Laboratory Focus March 2013

PHARMACEUTICAL

CLINICAL

CHEMICAL

FOOD

w w w. l a b o r a t o r y f o c u s . c a

Sample Preparation from FFPE Tissue Specimens Utilizing the Biomek FXP Liquid Handler

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ENVIRONMENT

MARCH 2013 Volume 18, Number 2

Bioanalytical Assays

for Biosimilar Monoclonal Antibodies

Page 10

R&D News.......................... 1 Appointments..................... 6 Pharma Notes..................... 7 New Products................... 15 Calendar........................... 17 Career Spotlight............... 18

NSERC AWARDS HIGHLIGHT RESEARCHERS IN THE NATURAL SCIENCES AND ENGINEERING

Photo: Cpl Roxanne Shewchuk, Rideau Hall

In a ceremony hosted by the Governor General David Johnston in Ottawa, researchers across disciplines, universities and levels of study were recognized for their achievements and discoveries in the natural sciences and engineering by the Natural Sciences and Engineering Research Council (NSERC).

For a comprehensive list of winners and their awards, go to www. laboratoryfocus.ca.

NSERC Gilles Brassard Doctoral Prize for Interdisciplinary Research

• Melanie Mastronardi, University of Toronto (U of T) is working to make nanotechnology more environmentally friendly by exploring ways of using silicon-based nanomaterials to find a greener and less expensive option than the heavy metals currently used.

E.W.R. Steacie Memorial Fellowship

• Aneil Agrawal, U of T, investigates how harmful genetic mutations enter populations and may then be removed by different forms of selection, adding to our understanding of the evolutionary consequences of such mutations and promising practical benefits in medicine.

Publications Mail Registration Number: 40052410

L to R: Greg Scholes, John C. Polanyi, Governor General David Johnston at the NSERC Awards.

• Paul W. Ayers, McMaster University, leads a research group that is developing computational and conceptual methods for predicting, interpreting and quantifying chemical phenomena by developing new machine-learning methods for predicting the prop-

erties of molecules and materials, deciphering complex chemical reactions and designing new drugs.

• Christophe Caloz, École polytechnique de Montréal, is developing a new generation of artificial semiconductor substances engineered to perform functions not possible with naturally occurring materials.

• Warren Chan, U of T, is researching the use of quantum dots in biomedical applications. He is leading the development of hand-held devices capable of screening for molecules that indicate the presence of pathogens, including HIV, Hepatitis B

and C, malaria and syphilis.

• Kevin Resch, University of Waterloo, is a contributor to many major quantum information science experiments including the Danube experiment, projects on quantum entanglement and chirped laser pulses, carefully stretched pulses of light, in single-photon nonlinear optics and medical imaging.

• Yu Sun, U of T, is developing robotics and automation technologies for manipulating biomaterials. His research into automated processes for biological cell manipulation is refiguring how genetic studies, cancer research and clinical cell Continued on page 3

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March 2013 Laboratory Focus www.laboratoryfocus.ca

NEWS GAINS MADE TOWARDS TREATMENT OF RARE BONE DISEASE Researchers have identified an elusive substrate protein in the most common form of heritable rickets. Diagnosed in toddlers, X-linked hypophosphatemia (XLH) is the most common form of heritable rickets, in which soft bones bend and deform, and tooth abscesses develop because

infections penetrate soft teeth that are not properly calcified.

Researchers at McGill University and the Federal University of Sao Paulo have identified that osteopontin, a major bone and tooth substrate protein, plays a role in XLH. Their discovery may pave the way to effectively

treating this rare disease.

The findings were made by the laboratories of Marc McKee, a professor in the Faculty of Dentistry and the Department of Anatomy and Cell Biology at McGill University, and of Nilana M.T. Barros, a professor at the Federal University of Sao Paulo. The team

built upon previous research that had shown that mutations in the single gene PHEX are responsible for causing XLH. The results of this latest research by Drs. McKee and Barros will be published in the March issue of the Journal of Bone and Mineral Research.

Continued on page 4

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www.laboratoryfocus.ca Laboratory Focus

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March 2013

NEWS Continued from page 1 surgery and diagnostics are performed.

HEART ARRHYTHMIA’S NATURE REVEALED USING X-RAYS AT CLS

Synergy Award for Innovation

• Category 1: Small and Medium-Sized Companies - J. Paul Santerre (U of T) and partner Interface Biologics • Category 2: Large Companies - Yusuf Altintas (UBC) and partner Pratt & Whitney Canada • Category 3: Two or More Companies - Derek Gray (McGill University) and partners FPinnovations, ArboraNano and CelluForce Inc. • Leo Derikx Award - Arthur Pelton, Partice Chartrand and Christopher Bale (École polytechnique de Montréal) and In-Ho Jung (McGill University) have created diverse chemistry simulation software called FactSage that allows users to perform complex chemical equilibrium calculations using the database, saving time and costs associated with physical experimentation.

John C. Polanyi Award

• Greg Scholes Greg Scholes (U of T) has shown that has shown that quantum mechanical effects are involved in the capture and distribution of the sun’s energy during photosynthesis.

By demonstrating the connection between biology and quantum physics, his work raises significant questions on how far quantum laws reach and whether they influence complex living systems.

The Brockhouse Canada Prize for Interdisciplinary Research in Science and Engineering

• FORAC Research Team Group from Laval University: Jonathan Gaudreault, Sophie D’Amours, Luc Label, Nadia Lehoux, Mustapha Nourelfath, Robert Beauregard, Daoud Aï- Kadi of Laval University and from the École polytechnique de Montréal: Jean-Marc Frayret

Gerhard Herzberg Canada Gold Medal for Science and Engineering • Stephen Cook, U of T

Andre Hammer Postgraduate Prize

• Megan Engel, University of Alberta; • Thomas A. Fox, McGill; • Christina Natalie Nona, U of T; • Alma I. Ornes, University of Alberta; • Graham Carey, U of T

Howard Alper Postdoctoral Prize

• Christopher Moraes, University of Michigan To see this story online visit www.laboratoryfocus.ca/nsercawards-highlight-upcoming-andseasoned-researchers-in-the-natural-sciences-and-engineering/

Powerful X-rays from the CLS were used to simulate a heart arrhythmia and discover its cause. Photo: CLS Flickr Gallery.

Using powerful X-rays at the Canadian Light Source (CLS) synchrotron, scientists have reconstructed the scenario of heart arrhythmia in action, making critical progress towards preventing deadly conditions and saving lives.

The research was published in the

prestigious journal Nature Communications and presented at the 2013 annual meeting of the American Association for the Advancement of Science (AAAS) in Boston.

A 3D model was created using imaging results from the CLS that reveals, for the first time, how gene mutations affect the pathway in heart muscle cells that control its rhythm.

Arrhythmias are characterized by the heart beating too fast, too slow, or inconsistently, causing a decrease in blood flow to the brain and body that results in heart palpitations, dizziness, fainting or even death.

The article goes on to explain that the heart runs on calcium and every heartbeat is preceded by calcium ions rushing into heart muscle cells.

Then, a special protein opens the pathway for calcium to be released from compartments within these cells and in turn initiates the contraction.

Mutations to this protein have been linked to arrhythmia and sud-

den cardiac deaths in otherwise healthy people.

“We analyzed several disease mutant forms of a specific calcium channel that has been linked to cardiac arrhythmias,” said University of British Columbia molecular biologist Filip Van Petegem. “Thanks to the 3D reconstruction of these new mutant structures, it allows us to look at the detailed effects of each genetic disease mutation.”

Van Petegem said that many heart diseases cause much larger structural changes than he originally anticipated and that could directly explain their effect on calcium leaking into the muscle cell and causing arrhythmias.

He is hopeful that the research will lead to new ways of stabilizing the pathway to the heart and preventing deadly conditions to save lives.

To see this story online visit www.laboratoryfocus.ca/heartarrhythmias-nature-revealedusing-x-rays-at-cls-2/

RESEARCHERS HELP FIGHT ANTIMICROBIAL DRUG RESISTANCE A new potential target for battling disease-causing bacteria – especially deadly bugs that resist current antibiotics – may result from a study by University of Guelph researchers.

The study showed for the first time the workings of a common bacterial enzyme that may prove vulnerable to drug treatments, said professor Anthony Clarke, Molecular and Cellular Biology.

The enzyme could offer a new target for drug companies looking for new ways to fight antimicrobial drug resistance, a growing health threat worldwide, Clarke said.

The study was published in the online edition of the Journal of Biological Chemistry.

The paper’s lead author is John Pfeffer, who completed his PhD in late 2012 and is working this semester as a post-doctoral researcher in Clarke’s lab. Their co-author, Joel Weadge, completed his doctorate with Clarke in 2006 and is now a biology professor at Wilfrid Laurier University.

“This new research follows a study where we showed this enzyme might be a new target,” said Clarke. “If so, then the more information we have on how it works, the better placed we are to design or search for inhibitors.”

Bacteria have evolved many variations of defensive enzymes. Knock out

one target with an antibiotic, and the bug often deploys a different protein to elude treatment.

This particular enzyme, called Oacetylpeptidoglycan esterase or “Ape,” has little redundancy, Pfeffer explained.

Drugs might be more effective or doctors might be able to outwit the bugs longer – although he added that bacteria will eventually find a way around potential new treatments. “It’s an arms race essentially.”

The researchers studied bacteria that cause gonorrhea. Out of more than one million new gonorrhea infec-

tions in the U.S. in 2009, up to threequarters involve antibiotic-resistant strains, he said.

