome x

y

z

i. Transfer to Si substra ii. Remove the epoxy 2 m

Au nanowire Si

Figure 5

by these fabrication techniques will be of great use to researchers and will find industrial applications—particularly in cellular assays—as the technologies mature.

Full references are available on www.pharmafocusasia.com/magazine/

George M Whitesides is Woodford L and Ann A Flowers University Professor in the Department of Chemistry at Harvard University.

His research interests include physical organic chemistry, materials science, biophysics, complexity, surface science, microfluidics, selfassembly, micro- and nanotechnology, cell surface biochemistry and rational ligand design.

Michael D Dickey is currently a postdoctoral fellow in the Whitesides Group. He will be starting his own research group at N.C. State University in the Department of Chemical and Biomolecular Engineering this fall. His research interests include unconventional nanofabrication, micro and nanotechnology and materials science.

@ Nanofabrication

A uthors

forming nanostructures that could serve as sensors to probe living cells. The primary advantages of these unconventional fabrication techniques compared to conventional lithographic techniques are their simplicity and accessibility to researchers in biomedicine and their low cost. We believe that the components and devices produced

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R esearch & D evel o pment

Improving

Pharmaceutical R&D Using Lean Sigma Application of Lean Sigma to pharmaceutical research and development is a scientific process and requires high level of engagement from all key stakeholders.

Keith Russell Director, Enhancing Product Delivery, AstraZeneca, USA

T

he pharmaceutical industry now operates in a complex and dynamic global environment.

The pace of change is much faster and far less predictable than ever before. To survive and prosper in today’s business environment we need to urgently rethink about how we operate. Specifically, we must challenge the thinking that created our current state and develop new thinking to design our future. To have a secure future we must become capable of being much more responsive to the changing environment and be more efficient in our internal processes. We must become Lean and agile. Lean Sigma is a highly structured customer-focussed, and datadriven problem-solving approach that has been applied with great success to process and business improvement across many industries. Following is a brief of our new thinking developed from our recent experiences in the application of Lean Sigma to meet the challenges of pharmaceutical R&D improvement.

and guide our new product development towards our goal.

Current state of R&D

At AstraZeneca Pharmaceuticals, putting patients’ health first is the foundation for all we do. Our scientists share a common goal: getting new life-changing medicines to patients as quickly and safely as possible. To do this our R&D processes deliver value to patients through a professional project management organisation supported by key disciplinary functions.

These cross-functional project teams build

How value is created by pharmaceutical R&D – We are not Toyota!

As our scientists like to tell us “We are not Toyota!” It is true that application of Lean Sigma to the world of pharmaceutical R&D required us to think through how we adapt this approach, which in

The core DESIGN-MAKE-TEST R&D discovery process The Discovery Process Yes May be

Remake?

Development Cmpd?

Test

No

Design

Make

Figure 1

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CaseStudy

part emerged from its manufacturing roots in Toyota’s Production System.

To understand this, it is imperative to compare the pharmaceutical industry with the auto industry in terms of our R&D processes.

There are some clear differences as well as similarities between the R&D processes of the two industries. For example, the auto industry produces cars that deliver value to customers in terms of the functions they provide i.e. mobility, status, entertainment etc. The pharmaceutical industry delivers medicines to patients that provide value and improve health and ultimately the quality of their life.

In the auto industry value is created by building a “machine” (i.e. a car) using engineering design concepts. In the pharmaceutical industry value is created by developing new medicines using the process of scientific discovery.

The core R&D process in both industries is similar and may be described as DESIGN–MAKE–TEST (Figure 1).

In this three-step process, engineers or scientists begin their work driven by some perceived customer need to creatively craft (DESIGN) early versions or subcomponents of their product.

Building prototypes (MAKE) then allows them to see if their initial ideas worked as planned (TEST). The results from the tests provide learning that is fed back into DESIGN for a second cycle of the process. The process cycles until a potentially viable new medicine or a new car emerges for full-scale development.

Both industries work to meet or exceed the requirements of the relevant regulatory authorities prior to launching their products in their respective markets.

The nature of a “Design” is different in the two industries. In the auto industry, it can take the form of a blueprint of a car or an engine. In the pharmaceutical industry, a “Design” may refer to the chemical structure of the bioactive agent. It is at this level we begin to see the major differences in the nature of the R&D processes of the two industries. A car is designed to work within a relatively well-understood system

Comparison of the scientific process with the core process of lean sigma

The Scientific Process = DMADV (DMAIC) Define Question

Define

Observe/Measure

Measure

Interpret (add meaning)

Analysis

Construct Hypothesis

Design/Improve

Experiment/Study

Validate/Control

NOT a linear process ­– many feedback loops Figure 2

(described by Newton’s Laws of Motion) whereas a medicine is designed to work within a poorly understood system (i.e. a disease state of the human biological system). The former applies knowledge from Physics that is relatively predictable, the latter uses knowledge from Biology and Chemistry that is much less predictable. It is this difference in the level of accessible knowledge between the two industries that necessitates a modification of how we apply Lean Sigma.

Improving the flow of knowledge and understanding

Science is concerned with the acquisition of knowledge and it follows similar processes around the world. Interestingly, there are significant parallels between this process and the core processes of Lean Sigma (DMAIC – Define, Measure, Analyse, Improve, Control) and Design for Six Sigma (DMADV – Define, Measure, Analyse, Design, Validate).

Application of Lean Sigma to R&D is arguably simply an application of the scientific process to the core R&D processes. As such, we can see that one way of “improving” the science we use is to accelerate the acquisition of knowledge in the DESIGN–MAKE–TEST cycle.

Core process of R&D – The DESIGN–MAKE–TEST

When we apply Lean Sigma to the iterative DESIGN–MAKE–TEST process, one question we face is what is actually flowing in our processes? In an early example from our work, we mapped the complex cross-functional lead optimisation process. This is the last stage of our discovery process that generates a potential drug candidate for clinical development. At first glance we see that chemical compounds are synthesised and tested to profile their physiochemical and biological properties.

These compounds “flow” from chemists to biologists. Knowledge and information “flows” into our molecular designs and is also created in biological assays.

In both cases, we quickly begin to see that these “flows” are often significantly interrupted in practice leading to delays.

These delays lead to many issues. One significant consequence of these delays is to slow the pace of learning for our scientists.

For example, we could see from our process mapping work and process measurement activities that the compound “flows” were often interrupted because of depletion of compound supply.

w w w . p h a r m a f o c u s a s i a . c o m 49

R esearch & D evel o pment

Schematic illustration of the approach used to eliminate waste and create flow in the iterative DESIGN-MAKE-TEST process used in lead optimisation

Remove Waste – Achieve Flow Time

Current Process

Design Make Test

Remakes

R&D is not a machine

Improved Process

Figure 3

Chemical synthesis as commonly practiced does not allow material to be made on demand (without a significant lead time) and so small batches of material are made to support several assays before resynthesis is necessary. Making large batches will allow many assays to be done without interruption. Unfortunately, this leads to the wastage of material since most molecules synthesised do not make it past the first few assays. The key customers for these processes—our discovery project leaders—made it clear that what they wanted was sufficient material to progress to a key late-stage assay without interruption. This led to a new process target for the amount of compound made for the first time by our chemists.

It was improvements such as this and other associated improvements that led to a significant (more than 50 per cent) acceleration in the timelines of our lead optimisation projects. A reduction in the need for resynthesis gave our scientists more time for creative thinking.

The flow of value is in people’s heads!

It is very important to appreciate that in R&D people primarily create value both individually as well as collectively

50 P h a r m a F o c u s A si A

called SIPOC (Suppliers, Inputs, Process, Outputs, Customers), which attempts to represent the process being studied at a high level as a unidirectional flow of value towards the customer. In practice, this can only work well if there is a counterflow of information to the suppliers so that they can provide their process inputs reliably and accurately. In the lead optimisation work each component of our discovery system interacts with several other components, often bidirectionally.

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in their heads in the way they think. A very important aspect of this comes from interactions between team members in our multidisciplinary project teams to develop shared meaning. Our scientists are not simply observers of the “process”—they are the process! Lean Sigma emerged from a manufacturing environment where we are often dealing with processes in which machines and / or the movement of “widgets” is a major component. The complexity of the R&D environment necessitates that we incorporate Systems Thinking alongside the more common analytical thinking mode used in Lean Sigma work. Many of our processes are far from linear and may be best thought of as being complex systems in which processes provide a means of dynamic interaction between the components.

Systems Thinking – Focus on the interactions

One simple example of applying Systems Thinking to our Lean Sigma work is to recognise that cause and effect patterns are usually better described as being like a circle than a straight line pointing in one direction. A tool commonly used at the beginning of a Lean Sigma project is

The complex R&D systems required to deliver new medicines can be approached using a mental model of a “machine” in which we diagnose issues and problems as a mechanic would diagnose an engine problem. We then proceed to fix the problem as if we were external to the system. This way of thinking works well in highly controlled environments (e.g. by isolating small simple components of a larger process) but it may not be ideal to address some of the more important system-level issues. In the latter case, an alternative mental model is that of a biological (or social) system. Using this kind of thinking brings human behaviour to the fore in which people are a key part of the process—not outside of it.

This type of thinking also recognises the existence of complex non-linear processes driven by numerous process feedback loops. For example, small effects in such systems can be amplified over time by reinforcing feedback loops only to reach over time a limit imposed by another type of feedback (balancing).

Approaching such systems requires a more flexible approach than simply fixing problems. It requires that we think about planting the seeds of change and nurturing change in a productive direction. In this way one should often need to think more like gardeners than mechanics! A simple example of this way of thinking is to appreciate that people (unlike machines) always have a choice and so cannot be “controlled” as a machine can.

10,000 globally) in an efficient and effective way. We are faced with many highly variable and slowly recurring processes and a history of many initiatives and change programmes that have left many of our employees desensitised to new approaches. In addition, the rapidly changing external environment shows no sign of slowing down. Against these and many other challenges are some very exciting opportunities: • Highly qualified and experienced scientists who have begun to adopt Lean Sigma thinking in their work can dramatically increase the efficiency and effectiveness of their application

of science to bring new medicines to patients • Lean Sigma adapted to include a stronger Systems Thinking approach can be applied to address some of the “big issues” in R&D in a holistic manner including speed, quality, and cost • Focussing on patient’s needs provides a powerful aligning force to overcome silo thinking.

• In R&D, people are critical components of the process—what they say and what they do (i.e. their behaviour) determine how well the system works.

Key learning points Keith Russell leads Lean Sigma deployment across crossfunctional development projects at AstraZeneca. Previously a Director of CNS Chemistry and Discovery Project Leader, he is the author of over 30 peer-reviewed publications and is an inventor with over 25 patents. He holds a PhD from the Cambridge University, UK and a NATO post-doctoral fellowship (1984-1986).

A uthor

Our Lean Sigma journey has just begun and we see many challenges as well as opportunities ahead. One major challenge is how to spread improvements and new thinking among our R&D employees (numbering more than

BOOK Shelf

NANOTECHNOLOGY IN BIOLOGY AND MEDICINE: Methods, Devices, and Applications Description:

Editor: Tuan Vo-Dinh Year of Publication: 2007 Pages: 792 Published by: CRC Press ISBN-10: 0849329493 ISBN-13: 978-0849329494

This book describes nanosensors that track the biochemical processes and reveal the sub-microscopic structures of living cells, covers methods and applications for various nanostructures including nanotubes, quantum dots, nanoshells, and nanowires and discusses self-assembly, biomanufacturing, and other methods and considerations for designing nanomaterials. It explores nanocarriers as a drug delivery tool to specific targeted antigens and considers the application of nanotechnologies for stem cell research, gene diagnostics, tissue engineering, and other biomedical uses.

Nanotechnology in Biology and Medicine: Methods, Devices, and Applications integrates interdisciplinary research and recent advances in instrumentation and methods for applying nanotechnology to various areas in biology and medicine. Pioneers in the field describe the design and use of nanobiosensors with various analytical techniques for the detection and monitoring of specific biomolecules, including cancer cells. The text focusses on the design of novel bio-inspired materials, particularly for tissue engineering applications.

It provides a comprehensive forum that integrates interdisciplinary research to present the most recent advances in protocols, methods, instrumentation, and applications of nanotechnology in biology and medicine.

