Laboratory Focus November/December 2017 by Promotive Communications

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November/December 2017 Volume 21, Number 4

How to reduce the complexity of custom single-use assemblies Page 10

Essential strategies to optimize protein expression Page 9

R&D News.................. 1 Appointments............. 6 Pharma Notes............. 7 New Products........... 16 App Reviews.............. 18

Stem cell harvesting market poised to surpass US$10B by 2025 Mitochondria help cells survive

Mitochondria could hold clue to killing cancer cells The findings of a McGill University research team looking into how mitochondria are able to keep cells alive, could help provide the solution to how to kill cancer cells. McGill scientists Heidi McBride and John Bergeron, working with McGill professor Nahum Sonenberg have found the mechanism which allows mitochondria to prevent cells from dying even when cells have been deprived of nutrients. Funding for the research was provided in part by the Canadian Institutes of Health Research, the Canadian Cancer Society

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Research Institute, the Terry Fox Research Institute, and the National Sciences and Engineering Research Council of Canada. In earlier work years ago, Sonenberg discovered that mTOR also controls protein expression in all human cells. mTOR targets the selective synthesis of proteins destined for the mitochondria which are the structures in a cell that generate the energy needed for cells to grow and divide. The research by McBride, Bergeron, and Sonenberg has now shown that mTOR also controls Continued on page 2

Grand View Research Inc. has done the heavy-lifting and projects that the global cell harvesting system market will reach a value of US$10.17 billion by the year 2025. There is surging demand for stem cell-based therapies due to factors such as an aging population, and increased prevalence of chronic diseases. These two critical aspects may just well be the main contributors towards a large rise in lucrative market growth. The growing investment in the field of stem cell research is one of the high-impact drivers of the

demand for stem cells that are contributing to the growth of the cell harvesting system markets. Another major driver across the globe is the substantial rise in stem cell transplantation. Growth in autologous stem cell transplantation along with increasing stem cell banking is driving demand for cell harvesting systems. The new sources of stem cells, such as umbilical cords, adipose tissue, and embryonic stem cells, exhibit tremendous potential to unlockContinued on page 3

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November/December 2017

news Mitochondria Continued from page 1

PUBLISHER/EDITOR-IN-CHIEF Terri Pavelic Editor Nestor Arellano Writer Michelle Currie CONTRIBUTING WRITERS Michael Liss Geoffrey Cassell Axel Trefzer Arnd Dankesreiter Guy Matthews Hisashi Konaka Akito Sasaki GRAPHIC DESIGNER Elena Pankova CONTROLLER John R. Jones MARKETING MANAGER Melisa Sukhdeo CIRCULATION DIRECTOR Mary Labao mary@promotivemedia.ca Tel: 905-841-7389 OFFICE: 226 Edward St. Unit 1 Aurora, ON L4G 3S8 Phone: 905-727-3875 Fax: 905-727-4428 E-mail: laboratory_focus@ promotivemedia.ca SUBSCRIPTION INQUIRIES mary@promotivemedia.ca Fax: 905-727-4428 Laboratory Focus is published 4 times per year by Promotive Communications Inc. Legal Depository: National Library of Canada ISSN 40052410 Subscription rate in Canada $35/year; USA $60/year; other countries $100/year. All rights reserved. No part of this publication may be reproduced without written consent. Publications Mail Registration Number: 40052410 Return undeliverable Canadian addresses to circulation dept: 1-226 Edward Street, Aurora, ON L4G 3S8 E-mail: mary@promotivemedia.ca All opinions expressed herein are those of the contributors and do not necessarily reflect the views of the publisher or any person or organization associated with the magazine. If you would like to order hard copy or electronic reprints of articles, contact www.laboratoryfocus.ca

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Feds give U of A $23-M for new labs, equipment The Government of Canada is providing the University of Alberta more than $23 million to fund the building of new laboratories, the purchase of lab equipment and to help train the next generation of scientists. Minister of Infrastructure and Communities, Amarjeet Sohi, speaking on behalf of the Minister of Science, Kirsty Duncan, announced the investment through the Canadian Foundation for Innovation’s Innovation Fund to support research infrastructure at the university. The funding for the University of Alberta is part of the more than $554 million earlier announced by Duncan. The funding will support 117 new infrastructure projects at 61 universities, colleges and research hospitals across Canada. “Our government understands that scientists need to have the best labs and tools if they’re going to make discoveries that will pave the way to a brighter future for all people,” Duncan said. “That’s why today’s funding announcement is so important; it gives scientists and their students the op-

portunity to further their research in areas where Canada has a competitive advantage.” “This CFI funding strengthens the University of Alberta’s research capacity in a number of diverse areas — nanotechnology, Arctic research and the health sciences — where UofA research can improve the lives of Canadians and people around the world,” said Dr. David H. Turpin, president, and vice-chancellor, University of Alberta. The Innovation Fund supports initiatives that allow universities, colleges, and research hospitals as well as students at these institutions to build on existing areas of expertise such as artificial intelligence, quantum science, brain health and renewable energy. The investment of more than $554 million announced by Minister Duncan includes $127,098,512 awarded under the CFI’s Infrastructure Operating Fund, which assists institutions with the operating and maintenance costs associated with their new research infrastructure. To see this story online visit http://laboratoryfocus.ca/feds-giveu-of-a-23-m-for-new-labs-equipment/

the expression of proteins that alter the structure and function of mitochondria. This allows mitochondria to protect cells from dying, according to a report by Cynthia Lee, in the McGill news site. mTOR is being studied in clinical trials for cancer treatments. However, while treatments have been able to stop the growth and division of cancer cells, they have been unable to kill the cancer cells. This recent study shows that mitochondria help cells survive by blocking a point to the apoptosis or the central point in a cell death pathway. According to the researchers, this offers the hope that a combination of therapies could kill cancer cells by reversing the protection provided by mitochondria..

To see this story online visit http://laboratoryfocus.ca/ mitochondria-could-hold-clue-tokilling-cancer-cells/

Stem cells Continued from page 1 ing new cancer treatments and the development of regenerative products. However, there are issues related to the use of embryonic stem cells and unapproved stem cell therapies rendered by healthcare providers in unregulated regions, such as Latin America and Asia Pacific that may limit the growth of this market within the forecasted period. The growth of the stem cell harvesting systems across the globe can also be attributed to the potential use of stem cells in regenerative medicine; such as cancer, trauma, and congenital diseases. The rising prevalence of certain diseases, as in the case of cancer, is expected to sky-rocket the growth of this market in the forecasted future. Further key findings from the report:

• Umbilical cord, bone marrow, peripheral blood, adipose tissue, and other sources are the application segments analyzed in this study. • Hospitals held the largest share in the end-use segment and are expected to hold the largest share over the forecasted period, as the majority of stem cell harvesting and banking is done within hospitals. • Stem cell research will contribute to the rise of stem cell harvesting in academic institutes and R&D centres. • North America and Europe subjugated the cell harvesting market, contributing to a high rate of stem cell transplantation and research. • The aging population and increased prevalence of chronic diseases will boost the stem cell harvesting market growth, especially in North America and Europe.