Pfeffer further commented their work might help in treating other organisms, especially drug-resistant strains that pose a greater health threat for people in hospitals and long-term care facilities. “We have to come up with new ways to combat disease.”

To see this story online visit www.laboratoryfocus.ca/ researchers-help-fight-antimicrobialdrug-resistance/

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March 2013 Laboratory Focus www.laboratoryfocus.ca

NEWS CANGENE ADVANCES ALZHEIMER’S DISEASE RESEARCH WITH TECH DEVELOPED AT UBC

A truly made-in-Canada treatment for one of the world’s most devastating diseases could be closer to patients thanks to a new technology targeting Alzheimer’s disease developed at the University of British Columbia (UBC) and licensed to Cangene Corporation.

Following the successful research collaboration with the inventor, UBC’s Dr. Neil Cashman, Cangene recently obtained commercial rights to the technology platform and plans to advance the program in its pipeline to explore treatments for Alzheimer’s disease, a significant un-met medical need with an increasing burden on healthcare systems.

Cashman’s discovery will allow Cangene to advance an immune therapeutic treatment approach to Alzheimer’s by targeting the toxic form of amyloid-beta with the potential to directly impact the disease

mechanism of action. Current treatment options are limited and the number of individuals afflicted with this degenerative disease is growing rapidly as the population ages, currently estimated to be 25 million worldwide.

“We are pleased to have the opportunity to collaborate with Dr. Cashman who is an expert in misfolded protein diseases and help advance his ground-breaking research in Alzheimer’s disease,” said Dr. Laura Saward, Cangene’s chief scientific officer. “While this work is in its early stages, it is showing promise for the development of a novel immunotherapy to address this devastating disease and fits Cangene’s refocused strategy.”

The Canadian Institute for Health Research (CIHR) is sponsoring the program. The discoveries made

by Cashman were also supported by research funding through CIHR

and further advanced through a research collaboration between UBC, the federally-funded PrioNet Network Centre of Excellence and Cangene – UBC’s pharmaceutical industry partner.

UBC and Cangene continue to collaborate on the development of treatments for Alzheimer’s disease with support from CIHR’s Proof of Principle Program Phase 2. The joint research program to evaluate the therapeutic potential of the target as a vaccine or antibody therapeutic was successful in the latest competition and CIHR has committed to provide $300,000 in funding over a one year period further cementing this as a made-in-Canada solution that has the potential to address this global problem.

“This is an all-Canadian solution in terms of its discovery, development and advancement towards the clinic,” said Dr. J.P. Heale, associate director of UBC’s universityindustry liaison office. “The partnerships developed to advance Dr. Cashman’s outstanding research are an excellent example of how Canadian universities, funding agencies and industry partners can work together to tackle a devastating disease of national and global importance.”

To see this story online visit www.laboratoryfocus.ca/ cangene-advances-alzheimersdisease-research-with-tech-developed-at-ubcpreservation/

Continued from page 2

“XLH is caused in part by renal phosphate wasting, which is the urinary loss from the body of phosphate, an important building block of bones and teeth, along with calcium,” says professor McKee. “In pursuing other factors that might contribute to XLH, we used a variety of research methods to show that PHEX enzymatic activity leads to an essentially complete degradation of osteopontin in bones.”

Loss of osteopontin, a known potent inhibitor of mineralization (or calcification) in the skeleton and dentition, normally allows bones and teeth to mineralize and thus harden to meet the biomechanical demands placed on them. In XLH patients lacking functional PHEX enzyme, osteopontin and some of its smaller potent inhibitory peptides are retained and accumulate within the bone. This prevents their hardening and leads to soft deformed bones such as bowed legs (or knockknees) seen in toddlers.

While not life-threatening, this decreased mineralization of the skeleton (osteomalacia), along with the soft teeth, soon leads to a waddling gait, short stature, bone and muscle pain, weakness and spontaneous tooth abscesses.

The fact that these symptoms are only partially improved by the standard treatment with phosphate – which improves circulating phosphate levels – prompted the researchers to look for local factors within the bone that might be blocking mineralization in these patients.

“With this new identification of osteopontin as a substrate protein for PHEX,” says Barros, “We can begin to develop an enzyme-replacement therapy to treat XLH patients who have nonfunctional PHEX, much as has been done using a different enzyme to treat another rare bone disease called hypophosphatasia.”

This research was jointly funded by the Canadian Institutes of Health Research (Canada) and Fundação de Amparo Pesquisa do Estado de São Paulo (Brazil).

To see this story online visit www.laboratoryfocus.ca/gainsmade-towards-treatment-of-rarebone-disease/

www.laboratoryfocus.ca

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Laboratory Focus March 2013

NEWS

CHRONIC PAIN ALTERS DNA MARKING IN THE BRAIN

U OF A RESEARCHERS BAKE NEW GROUND IN FOOD PRESERVATION

A pioneering study reveals the association of chronic pain and broad epigenetic changes. According to Researchers at the University of McGill, injuries that result in chronic pain such as limb injuries, and those unrelated to the brain are associated with epigenetic changes in the brain which persist months after the injury. Epigenetics explores how the environment – including diet, exposure to contaminants and social conditions such as poverty – can have a longterm impact on the activity of our genes.

The team led by Laura Stone, a professor at the Faculty of Dentistry and the Alan Edwards Centre for Research on Pain, and Moshe Szyf, a professor at the Faculty of Medicine’s Department of Pharmacology and Therapeutics, have discovered a mechanism that embeds the memory of an injury in the way the DNA is marked in the brain by a chemical coating called methyl groups or DNA methylation.

The researchers (including co-authors Maral Tajerian, Sebastian Alvarado, Magali Millecamps, Pascal Vachon, Cecilia Crosby, and Catherine Bushnell) report in the journal PLOS One that if the symptoms of chronic pain are attenuated, the abnormal changes in DNA methylation could be reversed.

McGill research has previously shown that experiences and not solely chemicals alter the way genes are marked epigenetically, impacting our behavior and well-being. DNA methylation, an epigenetic mark on the gene itself, can therefore serve as a “memory” of an experience that will alter the way the gene functions long after the origi-

nal experience is gone. The crucial difference between “genetic” and “epigenetic” causes for disease is that genetic changes are inherited and fixed, while epigenetic changes in contrast are possibly reversible.

The study is the first to link chronic pain to genome-wide epigenetic changes in the brain.

“Injury results in long-term changes to the DNA markings in the brain; our work shows it might be possible to reverse the effects of chronic pain by interventions using either behavioral or pharmacological means that interfere with DNA methylation, says prof. Szyf. ”Our findings have the potential to completely alter the way we treat chronic pain.”

In this study, the researchers show that behavioral interventions that reverse chronic pain also remove differences in DNA methylation in the brain.

The team report alterations in global DNA methylation are observed in the prefrontal cortex (PFC) and amygdala

of mice many months following injury to a nerve, and that environmental enrichment reduces both the pain and the pathological changes in PFC global methylation. They also found that the total amount of global methylation in the PFC significantly correlates with pain severity.

“These results suggest that epigenetic modulation mediates chronic pain-related alterations in the central nervous system (CNS), forming a “memory trace” for pain in the brain that can be targeted therapeutically, says Stone.

Since epigenetics respond to environmental changes, these mechanisms represent a mind-body link between chronic pain and the brain at the genomic level. “The implications of this work are wide reaching and may alter the way we think about chronic pain diagnosis, research and treatment.”

To see this story online visit www.laboratoryfocus.ca/ chronic-pain-alters-dnamarking-in-the-brain/

University of Alberta (U of A) researchers have found a way to replace artificial preservatives in bread, improving its taste and texture.

After analyzing strains of mould fermented in sourdough bread, Michael Gänzle, professor and Canada Research Chair in the Department of Agricultural, Food and Nutritional Science, PhD candidate Brenna Black and collaborator Jonathan Curtis, a professor of lipids research, were able to isolate natural compounds that can help keep bread fresh without altering its flavour.

Preservatives currently added to store-bought bread are safe to eat and extend shelf life, but change the taste and have a distinctive odour, Gänzle said.

“The scientific community has always known that sourdough bread has a longer shelf life than conventionally leavened bread, but we weren’t sure why, and this study has been able to uncover a large part of that.”

The U of A research is the first to link certain compounds—hydroxy fatty acids— to antifungal activity and to show that these compounds are formed in the production of fermented foods. “We were able to put known

compounds in quite a new context,” Gänzle said.

The findings also have the potential to be used in replacing or supplementing fungicides used in treating crop seeds and protecting crops.

The results appear in the March issue of Applied and Environmental Microbiology.

The study was funded by the Natural Sciences and Engineering Research Council of Canada and

by Ernst Böcker GmbH, a sourdough company based in Germany. The research was also supported by the Canadian Wheat Board, the Canada Research Chairs program and the NSERC Discovery program.

To see this story online visit www.laboratoryfocus. ca/u-of-a-researchersbake-new-ground-infood-preservation/

Great People.

Great Chemistry.

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March 2013 Laboratory Focus www.laboratoryfocus.ca

APPOINTMENTS

CO2 Solutions Inc. has appointed Dr. Louis Fradette as chief technology officer and senior vice presi-

dent, Process Engineering. Dr. Fradette has over 25 years’ experience in chemical engineering, engineering development and project management. He has worked in R&D in both the public and private sectors, including the petrochemicals, oil and gas industries. In his new role, Dr. Fradette will lead development and application of CO2 Solutions’ carbon capture technology for the

company’s Alberta oil sands project.