For more books, visit Knowledge Bank section of www.pharmafocusasia.com

w w w . p h a r m a f o c u s a s i a . c o m 51

CaseStudy

They can however be influenced and strong leadership and the understanding of human behaviour becomes an essential component of successful Lean Sigma projects.

We have found that success in our Lean Sigma work requires a high level of engagement of all key stakeholders in a process. Interestingly, scientists are often trained to be highly independent and sceptical of new approaches to improving R&D, but once they experience Lean Sigma they begin to see the parallels with the scientific process. At this point they can become powerful forces for spreading this powerful improvement approach.

M anufacturing

Cell-based Potency Assays

Adhering to GMP standards

The means of measuring the biological activity of a new drug is of critical importance to its release. The process through which a manufacturer can achieve this measure should be clearly defined before pre-clinical studies are initiated. The quality and validation requirements of these studies are defined in a number of regulations but experience of interpretation of these guidelines is essential in establishing a regulatory-compliant assay.

A

biological measurement of the activity of a drug is perhaps the most critical step in the series of tests required for product release for both clinical trials as well as the market and plays an important role in the stability assessment of drug candidates. All drugs require a clearly defined specification for release; wherever possible, this should be in place at the pre-clinical stage in a basic format and made more stringent throughout the product life cycle leading to a clearly defined set of release tests that constitutes the marketing authorisation. Some of these assays are generic and can be very simple, such as a

measurement of the pH of the material or the product appearance. However, others are product-specific and can be problematic and require significant scientific insight and thorough validation; the cell-based potency assays fall into the latter category.

Animal studies

Biological drugs seldom lend themselves to enzymatic reactions as a measure of biological activity. Insulin is perhaps the only biological which measures potency by enzymic reactions, therefore in vivo or in vitro measures of activity are the most popular choice. Animal studies have a

Measurements of biological activity can be performed in the following three ways: Animal studies in which a defined animal model demonstrates a measurable, physiological change in response to application of the drug.

Cell-based assays that use a specified cell system, which on addition of the drug, demonstrate a measureable biological response.

Enzymatic reactions where the biological activity of the drug can be measured by the accumulation of product following the chemical reaction facilitated by the drug.

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number of distinct disadvantages when considering product release within a Good Manufacturing Practice (GMP) environment, not withstanding the industry’s ongoing commitment to reduce the use of animal experimentation. Fundamentally, animal studies are typically performed to ascertain the Good Laboratory Practice (GLP) level of quality, rather than that of the GMP level demanded by the industry for other release tests. Animal studies are costly and time-consuming and are known to have a degree of variability raising questions about their effectiveness.

Cell-based potency assays

Another method to measure the biological activity of a drug that is proving to be of interest to the majority of manufacturers is cell-based potency assays or bioassays.

Cells are living entities, representing biological systems that possess many of the important in vivo characteristics that make them useful for measuring biological activity.

Cell-based potency assays possess a number of advantages over animal models. The most obvious is the cost, with cell-based assays typically reducing cost by 80 to 90 per cent when compared to

Daniel N Galbraith, Head, Operations Andrew Upsall, Head, In vitro Services BioOutsource Ltd., UK

equivalent animal studies. Initiation of cell-based assays can be achieved with greatly reduced time frames and the variation associated with animal studies is largely removed as the cells used are usually clonally derived and maintained within strict parameters. Therefore, the variability of response is reduced when compared to in-bred animal groups as well. For all these reasons and many more, cell-based potency assays have been the method of choice. However, these assays do require careful consideration when preparing them for use in the release of a drug product.

When considering a bioassay, the cells are universally regarded as the most important feature; without the cells responding in the characterised manner, the assay will never be of use for the release of the product. Cell-based potency assays typically start their life in the research and development section of a large pharmaceutical organisation or in an independent research organisation such as a University or Government Department.

These facilities often do not comply to recognised quality system such as GLP or GMP and this presents a significant problem. Cells from these institutions

may not have sufficient provenance to demonstrate the appropriateness of their usage for GMP and this can be a timeconsuming endeavour to undertake.

Ultimately, these tests will need to meet standard GMP requirements for product release for clinical trials (in the European Union) and for marketing authorisation in the rest of the world.

The next problematic feature that can hamper assay development and validation is the availability of information regarding the characterisation of the reference standard. Cell-based potency assays generally report a value relative to a standard drug batch, with every new batch measured against it. In this way each batch can be shown to be consistent with previous batches and the reference standard. The provenance of the reference standard is critical to every batch released and characterisation can be an arduous process. A successful approach employed by most manufacturers is to use one of the early production batches as the reference standard and obtain information during initial assay development. Issues can arise when, following the initial clinical trials, a number of process and formulation changes and improvements to the manu-

facturing process are implemented. This results in the reference standard no longer being an identical composition to the new batches of the product. Significant effects on the results of the bioassays can ensue and a new reference standard may be required to be established for the new product design. Whenever a new reference standard is created, a bridging study to show the comparability of the new and old molecules is required. This again can be a time-consuming and expensive work and may lead to project delays if not managed effectively.

Validation of assays

The next major hurdle, once the cells and reference standards are in place, is the validation of the assay to an International Conference on Harmonisation (ICH) standard that will also meet GMP requirements of the regulatory authority. The ICH sets out specific guidelines for all tests to be followed if they are to be used for the release of drugs. To meet GMP requirements, appropriate ICH Topic Q2 (R1) Validation of Analytical Procedures: Text and Methodology. NOTE FOR GUIDANCE ON VALIDATION OF ANALYTICAL PROCEDURES: TEXT AND METHODOLOGY (CPMP/ICH/381/95).

1

w w w . p h a r m a f o c u s a s i a . c o m 53

M anufacturing

A uthors

specifications and control of critical over time, can also be transferred to trained and it is often considered to be reagents and equipment must also be these organisations. A major advantage prudent for operators from the expert given full consideration. For example, of transferring an assay to a CTO is the laboratory to conduct training for analysts the incubators used to grow the cells as provision of a back-up facility providing of the receiving laboratory. Upon successwell as the reagents used in the culture a critical aspect of the expected business ful completion of these studies, a formal of the cells all require qualification and continuity programme that can be utilised protocol should be initiated to estabcontrol. Reagents, such as foetal calf in the event of problems arising with the lish conclusively that consistent results serum, should be considered as critiin-house testing programme. can be obtained between the laboratocal and even plasticware must be kept Transfer of an assay to a CTO requires ries. A technology transfer will then be as consistent as possible to ensure the several stages to be considered. Quality performed, if transferring a validated assay desired performance of assay. and technical agreements need to be or a validation study can be performed To meet all the specifications in the prepared where the individual responat the CTO. validation guideline, cell-based potency sibilities of both parties are defined and assay may present many obstacles and also identified. Experience has shown that the The way forward require considerable time and resources. important part of the agreements relate Cell-based potency assays are clearly The ICH Q2 guidelines describe a to critical GMP requirements such as seen as the way forward as methods to bioassay as a “Quantitative set the specifications for new drug test of the active moiety in products. These assays provide a samples of drug substance or clear and reliable indication of the drug product or other selected activity of the drug and provide a The aim of the validation data component(s) in the drug prodconsistent means of measuring this set is to provide evidence for the uct”. The validation characterisover time. These assays do however appropriateness of the assay to have tics which should be considered require a commitment of time and utility in the release of product. are accuracy, precision, repeatresources which may necessitate the ability, intermediate precision, use of outsourcing the work to a specificity, limit of detection, CTO. This endeavour should be linearity and range. carefully considered and the right The scope of the validation should responsibility for Out of Specification partner to assist should be selected with include a number of assay runs to be Results and Deviations to Protocols and care, ensuring that sufficient experience performed by several operators to provide these requirements should be clearly and resources are available. As biologic sufficient data from which appropriate defined. Following agreement on docudrugs become widely used, the demand for conclusions can be drawn. The validation mentation, “proof of concept” studies efficient release of drug to the market will process should also focus upon critical are performed to demonstrate that the also increase. Planning for this in a timely assay parameters in all robustness expericells used in the assay grow and respond manner is a challenge to all manufacturers. ments. Overall, the aim of the validation in a consistent manner with the original However, the failure to do so will result in data set is to provide evidence for the (expert) laboratory. GMP compliance inevitable delays to release and eventually appropriateness of the assay to have utility requires that the operators are suitably add cost to the bottom line. in the release of product.

Many organisations find that they have insufficient in-house resources to Daniel Galbraith heads the Operations for BioOutsource Ltd., a undertake the task of performing validaGMP testing organisation which supports batch release of vaccines tion or batch release testing of product and biologics. Daniel comes to BioOutsource following experience heading departments in Covance Laboratories and MedImmune. using these complex biopotency assays and often consider outsourcing to a Contract Testing Organisation (CTO).

CTOs typically have experience, qualAndrew Upsall leads a team of scientists who support safety and ity systems and suitably qualified equippotency testing for new biologics and vaccines. Andrew comes to ment for successful assay validation and BioOutsource following experience in a number of positions in the release of product to a GMP standard. pharmaceutical industry.

Responsibility for stability studies, where the stability of the biological potency of several batches of the drug is measured

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M anufacturing

Single-use Bioprocess Containers Economics of usage in Asia

Extensive adoption of single-use products in manufacturing has shown considerable cost reductions by limiting initial capital costs, utilities required and cleaning validation etc.

Do they promise the same benefits in the Asian manufacturing scenario, where overall cost of production and labour are significantly lower than in the West?

Swapnil Ballal, Head, Biopharmaceutical Bulk Manufacturing, Intas Biopharmaceuticals Ltd., India

S

ingle-use disposables have become a common name in the biomanufacturing industry world over. All major companies related to equipment and filtration products are present in this domain.

They are increasing their presence by introducing more products in the disposable / single-use format. Some of the well-known benefits are: • Lower capital investments • Cost savings in cleaning and sterilisation • Elimination of cleaning validation requirements • Speed of deployment and batch changeover • Flexibility of operating scales.

Increasing implementation of single-use technologies clearly indicates that their benefits are attractive to the users.

Drivers of single-use technology

More than 1,000 biotech-based new drug entities are in various pre-clinical

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and clinical phases. A large number of such leads are being developed by startups and small sized firms with one to four lead candidates in their development pipeline. Since manufacturing plants take years to be built and costs run into hundreds of million dollars, most biotechnology companies are reluctant to make investments to support a therapeutic candidate still undergoing clinical trials. They rely on Contract Manufacturing Organisations (CMOs) for facilities and production expertise.

This allows them to focus their resources on product development and establish a proof-of-concept in clinical trials.

Meanwhile, CMOs executing shortterm production requirements benefit from the fast turnaround and flexibility offered by the single-use products while limiting allied activities like changeover and cleaning validation.

The operating dynamics in Asia are quite different from the developed markets. This is largely due to the business model of the Asian biopharma-

ceutical industry which is primarily based on biogeneric products, lower labour costs and low-cost operations. In a scenario where the CMOs not catering to the local market and very few innovations taking place in biotechnology, the benefits of single-use technology using bioprocess containers as a model in Asian scenario can be reconsidered.

Capital investments

Asia benefits from having a well-developed steel fabrication set-up for the production of customised GMP vessels and related fabrication. A large number of EMEA and US FDA plants in India and in other parts of Asia make use of such indigenous fabricators.

A 200−500 litres mixing tank for buffer preparation costs around US$ 50,000 in India, which includes a jacketed pressure vessel with Clean-In-Place (CIP) / Sterilize-In-Place (SIP) pressure ratings, magnetic agitator, pH and temperature controls. Fabrication costs for similar units in the West are one and half to three times higher. The difference in cost is mainly due to the overall lower operating costs and low-cost skilled labour and not necessarily due to the usage of lower quality components.

M anufacturing

On the other hand, a 200-litre bag holder with jacket and magnetic stirrer would cost around US$ 40,000 to 60,000 depending upon the configuration selected. Additional accessories like tubing sealer, tubing cutter etc. would take the cost to US$ 90,000. pH, conductivity and temperature control requirements further increase the cost.

A. Sinclair & M. Monge showed that adopting a single-use Bioprocess Containers (BPC)-based manufacturing system could result in overall capital saving of about 20-40 per cent over the traditional Stainless Steel (SS) fixed tank set-up in US or Europe. The major difference in Cost of Goods (COG) is due to the reduction in capital charges, smaller size of utility systems and decreased manpower for Quality Assurance & Quality Control for a disposable-component-based concept plant.