Some of the major global companies that are expected to contribute to this growth are Argos Technologies Inc., Perkin Elmer Inc., Brand GmBh + CO KG, Arthrex Inc., Avita Medical, Tomtec, Terumo Corporation, Teleflex Inc., and Bertin Technologies. The report gathered by Grand View Research Inc. forecasts growth at global, regional and national levels to provide an analysis of the industry trends in sub-segments from 2014 to 2025. It has segmented the global cell harvesting system market based on application, end-use, and region. To see this story online visit https://biotechnologyfocus.ca/ research-is-in-stem-cells-expected-to-grow-to-over-us10b-industryby-2025/

November/December 2017 Laboratory Focus www.laboratoryfocus.ca

news

CRISPR base editing shows potential for fixing mutations A recently published study on CRISPR could potentially lead to breaking new ground in the use of genome editing to cure diseases. Many human diseases are caused by genetic mutations. Standard CRISPR editing employs gRNA and an enzyme, usually Cas9, to attach to a specific portion of a DNA base. Cas9 is used to snip the DNA at a targeted spot. Unfortunately, this method has

limited targeting abilities. The issue of off-target effects is important because there is the possibility that genome editing could inadvertently disable a tumour-suppressor gene or activate a cancer-causing gene. However, according to a study published in Nature, a new class of base editors could potentially be used to correct. The double helix is made up of four

bases which are identified by letters: A (adenine), G (guanine), C (cystosine), and C (thymine). They are arranged in a way that C pairs with G and A pairs with T. Unlike conventional CRISPR editing, base editing does not cut the double helix. Instead, this method uses enzymes to rearrange certain atoms in one of the four bases that comprise the DNA or RNA. This method converts the target base without changing the other bases around it. “The spontaneous deanimation of cytosine is a major source of C.G to T.A transitions which accounts for half of the known human pathogenic point mutations,” according to the study. “The ability to efficiently convert targets A.T base pairs to G.C could therefore advance the study and treatment of genetic diseases. Authors of the study said the method creates less off-target effects than Cas-9 and can install disease correcting or disease suppressing mutations in human cells. In another development Dr. Fen Zhang, core member of the Broad Institute of MIT and Harvard, described in Science, how he and his team developed a new CRISPR-based system called, RNA Editing for Programmable A to I Replacement or REPAIR for short.

With this system, they were able to alter single RNA nucleotides in mammalian cells in a precise manner. The researchers said REPAIR can reverse disease-causing mutations at the RNA level. The researchers searched the CRISPR-Cas13 enzyme family for a potential “editor.” Unlike Cas9 which targets DNA, Cas13 targets and cuts RNA. The team chose an enzyme called PspCas13b as an editor. The PSPCas13b was fused with a protein called ADAR2 which has the ability to alter the letters A to I in RNA transcript. Using the REPAIR system, Cas13b binds with the targeted RNA sequence and the ADAR2 converts the transcript without cutting it. “Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where diseaserelevant sequences can be rescued to yield functional protein products,” according to the study.”… REPAIR presents a promising RNA editing platform with broad applicability for research, therapeutics, and biotechnology.” To see this story online visit https://biotechnologyfocus.ca/ crispr-base-editing-shows-potentialfor-fixing-genetic-mutations/

Biomass burning emissions causes DNA damage: Study Researchers in Brazil have found that pollutants emitted by burning biomass can cause DNA damage and lung cell death. The researchers exposed human lung cells in a laboratory to pollution levels similar to those found in the Amazon region during the forest and crop burning seasons. In a study published in the journal Scientific Reports, the research team reported that after 72 hours of exposure to the pollutant retene, a chemical compound that belongs to the class of polycyclic aromatic hydrocarbons (PAHs), more than 30 per cent of the cells had died. Most of the studies on air pollution focus on emissions from fossil fuel burning in urban centers, according to the study which was authored by Nilmara de Oliveira Alves Brito and colleagues. “However, approximately half of the world’s population is exposed to air pollution caused by biomass burning

emissions,” the study said. “In the Brazilian Amazon population, over 10 million people are directly exposed to high levels of pollutants resulting from deforestation and agricultural fires. “ The study is the first one to present an “integrated view” of the effects of inhaled particles found in emissions of biomass burning. When human lung cells are exposed to particulate matter smaller than 10 µm (PM10) there is a significant increase in the level of reactive oxygen species (ROS), inflammatory cytokines, autophagy, and DNA damage, the team found. They said that retene is a potential compound that causes cell death. Studies show that people living in the Amazon’s deforestation arc breathe air with high concentrations of particulate matter smaller than 10 µm. The situation is exacerbated during the dry seasons when concentrations of PM10 have been measured (ranging from 400 up to 600 µg.m−3). That

exceeds by eight to 12 times, the upper limits of (24 h exposure to PM10 – 50 µg.m−3)which have been set by the World Health Organization.

To see this story online visit http://laboratoryfocus.ca/biomassburning-emissions-causes-dnadamage-study/

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UCalgary research project seeks faster diagnoses of rare genetic childhood diseases A project that is studying how precision medicines can help diagnose and determine treatments for rare genetic childhood diseases is among the six University Calgary projects being funded by the Canadian Foundation for Innovation. Studies have indicated that one-infour children admitted to hospitals are there because of illness that could be traced to genetics. Delays or failure to diagnose genetic diseases in children are common and often result in children going through useless and even harmful treatments. Part of the grant received by Prof. Jay Cross of the Communing School of Medicine and Faculty of Veterinary

Medicine, will be used to acquire innovative laboratory equipment in the areas of genomics and imaging technology, according to the UCalgary news site. Cross, who is also a member of the Alberta Children’s Hospital Research Institute (ACHRI), said that developments in technology have allowed researchers to gather genetic information more quickly an at a lower cost than before. The ACHRI team taking part in the grant is bio-engineering model organisms such as zebrafish. They intend to create a model organism with diseases which scientists can experiments on at a cellular level.

Through this approach, they believe scientists can better understand which genes have a role in causing certain conditions. The hope is that through the knowledge acquired, scientists can develop new genetic screening tests that would quickly identify the disease in patients. Through better understanding of the disease, scientists can also screen drugs that would lead to better treatments. To see this story online visit http://laboratoryfocus.ca/ucalgaryresearch-project-seeks-fasterdiagnoses-of-rare-geneticchildhood-diseases/

Top 10 research priorities for neurodevelopmental disorders set Care providers working with health professionals in Ontario have come up with a shortlist of the top 10 questions around research covering neurodevelopmental disorders. Participants in the project hope these questions will help prioritize and guide the direction of future research into brain disorder research. The Ontario Brain Institute (OBI), a provincially funded not-for-profit, conducted Canada’s first Neurodevelopmental Disorders Priority Setting Partnership workshop in Toronto in partnership with POND (OBI’s research program on neurodevelopmental disorders) and the James Lind Alliance (JLA). “The priority setting process will set the tone for future research of neurodevelopmental disorders,” said Dr. Tom Mikkelsen, president & scientific director at Ontario Brain Institute. “To fully capitalize on our learnings, OBI will methodically communicate the top 10 questions to broader neuroscience research community and work with researchers, funders, and policy makers to help ensure the priorities are adopted, addressed and translated into real world impact.” The priority setting process gathered over 1200 questions from 300 respondents across Ontario. Patients, caregivers, advocates, and health/education professionals consolidated, prioritized and ultimately distilled these questions into a shortlist of 10 research priorities. The 10 questions are: 1. What are the most effective treatment options/plans (e.g., timing,

9. Which are the most effective pharmacological and non-pharmacological intervention(s) to reduce anxiety in individuals with neurodevelopmental disorders? 10. Which interventions are most effective to help individuals with neurodevelopmental disorders improve their social skills and develop and maintain social relationships?

frequency, duration, type, intensity or dosage) for individuals with neurodevelopmental disorders for both short and long-term benefits? How can system navigation be organized in a manner that enables coordinated services and supports across the lifespan for individuals with neurodevelopmental disorders and their families? Which biological treatments (including medications, gene therapy, stem cell therapy, etc.) are effective for neurodevelopmental disorders and associated symptoms? Which child and family-centred interventions or approaches promote optimal individual and family functioning? Which interventions best help individuals with neurodevelopmental

disorders develop emotional and behavioural regulation (including increasing impulse control and reducing compulsive behaviour)? 6. Which resources are needed to more effectively address the health, social and emotional needs of families or caregivers of individuals with neurodevelopmental disorders? 7. How can treatment decisions for individuals with neurodevelopmental disorders be more precise (i.e., based on the diagnosis, age, functional need of the individual)? 8. Which are the most effective pharmacological and non-pharmacological treatments for aggressive and selfinjurious behaviour in individuals with neurodevelopmental disorders?