Dr. Fradette has a Master’s degree in Chemical Engineering from Laval University in Québec City, a PhD in Process Engineering from l’Institut National Polytechnique de Lorraine in France, and a PhD in Chemical Engineering from the École polytechnique de Montréal, where he is currently an associate professor in Chemical Engineering.

Imaging Dynamics Company, Ltd. announces the appointment of Anna Lentz, CGA, MBA, as chief financial officer. Lentz has broad experience in the financial management of private and public companies in the oil and gas, mining, engineering, and manufacturing sectors. Currently, she is the principal of a privately owned financial consulting firm that provides the financial management and required controls for the success of its small to mid-sized clients. Lentz replaces John Choy who acted as interim CFO for the company.

CHU Sainte-Justine appoints Dr. Alain Moreau as its director. Over the past 12 years, Dr. Moreau has

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cumulated a number of achievements under the various key positions he has held, including owner of the Technopôle rehabilitation project (to open in 2015), associate director of Academic Affairs at the Research Center, research scientist in musculoskeletal disorders, and co-director of the CHUSJ’s Department of Dentistry. Dr. Moreau is a professor at the Université de Montréal, in the Department of Stomatology at the Faculty of Dentistry and in the Department of Biochemistry at the Faculty of Medicine.

He holds a Bachelor of Science in Biology from the Université de Montréal, a Master’s of Science in Applied Microbiology from the Institut Armand-Frappier, and a PhD in Microbiology and Immunology from the Université de Montréal.

Xenon Pharmaceuticals Inc. has appointed Gary J. Bridger, PhD, as executive vice president of Research and Development. Dr. Bridger has spent over 20 years in the life sciences industry including positions at Johnson Matthey, AnorMED and Genzyme. Dr. Bridger was a primary member of the team that managed AnorMED’s acquisition by Genzyme for US$580 million. Previously, he was a partner with Ventures West Capital Management and he currently serves on the Scientific Advisory Board of Alectos Pharmaceuticals. He has co-authored over 90 peer-reviewed publications and 40 granted US patents, mainly in the chemokine receptor antagonists field. Dr. Bridger received his PhD in Organic Chemistry from the University of Manchester UK and completed a post-doctoral fellowship at Boston College.

Dr. Rachael Ritchie has been appointed director of Business Development at Genome BC. Over the past 20 years, Dr. Ritchie has held numerous positions for various

academic and government institutions. She began as a molecular geneticist for the Food, Fisheries and Aquaculture Department Research and Productivity Council in New Brunswick, where she then became department head. From there she went to project manage the International Chair in EcoInnovation at the UniverSUD Paris/ Université de Versailles in France.

Previously, Dr. Ritchie was a policy analyst in the Science Technology Policy Department at the Organization for Economic Co-operation and Development (OECD). Dr. Richie has a Bachelor of Science in biochemistry from UBC, a PhD in clinical

medicine from Oxford University and completed a post-doctoral fellowship at Harvard University at the Boston Children’s Hospital.

The University of British Columbia (UBC) Faculty of Pharmaceutical Sciences announces the appointment of Dr. Corey Nislow to the faculty as Associate Professor. Dr. Nislow has both academic and corporate experience, being the first to identify and the first to clone a human mitotic kinesin during his PhD and spending five years at three different biotechnology companies in the San Francisco Bay area. His current research includes using model organisms to understand the mechanism of action of human therapeutics, using high-throughput cell biology to investigate drug synergy, and developing technologies for understanding the relationship between chromatic structure and transcriptional regulation. Dr. Nislow will be joining UBC from the University of Toronto where he served as Associate Professor with the Banting and Best Department of Medical Research and as Director of the Donnelly Sequencing Centre.

www.laboratoryfocus.ca

Laboratory Focus March 2013

PHARMA NOTES

Lab Bio-Medic (Montréal, QC) and Algorithme Pharma (Laval, QC) have signed a partnership agreement for the delivery of biomedical analysis required as a part of clinical research projects conducted for pharmaceutical and biotech customers. Under the terms of the two-year renewable agreement, Algorithme Pharma will have privileged access to the biomedical and logistics expertise of the Lab Bio-Medic Team.

OncoGenex Pharmaceuticals, Inc. (Bothell, WA and Vancouver, BC) has announced plans for the initiation of its Borealis-2 clinical trial. Borealis-2 is an investigator-sponsored, randomized, controlled Phase 2 study evaluating OGX-427 in patients with advanced or metastatic bladder cancer who have disease progression after initial platinum-based chemotherapy treatment.

CDRD Ventures Inc. (CVI), the commercialization arm of The Centre for Drug Research and Development (CDRD) (Vancouver, BC), and Pfizer Inc. have entered into a strategic collaboration that will help advance Canada’s health technologies towards commercialization. Under the agreement, Pfizer will support mutually-selected CVI R&D projects with the aim of commercializing the programs and the resulting intellectual property. This new collaboration follows previous investments by Pfizer Canada Inc. into CDRD whereby a PfizerCDRD Innovation Fund was first established in 2007 to support innovative projects with therapeutic potential stemming from academic discovery.

Atrium Innovations Inc. (Québec City, QC) has entered into a joint venture contract with Fosun Industrial (Hong Kong), wholly owned of the Shanghaibased Fosun Pharma, for

the purpose of distributing Atrium brands in China.

The new joint venture will operate under the FOSIUM Innovations name. FOSIUM Innovations will initially import, market and sell Pure Encapsulations® products in the Chinese mainland market. The products will continue to be manufactured in the U.S. and other existing Atrium brands will subsequently be introduced.

Atrium will make an initial investment in the venture of $1 million for a participation of 49 per cent.

Bioniche Life Sciences Inc. (Belleville, ON), announced that it is terminating its exclusive global veterinary license agreement with Trophogen Inc. (Rockville, MD) effective immediately. The agreement was originally entered into in June, 2010 to provide commercial access for Bioniche to a patented, proprietary superagonist hormone technology platform developed

by Trophogen (originally

licensed from the National Institutes of Health) in the veterinary field.

Thallion Pharmaceuticals Inc. (Dorval, QC) and the France-based LFB Biotechnologies have reached an agreement on the termination of their Development and Licensing Agreement related to Thallion’s Shigamabs® program.

All rights to the program will revert back to Thallion, including all data, materials and know-how developed by and for LFB during the collaboration. LFB will cease having any rights to the Shigamabs® program and all future quarterly payments due to Thallion by LFB for Shigamabs® development will terminate. LFB will pay for all outstanding and accrued costs related to product manufacturing and Thallion will be responsible for all remaining costs associated with the completion of the SHIGATEC Phase 2 clinical study.

Immunovaccine Inc. (Halifax, NS) announces the signing of an investigatorinitiated study agreement for the ongoing evaluation of its DPX-0907 cancer vaccine in patients with breast and ovarian cancer at the Busto Arsizio Hospital, Italy.

Immunovaccine expects the Phase 1/2 study to be initiated during the fourth quarter of 2013.

Valeant Pharmaceuticals (Montréal, QC) has acquired the U.S. rights for Targretin® (bexarotene) capsules and Targretin® gel 1per cent from Eisai Inc. (Woodcliff Lake, NJ), the U.S. pharmaceutical subsidiary of Eisai Co. Ltd. for $65 million upfront, plus potential contingent payments based on certain milestones. As part of the transaction, which takes effect immediately, Eisai has transferred the New Drug Application (NDA) for Targretin to Valeant, with Valeant assuming responsibilities for all regulatory

7

obligations associated with the product in the U.S. while Eisai will retain its rights to Targretin outside of the U.S. and continue to meet the needs of its distribution partners outside the U.S. Stem Cell Therapeutics Corp. (Toronto, ON), announces the execution of a Letter of Agreement with Trillium Therapeutics Inc. (Toronto, ON), under which Trillium would be merged into Stem Cell Therapeutics Corp. (SCT) by way of a three-cornered amalgamation or plan of arrangement with a newly-created SCT subsidiary. In addition, SCT announces its Board of Directors has authorized the implementation of a share consolidation approved by its shareholders at the special meeting held on Dec. 20, 2012 at a ratio of one post-consolidation common share for 10 pre-consolidation common shares.

Important Notice:

Avis important :

Human Pathogens and Toxins Act

Loi sur les agents pathogènes humains et les toxines

Attention: Persons handling human pathogens and toxins

Destinataires : Personnes manipulant des agents pathogènes humains et des toxines

The Human Pathogens and Toxins Act is designed to protect the health and safety of the public against risks posed by the accidental or deliberate release of human pathogens and toxins from research or other facilities.

La Loi sur les agents pathogènes humains et les toxines vise à protéger la santé et la sécurité de la population contre les risques associés au rejet accidentel ou délibéré d’agents pathogènes humains et de toxines par un centre de recherche ou un autre établissement.

Under the Human Pathogens and Toxins Act, if you are handling human pathogens and toxins, you may be subject to certain obligations. The best way to know if the Act affects you is to register with the Public Health Agency of Canada on our website.

Selon la Loi sur les agents pathogènes humains et les toxines, si vous manipulez des agents pathogènes humains ou des toxines, vous pourriez avoir à remplir certaines obligations. La meilleure façon de savoir si la Loi s’applique à vous consiste à vous inscrire auprès de l’Agence de la santé publique du Canada en consultant notre site Web.