With products like SS tanks, CIP / SIP skids and other customised engineering products and piping available in Asia at a cost that is less than 25-50 per cent that of the US and Europe, the difference in capital investment between a plant with BPC set-up and with fixed SS tank decreases to a near equal level, if not to a lower one.

Operating cost

The cost of cleaning and sterilising a SS fixed tank is directly related to the cost of utility required. While the reduction in absolute quantity of utility may look very significant in terms of volume, the cost of CIP and SIP operation may actually be only a fraction of the cost of the bag itself. The cost of generating purified water / WFI is in the range of US$ 6 to 10 for 1,000 litres. For the cleaning of a 200-litre tank with a highly unoptimised CIP cycle, requiring 1,000 litres of water, one would spend less than US$ 10 of utilities in each run, while a single 200-litres bag alone costs above US$ 250. A process using five bags each day working 150 days in a year would require US$ 187,000 worth

of bags alone against US$ 7,500 worth of utilities for a fixed tank.

EU/US

Elimination of cleaning validation requirement

Cleaning validation is portrayed as one of the biggest cause of stress for bio-manufacturers. The gap between practice and regulatory expectation has been decreasing over the years. As per ranking of total GMP deficiencies by EMEA between 1995 and 2005, cleaning validation comes at 23rd place with only 1.3 per cent deficiency attributed to the category of cleaning validation and only one critical deficiency out of 193 deficiencies cited during the period, indicating that regulators are satisfied with the approach and extent of cleaning validation.

Cleaning validation of buffer and process intermediate tanks consume additional resources over the set-up based on BPC. The cleaning of buffer tanks in itself is easier to validate since these are not expected to contact and contaminate the product and product carryover issues are minimal. Cleaning validation of intermediate holding tanks can be reduced by intelligent and judicious selection of worst case scenario and bracketing to minimise the number of runs required for validation.

The point being emphasised here is that the science of cleaning validation is well established and since one would require cleaning validation of other plant equipment like fermenters, chromatography systems and columns, the set-up for cleaning validation would anyway exist and, therefore, it would only be the case of increasing the scope of the cleaning validation.

On the other hand, the use of bags is most practical for contract manufacturers since it eliminates cleaning validation studies for a different product each time.

Additional validation requirements

Cost of goods comparison Traditional Disposables Concept Labour

200.1

154.4

Material

61.5

57.0

Indirect Material

82.2

74.2

Consumables

40.8

76.3

Capital

149.1

83.7

Total

534.0

445.6

0%

17%

Savings

Adapted from: A Sinclair and M Mongo, Bioprocess International 3(9): S51-55 (October 2005) Table 1

Contribution of individual cost head to cost of goods Traditional

Disposables Concept

Figure 1

The use of single-use disposable systems brings a whole new set of validation

w w w . p h a r m a f o c u s a s i a . c o m 57

M anufacturing

ASIA Cost of goods comparison Disposables Concept Labour*

50.0

38.6

Material

61.5

57.0

Indirect material

82.2

74.2

Consumables

40.8

76.3

Capital**

104.4

71.1

Total

339.0

317.0

0%

6.4%

Savings

* Considering labour cost in Asia is 25% of that of US/EU ­** Considerig 30% lower capital for traditional and 15% lower capital for disposable plant in Asia Table 2

Contribution of individual cost head to cost of goods Traditional Plant

Disposables Concept Plant

Figure 2

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requirements, which are at times more complex in nature than cleaning validation, largely due to the limited knowledge base and regulatory exposure. These are compatibility testing, extractables and leachables testing, mixing efficiency testing, leak testing, endotoxin, short-term and long-term stability testing and sterility validation.

These are generally product-specific.

Though reduceable for similar buffers, they still require product-specific testing for each product and intermediate.

The analytical methods involved range from pH, conductivity to LC-MS, GC & GC-MS based analytical tools. The cost of carrying out the same also needs to be factored. Moreover, major bag manufacturers use different resins and probably different moulding procedures and chemicals. Simple substitution is not possible as in the case of stainless steel tanks, thereby increasing the reliance on a single bag manufacturer.

Cost of Goods analysis

The Cost of Goods (COG) for any manufacturing process plays a decisive role in acceptance of the technology.

As shown in Table 1, COG comparison of a traditional steel vessel-based plant and a disposable concept plant indicates a saving of 17 per cent using disposable technology. Contribution of each major head is shown in Figure 1.

Considering a 30 per cent lower capital cost in Asia for traditional plants and 15 per cent for disposable plant (since disposables set-up is to be imported), it is seen that the savings using a disposable set-up are reduced to about 6.5 per cent (Table 2 and Figure 2). If import duties on the disposable components are added at the current rate of 30 per cent in India, the COG for a traditional plant would be lower than that of the disposables.

With the continuous increase in price of crude oil and that of the base plastic resin, operating cost of plants using plastics may keep on increasing, while a

steel set-up once installed would not add significantly to operating expenses.

Decreasing the disposable products cost by about 30 per cent and eliminating import duties would make the scenario for adopting disposable products and technologies favourable. This cost reduction can be brought about by offering better pricing for Asian markets and by exploring the possibility of manufacturing such units in Asia itself.

Drivers for adopting single­-use disposable containers in Asia

Users from the US and Europe whose revenue came from contract manufacturing were the early adopters of disposable technology. Adopting the technology brought immediate reduction in capital with quick turnaround time.

However, in Asia, the focus is still on the manufacturing of biologics for captive use. Since the market for such products is highly cost-sensitive, increase in the overall cost of the finished product is critical. Since many of these plants operate mono-product lines / set-up, the need for adopting BPC set-up may not be very high.

In situations where living organisms are handled, single-use products can ensure the safety of the product as well as the operators, and thus would find early acceptance.

Contract manufacturers in Asia would also find single-use products useful, though such manufacturers are limited in number as of now. Single-use products would also find their use in scale-up and development labs, which have limited set-up for tank processing, allowing them to process on a larger scale in an R&D set-up.

One of the areas where singleuse disposables set-up can be of great interest is finished dose manufacturing. The existing fill-finish facility can be adapted to single-use set-up without disturbing the existing set-up, allowing manufacturers to have a greater degree of assurance as well as flexibility of scale.

M anufacturing

Conclusion

In the European and US scenario, there is possibly a clear capital investment benefit in adopting the single-use bags and tanks. The same may not hold true in most Asian countries, where steel fabrication and labour costs are low.

The decrease in efforts in carrying out cleaning validation of fixed system needs to be evaluated critically against the cost of additional validation related to compatibility of plastics with process fluids.

Potential adopters of single-use bioprocess vessels need to carry out

a critical evaluation of economic benefits while selecting between fixed tank system and single-use system.

They should also perform a thorough cost-analysis on their own before singling out one option.

Scenario of a hybrid system with a fixed non-product dedicated set-up like buffer preparation, transfer and hold with single-use product contact can also be a good compromise between cost and

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In addition to the above, facilities which are limited in capacity due to utility limitations like Water For Injection / clean steam can benefit from use of the single-use bioprocess bags.

flexibility while making full use of the advantages of single-use systems.

Acknowledgements: I wish to thank Mahesh Kodilkar of Intas Biopharmaceuticals Ltd. and Vishal Wagh, Director, adam fabriwerk, Mumbai for their valuable contributions.

Full references are available on www.pharmafocusasia.com/magazine/

Swapnil Ballal is currently heading India’s only EU-GMP approved biopharmaceutical plant at Intas Biopharmaceuticals Ltd. Being associated with the Indian biotechnology industry for the past 12 years he is presently overseeing manufacturing of 4 bio-therapeutics products along with contract manufacturing projects. He joined Intas in 2000 and played key roles in the set up of the biotechnology and R&D divisions and the manufacturing plant for biologics.

Prior to Intas, he worked for Wockhardt Research Centre. He received his MSc in Marine Biotechnology from Goa University and holds professional membership at PDA and ISPE.

w w w . p h a r m a f o c u s a s i a . c o m 59

M anufacturing

Large-scale Biochromatography

What lies ahead?

Many new approaches have been considered for large-scale chromatography including membrane adsorbers. The benefits and drawbacks of these devices and their potential impact on the biopharmaceutical industry are discussed.

Uwe Gottschalk, Vice-President, Purification Technologies, Sartorius-Stedim Biotech, Germany

U

pstream productivity in the biomanufacturing industry is not being matched by improvements downstream. For processes such as packed-bed chromatography that cannot be scaled up indefinitely, the future is starting to look bleak. Manufacturers around the world are approaching the limits of productivity and scalability, and innovative downstream processing (DSP) solutions are required to address this challenging issue.

Many new approaches have been considered including the re-evaluation of simpler separation technologies and the development of high-technology solutions such as membrane adsorbers.

Crucial chromatography

In the biopharmaceutical industry, downstream processing is completely dependent on chromatography. Different resins exploit different principles, so that the purification of specific proteins, such as antibodies, from cell culture broth is achieved by using two, three or even more types of chromatography in series.

There are three typical steps in the

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purification of antibodies: one based on Protein A affinity chromatography for initial product capture, followed by two steps of polishing. In many cases, one is based on Anion Exchange (AEX) operating in flow-through mode to retain negatively-charged impurities such as Host Cell Protein (HCP), host cell DNA / RNA, leached Protein A and

In the biopharmaceutical industry, downstream processing is completely dependent on chromatography.

endotoxins, and the other is based on either Cation Exchange (CEX) or hydrophobic interactions to resolve the intact dimeric antibody from aggregates and degradation products.

The crunch

Although packed-bed chromatography is a successful technology that dominates the biopharmaceutical industry, all is not well in the world of antibody manufacturing. There is all-too-frequent

talk of bottlenecks in DSP. The success of antibody-based products coupled with pressure on manufacturers to increase their yields while reducing the cost of goods has driven them to focus almost exclusively on the upstream phase of production. Therefore, 5 g/L titres can now be achieved routinely in mammalian cells, representing a 100-fold increase over the last two decades. At the same time, upstream manufacturing capacities have increased dramatically, with many manufacturers choosing to operate several 10,000-L bioreactors simultaneously. In contrast, little attention has been paid to DSP, and the same standard technologies that were developed to handle titres of 50-100 mg/L are still in use today.

The bottleneck in processscale chromatography negates any advantages of scaling up earlier process units, since bind-and-elute chromatography steps are driven by mass rather than volume. This means that savings made upstream do not translate into increased productivity during purification. Larger columns also impact directly on facility layouts, costs and infrastructure because the space and buffer volumes for all steps also increase. As a consequence, pool and buffer volumes act as serious limitations when it comes to the introduction of high-titre processes into existing facilities.

M anufacturing

Principal solute transport mechanisms

a. Packed-bed

b. Membrane Chromatography

Thick arrows represent bulk convection, thin arrows represent film diffusion and curly arrows represent pore diffusion Figure 1

There are also operational problems with columns operating in flow-through mode. In resin-based AEX media, the transport of solutes to their binding sites relies on pore diffusion, but the contaminants are often large molecules—DNA and viruses—which do not readily diffuse into the pores. This causes mass transfer resistance and lowers the column efficiency because large molecules can only bind to the outer surface of the bead and longer residence time is required to find binding ligands inside the resin particles. The only solutions are to use a greater column bed height and / or to reduce the linear flow rate, both of which will have an impact on the overall productivity. Therefore, to keep up with the process demand, most traditional polishing steps operate at a flow rate of between 100 and 150 cm/h and use significantly oversized columns to accommodate this.

Apart from productivity and economic issues, large columns also suffer from scale-related packing problems such as hysteresis, edge-effects and resin compression, which result in unpredictable fluid distribution and pressure drops.

Low-tech and high-tech solutions

The shift in perspective from upstream to downstream processing has become more evident in the last few years as experts have begun to acknowledge that improved productivity cannot rely on more efficient upstream production alone, and that downstream improvements must consider the entire production train and not just single operations to avoid shifting of the bottleneck from one operation to another. Various alternatives have been put forward either to replace column chromatography or to reduce the load of impurities in the feedstream so that one or more chromatography steps can be eliminated.

Many of these employ low-technology methods that are inexpensive but are applicable on a large scale. For example, techniques such as flocculation, precipitation and multiphase extraction can be used in combination with conventional cell separation techniques such as centrifugation and microfiltration to remove residual particulates and soluble impurities that might otherwise increase the burden on downstream polishing steps.