To maintain transparency and accuracy throughout the priority setting process, OBI and POND formed a Steering Group that included members from Holland Bloorview, Autism Ontario, Community Living Toronto, Children’s Hospital London Health Sciences Centre and The Centre for Addiction and Mental Health to help shape the process and eliminate any bias in the course. With this priority setting workshop, OBI along with its community partners will be able to accelerate towards a common goal of improving the lives of people living with neurodevelopmental disorders. “Asking the right questions is an integral part of research, hence setting priorities in partnership with the patient community is central to ensuring our work has direct impact,” said Dr. Evdokia Anagnostou, senior clinician scientist, Holland Bloorview Kids Rehabilitation Hospital and chair of the Steering Group. To see this story online visit https://biotechnologyfocus.ca/top10-research-priorities-forneurodevelopmental-disorders-set/

November/December 2017 Laboratory Focus www.laboratoryfocus.ca

Appointments

BiOasis Technologies Inc. has appointed Christopher P. Lowe, MBA, as chief financial officer and board advisor. Lowe will report to Mark Day, president and chief executive officer of biOasis and will be responsible for the company’s corporate and financial strategy that includes transactions and fundraising. Lowe has over 15 years of senior management experience as president, chief building officer and chief financial officer of various private and public life sciences, medical technology and technology companies. More recently, Lowe has been a partner at FLG Partners, a leading CFO service and board advisory firm in Silicon Valley. Prior to being a partner at FLG, he served as a strategy, financial and management consultant to several public and private companies. He has also served as a member of the board of directors to both public

Christopher P. Lowe

and private companies, and has chaired audit and compensation committees. Lowe contributes to companies by assisting them with corporate and financial strategy, successive fundraising, public company advising, Wall Street communications, licensing transactions, mergers and acquisitions. Lowe has successfully raised over $5 million in private and public financings, completed over $1 billion in merger and acquisition transactions, negotiated over $300 million in debt instruments for companies, and has overseen three SOX-404 implementations. Montreal-based BioAmber Inc. has appointed Richard P. Eno as its new chief executive officer and member of the board. Eno brings over 30 years of related management experience to the table from

Richard P. Eno sectors such as, energy, chemical, biotechnological and material industries. He was a president, CEO, and board member of a publicly traded industrial biotechnology company for over five years. Eno began his career in Chevron’s chemical operations with roles in engineering, manufacturing, and construction operations. Later, he began management consulting where he specialized in addressing critical strategic and operational issues and company leadership in the chemical and energy industries. Additionally, he had a leadership role in biotechnology company, Metabolix, and was a senior partner at a global management consultancy. “The board is pleased to have found an individual with an ideal industry background as well as public company experience to assume leadership of BioAmber. Rick is uniquely qualified to lead BioAmber successfully into the future,” said Ray Land, BioAmber’s chairman of the board. Prime Minister Justin Trudeau has named Mona Nemer to be

Mona Nemer

Canada’s new chief science advisor. She is currently vice-president of research at the University of Ottawa, where she oversees research strategies, infrastructure and commercialization; and is also a professor in the faculty of medicine and director of the molecular genetics and cardiac regeneration laboratory. Nemer’s office will have a $2 million budget, and she will report to both Trudeau and minister of science, Kirsty Duncan. Her mandate will include providing scientific advice to government ministers, helping to keep government-funded science accessible to the public, and protecting government scientists from being kept quiet. She will also deliver an annual report to the prime minister and science minister on the state of federal government science.

versity in Quebec City and holds a graduate diploma in clinical pharmacy from the same university. As the medical director for Akcea Therapeutics Canada, Komari will lead the company’s medical affairs function and work directly with the Canadian healthcare community to improve patient care for Canadians suffering with lipid disorders. She will be responsible for the development and implementation of the programs, be the link between Akcea and Canada’s medical community, as well as work with leading healthcare providers involved in the treatment of patients with lipid disorders. Emerald Health Therapeutics appointed Chris Wagner as new chief executive officer and a director of EHT. Wagner has spent over 25 years in marketing for pharma-

Akcea Therapeutics Canada appointed Nelly Komari B. Pharm., MSc., as medical director. Bringing almost 20 years of biopharmaceutical experience and knowledge, Komari has an extensive knowledge of the cardiovascular and throm-

Chris Wagner

Nelly Komari

bosis systems. Akcea Therapeutics Canada, the Canadian subsidiary of Akcea Therapeutics, Inc., an affiliate of Ionis Pharmaceuticals, Inc. is focused on developing and commercializing drugs to treat patients with serious cardiometabolic diseases caused by lipid disorders. Prior to joining Akcea, Komari held several strategic positions at Sanofi, including medical advisor, and was a member of Sanofi’s global medical launch team. Most recently, Komari was involved in the Canadian launch of the cholesterol fighting drug, Praluent, a PCSK9 inhibitor. She received her pharmacy degree from Laval Uni-

ceutical products and the building of biotechnology companies. Wagner will replace Huang, who will continue to focus on the management and growth of one of the company subsidiary’s. He worked with Eli Lilly for 10 years, mounting to global team leader and working in Europe, North America and Asia. He worked on the global marketing of several notable products, including Prozac, Cialis, and Zyprexa. He went on to become vice-president of business development and global marketing at Aspreva Pharmaceuticals Inc., where he aided in the negotiations of global alliances with Roche and Chugai. Most recently, he served as chairman, president and CEO of Contextual Genomics Inc, a molecular bioinformatics company that has worked with AstraZeneca, Pfizer and Sanofi to develop and commercialize genomic cancer diagnostic products.