To register or to obtain more information about laboratory biosafety in Canada, please visit

Pour vous inscrire ou pour en savoir plus sur la biosécurité en laboratoire au Canada, veuillez consulter le site

www.publichealth.gc.ca/pathogens

www.santepublique.gc.ca/pathogenes

8

March 2013 Laboratory Focus www.laboratoryfocus.ca

FEATURE

BY: BEE NA LEE PhD., MICHAELA BOWDEN PhD., ASHLEY ROSSI, SARAH SIMONS, SVITLANA TYEKUCHEVA, ED STACK PhD., MASSIMO LODA MD

Automated TruSeq RNA Sample Preparation from FFPE Tissue Specimens Utilizing

The Biomek FXP Liquid Handler Overview This article describes a TruSeq (Illumina, Inc. San Diego, CA) RNA library preparation method for FFPE samples that uses the Biomek automation platform (Beckman Coulter Life Sciences, Indianapolis, IN) in which 96 libraries are constructed in seven hours. The Biomek platform provides a solution for high throughput library construction, producing sensitive and reproducible sequencing data, which facilitates biomarker discovery in archival FFPE tissues.

Figure 1

The data presented here benchmarks an automated approach that uses the Biomek FXP Liquid Handler against a standard manual preparation. Archival FFPE tissue specimens have lower RNA yield and are highly degraded, which represents a major challenge in developing robust methodologies for sequencing. The findings of the study highlighted that preparation on the Biomek automated platform provided researchers with a more reliable, more streamlined approach for generating libraries from precious clinical samples with a faster turnaround time.

Methods and Results

Introduction Next Generation Sequencing (NGS) technology facilitates high throughput, high speed and cost-effective sequencing of DNA and/or RNA. RNA library construction from archival FFPE tissue specimens can be automated utilizing the Beckman Coulter Biomek FXP Liquid Handler. RNA is extracted using the Beckman Coulter Agencourt FormaPure kit and the library preparation method is based on the Illumina TruSeqRNA Sample preparation protocol.

The Biomek/TruSeq RNA method is comprised of three parts: 1. mRNA purification, fragmentation and cDNA synthesis

2. cDNA library construction (end repair, A-tailing and adaptor ligation) 3. PCR amplification and product purification

Representative RNA QC plots with associated elution bands on the left for Tumor/Benign pair, A. RNA concentration was determined by RiboGreen Assay (Life Technologies, Carlsbad, CA). Concentrations ranged from 24.4 – 61.1 ng/μl, of which 500 ng of total RNA was utilized for each library as the input amount.

Figure 2

Example of QC plots of Pre-and Post-Cleanup Library Preparations for Tumor/Benign pair A.

RNA Preparation Three tumour/normal pairs of archival FFPE prostate tissue specimens were selected and scored. Total RNA was extracted according to the protocol of the Agencourt FormaPure kits (Beckman Coulter Life Sciences). Each sample was eluted in 55 μL dH2O. RNA quality was determined on the Bioanalyzer 2100 system (Agilent Technologies Inc., Santa Clara, CA). Figure 1 shows the typical degraded profile of RNA isolated from archival FFPE tissue. The RN number was ≤ 2.4, which indicates the high level of degradation associated with these tissues.

TruSeq cDNA Library Preparation Fragmented RNA was reverse-transcribed to make the first strand cDNA. The first strand cDNA was then used as a template to generate double stranded DNA. The dsDNA template was blunt-ended and adenlyated at the 3’ ends to enforce strict ligation of the adapters. The adapter modified templates were subjected to 15 rounds of amplification to enrich and selectively amplify DNA containing adapter molecules. After the final clean up step, the purified DNA samples were eluted in 30 μL of Resuspension Buffer. 1 μL of the library was used to assess the quality of the library generated using Agilent High Sensitivity DNA kit on the Bioanalyzer 2100 system (Agilent Technologies). cDNA Library Quality Cleanup Initial quality review showed an average peak size for the amplified cDNA library at approximately 260 bp, however, there was a 120 bp non-specific adaptor amplified peak also present in all 12 samples. This represents adaptor-dimer contami-

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9

Laboratory Focus March 2013

FEATURE

Figure 3

ticular, the poor library quality of tumor/normal pair C is verified in the corresponding Pearson correlation coefficients.

A wide ranging panel of endogenous controls was found to show good concordance between the manual and Biomek sequenced libraries, with a Pearson coefficient of 0.99 calculated. The insets are plots of the average Log2 [FPKM] values of all samples for each library preparation method, which highlights the stability of these endogenous controls across all sample types, irrespective of preparatory method or tissue morphology.

Conclusion

Scatter plots comparing method preparation for each individual sample. Log2 [FPKM] values were utilized to make the correlation. and the supernatant treated with a 1.1X volume of AMPure beads to rebind DNA fragments between 150 bp - 400 bp. Figure 2 shows the overlaid QC plots of the pre- and post-clean up libraries for tumor/benign pair A with a side-by-side comparison of the Biomek and manual libraries.

Figure 4

Scatter plots of panel of endogenous control as measured in Biomek and manual sequenced libraries (Insets: Log2 [FPKM] values plotted for low, medium and higher abundance endogenous controls, G6PD, OAZ1 and GAPDH, respectively. nation, which can lead to junk reads when sequencing. In some cases, an additional fragment greater than 400 bp was also detected, which could represent single-stranded library products that have self-annealed.

In order to improve library quality, a two-step size selection clean-

up protocol was implemented. In the first clean-up step, a 0.7X ratio of AMPure XP bead solution (Beckman Coulter Life Sciences) was added to each sample. Under this condition, only larger fragments greater than 500 bp DNA were bound to the beads.

The bead-bound DNA was discarded

Single End Sequencing Performance Sequencing was performed on the Illumina HiSeq platform. 50 bp single reads were mapped using the TopHat program (Institute of Genetic Medicine, Johns Hopkins University Center for Bioinformatics and Computational Biology (CBCB), Baltimore, Md.) and transcript abundance in FPKM units (fragments per kilobase of mRNA per 106 reads) were calculated using Cufflinks (CBCB). Multiplexed sequencing was performed such that each lane contained three samples. RNASeq library construction/sequencing was successful in 12/12 samples with aligned reads ranging from 21 per cent to 72 per cent relative to the total number of reads.

Reproducibility was measured by calculating the Pearson Correlation from transcript abundance values, presented as Log2 [FPKM] values.

TruSeq libraries prepped both by the Biomek automated platform and manually on the bench, exhibited good concordance, as evidenced in the scatter plots depicted in Figure 3.

The total number of transcripts is also displayed in Figure 3. In par-

This study shows that RNASeq library preparation carried out on the automated high throughput Biomek platform, results in sensitive and reproducible sequencing data, which facilitates biomarker discovery in archival FFPE tissue. Going forward it is envisaged that new coding and non-coding transcripts, as well as gene signaling networks that strongly associate with prostate cancer progression will be identified.

The Biomek automation platform, capable of constructing 96 libraries in seven hours, offers a viable high throughput alternative to traditional manual bench preparation of RNA libraries for sequencing. The Biomek FXP automation platform provides a high-throughput workflow and a more reliable way to generate libraries from precious clinical samples with faster turnaround time. In addition, the same method can be used with fresh tissue or other sample types for library construction.

Reference 1. DeJonge, HJM et al., Evidence Based Selection og Housekeeping Genes, PLoS One, 2007, 2(8), e898.

Beckman Coulter, the stylized logo, Biomek, Agencourt, AmPure XP, and FormaPure, are registered trademarks of Beckman Coulter and are registered in the USPTO. The Biomek FXP is registered as a medical device in S. Korea, and is not intended for diagnostic applications in other countries. All other trademarks are the property of their respective owners.

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March 2013 Laboratory Focus www.laboratoryfocus.ca

FEATURE

BY: JAMES D. HULSE, PH.D.

Effective In Vitro Bioanalytical Assays for Comparing Pharmacodynamics of Biosimilar Monoclonal Antibodies ABSTRACT The rise in the development of biosimilar therapeutics has caused increasing demand for new approaches for precise, sensitive and accurate bioanalysis. In this white paper, we describe how a combination of the biopharmaceutical market, regulatory guidelines and the molecular characteristics of biologics has inspired the development of novel ligand-binding assays (specifically, flow cytometry and surface plasmon resonance) that may streamline evaluation of biosimilars.

Introduction The coming year faces the expiration of the first patent of a monoclonal antibody (mAb) biotherapeutic—the 6.8 billion-dollar-a-year drug, rituximab (Rituxan® as marketed by Biogen Idec/Genentech), will lose European patent protection in November, 2013.

This expiry represents just the first in a series of upcoming patent expirations for biotherapeutics, including, but not limited to, trastuzumab (breast cancer), cetuximab (colorectal, head/neck cancers) and natalizumab (for multiple sclerosis and Crohn’s disease).1 In anticipation of markets for these therapeutic areas opening up upon patent expiration, drugmakers have ramped up the development of biosimilars.

The European Medicines Agency (EMA), having approved its first biosimilar in 2006, has already begun reviewing its first application for ap-

proval of a biosimilar mAb – a version of infliximab, originally developed by Janssen Biotech, Inc., a subsidiary of Johnson and Johnson. The United States Food and Drug Administration (FDA) has received nine biosimilar investigational new drug applications, and some copy biologics have already been approved in China and India.1 Specifically, with several antidiabetes drug patents expiring in 2015, some insulin copy biologics and insulin analogues are already available in India.2 Investments in biosimilar development are expected to pay off — in Europe, revenue from the sale of biosimilars reached $172 million in 2010, and may be as high as $4 billion by 2020.3 However, developing a biosimilar therapeutic can be expensive, with costs reaching 80 per cent of the cost of developing an innovator biologic

drug and about 20 times as high as for developing a small molecule generic.2 Each new biosimilar faces the challenge of proving that any differences in potency and safety from the innovator drug are not clinically significant.