Some engineering-based solutions have also been implemented, including simulated moving bed chromatography, which allows higher throughput without increasing buffer consumption. Hightech solutions to the capacity crunch in downstream processing offer innovative replacements for traditional packed-bed chromatography. Examples include monoliths, charged ultrafiltration membranes and membrane adsorbers, all of which carry out analogous functions at reduced cost.

Membrane adsorbers – The advantages

Membranes are already integral to many bioprocesses because disposable filters have proven to be inexpensive and convenient while matching the performance of their fixed, stainless steel counterparts. For many unit operations, particularly filtration and media / buffer storage, disposable devices are now so commonly used that they are regarded as a normal practice. The industry is keenly aware of the advantages they provide in terms of reduced down-time, flexibility in process

w w w . p h a r m a f o c u s a s i a . c o m 61

M anufacturing

development / scaling, and the elimination of cleaning and validation costs, especially where multiple products are manufactured in the same facility.

Interest in membrane chromatography is growing because of the success of disposable membrane filters, yet there is a lack of appreciation of the many advantages they offer in downstream processing. Not only do they share all the economic and versatility benefits listed above for filters, but they also have specific functional advantages over equivalent packed-bed columns.

These are: Convenience – Membrane adsorbers are disposable modules. They are supplied ready to use and can be discarded once exhausted. There is no cleaning or validation, no need for life-time studies, no packing, re-packing and recycling. Fouled modules can be replaced with new ones more conveniently and cheaply than packed resins.

Scalability – Manufacturers offer devices of various sizes that can be plugged into existing process trains, thereby allowing required rapid scale-up or scale-down. Importantly, as shown in Table 1, there is linear scale-up for important parameters such as frontal surface area, bed volume, flow rate and static binding capacity, while normalised dynamic capacity remains fairly constant at 10 per cent or complete breakthrough.

Small footprint – Membrane adsorbers are typically available in a cartridge format wherein 10 to 15 membranes are stacked. The consequences in terms of buffer usage are impressive, reducing buffer consumption to less than 1 per cent of the amount required for an equivalent packed column. The impact of this is felt not only in the reduced buffer usage, but also in the facility requirements in terms of buffer storage, preparation time and waste management. The savings made here more than offset the cost of the modules themselves.

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Operational performance – In flowthrough applications such as AEX in antibody polishing, membranes outperform columns because the transport of solutes to their binding sites occurs mainly by convection, while pore diffusion is minimal (Figure 1). Because of these hydrodynamic benefits, membrane adsorbers can operate at much greater flow rates than columns, reducing buffer consumption even more and shortening the overall process time by up to 100fold. The use of membrane adsorbers can be viewed as the equivalent of shortening traditional columns to near zero length, allowing large-scale processes to run with only a small pressure drop at very high flow rates. For example, polish-

Membrane adsorbers are typically available in a cartridge format wherein 10 to 15 membranes are stacked.

ing with an anion exchange membrane can be conducted with a bed height of 4 mm at flow rates of more than 600 cm/h, providing a high frontal surface area to bed height ratio. Small-volume disposable membrane chromatography devices can now handle more than 10 L per minute/bar/m2.

Virus clearance – All biomanufacturing processes involving mammalian cells must include steps to clear and inactivate viruses. The efficiency of these steps must be demonstrated via spiking studies using a range of model viruses with properties representing possible contaminants.

Multiple studies have now demonstrated that membrane adsorbers are at least as efficient at virus removal as equivalent columns, and due to the hydrodynamic benefits mentioned above often exceed columns in terms of virus clearance.

Log reduction values of >5 are routinely achieved for most model viruses. The performance studies corroborate the fact that packed columns and membrane

chromatography devices are both capable of removing trace contaminants and clearing viruses in polishing applications. Apart from the handling concept, the main difference between the two formats is the load capacity at flow rates acceptable for large-scale manufacturing. Multi-layer Q membranes achieve much greater productivity compared to equivalent volumes of resin with no loss of performance in contaminant and virus removal. For this reason, the volume of a disposable membrane device is typically 5 per cent of that required for a conventional column, which needs to be oversized to accommodate the volumetric flow rate.

Integrity – Functionality and integrity tests demonstrate that even with bed heights of less than 1 cm, membrane stacks are robust and reliable.

Economy – Several cost comparisons have been carried out for membrane adsorbers and resins used for flow-through polishing of antibodies. These indicate that column chromatography is overall more economical for low loading rates whereas membranes break even with them at a load of 2 kg/L. At higher loads, membranes become more economical, which means that as the productivity of a process increases membranes become the better choice for polishing. The models were comprehensively researched and they took into account factors such as capital charges (higher for columns due to the need for capital equipment purchase), labour (higher for columns due to the time required to prepare buffer, pack and re-pack the columns, and time for cleaning and validation studies), consumables (higher for membranes because of the cost of each membrane device, but offset to an increasing extent at higher scales by the increased usage of buffer and water for injection in column chromatography) and material costs (including utilities, spare parts and the resin, which is significantly higher for columns).

w w w . p h a r m a f o c u s a s i a . c o m 63

M anufacturing

Comparative results from a cost model* comparing packed-bed and membrane AEX chromatography in flow through mode 100 90

100 90

80

80

70

70

60

60

50

50

40

40

30

30

20

20

10 0

10 0

At a load capacity of 2 kg/L, the overall costs of membranes and columns are equivalent.

Labour

Material

At 10 kg/L membranes cost less than 30 per cent per batch as compared to running a column

Consumable

Capital

*Each component is represented as a percentage of the total cost of column chromatography (fixed arbitrarily at 100 per cent) Figure 2

The outcome of the cost analysis can be seen in Figure 2.

Membrane adsorbers – Drawbacks

Early membrane adsorbers suffered from problems related to both adsorptive capacity and device performance, e.g. low loading capacity, membrane fouling, leaching and suboptimal fluid distribution leading to a substantial performance loss during scale-up. Many of these issues have been addressed by the development of more suitable matrices, better device housings and improved surface chemistries. For flow-through applications where trace impurities are

retained, membrane adsorbers now outperform columns under most conditions and therefore are adopted by more and more manufacturers. Performance tests routinely show reduction of HCPs and DNA below detection levels.

Currently, the greatest limitation of membrane adsorbers is in the bind-and-elute operations for small molecules, where the binding capacity is lower than equivalent columns. Thus, packed-bed chromatography is still the preferred unit operation for capturing molecules of <200 kDa, especially when peak cutting, gradients etc. are required for the separation of closely related

species. Membrane chromatography, on the other hand, is more suited to simple on-or-off binding scenarios such as polishing and the capture of large molecules from diluted feed streams.

Even so, membranes are beginning to encroach on bind-and-elute operations, the traditional strong-hold of column chromatography. It is now possible to use both AEX and CEX in antibody polishing, the latter to purify the antibody from product-related impurities such as aggregates and degradation species, as well as positively-charged HCPs. Here, the first polishing step plays to the strengths of membrane chromatography—it is carried out in flow-through mode to retain large, negatively-charged contaminants such as DNA, RNA, leached Protein A, HCPs, endotoxins and viruses—whereas the second step exploits the fact that the feedstream is relatively pure at this stage, and therefore the binding capacity of the membrane is unlikely to be challenged.

The way forward

There is no doubt at all that pressure on downstream processing will increase over the next decade. There are more products in the pipeline, a greater demand for drugs, more awareness of regulatory issues and greater acceptance of biotechnology, plus lots of biotechnology-derived drugs coming off patent leading to an increase in the manufacture of biogenerics. The outlook for biochromatography is therefore challenging because today’s processes based on the

Scale-up with Single Sep Q membrane chromatography (assuming constant bed height of 4 mm) Frontal surface area (cm2)

Scale-up factor for flow rate

Rec. flow rate (l/min)

Bed volume (ml)

Min. static binding capacity(g) (Release test)

Dynamic capacity at 10% (mg/ml)

Dynamic capacity at 100% (mg/ml)

nano

2.4

1

0.03

1

0.03

22.5

39.0

5"

160

66

1.90

70

2.00

19.5

30.0

10"

450

187

5.00

180

5.30

20.5

29.5

20"

900

375

10.00

360

10.50

20.5

35.0

30"

1350

562

15.00

540

15.80

20.5

37.5

mega

4050

1687

45.00

1620

47.00 Table 1

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M anufacturing

multiple products with minimal turnaround times, maximising the use of resources and minimising the use of space. However, the main bottleneck in DSP will always remain at the initial capture step, since this is driven by mass rather than volume and has a knock-on effect throughout the facility.

Membrane adsorbers are unlikely to replace Protein A columns for the foreseeable future unless there is a revolutionary leap forward in the technology to allow more efficient capture. Therefore, the industry faces the prospect of further parallelisation (more columns per

A uthor

use of oversized columns stuffed with expensive resins are becoming increasingly uncompetitive. The continual scaling up of processes is unsustainable, and needs to be given a serious thought.

More and more companies will look at adopting membrane chromatography for polishing, since flowthrough operations are considerably less expensive and more convenient when membrane adsorbers are used.

Any cost savings in processing can lead directly to a reduced cost of goods, and this can only be good for the industry and, ultimately, its customers. The convenience of membrane adsorbers is possibly more important than the economic benefits, since the adoption of a fully disposable process train with plastic buffer and media bags, disposable bioreactors, single-use filters and single-use adsorbers will eliminate the need for cleaning and validation. It would also help the facilities to produce

process) or the adoption of other technologies, e.g. innovative resins with mimetic ligands, or the replacement of Protein A with other chromatography modes that are cheaper and more scalable. If no replacement for Protein A can be found, the problem could be circumvented in a radical and unorthodox manner by simply removing the capture step all together. It is not inconceivable that processing in the future may be based almost entirely on polishing and removing contaminants from the feedstream rather than capturing the product.

Uwe Gottschalk has a PhD in Chemistry from the University of MĂźnster, is Group Vice President, Purification Technologies, with a global responsibility for bioseparation products at Sartorius Stedim Biotech. He worked in different capacities for Bayer Health Care from 1991-2004 and became head of the GMP protein purification facility in Wuppertal (Germany). He was responsible for the production of monoclonal antibodies and other recombinant proteins using various expression systems.

w w w . p h a r m a f o c u s a s i a . c o m 65

CLINICAL TRIALS

Retention of Subjects The question of informed consent

Although the factors that lead to the loss of subjects in clinical trials are poorly understood, “lost to follow-up” is still too frequently recorded as an outcome. This article explores the issues involved in the planning and conduct of clinical trials, and suggests a number of practical and relatively simple steps that may be taken to enhance the retention of clinical trial subjects.

J Findlay Walker Vice President, Administration, Daiichi Sankyo Development Ltd., UK

T

hese days, pharmaceutical development is increasingly becoming an area where “hightech” solutions exist for every difficulty encountered. Genomic analyses and biomarkers are required for enrolment in an ever increasing proportion of studies. Contract Research Organisations (CROs) have electronic files of potential investigators and patients for many diseases, and Electronic Data Capture (EDC) and electronic filing are the modern way to go. For all of the seeming sophistication, one issue, the retention of subjects in clinical trials, has not changed much in decades. The abbreviation LTFU (lost to follow-up) is still used all too frequently!

In addition to patients being entirely “lost” to follow-up, there is another group who will have their assigned medication discontinued for known or unknown reasons, but who can still be located to ascertain their vital status. In a study of say, three to five years’ duration, it is likely that 25 per cent or more of

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It is a good practice to have the prospective subject review the document with a study coordinator to answer any questions. The study personnel must be fully aware of the details of the study and must answer all questions fully and honestly.

patients will fall into this category. The actual proportion will vary depending on the initial indication: life-threatening conditions, like acute myocardial infarction attract better persistence with therapy than perceived low-risk diseases like hypertension.

There are both scientific as well as financial benefits of retaining subjects after randomisation. Scientifically, the answer to the question being asked in the specific study may be jeopardised, but the credibility of the company involved, the generalisability of the results generated and their regulatory persuasiveness are also at stake. Financially, the cost of qualifying a replacement can be high, especially if that requires diagnostic interventions, genomic analyses or the validation of costly biomarkers.

Over a number of years, various strategies have been employed to enhance compliance and persistence with assigned medication. These have included automated and manual telephone reminders, as well as medication containers

CLINICAL TRIALS

Ways to retain study subjects or retaining study subjects

Ensure that each visit is a pleasant experience Ensure that punctuality is maintained Make certain that appointment times are suitable to the subjects Call the subjects personally to remind them about their upcoming visits See that the number of study personnel is limited Make sure that questions posed by subjects are answered honestly Ensure that the personnel are always available to subjects

w w w . p h a r m a f o c u s a s i a . c o m 67

CLINICAL TRIALS

Unacceptable practices

Before dealing with ways to enhance retention, some unacceptable ways of patient retention are considered briefly.