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Laboratory Focus November/December 2017

Pharma Notes UofT M oncology start-up receives $22-M funding A biopharmaceutical start-up which was spun out of the University of Toronto Mississauga has snapped up $22 million in funding from Medicxi, a venture capital firm based in Europe. The massive cash infusion launched Janpix Inc. The company’s research is focused on small molecular inhibitors known as signal transducer and activator of transcription (STAT) protein. Studies have shown evidence that STAT proteins are involved in the development and function of the immune system and play a role in maintaining immune tolerance and tumour surveillance. With the funding from the GlaxoSmithKline and Johnson & Johnson-backed Medicixi, Janpix will be opening its new headquarters in Massachusetts. Roman Fleck, a former scientist who became a venture capitalist, is chief executive officer of Janpix. STAT 3 and STAT 5 proteins play a crucial role in regulating cell cycle, apoptosis, and proliferation. Until now, STAT proteins “remained a hard-to-crack molecular target” because intracellular protein-protein interactions are extremely difficult to inhibit with small molecules, according to a press release issued by Janpix. Janpix has made great progress in developing tractable compounds that inhibit these difficult-to-target proteins, said Giovanni Mariggi, a principal at Medicxi. Intellipharmaceutics announces 3.64M share direct common offering Novel and generic drugs specialists Intellipharmaceutics International Inc., has entered into a securities purchase agreements with institutional investors for the purchase of 3,636,364 of the company’s common shares at a purchase price of US$1.10 per share in a registered direct offering. The total gross proceeds of the transaction are approximately US$4 million. In a press release, the company indicated that there are plans to use the

net proceeds of the offering for general corporate purposes. This may include working capital, capital expenditures, research and development, accounts payable and other commercial expenditures.

The Toronto-based biotech company specializes in the research, development, and manufacture of novel and generic controlled- and targeted -release oral solid dosage drugs. Its patented Hypermatrix technol-

ogy is a multidimensional controlled-release drug delivery platform that can be applied to a wide range of existing and new pharmaceuticals. Intellipharmaceutics has developed several drug delivery systems based

on this technology platform, with a pipeline of products (some of which have received United States Food and Drug Administration approval in various stages of development.

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November/December 2017 Laboratory Focus www.laboratoryfocus.ca

feature

B y Micha el Liss , Geof f rey C a ss ell , Axel Trefzer , and Arnd Dankesreiter

Essential strategies to optimize

protein expression Many scientific research projects are based on the investigation of either transiently or stably expressed proteins. Achieving detectable and reliable amounts of recombinant protein may be challenging, especially in heterologous expression systems or while expressing highly regulated proteins. In this white paper, we summarize several different technologies that can be applied to maximize the success of your protein expression experiments.

Gene Op�miza�on

Vector Op�miza�on

Cell Culture Op�miza�on

Transfec�on Op�miza�on

Figure 1

Gene optimization After you have chosen your “favourite” expression system (e.g., mammalian, bacterial, or many other systems) there are several ways to obtain the DNA starting material for your expression experiments. Gene synthesis offers the utmost exibility in realizing individual sequence requirements like functional motifs, cloning sites, and detection tags. Since codon usage is diverse in different organisms, a good starting point is to optimize the DNA sequence for expression in your host and obtain your gene by de novo synthesis. By using our GeneOptimizerTM algorithm, you not only adapt the gene to the codon usage

of your host system, but you also remove elements that potentially inhibit expression (e.g., killer motifs, splice sites, and RNA secondary structures). Overall, the GeneOptimizer algorithm takes more than 50 parameters into account in order to determine the optimal gene sequence for more reliable and higher- level protein expression without altering the protein sequence. Figure 1 shows an example of increased expression by optimized gene sequences in different host cells [1].

Vector optimization The coding sequence of a gene is not the only factor that needs to be con-

Gene optimization effects are not restricted to a distinct mammalian cell system. Western blot analyses of ZNRD1 protein transfected using three independent plasmid preparations (PP) into HEK 293T and CHO-K1 cells, or two independent plasmid preparations into Sf9 cells. Right: the fold increase in expression of the optimized gene.1

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Laboratory Focus November/December 2017

feature

Figure 2

External collaborator results

The expression levels of 98 proteins was examined by 22 labs using the Expi293 Expression System. An average increase of 4.6-fold was observed.

The coding sequence of a gene is not the only factor that needs to be considered when optimizing a construct for expression.

sidered when optimizing a construct for expression. It is also recommended to optimize the surrounding noncoding DNA elements. Tuning the expression level by choosing the optimal promoter and terminator combination could also be an essential part of an expression project, as high expression levels of foreign protein driven by a strong promoter or insufficient termination by a weak terminator can lead to growth inhibition of the host. The copy number of the vector, which is determined by the origin of replication, also has a significant influence on the expression level of a foreign protein [2]. There is a broad range of commercially available, predesigned vectors optimized for various expression systems. You can use our Vector Selection Tool available at thermofsher.com/vectors to see if one fits your research purposes. In some cases you might need a vector that is not commercially available. The GeneArtTM ElementsTM Vector Construction service provides you with individually designed vectors, serving your personal experimental needs. Example applications that might require tailored vectors are gene therapy where the on/off regulation of a gene could be a major goal [3] or DNA vaccines where the presence of CpG motifs in plasmid DNA [4] plays an important role.

sion. Options include stable cell line expression systems and transient expression systems. Factors that can be optimized in transient expression systems include cell density, the expression host, and transfection efficiency. For expression in mammalian cells, we have developed the Expi293TM Expression System that optimizes all three of these factors. In collaboration with 22 labs, expression levels of 98 different proteins were tested using the Expi293 Expression System (Figure 2). The following results were obtained: • 87 per cent of proteins demonstrated increased expression in the Expi293 system compared with the user’s current system • 4.6x average increase for all proteins (n = 98) • 4.0x average increase for mAbs (n = 54); highest level was 826 mg/L • 5.3x average increase for nonmAbs (n = 44); highest level was 790 mg/L

Cell culture and transfection optimization

Resources:

The choice of expression system is of further importance for getting optimal and reliable protein expres-

Conclusions We have summarized various factors influencing protein expression. These factors, while not comprehensive, are of considerable importance for achieving reliable and high-level protein expression for your research. Please see the following resources for additional information on the technologies presented here.

thermofisher.com/genesynthesis thermofisher.com/expi293 thermo sher.com/elementsvc

References: 1. Fath S, Bauer AP, Liss M et al. (2011) Multiparameter RNA and codon optimization: a standardized tool to assess and enhance autologous mammalian gene expression. PLoS One 6:e17596.   2. Da Silva NA, Baily JE (1991) In uence of plasmid origin and promoter strength in fermentations of recombinant yeast. Biotechnol Bioeng 37:318–324.   3. Goverdhana S, Puntel M, Xiong W et al. (2005) Regulatable gene expression systems for gene therapy applications: progress and future challenges. Mol Ther 12:189–211.   4. Chen YS, Hsiao YS, Lin HH et al. (2006) CpG-modi ed plasmid DNA encoding agellin improves immunogenicity and provides protection against Burkholderia pseudomallei infection in BALB/c mice. Infect Immun 74:1699– 1705.

Michael Liss is senior manager of R&D; Geoffrey Cassell is market development manager at GeneArt Cloning and Gene Synthesis; Axel Trefzer is R&D team lead; and Arnd Dankesreiter, is senior product manager Gene Synthesis, all from Thermo Fisher Scientific.

To see this story online visit http://laboratoryfocus. ca/essential-strategies-tooptimize-protein-expression/

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B y Gu y Mat thews

How to reduce the complexity of

custom single-use assemblies

A guide for bio manufacturers on how to avoid unnecessary customizations

f more than 85 per cent of your single-use assemblies is customized, you are likely over customizing at the expense of time, performance, and quality. Excessive customization is a key challenge of implementing single-use technology (SUT) in bio manufacturing. Customizing adds six to sixteen weeks or more to lead time, increases costs and potential for quality issues, adds complexity to inventory control and compromises supply chain security. Industry experts estimate 80 per cent of assemblies for new processes is customized — a number that has the potential to be reduced to approximately 15 per cent.