Especially in the case of monoclonal antibodies, which are large and complex, chemical differences between biosimilars and innovators may be numerous. Such cases require rigorous demonstration of biosimilarity as a proxy for therapeutic “interchangeability,” the ultimate (though probably unprovable) standard.

Therefore, there is a critical need for increasingly accurate and precise nonclinical, in vitro assays for measuring

Figure 2

drug potency, as these are the cornerstone of quality control of manufactured therapeutics. A recent survey showed that 32 per cent of drugmakers declared that innovations in assay technology were required to meet the demands of proving biosimilarity.4 Such assays can better determine lotto-lot variability in the manufactured product, assess the impact of process changes on drug quality, assess drug stability, and more. Therefore, increasing the precision of an assay improves the assay’s statistical power, facilitating the comparison between biosimilars and innovators.

Although regulatory agencies are considering biosimilars on a caseby-case basis, they have issued some guidelines on what types of in vitro studies should be performed in the evaluation of biosimilarity. Of note is the European Medicines Agency (EMA)’s draft guidance, titled “Guideline on Similar Biological Medicinal Products Containing Monoclonal Antibodies,” published in November 2010.5 The EMA’s guidance recommends measuring, among other parameters, binding to the target an-

Principle of the SPR system.

Figure 1

Using a fluorescently labeled detection antibody (black )specific to the mAb of interest, flow cytometry can be used to assess binding of a mAb (blue) to Fc receptors (red) expressed on the surface of a transfected cell.

A sensorchip is interrogated by a wide angle laser beam which is internally reflected from the sensor surface. At one particular angle (the resonance angle) light is absorbed at the surface and the signal is relatively dimmed (green line). This angle depends on the refractive index difference at the surface. Drug is attached to the surface and serum, containing a variety of antibodies flow across. If an antibody binds to the drug, the refractive index changes slightly, and this causes a change in the resonance angle. The response is nearly proportional to the mass of the substance binding to the chip.

www.laboratoryfocus.ca tigen and binding to all Fcγ receptors, FcRn and complement. Binding to Fc receptors and complement can result in cytotoxicity and safety concerns. The recent FDA draft guidance, “Scientific Considerations in Demonstrating Biosimilarity to a Reference Product,” is less specific in its recommendations, but indicates a strategy for evaluating biosimilarity based on “totality of the evidence.”6 In other words, comparing a biosimilar with an innovator using multiple, orthogonal assays is like matching fingerprints-the more multivariate the fingerprint, the more likely that a match is predictive of clinical biosimilarity. Most recently, the Indian regulatory agencies, the Department of Biotechnology and the Central Drugs Standards Control Organization issued its own guideline on the development of similar biologics, in part to attract investment from global pharmaceutical companies.7 Innovations in in vitro assay development are being welcomed by regulatory agencies, who are championing the “risk-based,” or “stepwise,” approach to evaluating biosimilarity, suggesting that the results, very sensitive, highly predictive nonclinical assays, can help shape the direction of further testing [FDA guidance]. For example, appropriate pharmacodynamic (PD) markers can be a very sensitive indication of potential clinical differences between two drugs. Regulatory agencies have identified immunogenicity testing as an area enhanced by in vitro ligand-binding analysis. Regulatory guidance (ICH Q6B, 1999) states: “When an antibody is the desired product, its immunological properties should be fully characterized. Binding assays of the antibody to purified antigens and defined regions of antigens should be performed, as feasible to determine affinity, avidity and immunoreactivity (including cross-reactivity).”

In this white paper, we describe two bioanalytical approaches involving measurements of ligand binding that have recently been applied to comparing PD of biosimilars.

Specifically, we show the use of surface plasmon resonance (SPR) and flow cytometry to quantify the binding between therapeutic mAbs, alemtuzumab (and its variants) and infliximab, to molecules mediating cytotoxicity.

11

Laboratory Focus March 2013 Given the high cost of advancing from in vitro assays to preclinical studies, there is high interest in establishing assays that provide valuable pre-clinical data about the properties of drugs and their physiological interactions without the need for animal experiments. SPR and flow cytometry are ideal techniques to use for this type of study.

Flow Cytometry: How It Works Flow cytometry is an essential tool for in-depth cell analysis.

In a traditional flow cytometer, cells in a liquid stream pass through a laser beam, which excites any fluorescent molecules on the cell. Emitted fluorescence is then measured by detectors tuned to specific wavelengths. With the capac-

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ity to simultaneously measure multiple parameters on hundreds of individual cells per second, flow cytometry is a powerful technology with a wide variety of applications in pharmaceutical development.

As shown in Figure 1, flow cytometry can be a sensitive, information-rich method for measuring mAb binding to Fc receptors on the cell surface.

FEATURE When developing a flow cytometry assay for PD assessment of a therapeutic, factors to consider include: • Sample type (e.g., whole blood, separated PBMCs, serum, cultured cells) • Stability of marker(s)/

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FEATURE

analyte(s) being measured • Appropriate fluorochromes, dyes and conjugates to get the clearest data from samples • Incubation temperatures and periods most suited to the matrix and/ or analytes • Most suitable controls to monitor assay performance • Appropriate quality control checks on instrumentation to ensure cytometers are performing reproducibly

Surface Plasmon Resonance: How It Works Surface plasmon resonance (SPR) is a powerful technique for measuring the binding of any pair of interacting molecules, including drugs and targets, and antibodies and antigens. Interactions are measured in real time, enabling the determination of kinetic parameters. The SPR signal is proportional to the mass of analyte and does not require any type of label. Figure 2 shows the principle by which the SPR signal is generated.

SPR is a very versatile platform with many different applications throughout pharmaceutical development. The measurement of the kinetics of critical molecular interactions between the drug and its target and other key receptors (e.g., Fc receptors in the case of antibodies), can increase the precision and accuracy of comparisons between biosimilars and innovators.

Results Effect of differential glycosylation of alemtuzumab (Campath®-1H) on binding to FcγRIII: SPR and flow cytometry assays Although alemtuzumab can be produced in suspension CHO cells with much greater yield than in the traditional rat cells, alemtuzumab from CHO cells is improperly glycosylated compared to that from rat and human cells.

Figure 4

Glycoengineered alemtuzumab, made in CHO cells transfected with glycosyltransferases, showed higher antibodydependent cell-mediated cytotoxicity compared to the wild type drug.

In order to determine the mechanism of cytotoxicity, we used SPR and flow cytometry binding assays to measure the binding of alemtuzumab and its glycosylated forms to several Fc receptors.

To demonstrate that the assays could detect differences in the measured binding, the assays were first qualified by adjusting the concentration of alemtuzumab to 50 per cent, 70 per cent, 80 per cent, 100 per cent, 120 per cent, 130 per cent and 150 per cent of the reference alemtuzumab concentration.8 Determination of the relative potency was performed using a 5-parameter logistic parallel line model using Statlia software. The curves of the different starting concentration samples were all determined to be parallel to the reference curve with p>0.05. The assay had good linearity with a correlation coefficient of 0.993 and measured potencies in the range of 91.4 to 100.6 per cent.8 The curves showing binding between the various forms of alemtuzumab and FcγRIII (CD16a) are shown in Figure 3. A negative control antibody, engineered to have no CD16a binding, showed no response in this assay. The glycoengineered antibodies Glyco1 and Glyco 2 showed enhanced CD16a binding compared to the wild type alemtuzumab with relative potencies of 300 per cent and 411 per cent respectively. The glycoengineered forms of alemtuzumab did exhibit small deviations from strict parallelism [p=0.017 and p=0.038 respectively at p>0.05]. These data correlated with the increased ADCC activity previously observed.8 Precision between replicates at each concentration was generally less than 5 per cent.

Figure 3

Binding of glycoengineered and control antibodies to human FcγRIII (CD16a) measured by SPR.

The starting concentration of the test samples was adjusted to simulate samples of different potency. The standard deviations of the triplicate measurements were smaller than the plotted symbols.

Similar analyses were performed for CD32a and CD64 although the magnitude of the signal obtained was markedly different, reflecting the strength of binding to the different forms of the receptors. Unlike CD16a, CD32a and CD64 bound to wild type alemtuzumab with higher affinity than to its glycosylated forms (data not shown).

The assay was also tested for other therapeutic monoclonals—namely, bevacizumab (Avastin®), rituximab (Rituxan® or MabThera®) and eculizumab (Soliris®). Eculizumab, a monoclonal antibody against C5 which has a hybrid Fc domain of

Figure 5

IgG2 and IgG4 and hence no Fc binding, was used as a negative control in the assay. Although no absolute measurements of potency were determined for these therapeutic monoclonals, their relative binding to CD16a was as follows: alemtuzumab bound CD16a with the strongest affinity, followed by rituximab and then bevacizumab. Eculizumab, as expected, exhibited no measurable binding.

To support our SPR observations using recombinant alemtuzumab and Fc receptors, we used flow cytometry to measure the binding of alemtuzumab to Fc receptor-expressing cells. Sus-

Binding of glycoengineered and control antibodies to human FcγRIII (CD16a) measured by flow cytometry.

Binding of different antibodies to FcγRIII (CD16a) by SPR.

The starting concentration of the test samples was adjusted to simulate samples of different potency. The standard deviations of the triplicate measurements were smaller than the plotted symbols.