The first of these is financial coercion. While the payment of expenses incurred by the patient in participating is permitted (e.g. actual travel costs and reimbursement of lost wages), higher payments are not. One has to bear in mind that a payment that is not objectionable in the US or Western Europe (say US$ 50) may be considered coercive if offered to an impoverished subject in India or elsewhere.

In a recent case reported by the US FDA, a psychiatric investigator was cited for keeping a patient in a locked ward against her will, an obvious infringement of acceptable practice (except under specific circumstances in which the patient may be “sectioned” under the Mental Health Act).

Making continued treatment dependent upon return to the investigator is also unacceptable, but this prohibition should be a part of the informed consent procedure. Using family members to persuade patients to continue in study may also be questionable, as it may undermine the view of a subject as a “free agent”.

Acceptable practices

The acceptable ways to enhance retention are mostly “low-tech” and may be considered “boring”, since they incorporate practices that have been widely used

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for many years by experienced investigators and their research staff. For the patient, the prospect of participating as a subject in a clinical trial may be a little daunting, and the first step to take is to make the patient feel comfortable in the new situation.

How is the informed consent procedure handled? Problems with the adequacy of informed consent are among the most commonly identified issues during regulatory audits by the FDA and other agencies. Sometimes it seems as if the signing of the Informed Consent Form (ICF) is a formality to be dispensed with as quickly as possible, so that the real business of the investigator can be engaged: physical examination, special diagnostic testing and bloodletting!

For the average patient, for whom this may be a first experience as a subject, the ICF is a complex document, incorporating as it does, the details of the study, frequency of evaluations, consequences of non-participation, likelihood of adverse events, availability of care for any adverse event, as well as details of indemnification for resulting in any injury. It is a good practice to have the prospective subject review the document with a study nurse or coordinator to answer any question. The study personnel must be fully aware of the details of the study and must answer all questions fully and honestly.

Sometimes it may take more than one visit before the patient feels sufficiently informed to give consent.

Once the patient is enrolled, there are a number of simple steps that can be taken to reduce the likelihood of drop-out. A telephone reminder of each

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that emit an alarm or a vocal message reminding the subject that the dose is due or that a follow-up visit is pending.

Some people have mixed feelings about being reprimanded by a pill bottle. With the widespread availability of Internet access and cell phone technology, these tools are increasingly in use despite the limited geographic penetration, especially in non-traditional sites (e.g.

India, South-East Asia, etc.) where drug development studies are now increasingly being conducted.

visit is recommended, preferably not by an automated calling system, since a personal call can positively motivate the patient who is equivocal about continuing. As far as possible, the subject should be seen by the same study personnel at each visit so that he / she may build trusting relationships that will facilitate the solving of any problem that arises in the course of the study.

Additionally, the waiting area for appointments should be pleasant, although, little used, since appointment times will be punctually adhered to. The timing of available appointments may also be critical with regard to the subject’s willingness to participate and comply. A 7.00 a.m. appointment may be ideal for a subject in full-time employment, but may be unattractive to a retired person who may prefer to wake up late. Unless the protocol calls for a trough reading of blood pressure, pharmacokinetic or other variables, such early morning appointments may be counter productive in some cases.

Finally, the subject should be given the name and contact details of the appropriate contact person(s) if difficulties or questions occur in the course of the study or if suspected adverse effects of treatment arise.

In summary, the subject is more likely to continue in the study if the overall experience of participation is positive, if relationships with study personnel are open and honest and if the subject is treated with dignity and fairness in all respects.

May LTFU be banished from the clinical study lexicon!

Findlay Walker is the Vice President and Managing Director of Daiichi Sankyo Development Ltd. He graduated in Medicine from the University of Aberdeen, Scotland. He worked for Merrell National Labs and was employed with Merck and Co, Inc., for over 20 years. He developed drugs like amiloride, timolol, enalapril and simvastatin. Taking early retirement from Merck, he worked as an independent consultant for almost 3 years. He joined Sankyo Pharma Development as Senior Director of Clinical Development in April 2002 after which he became Vice President of Medical Affairs at Daiichi Sankyo Inc.

w w w . p h a r m a f o c u s a s i a . c o m 69

Clinical Trials in China Mark Engel Chairman, Excel PharmaStudies, China

What are the prospects for conducting clinical trials in China?

There are a number of driving factors that make China very attractive for conducting clinical trials. People are already aware of most of these factors—relatively inexpensive market in China, its growth potential and reasonably easy access to patients. Additionally, there has been an increased push from the US for Post-Marketing Surveillance (PMS). In this regard, China is perhaps the most attractive destination in the world for doing this sort of work for two reasons.

There are two reasons in addition to the ones normally sighted for conducting trials in China—lower costs and access to many patients. As we know, the China market is very robust for sales with more than 20 per cent growth per annum. However, lesser known is the fact that the cost of changing physician prescribing patterns at leading hospitals (which account for a majority of the sales of “western” products)

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is very expensive. The repetitive nature of PMS trials allows for a cost-effective manner of changing these patterns. In addition, most people do not understand that it is difficult for companies to costeffectively remove profits from China.

Therefore, companies are looking for alternative ways to “export” profits and one way is to conduct clinical trials and exporting data.

Is getting approval for a clinical trial application a time-consuming process in China? If yes, how will it affect the market and the players?

The simple answer is yes. However, what most pharma companies look at is not just approval time but, more importantly the timeline for the first patient enrolled.

Ethical Committee (the China equivalent for an IRB) approvals and hospital contracting, both normally take less than six weeks in total. So, although it takes a long time to get approvals in China, the time taken to get the first patient

enrolled is very less. When we consider this fact, China is not nearly as slow as people imagine.

What is the current status on intellectual property issues in clinical trials in China?

Today, we have as our client base 19 of the top 20 global pharma

E X P E R T TA L K

companies. Every one of them is utilising China as part of their global clinical trial activities. Today, the issue of IP is no longer a matter of great concern, at least among Big Pharma. The law was changed significantly few years ago to ensure enough protection to new intellectual property.

Do you feel that foreign CROs have an edge over the local companies in China?

We can classify ourselves as a local company with internationally experienced management. Such a combination of global expertise at reasonable pricing has proved to be the winning combination in China. We have been able to retain international management and knowledge and at the same time are able to navigate the idiosyncrasies of the China market.

Generally speaking, the purely local CROs do not have the expertise to service all the needs of the multinational pharma industry. Conversely, global CROs are quite expensive and have a mixed performance history. The typical local CRO works primarily for Chinese pharmaceutical companies.

What are the steps being taken by CROs to reduce the gaps between the international and Chinese standards for conducting clinical trials?

The difference between Chinese and global standards is relatively little. The difference is largely in the standard of thoroughness rather than the standards themselves. For example, if you look at a local CRF, it tends to be about half the length of a multinational CRF. So, although the same ICH GCP standard is applied to both CRFs, the amount of information that a local company needs is significantly less than what a global client might need. This is understandable since local companies primarily work with generic pharmaceuticals. Similarly, the amount of monitoring visits and quality review required for local trials is far less than that of their foreign counterparts with innovative products.

While the standards are basically the same, the primary difference lies in how robustly one follows those standards.

What are the new opportunities / areas for clinical trials in China?

The largest area of growth that we have seen in the last year is in PMS studies. In November 2007, a new law was enacted in the US called H.R. 3580, in which the FDA was given both the budget PROFILE

What is your opinion on the way the issues of informed consent, ethics and regulatory compliance in clinical trials are addressed in China?

There are about 250 hospitals in China that have been approved to conduct clinical trials by the government for the purpose of utilising the data for local registrations. These hospitals are carefully checked by the government before they are approved to conduct clinical trials.

Such checks include how they handle informed consent. So among these top hospitals today, there is a good record of being able to register true informed consent from patients. Having said that, we at Excel are willing, should our clients ask us to go one step further, to actually videotape the patientsâ&#x20AC;&#x2122; consent.

But generally speaking, informed consent among the top sites and top investigators is really a non-issue.

The number of trained staff to conduct the increasing trials as compared to the amount of work coming to China is far from sufficient.

and considerable enforcement powers to require companies to do large-scale PMS trials. The result is that many US companies are now struggling to find ways in which they can enroll large numbers of patients at a relatively low cost. Only China and India are able to meet this bill. The PMS studies have seen the biggest spurt in the business that we have seen in the last year or so.

What according to you needs to be done to give China an edge over competition from the other countries?

One important issue is the lack of qualified staff. The number of trained staff to conduct the increasing trials as compared to the amount of work coming to China is far from sufficient. And, probably this is going to be the biggest hurdle in the coming years and we need to find more and better ways to train people involved in clinical trials.

Any other area that you would like to focus on?

I think that we are seeing a lot of trials in the oncology area as well and the reasons are two-fold. One is the availability of patients and the other is relatively low-cost trials. However, a major factor that distinguishes China from other competitors such as India, Russia and Brazil is that there are many patients in China who can afford expensive products.

In fact, just about all of the multinational pharmaceutical companies have in-licensing groups that are particularly focussed on oncology. So, other than post-marketing trials, oncology trials are the other area where we continue to see a significant increase in demand.

Interview conducted by Bhamoti Basu, Editorial Associate, Pharma Focus Asia

Mark Engel is the co-founder of Excel PharmaStudies, the largest full service clinical research organisation in China. Mark is also the co-founder of several other medically related companies in China: (1) Haoyisheng, the leading health care information, education and database management company in China with about 400 employees; and (2) Tiger Medical Group, including Tiger Medical Products, Tiger Health Care Group, and Tiger Medical Sourcing.

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The discovery of a disease biomarker using mass spectrometry involves careful planning and strategy.

The article describes how a rheumatoid arthritis biomarker was identified and explains various steps in biomarker discovery.

Leticia Cano IRTA Postdoctoral Fellow, Laboratory of Applied Mass Spectrometry, National Heart, Lung and Blood Institute, National Institutes of Health, USA

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P

roteomics is an emerging field depleted synovial fluid was fractionated that is frequently used for cliniusing a Beckman ProteomeLab PF2D cal biomarker discovery. For Protein Fractionation System. Proteins this article, proteomics is defined as were fractionated in the first dimena mass spectrometry-based approach sion by chromatofocussing and in the used to identify proteins. The biomarsecond dimension by reverse phase High ker discovery process is distinct from Performance Liquid Chromatography. biomarker validation or the developThis protein fractionation strategy ment of a clinical test. The development increased the chance of identifying the of a clinical biomarker incurs heavy lower abundance proteins by separatexpenditure and also requires relevant ing them from the higher abundance expertise and resources. The biomarker proteins, which could mask their presdiscovery process consists of choosing ence. The second dimension fractions the best proteomic strategy, the right clinical samples, selecting the appropriate protein sepaA biomarker for RA was identified in the rations, performing the best mass spectrometry, analysing synovial fluid of patients with RA using the data and verifying results a modified strategy previously used to with another set of clinical identify cancer biomarkers. samples and assay. The strategy used to discover a biomarker in Rheumatoid Arthritis (RA) and the process of biomarker discovery were used to construct protein arrays on are discussed below. nitrocellulose membranes, which were used to test for the presence of autoantiRheumatoid arthritis biomarker gens by analysing differential reactivity of discovery RA and control sera. Only sera obtained A biomarker for RA was identified in the from RA patients is thought to contain synovial fluid of patients with RA using autoantibodies which bind to autoantia modified strategy previously used to gens. Fractions that tested positive when identify cancer biomarkers. An Agilent probed with RA serum but negative when immunodepletion column was used to probed with normal control serum were remove six abundant serum proteins found in the first dimension fraction elut(albumin, antitrypsin, haptoglobin, ing at pH 5.63-5.45. In order to obtain IgA, IgG, and transferrin) then the enough material for mass spectrometry

analysis, SF from nine different patients was pooled and used for a large-scale procedure. Fractions corresponding to the region that tested positive for RA serum binding in the protein array were digested with trypsin and analysed by LC / MS / MS. SEQUEST searches were performed using the SwissProt database limiting the search to tryptic peptides. From the protein array experiments, the candidate autoantigen specifically detected by the RA serum was estimated to be in fractions 20-22. Amongst other proteins, these fractions contained fibrinogen, a known autoantigen that can be citrullinated in vivo.