History of the customize everything mentality Since its introduction more than 20 years ago, the benefits of SUT have been well-recognized and adoption has been swift. What started out as a simple bag for holding buffers or cleaning solutions has evolved to thousands of single-use products, and entire processes that run in a singleuse format. The explosion of products is causing challenges; and the industry is calling for standardization. How did we get to this state of excessive customization? The message to end users for more than 20 years has been to customize a solution for every operation, every scale, and every product. That was, and still is, an

attractive proposition compared to the fixed stainless steel facilities SUT replaced. In the past, customization (modification of stainless steel plants) required time-intensive, costly engineering and revalidation work. With the introduction of SUT, customized designs became more affordable; but now customization has gone too far. Customized assemblies are being used when there is no need or benefit. There was a report of one biopharma organization having more than 2,000 different assemblies in use. Upon recognizing the need to standardize, the number of assemblies was reduced by approximately 80 per cent. However, standardizing on the back end

is not the solution. The decision to standardize or customize an assembly should be made on the front-end where upfront time and costs can be avoided.

Consequences of excessive customization Too much customization creates complexity. One challenge is the need to stock and control many low-volume items, including managing and writing-off expired stock. Lead times for products are increased due to low-volume production runs and the changeover processes between products. The potential for quality challenges increases in smaller production runs due to the complex as-

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Feature semblies and customized parts. Finally, costs are higher because of the time required for the design, testing, and management of single-use systems. All of these factors add up to longer implementation times, and potentially, higher cost of goods. Customization, on average, adds a minimum of 16 weeks to a project, compared with only 10 weeks for a configured device. Why is this important? Speed to market is critical when a manufacturer patents a drug — the clock starts ticking. If you have a blockbuster drug, every day of missed sales can be worth millions of dollars. How does customization lead to longer implementation times? Imagine a scenario in which an end user has a project and a need is identified. The solution is customized with a part that has not been used in an assembly before. The end user approaches a single-use systems provider. At a minimum, quality and safety issues must be met, and a number of issues should be resolved before the new device is built into the assembly. These issues take time, both for the vendor and the end user. The end user must be aware of the time requirements — time that may not be included in the project plan. For vendors, customization requires time to: • Ensure product safety and compliance • Verify supply chain reliability • Confirm fit with manufacturing capabilities • Manage parts and components supply timelines

For end users, customization requires time to: • Manage the design review process • Coordinate specialist knowledge or training • Plan ahead for any lead time issues for smaller quantities of customized items • Perform quality checks on a higher quantity of smaller incoming batches

When is a customized solution necessary? A customized solution should only be used when the process absolutely demands it. If other ways to run the process have been considered and nothing else works, then there is a case for customization. The decision should be driven by the process needs and quality requirements. What may seem to be a new process requirement to the process development engineer, has likely been encountered previously by the supplier. This expertise should be exploited as much as possible. The absolute need for a customized part is going to be rare. An example of when a customized assembly may be required could be for process reasons such as material compatibility issues or nonstandard quality requirements. Since customization adds time, cost, and complexity for end users, Parker recommends asking the following questions when considering a customized solution: • Is the decision to customize driven by a process or quality need? • Can the need be filled by a standard or configurable assembly? Have you asked your SUT supplier this question?

• What benefit does the customized option offer that the standard or configurable option does not? End users must have the discipline to resist customization when it is not absolutely necessary.

What are the alternatives to customized solutions? Standard solution: A standard solution is one your vendor already has on the shelf and is available immediately. It is one built to the vendor’s validated design specifications. This should be the first consideration for a solution. If a standard solution can meet the process requirements, it will be the most timely and cost-effective solution. Configurable solution: In the absence of a standard off-the-shelf solution, the second option is a configurable solution. Configurable solutions use parts from the vendor’s design space (described below). If the customer needs a design that is not met by the vendor’s standard offerings, a solution configured from validated parts in the toolbox will take six to eight weeks — a significant time improvement over the customized piece which can take on average a minimum of 16 weeks. Design space concept: What is a design space? The design space is a toolbox of validated parts and assemblies. The customer can build any device needed — as long as the validated parts are used. The toolbox provides various types of tubing, filters, and connectors and other parts necessary to create an assembly. It is similar to customized, but with a reduced num-

ber of choices. The materials used meet the quality profile with the right supporting documentation prior to being included in any design. This requires controls regarding how something is designed, built, and tested. The controls should ultimately lead to the development of the design space from which assemblies are built. Design space eliminates availability issues, offers faster implementation, improved control, documentation of components, and reduced workloads. With the design space, the SUT supplier is responsible for assembly design and ensuring the product fit not only from a performance and regulatory point of view, but also ensuring it can be manufactured robustly and consistently. Using the design space concept for a configured solution offers significant speed benefits. A good analogy of a configured design space is from the automotive industry. If you go online to design a new car, there are many options available. The options are defined by the car manufacturer. The car manufacturer offers choices and the customer feels like they are customizing their car to their specifications. The customer chooses a steering wheel, but in reality, there are only four models of steering wheels and not a whole universe of steering wheels. The customer can also choose a paint color from 12 options, again a limited number. What differentiates configuration from the standard is that the customer specified the design. Parker domnick hunter’s bioprocessing design space in Birtley, UK is similar. A toolbox of validated parts

A customized solution should only be used when the process absolutely demands it.

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feature

The decision to standardize or customize an assembly should be made on the front-end where upfront time and costs can be avoided.

has been tested for quality and performance. Specifications are known. The supply is backed-up and extra parts are in stock. Having a design space not only saves lead time, but improves quality, performance, and the reliability of the supply chain. The automotive industry analogy illustrates that hundreds of different cars are built on the same production line based on a standard, configurable package. The resulting car can be operated by many different people, and the people building it have the right skills, but they are not specialists. The process to build and maintain a car requires some training, but it is not at a specialist level requiring years of experience. A customized product would have much more complexity such as requiring specialist training for the operator and the people building it. As an industry, one way to simplify is to move away from customized solutions to configured solutions based around a validated design space. Making the compromise of choosing from a limited toolbox offers the benefits of time, performance, and quality.

Responsibilities of the vendor and end-user Defining roles when using the design space is also helpful. End users own their process and product and are responsible for the optimization of those. Vendors have industry-wide experience and expertise with building assemblies. Vendor expertise,

such as knowledge about how liquids flow through tubes and how to create assemblies is a different skill set than what customers typically have. End users who are accustomed to designing assemblies can gain time and expertise by deferring to their vendor for assembly design. It is easy for the end user to fall into the habit of wanting to design the assembly. Moreover, if vendors and end users each stick to their areas of expertise, time and results can be optimized.

How can standardization be achieved? While a substantial amount of work is still needed across the industry to standardize materials, connectivity, and testing, the design space is one way to achieve some benefits of standardization now. Customized assemblies are commonly used, but rarely needed. Configuring assemblies within a design space is a solution with many benefits, especially the six weeks gained in speed to market. What does a design space actually look like? It is, in effect, a large spreadsheet containing all the potential components. The number of components can appear daunting. For perspective, a design space could offer over sixty different tubing options alone. Yet, there would not be 60 different tubing materials, perhaps only four — but including different inner and outer diameter (ID/OD) dimensions, the options multiply. The design space is dy-

namic but controlled. Before something can be added to the design space, documentation to support both product specifications and regulatory requirements is needed. If an item is not being used in assemblies, it is taken out of the design space to maintain focus on ensuring the quality and supply chain reliability for the materials in use. The design space offers a number of benefits in addition to the speed to market. The supply chain is robust and reliable. The vendor audits the supply chain. Supply agreements are in place, including change control, to ensure long-term availability and quality of parts. The vendor has performance data available and knows how the part can be used in manufacturing. Having such a process allows vendors to design, test, and supply assemblies to customers quickly and efficiently while offering process flexibility. It also means the end user can stay focused on producing biopharmaceuticals. In summary, after 20 years, singleuse technology is here to stay. It is the key enabling technology for biopharma production today. A dynamic design space, with its rapid design, testing, and manufacturing of configured solutions, can improve the efficiency of single-use technology in bioprocessing. As the industry matures, standardization of systems, components, and designs, plus manufacturing and testing methods, is necessary to support continued growth. Stan-

dardization frees up the end user to concentrate on process development and production, rather than having to focus on being a design engineer for SUT systems.