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pension CHO cells were transfected with vectors directing the expression of various Fc receptors. These cells were incubated with the therapeutic monoclonal antibodies and the bound antibodies were detected using a FITC-conjugated goat Fab2 anti-human kappa light chain antibody. The level of binding was measured by flow cytometry with the labeled cells gated by forward and side scatter, and the median fluorescence intensity was measured.

As with the SPR assays, the assay was qualified for alemtuzumab using the same nominal concentrations to mimic different potencies of 50 to 150 per cent of the reference. The assay performance for CD16a was very similar to that observed using the SPR assay. The correlation coefficient was 0.99 with measured potencies in the range of 97.9 to 108 per cent.8 As shown in Figure 5, the glycosylated variants of alemtuzumab exhibited higher binding to CD16a than the wild type alemtuzumab. Relative potency values could not be obtained due to the lack of parallelism due to both Glyco1 and Glyco2 reaching a higher plateau of binding than the wild type reference.

Binding of infliximab to neonatal Fc receptor FcRn and complement component C1q The EMA draft guidance recommends testing biosimilars for their binding to the Fc receptor FcRn. The neonatal, intracellular FcRn receptor is responsible for transport of IgG across the placenta. FcRn binds to IgG and albumin at low pH but not at high pH. This receptor is responsible for “salvage” of internalized IgG or albumin and therefore endows these proteins with a long half life. Structurally, the molecule is similar to Class I MHC and consists of a specific heavy chain combined with β-2 microglobulin.

Although FcRn is not commercial-

Figure 6

13

Laboratory Focus March 2013 ly available, we obtained a supply of recombinant dimeric receptor suitable for SPR assays. We coupled the receptor directly to the chip surface and qualified the assay using the same protocol as for the alemtuzumab assays. Although the magnitude of the signal obtained was very low, the data were consistent and reproducible (Figure 6). In the qualification of the assay, the r2 value was 0.96 and the measured potencies were from 81 to 110.9 per cent.

The final assay that was developed to satisfy the requirements of the EMA biosimilar monoclonal antibody guidance was to measure C1q binding. C1q is the first component of the complement cascade, binds to IgM or IgG which is complexed with antigen.

A large hexameric molecule, its binding affinity in solution is extremely low. In fact, it has proved impossible to detect binding of IgG to C1q which was immobilized on an SPR chip. Although it is possible to measure binding of C1q to immobilized IgG, this assay will be difficult to convert for potency or comparability studies.

We therefore developed an ELISA to measure C1q binding. The therapeutic monoclonal antibodies were serially diluted and coated to microtitre plate surface, C1q was added and, following washing, the bound material detected using anti-C1q-HRP conjugate. The assay qualification data showed a correlation coefficient of 0.98 with measured accuracies of between 100.4 and 114.8 per cent (Figure 7).

data were amenable to potency determination by the parallel line method, and we could easily distinguish variant glycoforms and different antibodies. Results showed high correlation with results of antibody-dependent cell-mediated cytotoxicity (ADCC) experiments, without the variability due to biological complexity and statistical noise. ADCC experiments testing the effect of Fc receptor binding to alemtuzumab yielded more variable results, with CVs of individual triplicates in the range of 0.2-18.6 per cent (mean CV=5.4 per cent).8 These methods are accurate, robust, reproducible and currently in use within a GMP setting for batch release of drug lots. The assays are sensitive to changes in potency related to glycosylation, aggregation, concentration or protein modification. However, many changes can occur in biologics without affecting a potency readout, so careful physico-chemical characterization is still essential.

Our data also indicate that flow cytometry and SPR might be used to predict potential immunogenicity, induced cell proliferation, cell-mediated lysis, and receptor-mediated events (cAMP, cytokine release, antigen expression). A combination of such assays can provide a comprehensive picture of functional activity which is sensitive to small changes and provides reliable tools for quality control and for comparison of biosimilar with innovator products.

Conclusion

1. Mullard A. Can next-generation antibodies offset biosimilar competition? Nat Rev Drug Discov.

2012 Jun 1;11(6):426-8.

2. Nair, A. Pharmaceutical Technology. 2011 Feb; 35(2):18.

3. Frost & Sullivan. Analysis of European Biosimilars Market. Dec 2011, Pages: 223 [http://www.

Our studies of Fc receptor binding by monoclonal antibodies indicate that binding to soluble or cell-surface Fc receptors can be accurately measured by surface plasmon resonance or by flow cytometry. We observed excellent precision for replicates, typically less than 5 per cent CV. The

Qualification of SPR binding assay to measure affinity of infliximab for FcRn.

Assay was qualified by adjusting the concentration of infliximab to 50%, 100%, and 150% of the reference infliximab concentration.

References

FEATURE 4.

5.

6.

7.

8.

frost.com/prod/servlet/svcg.pag/ HCPD] 9th Annual Report and Survey of Biopharmaceutical Manufacturing, BioPlan Associates, Inc, April 2012, www.bioplanassociates.com European Medicines Agency.

Guideline on Similar Biological Medicinal Products Containing Monoclonal Antibodies. EMA/ CHMP/BMWP/403543/2010. Nov 2010.

United States FDA. Scientific Considerations in Demonstrating Biosimilarity to a Reference Product. Feb 2012.

India launches ‘similar biologics’ guidelines at BIO2012 http:// www.in-pharmatechnologist. com/Regulatory-Safety/Indialaunches-similar-biologics-guidelines-at-BIO2012 Harrison A et al. Methods to measure the binding of therapeutic monoclonal antibodies to the human Fc receptor FcγRIII (CD16) using real time kinetic analysis and flow cytometry. J Pharm Biomed Anal. 2012 Apr 7;63:23-8.

James D. Hulse, Ph.D. is Managing Scientific Director, Discovery & Development Solutions, EMD Millipore Corporation. He can be contacted at James.Hulse@ emdmillipore.com.

To see this story online visit www.laboratoryfocus.ca/effective-invitro-bioanalyticalassays/

Figure 7

Qualification of ELISA to measure binding of C1q by infliximab.

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March 2013 Laboratory Focus www.laboratoryfocus.ca

FEATURE

BY: THORSTEN VAMMEN

Designing out contamination for white biotech applications one being produced. If so the stronger bacteria will take over the environment and decrease the yield or in the worse case, ‘kill’ the one being produced.

Effective cleaning

Flush-mounted instruments with no dead legs Biotech applications are particularly susceptible to contamination. Some of the cells used in biotech processing are very sensitive to contamination from the outside; carrying cells from one batch to another too can be disastrous. So, designing a plant in the right way to minimize the opportunity for contamination is a primary requirement that can help prevent expensive mistakes. Here, Thorsten Vammen, director of GEA Liquid Processing in Skanderborg Denmark, looks at what can be done to avoid contamination from your white biotech plant at the design stage.

White biotech is the name given to that particular branch of biotechnology that is concerned with industrial processes. It uses living cells - from yeast, moulds, bacteria and plants and enzymes to synthesize products that are easily degradable, require less energy and create less waste during their production.

The equipment used for white biotech processing includes fermenters, separators, evaporators, freeze- and spray-dryers, valves, pipework, etc.

All of this equipment is designed for efficiency, efficacy and to minimize the opportunities for contamination.

So when designing a complete plant, it’s not the equipment itself that causes the problem when designing out opportunities for contamination.

It’s the way the whole system is put together and integrated that makes the difference.

Biotechnology is a relatively new science. Although it has been an accepted practice for decades it has only been relatively recently that the shortage of oil worldwide, and the environmental concerns relating to fossil fuel emissions, has seen the rise of biotechnology as an alternative to oil.

It is this driver that has largely been at the root of the recent rise in invest-

ment. However the meteoric rise in popularity has also meant that only the newest sites have been designed specifically to handle biotech products. Many existing plants were originally designed as chemical plants where Clean in Place (CIP) technology was not generally employed. Therefore, some of these plants that today are used for producing biotech products are not made for CIP, either because they used to be chemical plants or because people are not used to designing CIP’able systems.

One of the problems of not having proper cleaning is that ‘left overs’ from the product stay in the system. When sterilizing, the product that is left over will build up over time and be burned onto the equipment. This makes it difficult for the sterilizing steam to reach all the crevices and corners. Even if the left over material is sterile in itself, it creates a perfect environment for bacteria to grow. In addition, burnt-on product remains will, over time, produce particles which can easily clog and cause a malfunction in steam traps and small drain valves, etc.

Dead legs too are another challenge. These are areas, usually within pipework, that cause air pockets making it difficult for the steam to reach to the bottom of the dead leg area. These dead leg areas typically have seals at the bottom which can be notoriously difficult to clean.

Multi-purpose plants In today’s competitive world plants have to work harder. Many biotech plants are now multi-purpose, producing different products based on different types/strains of bacteria. In these cases it is very important that there is no cross contamination from the previous batch especially if the previous batch was based on a bacteria type that is ‘stronger’ than the

The part of the industrial biotech segment that are producing living bacteria, frequently require their cleaning and sterilization systems to achieve a very high efficacy level during product treatment at F0= log10 9 to log10 16.

To obtain this high level it is necessary to have a well-functioning CIP system combined with a well-designed steam sterilization system (SIP).

To achieve this level of efficacy many factors come into play. The physical system design, selection of best suited components, care in mechanical manufacturing and a well-designed control system will be of outmost importance. Even very small design flaws can lead to product losses. More importantly, perhaps, if there are faults, finding them can be very complicated, difficult and time consuming leading to lost production.