Fibrinogen alpha (FIBA_HUMAN, SwissProt Accession # P02671) was identified in fraction 20 with 9 unique peptide hits (15 per cent sequence coverage) and in fraction 22 with 18 unique peptides (24 per cent sequence coverage). Peptides were only found originating from the centre of the fibrinogen protein (amino acids 250-599) corresponding to the alphaC domain of fibrin (amino acids 221-610).

Careful examination of the mass spectra assigned to arginine-containing fibrinogen peptides led to the assignment of a citrullinated peptide corresponding to fibrinogen 259-287. The mass calculated from the mass spectrum was one Dalton higher than the calculated mass for the unmodified peptide.

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CLINICAL TRIALS

The lower resolution MS / MS spectrum obtained with the ion trap part of the LTQ-FT did not allow determination of the exact location of the modification.

However, a number of other fragments corresponding to parts of the peptide were observed in the spectra obtained with the Ion Cyclotron Resonance (ICR) analyser of the LTQ-FT, which provided precise mass measurements. There are four sites on the fibrinogen 259-287 peptide that could possibly be modified resulting in a mass shift of +1 Da. The peptide contains three arginines that can be citrullinated and an asparagine that can be deaminated to form aspartic acid. All fragments that did not show the expected tryptic cleavage at Arg 271 were one Dalton higher than the expected mass for the unmodified form.

Trypsin does not cleave after citrullinated Arg residues. The conversion of Arg 271 to citrulline is consistent with the failure of trypsin to cleave at that site.

As a final proof that the citrullination site was correctly assigned, the peptide corresponding to residues 259-287 was synthesised with and without the citrulline in position 271. Both the charge state distribution in the MS spectrum and the fragment masses in the MS / MS spectrum of the 3+ charge state of 271X matched spectral data obtained with the clinical sample.

To establish that the citrullinated fibrinogen 259-287 peptide was

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recognised specifically by RA patient sera, the two fibrinogen 259-287 synthetic peptides were tested in an ELISA. An additional citrullinated synthetic peptide, corresponding to profilaggrin 619-631 (FIL) with a citrulline substitution at Arg 625, was included as a control. The immobilised peptides were incubated with sera from RA, SLE, or healthy controls, followed by detection of bound antibodies

by HRP-conjugated anti-human IgG, IgA and IgM antibodies and a colorimetric assay. Of 18 healthy control sera tested,

antibodies in RA sera bind specifically to a fibrinogen peptide biomarker generated by substituting arginine to citrulline at position 271. This study proves that clinical biomarkers can be discovered using proteomics.

General guidelines for discovering more biomarkers are described in the next section.

Biomarker discovery process

The first and most important step of biomarker discovery is to plan the proteomic strategy. Each strategy needs to be custom-designed to achieve specific goals for each individual biomarker discovery Of the 12 RA sera tested, four project. There are many differreacted to the 271R peptide, 10 reacted ent proteomic approaches, to the 271X peptide and three reacted each with distinct advantages to the FIL peptide. Of the 10 SLE sera and disadvantages. The plantested, one patient reacted to all three ning should be made regarding the required amount, peptides. number, type and quality of the samples. An assessment of the available equipment, expertise, two reacted to the 271R peptide, two and computer resources has to be done. reacted to the 271X peptide and 1 reacted The details such as the amount of time to the FIL peptide. Of the 12 RA sera that can be dedicated to the project, tested, four reacted to the 271R peptide, the expected budget, and competing 10 reacted to the 271X peptide and three projects, if any, have to be considreacted to the FIL peptide. Of the 10 SLE ered. A lot of time needs to be spent sera tested, one patient reacted to all three on this step. peptides. The number of sera that reacted The selection of the clinical samples is exclusively to the 271X peptide, and not the second crucial step. Clinical proteomwith the 271R or FIL peptides, were 5/12 ics should use a team science approach RA sera, 0/18 healthy sera, 0/10 SLE with leaders from different disciplines. sera. These results provide evidence that Input is needed from each of the leaders

CLINICAL TRIALS

Mass spectrometry should be performed by an expert or in collaboration with a mass spectrometry facility.

There are many types of mass spectrometers. The type of mass spectrometer used for analysis should be discussed and the mass spectrometrist should be trained to use that particular instrument. The scientists of the instrument company can be approached for any advice. In using a core facility for a large number of complex samples that require expert handling and analysis, the scientists may be expected to ask for authorship. This is fair since their expertise will be important for the project.

Analysing the mass spectrometry data is the most time-consuming part of the project. Complex projects can result in hundreds of thousands of spectra. Protein identifications are performed by matching experimentally derived mass spectra to theoretically derived spectra obtained from a specific database. A statistical analysis is performed to rank the best matches.

Even the poorest spectra will be assigned a match and the software can match only what is there in the chosen database.

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and there needs to be an honest discussion amongst the group. The samples that are likely to contain biomarkers that can be identified using the available resources, have to be chosen. The necessary patient information required to complete the project has to be collected during this stage itself because the access to this information might not be available at a later stage. The entire process of collecting and processing the samples has to be monitored closely.

Serum or plasma are complex proteomes and are unlikely to yield biomarkers using shotgun approaches. Proximal fluids and tissues are more likely to contain biomarkers in relatively high concentrations. Using a biorepository to obtain all or a few clinical samples might be considered.

Protein separations should be designed to produce clean, pure and fresh samples for mass spectrometry. Separations and any sample processing should be performed in clean tissue culture room-type environments. One should be careful not to introduce any new contaminants (detergents, salts, and keratin) that might interfere with the analysis and be aware of the fact that high concentrations of sample may contaminate the instrument and interfere with subsequent runs. Sample carryover issues may become a problem in shared instruments. The proteins that bind non-specifically are a problem in every experiment and the same proteins will bind non-specifically in different sample sets.

Searches performed with different databases, search parameters or algorithms can yield slightly different results. It is important to estimate the probability that the correct peptide and protein have been identified.

The final step of biomarker discovery is verification. Generally this is an ELISAbased experiment performed with a different and larger sample set. The samples should include disease samples, health and disease controls. The disease controls will help identify markers of inflammation. The sample set should reflect the population seen in the clinic. The final point is to find people who are genuinely interested in studying the particular disease. Although the long hours spent to complete these enormous projects cannot be compensated, people need to feel proud that their work is meaningful.

People need to value the clinical samples and involve themselves in the process of innovation. Also, it requires a lot of time for planning the strategy.

Full references are available on www.pharmafocusasia.com/magazine/

Leticia Cano is currently an IRTA Postdoctoral Fellow in the Laboratory of Applied Mass Spectrometry at the National Heart, Lung and Blood Institute (NHLBI) of the National Institutes of Health (NIH). Her research focusses on identification of biomarkers in autoimmune disease and the development of novel protein separation strategies. She identified a biomarker for rheumatoid arthritis for her PhD dissertation at the City of Hope Graduate School of Biological Sciences. She is also a former MALDI-TOF MS Applications Scientist with Bruker Daltonics Inc.

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Eliminating Guesswork Automating PAT PM process control

Modern process analytical technology systems generate continuous quality monitoring and provide plant process controllers with highly useful process information to increase the productivity and adhere to higher quality standards.

Frank E Sistare, Principal PAT Scientist, CAMO Technologies Inc., USA

T

he words â&#x20AC;&#x153;process controlâ&#x20AC;? as related to Active Pharmaceutical Ingredients (APIs) production are interesting.

Process can mean anything from simple single-step reactions to very complicated chemistry, multi-step processes and continuous processing, while control implies some state of stability and knowledge of the process and process steps. Upon closer examination of current processes, it is apparent that a typical process control relies heavily on offline testing at the end of each step. The current testing pattern is not effective to control or characterise the chemistry or processes that generate much of the APIs produced. A much more effective control scheme, illustrated here, includes the use of automated PAT techniques. Additionally,

as we move through the 21st century, new API entities will be developed using Quality by Design (QbD) techniques generating a higher level of process stability and understanding.

This is accomplished in laboratories and small-scale production facilities in a research and development mode.

QbD philosophy can be applied to current processes but it entails great effort and expense making QbD ineffective for current production. Current large-scale commercial production of APIs pose their own difficulties and require techniques to ensure the processes continue to perform efficiently,

NaHSO3 reduction of excess bromine (Compound B)

Compound A

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Compound B+Br2 (>650 mv)

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Compound B+NaBr (<500 mv)

Figure 1

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Graphical representation of millivolt response

Millivolt response

Process â&#x20AC;&#x153;Redoxâ&#x20AC;? Monitoring

Sample#

thus PAT. Several PAT automation schemes are illustrated here describing traditional and current automation solutions.

Traditional PAT communications

Older PAT techniques such as ph and redox probes generate useful data that is easily interpreted in the form it was generated. Such values were traditionally available as a meter response measuring the electronic potential of a reaction.

The output as generated, i.e. millivolts, was easily sent to a computer or process controller via an analogue transmitter. The simplistic nature of the data and analogue transmission does not diminish the usefulness of the application data. The following example of a simple redox application to monitor and control a bromine reduction during critical processing highlights the process knowledge and control gained when using this simple PAT application.

In this reaction, (Figure 1) a molar excess of bromine is added to compound A in an acidic reagent to form the di-bromo compound B. The reaction is further treated with sodium meta bisulfite to reduce the excess bromine to a slightly positive endpoint. A slightly positive bromine endpoint is required to protect the dibromo compound from decomposing

Graph 1

to a mono or des bromo compound.

Once the reaction is complete, sodium meta bisulfite is added by theoretical weight to reduce the excess bromine to the desired endpoint. Several factors can affect the amount of sodium meta bisulfite required to complete the excess bromine reduction. These variables were observed but found not to be consistent from one batch to another.

Some variables that could affect the reduction were caused by operational differences such as temperature,

raw material weights and tank rinses between batches. For example, if the temperature of the reaction varied upward a few degrees from -10°C, bromine could be lost in the vapour phase. Less bromine would require less sodium meta bisulfite during the reduction reaction. The sodium meta bisulfite is added by standard weight based on the theoretical excess bromine.

If the amount of bromine is reduced by one of the variables, the addition of a standard weight of the reducing agent could mean over-reduction of bromine requiring a recharging of bromine and further repeating the sodium meta bisulfite reduction. Losses in efficiency and raw material add unwanted cost to the product. Earlier reaction monitoring was done visually, when the operator thought the reduction was complete and after a standard sodium meta bisulfite charge, a sample was taken for laboratory analysis. Through laboratory experimentation, the desired millivolt (mv) response to achieve the correct bromine endpoint was determined to be around 550 mv. The result of a successful reduction reaction using redox and analogue communications (Figure 2) is graphically illustrated (Graph 1).

Analogue Transmitter Through laboratory experimentation, the desired millivolt (mv) response to achieve the correct bromine endpoint was determined to be around 550 mv. The result of a successful reduction reaction using redox and analogue communications is graphically illustrated.

Figure 2

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The next generation

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Closed loop control of water in the reaction – Target 1.25% water NIR vs KF week 4 2.00 2.25 2.00 1.75 % water

PAT used as a tool to gain process knowledge has been firmly established.

As modern PAT and process automation became available, it was apparent that PAT could be included in the automation scheme. The theory was simple. Translate the process knowledge developed using PAT into a process control application to improve product quality, then reduce process variation, and increase productivity and product yield. A Near Infra Red (NIR) spectrometer was used to predict percentage of water in a continuous process (Figure 3). Water caused variation and with too much water a competing hydrolysis reaction took place resulting in a reduction of the product yield due to the excessive soluble product losses to the mother liquor. Too little water caused a crystal habit change (small crystals and a thick granulation slurry) causing operation shutdowns and system flushing resulting in a loss of productivity and low yields.

The PAT application used chemometrics to predict the water percentage. The water value (as given by Karl Fisher) was sent to an analogue input / output converter as a RS 232 signal.

The RS 232 signal was converted to a 4–20 millivolt signal and sent to the process controller. The analogue signal was then placed into a multiple linear regression algorithm to regenerate the predicted Karl Fisher (KF) value from the millivolt signal. The operator could then respond to the KF value as seen at the process controller. The application was further developed to eliminate the human factor. Once the predicted water value was regenerated it was automatically directed to the process controller code that automatically adjusted the span of the water valve (Figure 3). The water valve was effectively controlled by the NIR which predicted water values by automatically opening and closing the valve as required to maintain the correct water content of the reaction. The process

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control and stability gained (Graph 2) was phenomenal and resulted in higher yields and no productivity loss.