Guy Matthews has worked in the biopharma industry for the last 20 years starting his career as a scientist at a well-known CMO in the UK before moving to more commercial roles. Guy now works as market development manager (life sciences) for Parker domnick hunter.

To see this story online visit http://laboratory focus.ca/how-to-reducethe-complexity-of- customsingle-use- assemblies/

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Feature

B y H i s a s h i K o n a k a a nd A k i t o S a s a k i

Crystal structure analysis

from powder X-ray diffraction data using high-temperature attachment for capillaries Introduction The physical and chemical properties of a crystalline solid depend heavily on the conformations of the molecules and the arrangement of atoms and molecules, that is, the “crystal structure”, as well as on the composing elements and molecular structures. The single crystal structure analysis technique is used in many fields as a good tool to precisely clarify the crystal structures required to understand the mechanisms of developing physical properties of crystalline materials. In recent years, reports of the success of crystal structure analysis from powder X-ray diffraction data are increasing, particularly in regards to polycrystalline samples that were difficult for single crystal structure analysis. In addition to improvements in analysis methods and highperformance PCs, one of the major reasons is that very good diffraction data can now be obtained using inhouse powder X-ray diffractometers instead of synchrotron facilities due to improvements in X-ray optical devices and detectors. In particular, there has been increased interest in materials whose physical properties change due to an external field because these materials have applications relating to switching devices, sensors, memory materials, and so forth. For these external field active materials, solving the crystal structures is crucial to elucidating the mechanisms of physical property changes. So far, numerous single crystal analysis results have been reported for external field active crystals before the response to the external field. However, most of the crystal structures after response to the external field have not been solved because the single crystal state usually collapses during phase transitions brought about by the changes in physical properties. It is worth solving the crystal structures of these materials after response to the external field using powder X-ray diffraction data to elucidate physical properties and to obtain new knowledge about physical properties. This requires a method of measurement while the external field is maintained. The “high-temperature attachment for capillaries” is an attachment to

be used in powder X-ray diffraction measurement. This attachment has the following two features: (1) The sample can be heated. Powder X-ray diffraction data can be collected at a specified temperature. Diffraction data from the sample after phase transition or crystalline phases change by heating can also be collected. (2) The polycrystalline sample in a glass capillary can be measured while it spins. If you fill the indent of a glass sample holder with a polycrystalline sample, the intensity ratios between one diffraction and another often change due to preferred orientations, which makes crystal structure analysis difficult. The degree of preferred orientations is reduced by spinning the capillary samples; therefore, you will be able to obtain data of high enough quality for crystal structure analysis. The Ni(II) complex with the N,N-diethylethylenediamine (Et2en) ligand (Fig. 1) shows structural changes during heating. It has been known since 19631 that this complex exhibits colour changes based on hydration/ dehydration transitions and thermochromism depending on the selected anion.2–7 Many researchers performed detailed analyses of this complex. However, crystal structures of most of the complexes have not been solved after the structural changes. The only crystal structures that have been solved are in cases where the single crystals survived the phase change.7 In this article, from among the various Ni(II)-Et2en complexes, [Ni(Et2en)2(H2O)2]Cl2, [Ni(Et2en)2]Br2 and [Ni(Et2en)2](BF4)2 were selected, and hereafter are described as the results of crystal structure analyses using the high-temperature attachment for capillaries for the purpose of elucidating the transition behaviours based on the powder X-ray crystal structure analysis.

Fig. 1. Molecular structure of [Ni(Et2en)2]2

ture and to decide on the measurement temperature for powder X-ray crystal structure analysis, powder X-ray diffraction measurement was performed using the X-ray DSC attachment (Fig. 2). With this attachment, both DSC and X-ray diffraction data can be collected simultaneously while temperature and humidity are adjusted. The measurements for crystal structure analysis were performed using focusing beam optics,8 where the CuKα1 X-rays, monochromatized by the Kα1 unit, are line-focused on the detector window using the CBOE unit. A glass capillary was ﬁlled with a sample, which was then measured at a speciﬁed temperature using the high-temperature attachment for capillaries (Fig. 3). PDXL Integrated Powder X-ray Diffraction Software(9),(10) was used for the crystal structure analysis from powder diffraction data.

Hydration/dehydration transition of [Ni(Et2en)2(H2O)2]Cl2

As shown in Fig. 4, [Ni(Et2en)2(H2O)2] Cl2 (1a), light blue crystals, shows a dehydration transition during heating;2 1a turns to [Ni(Et2en)2(H2O)2]Cl2 (1b) in which chloride ions coordinate with the Ni(II) ions. While the crystal structure of 1a has been reported (11), the

structure after dehydration transition has not been reported yet. Figure 5 shows the simultaneous XRD & DSC measurement results of 1a under heating and humidiﬁcation. 1a showed an endothermic reaction at 361 K after heating from room temperature, and eventually gave the Xray diffraction patterns of a crystalline phase considered to be 1b. Since no other crystalline or amorphous phases were observed around the time of the reaction, it turned out that 1a underwent the crystal-to-crystal dehydration transition to generate 1b. After cooling the generated 1b down to 300 K, the surrounding humidity was changed from 5%RH to 70%RH at 300 K. As a result, the diffraction pattern of 1b changed at 25%RH with exotherm. The diffraction peaks of 1b eventually disappeared and the diffraction pattern returned to that of 1a. Thus, it was clariﬁed that 1b turned into 1a by exposing 1b to water vapour. Here, 1a′ is the crystalline phase generated by way of 1b. In In the middle of the transition from 1b to 1a′, two peaks were observed at 2θ10° and 13°, neither of which could be assigned to the crystalline phases, and the crystal structure could not be analyzed. Next, the crystal structure analyses of 1a, 1b, and 1a were performed from their powder diffraction data collected using the high-temperature attachment for capillaries. For 1b, the measurement was done at 390 K, after the endothermic reaction caused by dehydration was completed. Figure 6 shows the measurement data, calculated data and difference plot of each compound. In each result, the calculated data was in good agreement with the measurement data. While the crystal structure of 1a, which is already known, is mono-

Measurement and analysis All the powder X-ray diffraction measurements were performed using the SmartLab automated multipurpose X-ray diffractometer. In order to get rough information on structural changes with tempera-

Fig. 2. X-ray diffractometer equipped with an X-ray DSC attachment.

Fig. 3. X-ray diffractometer equipped with a high-temperature attachment for capillaries.

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feature Fig.4. Structural changes between 1a and 1b.