Frequently it is not the production systems that are the main culprits: utility systems such as water supply, steam generation, ventilation systems and gas and chemical supply systems can harbour bacteria if the cleanliness of these systems does not fall within design parameters. It is often these utilities that do not always receive the attention they require and, therefore, can be the starting point for serious problems.

Designing out contamination Effective cleaning is important, however designing systems that are not bacteria friendly is a vital part of a system. For example, all instruments should be flush mounted to avoid creating dead legs or crevices where bacteria can collect. Every piece of pipework must be designed in such a way as to ensure that the CIP fluid hits all surfaces at the correct velocity and temperature. Utility systems too should be designed to be cleaned and sterilized or if this is not required, a sterile barrier should be created at the point where the utility product meets the sterile process.

Air pockets can be a problem and should be designed out as far as possible. If this is impracticable, the air should be capable of being vented.

Air creates an isolation layer between the steam and the metal preventing the metal surface from reaching the

correct temperature for effective sterilization to take place.

It is also necessary to design out human intervention as much as possible to avoid contamination. Instruments also need to be placed correctly to ensure that they provide accurate readings of temperature and pressure to validate sterility and, therefore, verify that sterilization has been performed to the correct level.

The environmental gain Designing a plant to limit contamination and to make sterilization easy limits the amount of chemicals, water and power necessary during the sterilization process. In turn, this saves money and limits the effect of the process on the environment. The initial investment might be a little higher but the total cost of ownership will be lower, more than compensating for the additional up-front expenditure. Less use of power reduces fuel bills, avoids penalties for unacceptable emissions and saves resources.

Efficient use of chemicals and the clever use of water – including closed circuit systems – minimizes disposal costs. Good cleaning means less down time and can reduce the need to use preservatives.

Saving money and avoiding contamination with heat recovery Heat recovery systems save money and help the environment too so they make good commercial sense and can save up to 20 per cent on heating bills for chemical plants. Modern systems can also contribute significantly to reducing contamination in sanitary operations. Heat recovery systems re-use heat that has already been generated in the plant. One of the main opportunities for heat recovery is after CIP and SIP operations that inherently require high temperatures to be effective. By recovering this energy and using it for pre-heating of the next batch, it’s possible for plants to make a significant power saving.

Thorsten Vammen is the Director of GEA Liquid Processing in Skanderborg, Denmark.

To see this story online visit www.laboratoryfocus.ca/ designing-out-contaminationfor-white-biotech-applications/

www.laboratoryfocus.ca

Laboratory Focus March 2013

NEW PRODUCTS

CE Platform The new PA 800 plus nextgeneration capillary electrophoresis (CE) platform by Beckman Coulter Life Sciences provides automated, quantitative analysis of protein purity, charge isoform distribution and glycan structure. Originally designed with biopharmaceutical analysis in mind, the system can also be used in food and beverage quality studies for monosaccharide and protein analysis, and in the quantitative characterization of ions, organic acids and other charged or polar molecules of importance. The PA 800 will be launched alongside the Vi-Cell Series CELL Viability Analyzer; the Multisizer 4 COULTER COUNTER, a versatile and accurate particle sizing and counting analyzer; and the LS 13320 laser diffraction particle size analysis instrument.

Web: www.beckmancoulter.com.

Grinder RETSCH introduces its new CryoMill for grinding sample materials that cannot be processed at room temperature. An integrated cooling system ensures that the grinding jar is continually cooled with liquid nitrogen before and during the grinding process. Thus the sample is embrittled and volatile components are preserved. An autofill system provides liquid nitrogen in the required amount so that the user never gets into direct contact with the LN2 which makes operating the mill particularly safe. The new CryoMill achieves considerably improved grind sizes thanks to the increased oscillating frequency of 30 Hz. Another new feature is the possibility to store up to 9 SOPs. Further improvements include a reinforced housing, optimized liquid nitrogen duct and new accessories such as an adapter for six reaction vials and a 10 ml grinding jar. The mill can also be operated without cooling which makes it suitable for a vast range of applications.

Web: www.retsch.com.

Photoelectric sensors Baumer introduces qTeach, an easy-to-use, wear-free teach procedure initiated by touching the housing of O500 series photoelectric sensors.

O500 optical sensors with qTeach features no mechanical buttons or moving parts that can wear out, and are available in three models: diffuse sensors with background suppression, SmartReflect reflector-free light barriers and retro-reflective sensors for reflective surfaces. The sensors require no mechanical adjustment during setup and offer convenient teach-in process with a light touch to the housing using any ferromagnetic tool such as a screwdriver. A blue LED on the sensor provides clear optical feedback during the teach procedure, blinking to indicate when the user has activated teach mode and when the teach function has been completed. To prevent inadvertent reprogramming in the field, qTeach automatically locks out after five minutes of operation.

Web: www.baumer.com

Scales AirClean Systems PowderSafe Type B enclosures usher in a new era of powder weighing and containment technology. Equipped with a controlled negative pressure HEPAfiltered environment, PowderSafe Type B is fabricated with polypropylene, which is a chemically resistant, high mass polymer. The innate chemical resistance of polypropylene allows users to easily clean the enclosure without the worry of degradation, while the high mass construction alleviates the threat of vibration and balance disturbance during weighing. Furthermore, FlowSmooth™ technology provides even, horizontal air distribution throughout the enclosure, preventing turbulence. This laminar horizontal air movement is also key to operator protection from hazardous chemical and compounds being manipulated within the enclosure.

Web: www.aircleansystems.com

Thermal Cycler Techne has introduced a new Combi-Block for its widely used Prime Thermal Cycler range, providing instrument users with even more flexibility. The new Combi-Block has a total capacity of 66 tubes, either 33 x 0.2 ml or 33 x 0.5 ml volume.

There is an optional 25°C temperature gradient spread over the 11 columns. Multi-user laboratories and core facilities have a range of cycling requirements, and the use of different sized wells means a selection of consumables can be accommodated in the same instrument. Techne Prime Thermal Cyclers are suitable for a range of PCR applications, with the new Combi-Block adding to the existing 96-well (0.2 ml), 60-well (0.5 ml) and 384-well block options. Prime Thermal Cyclers can be purchased with or without a temperature gradient, with the option to upgrade later using a simple unlock code. The instruments are supported by Techne’s market leading four year warranty.

Web: www.techne.com

Sequencing kit The NEXTflex 16S V4 Amplicon-Seq Kit has been developed to simplify bacterial metagenomics studies using Illumina® HiSeq and MiSeq platforms. This kit allows users to go from sample to sequence in two hours. Using specialized NEXTflex primers that target the V4 region of the 16S subunit, a single PCR amplification simultaneously ligates the necessary sequencing and barcoded region for multiplexing. The entire workflow requires only one clean-up step maximizing recovery.

The kit is available with up to 48 barcodes, with higher degrees of multiplexing available on a custom basis.

Web: www.biooscientific.com

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NEW PRODUCTS Pipettes After nearly a year on the market, the flexible, accurate Mettler Toledo Rainin E4 XLS family of electronic pipettes are designed to be the most ergonomic electronic pipette available. Offering a comfortable grip, precise balance and a large full-colour screen, the pipette is made to enhance speed and prevent user fatigue. The E4’s joystick control and logical menus, available in single channel, multichannel and adjustable spacer formats, simplify switching between functions. The E4 XLS is also extremely versatile, allowing operators to gain its ergonomic benefits across a wide range of pipetting functions. These include titration, dilution, reversemode pipetting and sequential volume programming for complex protocols. A multi-dispense feature automatically calculates how many aliquots can be dispensed from a single pickup so operators quickly ascertain how many samples can be prepared.

Web: www.mt.com/e4

Photometer The new, Omega lightweight HHWT-13 series of handheld dip strip photometers are a more “green” and cost effective alternative to testing water for cyanide, iron, ammonia, phosphate, pH (five to 10) sulfide, fluoride, chloride and Quarternary ammonia. Instead of using a 10 ml water sample, this CE compliant product uses a four ml water sample, which uses up to 60 per cent less chemical per test. It features a simple three-button control, 140 test memory and an automatic countdown test time. This product is ideal for wastewater treatment and in all industries which require the monitoring of their process pH.

Web: www.omega.com

HPLC columns Protea Biosciences Group Inc. introduces its Amplus wide-pore core shell HPLC columns, a new line of liquid chromatography columns specifically designed for intact protein analysis. This new family of HPLC silica-based columns employ core shell technology combined with a specific porosity designed to facilitate protein separation, including those with high molecular weight.

Web: www.proteabio.com

PCR System Thermo Fisher Scientific Inc. introduces its new SureTect PCR System.

The system is ideal for food safety laboratories that are testing for the most common bacterial pathogens. With the new system, results for all targets and matrix types can be obtained in less than 24 hours of the food sample being received, leading to faster food release. The SureTect System combines a real-time PCR instrument with bespoke software and convenient pre-filled reagent components that together with a single PCR protocol for a range of common targets and sample types, streamline the test workflow. Assays for Salmonella species and Listeria monocytogenes are available now. Assays for Listeria species and Escherichia coli O157:H7 will be released later this year with further tests already in development.