Current (Modern) process

Modern process controllers use Object linked and embedded for Process Control (OPC) communication protocols to communicate with

and control process equipment. This two-way communication and process control infrastructure increases compatibility. The latest PAT instruments and software are programmed with OPC capabilities. OPC communications allow predicted values to be sent directly to the modern process controller as generated or predicted. The PAT appli-

M anufacturing

to review, statistically evaluate and model data while data collection is being done.

The whole system including the PAT instrument, ancillary device control and data evaluation elevates the PAT system to a true process control tool resulting in increased productivity and yield while reducing risk. The holistic system meets the FDA’s 21st century definition of PAT for Process Control.

Conclusion

Simple or complicated PAT instruments generate data that can be used to control processes by generating real-time feedback with the automated process

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cation is server-based residing on the local area network and can be accessed via the internet and client PCs with proper permissions. The use of PAT, OPC and the process controller results in a broad range of benefits allowing the developer to meet FDA, GMP and 21CFR part11 requirements, such as automated diagnostics and secure data storage thus eliminating the risk of having poor or loss of data.

System flexibility extends further to the operation of ancillary devices that may be required for efficient PAT instrument operations. Automation enables the process controller to receive reports concerning the health of the instrument as well as alarms reporting out-of-range results or instrument errors. Additionally, the application of independent software residing on the process controller such as the “Unscrambler software package” (CAMO Software) enhances the PAT system by providing an independent tool

controllers using IT and automation solutions thereby enhancing process knowledge and control. PAT generates continuous quality monitoring by measuring “Critical to Process” and “Critical to Quality” attributes (i.e. productivity, yield, over / under reaction and impurity formation) in situ. These facts give the PAT administrator and production supervisors the confidence to process forward prior to completing conventional in-process testing with little or no risk. The modern PAT process interface delivers all the needed elements for a complete, GMP-compliant, modern and effective PAT process control system.

Frank Sistare has over 33 years of experience in Pharma with over 23 years of experience across all aspects of PAT. His expertise includes PAT systems installation, IT / automation integration and PAT systems validations.

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Using Chemometrics in PAT Investigating data from the process

Processes of today are often equipped with sensors and measurement systems collecting large amount of data. Chemometrics is developed to handle large amount of data for overview (monitoring) and relations between raw materials, process parameters, instruments and Critical Quality Attributes.

Petter MĂśrĂŠe Director, Online Products, Umetrics AB, Sweden

I

ndustry of today is generating large amount of data. Data that comes from raw materials, excipients, media and the process. Most processes consist of a series of operations (steps) and are also equipped with instrumentations collecting parameters as the process is in operation. The chemometrical analysis of the numerous data collected from such processes provides characteristic patterns relating to groups (classes), trends, and other relationships. The chemometrics technique makes it possible to interpret these patterns and drill down to the possible root cause. The interpretation should when possible be compared with prior process-, chemical-, physical- and biological knowledge. A PAT example illustrates the approach.

Chemistry is primarily an experimental science, where most of the knowledge is based on experiments and measurements, i.e. data is collected under more or less well controlled conditions. Hence, the interpretation of measured data is central to the progress of any project and in any field of chemistry, from physical and inorganic, to biochemistry and molecular biology.

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Before the advent of user-friendly computers, around 1965, the only way to interpret these data, i.e. converting the data into easily interpretable knowledge, was to use simple concepts and models, such as mean values for different classes, ANOVA, linear regression and correlation. These were happy times because almost anybody could decide by means of a t-test whether two classes were significantly different or not. Therefore, they 110 could also judge whether a productâ&#x20AC;&#x2122;s quality property had changed 100 significantly with time (Figure 1), and could recognise if there is a significant linear relationship between, say the log 90 of a rate constant and 1/ temperature (in degrees Kelvin). However, the 80 situation has changed since 1970s.

Now, each experiment derives multiple data from 70 spectra, chromatograms, kinetic experiments,

gene arrays, and often combinations thereof. Therefore, the simple averages, t-tests, lines, and slopes do not work anymore.

Chemometrics

Chemometrics is specifically designed to handle many data. Around 1970, chemometrics was initiated by Kowalski, Massart, Wold, and co-workers to deal with the data-sets with many variables.

Tools such as Principal Components Analysis (PCA) and Partial Least Squares Projections to Latent Structures (PLS) were used to handle many experimental values for the same

a

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Figure 1

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Raw Material

Granulator

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PLS with Quality

Figure 2

types of questions as beforeâ&#x20AC;&#x201D;whether there are differences between classes of samples, and whether there are significant relationships between sets of numbers, e.g. in a synthesis between (i) experimental conditions (X) and (ii) resulting yield and impurities (Y). The tools of chemometrics are designed to give interpretable results, meaningful to the scientist / engineer. Class differences

are expressed as either the data profile related to the class difference, or the data profile for each class. Relationships are expressed in profiles of variation, i.e. which combinations of data are related to each other, how, and in what strength, all with measures of uncertainty such as statistical confidence intervals, and diagnostics for data quality. This makes the interpretation of the results very similar to that of the old days, except that the entities plotted and evaluated are no longer single measurements but are combinations of many. These combinations (called scores) are calculated to maximise their information content with regard to the stated questionsâ&#x20AC;&#x201D;classification or relationships. The weights (loadings) with which the individual measurements are combined to form the scores indicate their relative Figure 3

importance in the measurements, and the correlations between them. An illustration of the basic tools of chemometrics is given by a PAT project run at Novartis, Suffern, USA. PAT rests on good data, data management and data analysis

The term Process Analytical Technology (PAT) has at least two meanings. First, PAT is a regulatory framework of the FDA for enabling pharmaceutical manufacturers to develop and implement new and efficient analytical tools for use during development, manufacturing and quality assurance to maintain or improve the current level of quality assurance. Second, to the technically oriented pharma community, PAT signifies the technology to achieve real-time quality control in pharma manufacturing, without any decrease in the quality level. This technology includes the instrumentation and sensor arrays providing the data that characterises raw materials, intermediates, products, and the manufacturing process itself, plus the tools to relate the data to the critical quality issues (Figure 2).

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Figure 4

Figure 5

Projections to Latent Structures

With PLS, large datasets containing measurements from raw material quality and manufacturing process conditions can be combined into a single model to identify the cumulative influence of the production environment on the product quality. PLS identifies the sources of variation from the process (X) that correlate with quality (Y). PLS can handle a highly correlated X space retaining a high level of interpretability for interrogation of the model itself, which is essential for development of an understanding of the process and identification of key influences. The hierarchical nature of PLS allows for modular model development, meaning that separate models can be built for raw materials and each unit operation, which later can be combined to form a global view. This allows the starting point for PAT activities to development e.g. end-point detection for a dryer or granulator.

Example of PAT

The pharmaceutical manufacturing process of Novartis, Suffern, New York is discussed here as an example

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of the approach. The objective of the analysis is to find reasons for a recent change in the process quality, particularly the dissolution rate of the final tablets which often was too low. This is a secondary production process.

Drying and compacting were thought to be the steps critical for the dissolution rate of the final product. Each process step is batch-type (non-continuous). On each step, a set of variables (K between 7 and 50) is measured from beginning to the end at regular intervals. In addition, “one shot” point data (characteristics of seven raw materials) and final quality of the batches are measured. The data comprise N = 314 Batches (approximately two years of production).

First, an offline analysis

The analysis is based on the ability of PLS to summarise higher order data (3-way dynamic data of batches, i.e. measurements × time × batches). Here, only the data collected from the drying steps (two steps) are included in the first analysis of the 3-way data. Summary data, average and standard deviation from first step is used and modelled. The second

drying step is dynamic and needs 3 PLS components (y = local time), modelling 50 per cent of the X-variation (the process data). The static and dynamic results are thereafter, together with raw material characteristics, used as X-variables in a second (upper) PLS model, with dissolution rate used as Y in the PLS modelling (Figure 2). A significant relation (3 PLS components) is obtained at the upper level, giving the scores shown in Figure 3. These are coloured by dissolution rate (red is high, blue is low), showing the relationship between the joint score values and the dissolution rate. The corresponding PLS-weights in Figure 4, identify dominant variables (at the lower left and the upper right in the plot). The moderate explanation of y = dissolution rate is seen in Figure 5 which is coloured by time of observation. Recent batches (red = large time values) are seen to have a tendency to have low dissolution values.

The coefficients clearly identify the raw materials as dominant factors relating to the variation of the dissolution rate (left half of Figure 6), but a few coefficients of the two dryer phases are also being somewhat important (right half of Figure 6).

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Second, online modelling and analysis

The model coefficients were used to modify the control strategies for the dryer also tighter control of the raw materials was implemented. An online monitoring system based on multivariate modelling was then installed, and additional data were included in the modelling covering more of the total process (Figure 2).

The results were very encouraging. The dissolution rate stabilised at a higher level, with very little or no inferior product being made. The upper level model was able to have a better accuracy between observed and predicted for dissolution rate and other CQAs, enabling early fault detection and process monitoring.

Conclusion

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Chemometrics addresses the data explosion and its consequences. The

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data emerging from chemical laboratories, product development, and manufacturing are nowadays too numerous for traditional statistical tools, and chemometrics has been developed to face this emerging situation. Checking that a process is doing what it is supposed to, is of critical importance in manufacturing (PAT), ensuring that the critical quality attributes are met on the product and the process. Determining the functional relationships that link material attributes and process parameters to product Critical Quality Attributes (CQAs).

These results can serve for control strategies, if monitoring and prediction is achieved and understood giving improved process knowledge and process understanding.

Full references are available on www.pharmafocusasia.com/magazine/

Petter Mörée is currently Director of On-line products for Umetrics.

Petter is also responsible for sales to large Pharmaceutical companies in the Germanic market. In both of these areas Petter’s strong technical background with a MSc in technical chemistry with a specialisation towards chemometrics is a large benefit. After his MSc Petter joined Umetrics as an application specialist and has for the last five years worked towards PAT with increased responsibilities.

Editor: Vladimir P Torchilin Year of Publication: 2008 Pages: 474 Published by: Springer ISBN: 978-0-387-76551-8 Description: Various pharmaceutical nanocarriers, such as nanospheres, nanocapsules, liposomes, micelles, cell ghosts, lipoproteins and some others are widely used for experimental (and already clinical) delivery of therapeutic and diagnostic agents.

The use of nanoparticulate pharmaceutical carriers to enhance the in vivo efficiency of many drugs well established itself over the past decade both in pharmaceutical research and clinical setting. Looking into the future of the field of drug delivery, we have to think about the development of the next generation of pharmaceutical nanocarriers combining the whole variety of properties and allowing for the simultaneous performance of multiple functions.

Surface modification of pharmaceutical carriers is often used to control their properties in a desirable fashion and make them to simultaneously perform several different functions. This book is all about these “futuristic” multifunctional medicines.

For more books, visit Knowledge Bank section of www.pharmafocusasia.com

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M anufacturing

PLM solution in packaging and labelling The pharma industry can add value, reduce costs and improve the overall quality of its products by improving packaging and labelling processes, enabled by integrated Product Lifecycle Management solutions.

Marc Sluijs EMEA Business Development Director Life Sciences Oracle Healthcare & Life Sciences Europe, Middle East & Africa

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ffective package management is an increasingly critical capability for pharmaceutical companies.

Pressure to maximise the potential of current product portfolios due to thin pipelines has now propelled several companies to enhance packaging to add more value to the consumer, as well as to reduce Cost of Goods Sold (COGS).

Better packaging brings benefits not only to the patient, but also to nurses, pharmacists, doctors and manufacturers. Packaging can reinforce brand preference, improve compliance, facilitate consumption, limit dosing errors and help prevent counterfeiting.

Adding value to packaging includes anything that can be improved that will support the following four core objectives: administration of the right medicine, at the right dose, at the right time and in the right conditions.

Adoption of these concepts will enable companies to add considerable value for the patient. In order to accelerate this, pharmaceutical companies can—and should—benefit from the relevant experience of consumer goods companies. This industry has proven that packaging innovation has a big impact on brand preference and profitability. One of the actions to consider is hiring more people with relevant experience in consumer goods

industries to bring in new ideas and drive a change in culture, desperately needed to become more consumer-focussed.