Fig.5.

clinic, P21/n, that of 1b is triclinic, P-1. The crystal structure of 1a′ was conﬁrmed to have the same unit cell parameters and the same space group as 1a. Figure 7 shows the results of crystal structure analysis from powder X-ray diffraction data. The water molecules in 1a are coordinated in the axial direction of the [Ni(Et2en)2]2+. Adjacent complex molecules were connected by hydrogen bonds via free chlorides and formed two-dimensional sheet structures. Here, the amino groups of the Et2en ligands were not involved in hydrogen bond formation. Chlorides were coordinated to the Ni atoms in the crystal structure of 1b, which was obtained by heating and dehydration. The complex molecules formed two-dimensional sheet structures through hydrogen bonds between amino groups and chlorides of adjacent complex molecules. As shown at the bottom-right of Fig. 7, a disordered structure was found in the conformation of the Et2en molecules in 1b. The site occupancies of the two types of conformation were 64.9% and 35.1%, respectively. 1a, obtained by hydration of 1b, was conﬁrmed to have the same crystal structure as 1a.

Hydration/dehydration and structural phase transition of [Ni(Et2en)2]Br2

As shown in Fig. 8, it is known that [Ni(Et2en)2]Br2

(2a) undergoes a hydration transition to result in [Ni(Et2en)2]Br2 (2b), where water molecules coordinate to Ni(II) by humidiﬁcation(2). Although the crystal structure of 2a has already been reported(12), the crystal structure after the hydration transition has not been reported yet. Figure 9 shows the results of XRD and DSC measurements of 2a during heating and humidifying. The diffraction pattern of 2a converted to that of a different complex considered to be 2b at 30%RH with exotherm is a result of humidifying the sample from 5%RH to 70%RH at 300 K. Subsequent heating of 2b from room temperature caused an endothermic reaction at 344 K to provide a new crystalline phase 2c with a different diffraction pattern from that of 2a. Further heating returned the diffraction pattern of the new phase 2c to that of 2a, without a big change in the DSC chart in the temperature range from 390 K to 400 K, although there was a big change in the diffraction pattern. This clariﬁed that 2c underwent a structural phase transition during heating. Considering that no crystalline or amorphous phases other than 2a, 2b, 2c, and 2a were observed around the time of each reaction, 2a, 2b, and 2c turned out to undergo the crystal-to-crystal hydration/dehydration/structural phase transitions, respectively. Next, the crystal structure analysis of 2a, 2b, 2c, and 2a were deter-

mined from powder X-ray diffraction data as described in the previous section (Fig. 10). The diffraction measurements were made for 2a and 2b at room temperature, 2c at 350 K, and 2a at 400 K. Also, when 2c was prepared by heating and dehydrating 2b, about 26% of 2c turned to 2a by a structural phase transition. However, we succeeded in the structure analysis of 2c by taking into account the fact that the diffraction pattern was obtained from a mixture of 2a and 2c. While the crystal structure of 2a is monoclinic, C2/c, the space group of 2b was P21/n. The phase 2c obtained by heating and dehydrating 2b caused a structural phase transition without changing space groups. Furthermore, although the unit cell became a little larger because the measurement was made at high temperature, the diffraction pattern of 2a obtained by heating 2c was very similar to that of 2a and the space group returned to that of 2a. The results obtained by powder X-ray crystal structure analysis are shown in Fig. 11. The Ni2ions of 2a form a square planar structure that has two Et2en molecules coordinated but no bromides coordinated. Adjacent complex molecules were connected through hydrogen bonds via free bromides and formed one dimensional chains. 2a, generated after hydration transition by humidiﬁcation, formed an octahedral structure where water molecules coordinated to the square planar Ni-Et2en complex from the axial

Fig.7.

Fig.8. Structural changes between 2a and 2b.

direction. 2b had a similar packing structure to that of 1a, where the amino groups of the Et2en ligands were not involved in the hydrogen bonds but the water molecules and bromide ions formed hydrogen bonds. 2c, obtained by dehydration transition of 2b, had a similar molecular structure to that of 2a, but caused a very different hydrogen bond formation to produce a two-dimensional sheet structure. The crystal structure of 2a was the same as that of 2a.

Structural phase transition of [Ni(Et2en)2](BF4)2

[Ni(Et2en)2](BF4)2 (3a), orange crystals at room temperature, shows thermochromism when the orange crystals suddenly turn to red crystals upon heating as shown in Fig. 12.1,3 Details of the thermochromism of 3a have been clarified by many researchers, but the crystal structures of the low-temperature phase 3a and the high-temperature phase 3b have not been solved because it was not possible to prepare any single crystals large enough to measure. Figure 13 shows the results of the simultaneous XRD and DSC measurements of 3a under heating and cooling conditions. As reported by S. Hayami et al.(7), we confirmed that the endothermic and exothermic reactions occurred at 376 K and 374 K when heating and cooling, respectively. Since no crystalline or amorphous phases other than 3a and 3b were observed, this reaction turned out to be the crystal-to-crystal structural phase transition. As in the previous report, the crystal structures of 3a and 3b were analyzed based on their powder diffraction data (Fig. 14). The diffraction pattern of 3a was very similar to that of [Cu(Et2en)2](BF4)2.13 The powder diffraction pattern of 3b was obtained from the measurement at 400 K. The analysis result elucidated that the unit cell volume was 1646 Å3, and that the crystal system was trigonal or hexagonal. Figure 15 shows the result obtained from the powder X-ray crystal structure analysis of 3a. The space group of 3a was triclinic, P-1, and the Ni atoms were located on the symmetric centers. The complex molecules were connected by two molecules of tetrafluoroborate through hydrogen bonds and

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Feature

Fig.10. Collected XRD profiles, calculated profiles, and determined unit cell parameters of 2a, 2b, 2c and 2a.

Fig.12. Molecular structure of 3a and a demonstration of its thermochromism.

Fig.13. Results of XRD & DSC measurements of 3a during heating and cooling.

formed a one-dimensional chain structure. This crystal structure was isomorphic with that of [Ni(Et2en)2] (ClO4)26, [Cu(Et2en)2](ClO4)214,15, and [Cu(Et2en)2](BF4)2. In the crystal structure analysis of 3b, structure determination was attempted with several possible space groups based on the systematic absences in the diffraction patterns. As a result, the measurement data was in good agreement with the data calculated when trigonal, R-3 was selected. The asymmetric unit volume is one-eighteenth of the unit cell volume in the space group R-3. The unit cell volume was almost equivalent to that of three complex molecules; in other words, one-sixth of the complex molecule existed in the asymmetric unit. It was reported that the chelate ring composed of Ni and Et2en of 3b caused – puckering, based on the measurement results of solid-state 2H NMR(6). Therefore, the crystal structure of 3b was determined and refined by assuming that a disordered structure between the forms could exist. The result showed that the Ni atoms were located on the symmetric centers and three-fold axes, and that the complex molecules were packed in three directions randomly (Fig. 16). The tetrafluoroborate molecules were located on the same three-fold axes as [Ni(Et2en)2]2+ such that [Ni(Et2en)2]2+ was positioned between the tetrafluoroborate molecules. There were hydrogen bonds between the amino groups in the Et2en ligand and tetrafluoroborate molecules, and they formed two-dimensional sheet structures parallel to the ab plane (Fig. 17).