Web: www.thermofisher.com

March 2013 Laboratory Focus www.laboratoryfocus.ca

Data logger The RTR-574-H high precision wireless 4 channel light logger achieves a temperature measurement accuracy of ±0.3°C (between 10 to 40°C) with an overall range of -30 to 80°C while the humidity measurement accuracy is ±2.5 per cent (at 25°C, 10 to 85 per cent RH) with an overall range of 0 to 99 per cent RH. Other features include a TR-574-H illuminance UV recorder that can simultaneously measure and record four parameters: illuminance, ultraviolet light (UV), temperature and humidity. In addition to these parameters, the TR574-H is also capable of displaying cumulative illuminance and cumulative amount of ultraviolet light in the LCD display. The wide luminosity range covers 0 to 130k lx and low light resolution to 0.01 lx and the UV range covers 0 to 30 mw/cm2. Internal accumulation of exposure is for both light and UV. This compact, lightweight unit is approximately 2” X 3” and operates on one AA battery. The TR-74Ui-H has a large data capacity which can store up to 8,000 readings times four channels for a total of 32,000 readings in one-time or endless recording mode. This unit is compatible with any RTR-500 series wireless data collector and can be used for automatic downloading of logged data, real time monitoring and warning notifications by email or text to cell phones.

Web: www.tandd.com

COMPANY & ADVERTISER INDEX COMPANY

PAGE

WEBSITE

Atrium Innovations.............................. 7...............www.atrium-innovations.com Baumer............................................ 15.............................. www.baumer.com Beckman Coulter................................ 8................... www.beckmancoulter.com Bioniche Life Sciences Inc.................... 7.............................. www.bioniche.com BIOO Scientific.................................. 15...................... www.biooscientific.com Caledon Labs.................................... 5......................www.caledonlabs.com Cangene............................................ 4..............................www.cangene.com CDRD Ventures Inc............................. 7....................... www.cdrd.ca/cvi-home/ Chemical Institute of Canada............. 6................ www.cheminst.ca/profdev Children’s Miracle Network............. 4.... www.childrensmiraclenetwork.ca CO2 Solutions.................................... 6.............................. www.co2solutions EMD Millipore................................... 10......................... www.emdmillipore.ca Eppendorf........................................ 20.......................... www.eppendorf.ca GEA Liquid Processing........................ 14........................... www.gea-liquid.com Government of Canada...................... 7................... www.publichealth.gc.ca Imaging Dynamics Company Ltd............ 6..................www.imagingdynamics.com Mandel............................................ 11...............................www.mandel.ca Mettler Toledo.................................. 16................................www.mt.com/e4 Oncolytics Biotech.............................. 7.................www.oncolyticsbiotech.com Protea Biosciences Group Inc............. 16...........................www.proteabio.com RETSCH............................................ 15............................... www.retsch.com Stem Cell Therapeutics Corp................ 7......................www.stemcellthera.com Techne.............................................. 15...............................www.techne.com Thallion Pharmaceuticals..................... 7............................... www.thallion.com Thermo Fisher Scientific..................... 16...................... www.thermofisher.com Valeant Pharmaceuticals..................... 7............................... www.valeant.com VWR................................................. 2.................................. www.vwr.com Xenon Pharmaceuticals....................... 6..................... www.xenon-pharma.com

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APRIL 2013 April 6-10

March 2013

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Fax: 203-840-9648 Email: inquiry@interphex.com Web: http://www.interphex.com/

MAY 2013

1 12/19/2012 10:01 AM Page 1 AACR Annual RC_lab_new:Layout Meeting 2013 April 26-27 Venue: Washington, DC 11th Annual Global Biomarker Tel: 215-440-9300 Conference Fax: 215-440-9313 Venue: Toronto, ON Email: aacr@aacr.org Email: info@ Web: www.aacr.org globalbiomarkerconference.org Web:www.globalbiomarkerconference.org April 7-11

May 5-9

May 5-8 Wave Conference 2013 Venue: Lake Louise, AB Tel: (780) 468-2443 Email: kbrizel@acamp.ca Twitter: @acampmnt Web: www.wave2013.com

ARVO 2013 Venue: Seattle, WA Tel: 240-221-2900 Fax: 240-221-0370 Email: arvo@arvo.org Web: http://www.arvo.org/ Annual_Meeting/

245th ACS National Meeting & Exposition Venue: New Orleans, LA Tel: 202-872-6061 Email: nationalmeetings@acs.org Web: portal.acs.org/portal/acs/corg/ content

SRC103

April 9-11 Design of Medical Devices Conference Venue: Minneapolis, MN Tel: 612-626-5642 Email: johnsont@me.umn.edu Web: www.dmd.umn.edu

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World Orphan Drug Congress USA Venue: Washington, DC Tel: +1 212 379 6322 Fax: +1 212 379 6319 Email: enquiry.us@terrapinn.com Web: www.terrapinn.com/ conference/world-orphan-drug-congress-usa/index.stm

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April 13-16 ACRP 2013 Venue: Orlando, FL Tel: 703-254-8100 Fax: 703-254-8101 Email: office@acrpnet.org Web: http://acrp2013.org/

April 20-24 Experimental Biology 2013 Venue: Boston, MA Tel: 301-634-7075 Fax: 301-634-7008 Email: eb@faseb.org

April 22-25 2013 Bio International Convention Venue: Chicago, IL Tel: +1-202-962-6655 Email: convention@bio.org Web: www.convention.bio.org/

April 22-26 12th International Symposium on Mutation in the Genome Venue: Lake Louise, AB Tel: +61 (0) 3 8344 1831 Fax: +61 (0) 3 9347 6842 Email: rania@variome.org Web: wired.ivvy.com/event/ MPZ4L2/

April 23-25 INTERPHEX 2013 Venue: New York, NY Tel: 203-840-5648

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Laboratory Focus March 2013 www.laboratoryfocus.ca

Readers want more basic research funding We recently ran a survey asking readers to respond with their thoughts and feelings about Canada’s biotechnology and life science industries.

Through your answers, we put together an industry trends report for 2012, which you can read online at www.laboratoryfocus.ca.

We asked you about the state of research and innovation in Canada, what issues you want raised before government and what needs to change for Canada to be a world leader in your field.

For a clearer picture, we divided the survey into two streams: business and research. The answers highlighted below are from the survey for researchers, innovators, lead investigators, scientists, research organizations and academia.

In answer to the question do you feel government is listening to what the life science field is telling it, a resounding 85.7 per cent of you said ‘no,’ with only 14.3 per cent saying ‘yes.’

Top among the concerns you want raised to government was the lack of funding for basic discovery-based research. Respondents called for more funding agencies (or simplified application methods) for new discoveries that require risk – innovation – to really take off. You also wanted funding to be awarded based on quality instead of commercial potential. While traditionally Canada’s weak spot has been getting innovations commercialized, respondents cautioned that collaborations created with the sole intent of commercialization can curb true innovation.

Meanwhile in response to the question is Canada a leader on the world stage in your field, just 41.2 per cent said ‘yes,’ while 64.7 per cent answered ‘no.’

However in your individual comments, you credited Canada’s strongest attributes as the intelligence and creativity of its researchers and the excellence of our academic institutions. Results that are reflected in your feedback concerning the state of research and innovation in Canada: 44.4 per cent rated it above average (more so on the research side), 44.4 per cent called it average and 11.1 per cent below average.

Concerning funding for the industry, a whopping 83.3 per cent of respondents found granting and financing opportunities harder to come by, while 16.7 per cent found them the same.

Many also noted that a small group of individual researchers are getting increasingly larger grants, while less funding is available for the majority of researchers and students. Similarly, respondents mentioned they would rather see money being distributed amongst more researchers instead of going towards “big stars.”

Lastly, many respondents commented on the plight of students and recent graduates. As research funds and post-graduate scholarships decline, many are concerned that we could enter another brain-drain period if our grads cannot find employment in Canada. Respondents said it’s time to think about a national strategy for funding graduate student training.

We thank you for participating in our survey and Happy March!

CAREER SPOTLIGHT Bio-economy Career Profile

Compiled by BioTalent Canada Position: Assistant Plant Manager Name: Mike Fletcher Company: Iogen Corporation Salary Range: $80,000 to $100,000 per year

What I do:

My role is assisting in all operational aspects, ensuring the facility reaches its goals. I ensure the health, safety, and environmental issues are taken into account each day. The Assistant Plant Manager role is a mixture of short-term problem solving and longer-term planning, including building teams and defining employee roles. Hiring people is part of the process, as we are a growing company and always looking for good people. Other duties include assisting in marketing the commercial enzymes that the plant produces for food, pulp and paper, and textiles.

What education and skills do candidates need for this position?

People in the position tend to have a bio-science, chemical engineering, or biology background. However, plant managers are from all sorts of disciplines; as long as you are someone who is willing to learn on the job, and you come to the position with core competencies. Essential skills include the ability to adapt to changing circumstances in order to take advantage of opportunities that may arise to better the company. There is also a need to effectively troubleshoot, and keep an open mind to learning new things on the job. People skills are also important, as the Assistant Plant Manager spends a good part of the day on the plant floor speaking with employees and listening to their concerns.

What are the best parts of your job?

The best thing about this job is having the ability to take some risks in trying to improve the operation of the plant. It is good for morale and good for the company’s bottom line when improvements pay dividends. I encourage people interested in the industry to remain open-minded about the course of their career, and remember that you can learn from anybody. As well, it’s important to not be too easily discouraged. You can always work your way up in the bio-economy sector.

For more Bio-economy Career Profiles visit www.biotalent.ca/careerprofiles-list-en

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Think outside the bag New Eppendorf New Brunswick CelliGen® BLU 50 L single-use vessels The Eppendorf New Brunswick CelliGen BLU portfolio now features interchangeable pitched-blade, single-use vessels in 5, 14 and 50 L capacities. An ideal setup for time sensitive R+D labs, cGMP production and BL-2 and higher facilities.

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