At the same time however, adoption of these concepts will further increase the—already great—complexity in the product portfolio due to an increasing number of packaging components and technologies used.

Managing complexity

Packaging and labelling (as percentage of COGS) constitutes one of the main components of the cost of a drug. The management and physical costs of the packaging can add up to 75 per cent of COGS. Management of packaging components is a complex, multi-stage operation requiring attention to detail and accurate communications at every stage.

To illustrate the management challenge, consider a 100 mg tablet in a blister pack with an associated lidding foil, insert / leaflet, carton and printed dispatch label for each market. If sold in 40 countries, this can add up to 160 different printed packaging components. If the product is available in three strengths, this total could rise to 480.

When this figure is extrapolated to all products in a company portfolio of 40 products, this totals 19,200 items.

Complexity related to packaging and labelling are due to: Mergers and acquisitions, leading to large, often not fully integrated portfolios and supply chains Increasing involvement of third parties for design, manufacturing and packaging The traditional “Silo” mentality, obstructing effective communication and collaboration Multiple affiliates, countries and authorities, various numbers of plants Multiple versions of processes and approaches (for as many affiliates and plants) No real structured and targeted information flow Reactive inventory management No central repository for visibility of current packaging components No agreed standard timelines for key milestones and lack of control of bottlenecks Timing of pack development scheduled very late in product development process Lack of transparency of information over the complete process (end to end)

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M anufacturing

• Improved time to complete packaging changes ( a 50 per cent reduction in cycle time).

Packaging can add value in the following ways Improving user-friendliness by ensuring (and testing with consumers) that the product is easy to handle, with simple and intuitive operation to extract the drug for consumption Implementation of new standards for child resistance and Braille readability Reminder / calendar functionality Folding blister packs with instructions incorporated Discrete packaging which is easy to carry and use, such as blister / other unit dose formats Combination therapies (packaged together in one blister) User-oriented, relevant, understandable information in patient leaflets, integrated in the pack Anti-counterfeiting measures (overt, covert) and application of e-pedigree / tracking & tracing technology, such as the European Federation of Pharmaceutical Industries Associations (EFPIA) 2D Matrix.

Solutions – Integrated Product Lifecycle Management

The issues listed above can be effectively addressed by a Product Lifecycle Management (PLM) solution that manages the printed packaging component development and package management process that can result in faster throughput of changes, increased reuse of intellectual property and packaging components and reduced risk for errors.

The solution should be focussed on providing collaboration, workflow, communication and audit trail capabilities for the management of artwork, packaging components, change requests and approvals, and enforcement of policies and procedures to enable compliance with

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• Five per cent lower material costs due to reuse and component standardisation • Twenty per cent lower scrap rates due to consistency of product specification and coordination of component changes • Forty per cent greater packaging change throughput with existing organisational resources • Reduction in Total Cost of Ownership (TCO) of IT infrastructures by replacing multiple legacy systems with one central solution.

Improved compliance

regulatory requirements and company quality policy guidelines.

Such an integrated PLM solution is an essential component for packaging management, resulting in shorter processes, end-to-end audit trails and completion tracking, coupled with relevant management metrics, and document management promoting global standards that drive quality and cost-efficiencies.

The value of these benefits will differ for each company based on its size, structure and product portfolio but are generally grouped into the following categories: Reduced process lead time

• Speed up introductions (reduce packaging errors for New Product Introduction (NPI) to less than 10 per cent)

A uthor

The resultant large number of packaging components must be managed effectively and also requires the infrastructure to create, purchase, store and distribute the components before packaging. Additionally, many companies now require the ability to fill and pack products in more than one location—a very complex operation to manage given the above scenario.

Reduced product and operational costs

• Up to 75 per cent fewer errors in product information • Consistent policies and procedures among geographically dispersed groups • Greater compliance with regulatory requirements, and significant reduction in audit effort.

The integrated PLM solution should incorporate the following business functionality in support of package management: • Stage / gate process and workflow management • Document management with built-in compliance (21 CFR Part 11) • Document and artwork collaboration • Project and portfolio management • Tracking, reporting and analytics • Electronic signatures, security and audit trail.

Marc Sluijs is Business Development Director in Oracle’s EMEA Healthcare and Life Sciences Industry Business Unit. Marc has spent the main part of his career in Management Consulting with Accenture’s European Life Sciences practice; additionally Marc has worked for L’Oréal, IMS Health, and Merck Serono. He holds a degree in Business Administration from the Erasmus University Rotterdam with a major in marketing management.

M anufacturing

GlaxoSmithKline (GSK) has implemented a thorough re-engineering project called Global Pack Management. Like other companies, GSK has struggled with increasingly complex product portfolios and processes. As a consequence, GSK now runs a process of optimising the product and component portfolio on a continuous basis. As pressure on margins is ever increasing, opportunities for packaging cost reduction are a key focus area too.

To enable management of packaging specifications, graphics and artwork changes, GSK developed Global Pack Management. GSK is using an industryleading complete PLM suite. The software gives GSK flexibility in how processes and information are managed and who has access to what—without need for customisation. The aim was to ensure that GSK had a flexible solution allowing evolution according to the evolving business needs, as well as meeting all regulatory requirements.

Compliant with 21 CFR Part 11, Global Pack Management has four components: Standardised change processes GSK has implemented a uniform global process for managing packaging changes. This improves efficiency because the work can get done either faster or with fewer people.

It also helps ensure regulatory compliance, which was a concern for two reasons. One, artwork errors are the most frequent cause for product recalls. Secondly, because of the mergers and market conditions, GSK was driving through a massive amount of labelling changes.

Establishing a central Pack Catalogue All employees have access to a complete catalogue of current GSK’s packages; so, if someone in Brazil wants to launch a new product he can see what is already available within the organisation. The purpose of the

Conclusion

Packaging and labelling is an area that receives increasing attention in the pharma industry to deliver added value to consumers and reinforce competitive advantage. Next, the area still offers many opportunities to reduce cost and compliance risks by establishing more structured, consistent processes across the organisation. In order to enable this, the introduction of a

CaseStudy GlaxoSmithKline “There’s no point spending £800 million developing a drug with great clinical indication if the patient can’t use it because it’s either difficult to handle, difficult to get out or the package doesn’t give us tight, regulatory control.

Packaging is an absolute integral, fundamental part of what we’re trying to do… The development of a pack is just as important as the development of a drug or a product line extension.”

David Pulman President, Global Manufacturing and Supply, GlaxoSmithKline catalogue is to share ideas and best practices worldwide, while promoting packaging component reuse and optimisation, as well as rationalisation of the supplier base.

Centralised artwork production GSK has grouped packaging artwork management for all products in four regional service centres (instead of the previous 250 sites). Establishing dedicated art work centres of excellence has enabled GSK to maximise resources and expertise, including language skills. Together, the centres handle languages for all GSK’s markets.

GSK can monitor a task’s progress, providing real-time insight down to task and deliverable level. Also, replacing multiple legacy tools with one central solution has had a positive impact on the total cost of ownership of the IT infrastructure.

GSK strongly benefits from the collaborative review and approval capability.

Regardless of where they are geographically, people will be looking at exactly the same version of a document at the same time and making updates in real time. Monitoring capability also let GSK assess the performance of operating units based on project timetables and customer expectations.

GSK has realised excellent results following the successful implementation of Global Pack Management, by increasing process speed and efficiency, reducing cost, errors and compliance risks. Some examples of the outcomes are: • Fifty per cent reduction in effort to develop new packaging, allowing shorter times to market for packaging changes and introductions of new products • Reduction in process cycle times by 60 per cent: from 13 iteration loops to 4 • Seventy per cent reduction of errors in packaging development • Five per cent lower materials costs • Harmonisation and simplification of IT infrastructure due to the replacement of multiple legacy tools with one central solution.

Standardised and centralised information technology With one centralised IT system, employees around the world are all using the same application. Not only did this allow the company to create its pack catalogue, but it also helps increase the workflow capability because

Total yearly savings have been estimated to be an eight-figure number, demonstrating the strong business case for Global Pack Management, as well as highlighting the ROI and short payback time for the investment.

GSK also started improving other core business processes enabled by this solution.

packaging management system based on an integrated PLM solution is an essential component in addition to thorough process re-design and change management.

As proven by several pharmaceutical companies, one of them being GSK, they can expect to achieve significant— quantifiable—benefits by deploying the PLM solution for packaging management. In addition, the integrated

PLM platform will allow companies to continue to integrate information and processes across their organisation and across the product lifecycle. This has the potential of delivering even bigger benefits in key areas such as reduction of process lead time, reduction in time to market, increase in process efficiency and productivity, reduction of cost, errors and compliance risks.

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Page No.

Strategy Frost & Sullivan ................................................................................

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Health Protection Agency . ..........................................................

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Research & Development Aseptic Projectss ............................................................................ IFC Clinical Trials Aseptic Projectss ............................................................................ IFC Manufacturing Health Protection Agency . ..........................................................

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NK Pharma Industries ................................................................... OBC Sovereign Pharma Pvt. Ltd. .......................................................

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Stamfag . ............................................................................................. IBC

SuppliersGuide

Products&Services

Company

Company

Aseptic Projectss ............................................................................ IFC www.asepticprojectss.com Frost & Sullivan ................................................................................ www.frost.com

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Health Protection Agency . .......................................................... www.hpa.org.uk

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NK Pharma Industries ................................................................... OBC www.nkpharmaindustries.com Sovereign Pharma Pvt. Ltd. ....................................................... www.sovereignpharma.net

PharmaEvents

Jul 04 - 2008 Public Private Partnership Summit Venue : Hotel Shangri-la, New Delhi Organisers : Frost & Sullivan Email : remi.chaterjee@frost.com Web Link : http://www.frost.com Jul 22 - 23 2008 2008 BioBusiness Asia Conference Location : Taipei, Taiwan Organisers : Industrial Technology Research Institute Email : stacylee@itri.org.tw Web Link : http://www.biobusiness-asia.com Jul 25 - 26 2008 DCC India 2008 Location : Mumbai, India Organisers : MMG Worldwide Email : dccindia@mmgworldwide.biz Web Link : http://www.mmgworldwide.biz Jul 29 - 30 2008 Asia GMP Summit 2008 Location : Singapore, Asia Organisers : IQPC Email : enquiry@iqpc.com.sg Web Link : http://www.iqpc.com Jul 31 - Aug 02 2008 The 43rd New Drugs China Venue : Yantai International Exhibition Centre, China Organisers : Reed Exhibitions Email : enquiry@reedexpo.com.cn Web Link : http://www.reedexpo.com.cn

IFC: Inside Front Cover IBC: Inside Back Cover OBC: Outside Back cover

Aug 31 - Sep 06 2008 67th Congress of Intl. Pharmaceutical Federation Location : Beijing, China Organisers : FIP Email : congress@fip.org Web Link : http://www.fip.org

September 2008 Sep 03 - 05 2008 China International Pharmaceutical Summit 2008 Location : Beijing, China Organisers : DUXES Business Consulting Email : info@pharmasummit.com Web Link : http://www.chinapharmasummit.com/ Sep 11 - 12 2008 3rd Annual China Pharma Marketing Conference Location : Shanghai, China Organisers : IBC China Email : register@ibcchina.com.cn Web Link : http://www.ibcchina.com.cn/ Sep 15 - 17 2008 Asia Antibody Congress 2008 Venue : Grand Hyatt, Singapore Organisers : Terrapinn Email : lynn.chew@terrapinn.com Web Link : http://www.terrapinn.com

August 2008

Sep 16 - 18 2008 CHIâ&#x20AC;&#x2122;s Drug Development China Venue : The Fairmont Hotel, USA Organisers : Cambridge Healthtech Institute Email : mlieberman@pharmaseries.com Web Link : http://www.healthtech.com

Aug 28 - 30 2008 Interphex India 2008 Venue : Hitex Exhibition Centre, Hyderabad, India Organisers : Reed Exhibitions India Email : shammi@saffronmedia.in Web Link : http://www.interphexindia.com/

Sep 30 - Oct 02 2008 The Japan Biotech Meeting Location : Tokyo, Japan Organisers : Burrill & Company Email : events@b-c.com Web Link : http://www.burrillandco.com

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Stamfag ............................................................................................. IBC www.stamfag.ch

To receive more information on products & services advertised in this issue, please fill up the "Info Request Form" provided with the magazine and fax it, or fill it online at www.pharmafocusasia.com by clicking "Request Client Info" link.

July 2008

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