Conclusion We could demonstrate that the crystal structure analysis technique from powder diffraction data is very useful and effective when the single crystal state cannot be maintained due to structural phase transitions, etc. as well as when a certain size and quality of crystals cannot be prepared. We also collected high-quality data

for crystal structure analysis at temperatures above room temperature using the high-temperature attachment for capillaries. This attachment is very useful for structure analysis after the changes in crystalline phases by heating. By using the X-ray DSC attachment, understanding the outline of the structural changes played an important role in the crystal structure analysis. The crystalline phase transition from 2b to 2c, in particular, was not elucidated in previous papers and articles because heating and dehydrating 2b leads ultimately to sample 2a without much evidence of another phase in the DSC measurements. Therefore, the existence of 2c went undetected, so its structure was not determined until now. As described above, powder X-ray crystal structure analysis is a very powerful tool; however, you have to bear in mind that this technique is not versatile/perfect as is the case in other analysis methods. For instance, there was a trial and error approach in determining the initial structures with several space groups. The results of the solid-state 2H NMR had to be used for the structure reﬁnement. Thus, trial and error methods and results obtained from other analysis tools sometimes have to be combined with the structure analysis from powder diffraction data. Last but not least, crystal structure analysis results are always inevitable in elucidating physical properties from the structure point of view. We hope to gain a much better understanding of the relations between crystal structures and physical properties by solving unknown crystal structures under various measurement conditions in future. * XRD Application & Software Development, X-ray Analysis Division, Rigaku Corporation.

References 1. D. M. L. Goodgame, L. M. Ve-

nanzi: J. Chem. Soc., (1963), 616–627. 2. R. Tsuchiya, S. Joba, A. Uehara, E. Kyuno: Bull. Chem. Soc. Jpn., 46 (1973), 1454–1456. 3. L. Fabbrizzi, M. Micheloni, P. Paoletti: Inorg. Chem., 13 (1974), 3019–3021. 4. J. R. Ferraro, L. J. Basile, L. R. GIneguez, P. Paoletti, L. Fabbrizzi: Inorg. Chem., 15 (1976), 2342– 2345. 5. S. Mitra, G. De, N. R. Chaudhuri: Thermochimica Acta, 66 (1983), 187–195. 6. R. Ikeda, K. Kotani, H. Ohki, S. Ishimaru, K. Okamoto, A. Ghosh: J. Mol. Struct., 345 (1995), 159– 165. 7. S. Hayami, D. Urakami, S. Sato, Y. Kojima, K. Inoue, M. Ohba: Chem. Lett., 38 (2009), 490–491. 8. A. Nedu: Rigaku Journal (English version), 28 (2012), No. 2, 5–10. 9. Rigaku Journal (English version), 26 (2010), No. 1, 23–27. 10. A. Sasaki, A. Himeda, H. Konaka, N. Muroyama: Rigaku Journal (English version), 26 (2010), No. 2, 10–14. 11. Y. Ihara, Y. Satake, Y. Fujimoto, H. Senda, M. Suzuki and A. Uehara: Bull. Chem. Soc. Jpn., 64 (1991), 2349–2352. 12. S. J. Ferrara, J. T. Mague and

J. P. Donahue: Acta Cryst., E67 (2011), m48–m49. 13. B. Narayanan, M. M. Bhadbhade: J.Coord.Chem., 46, (1998), 115–123. 14. I. Grenthe, P. Paoletti, M. Sandström and S. Glikberg: Inorg. Chem., 18, (1979), 2687–2692. 15. P. Naumov, K. Sakurai, T. Asaka, A. A. Belik, S. Adachi, J. Takahashi and S. Koshihara: Inorg. Chem., 45, (2006), 5027– 5033.

Hisashi Konaka works in the Application & Software Development Department, X-ray Instrument Division at Rigaku Corp. Akito Sasaki is senior manager, XRD Application & Software Development Dept., X-ray Instrument Division at Rigaku Corp.

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Fig.14. Collected XRD profiles, calculated profiles, and determined unit cell parameters of 3a and 3b.

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EVOS FL Auto 2 imaging system The Invitrogen EVOS FL Auto 2 Imaging System has been designed to bring a high-level of performance with more advanced capabilities and to simplify demanding cell-based imaging applications, including livecell imaging, image tiling, and z-stacking. The new instrument can scan a 96 well plate in three fluorescent channels in less than five minutes making it a quick and efficient model. The imaging system can be customized to meet a variety of needs with over 20 user-friendly LED light cubes, dual monochromatic and colour cameras, sports a variety of objectives ranging from 1.25x to 100x, and has multiple vessel holders. The time-lapse live-cell imaging options allows for precise control of temperature, humidity and gases for normoxic, or hypoxic conditions, and the area view of the imaging system moves rapidly from low to high magnification to easily define and capture the area of interest. These time-saving features such as autofocus, rapid stage movement, and automated routines reduce time spent on experiments while still acquiring high-quality data.

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New gasket and braided hose range from BioPure The Watson-Marlow Fluid Technology Group (WMFTG) has added additions to the BioPure range. BioPure has innovated a range of high purity platinum-cured silicone gaskets and platinumcured silicone braid hoses to add to the current collection of fluid path components. The gaskets and hoses have been designed to meet the exacting requirements of biotechnology and pharmaceutical single-use applications with the highest level of validation available and extractable data in line with BioPhorum Operations Group (BPOG) guidelines to enable users to simplify and expedite validation procedures.The silicone, platinum-cured gaskets have been designed for bioprocessing applications where fluid paths are required. Each gasket has been engineered to achieve a smooth bore, contaminationfree fluid path under clamping compression. They are designed in accordance to standards of the ASME-BPE and are ISO14644 Class 7 cleanroom manufactured and packed. The gaskets are available in ten varied sizes, ranging from ½” to 8” unflanged, and in eight flanged size options from 1” to 8”. The silicone platinum-cured braided hose offers another biotech and pharmaceutical fluid path option. These single and double-braided flexible hoses have been designed for high pressure situations. The hoses offer an immense bending radius and the continuously extruded silicone bore assures the validity of the contacting fluid. They are also built to withstand repeated autoclave and sterilisation processes. BioPure platinum-cured silicone braided hoses are manufactured and packed within an ISO14644 Class 7 cleanroom environment to thereby give users full batch traceability to raw material. There are ten inner diameter sizes for single braided hose are available from 0.125” to 1”, while double braided hoses come in ½”, ¾” and 1” internal diameters. BioPure’s silicone hose is similarly supported, as is the gasket, by the highest level of validation and extractable data.

Web: http://www.watson-marlow.com/us-en/range/

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Laboratory Focus November/December 2017

Consistent type three pure lab water MilliporeSigma’s Milli-Q HR 7000 has designed a powerful, versatile and modern central pure water station. Able to purify up to 13,000 litres of water a day for small laboratories or entire research facilities with clean, type three laboratory water. The Progard cartridges placed inside the purification system efficiently remove particles, colloids, free chlorine and hardness; and the advanced reverse osmosis then removes up to 99 per cent of ions, and 99 per cent of all dissolved organics, microorganisms and particles. The Milli-Q HR 7000 takes less hands-on time to maintain and reduces water waste of up to 50-percent. The equipment can be integrated into almost any lab or building and is capable of driving and controlling all ancillary equipment needed for a complete installation including distribution pumps and loops, alarms, monitors, UV lamps and sanitization modules. They are outfitted with a large, colour touchscreen display panel to facilitate maintenance control and access stored data. Authorized users can securely access the monitor and data from any location via laptop or smartphone 24/7. The Milli-Q HR 7000 system makes it easier to manage and keep records of all running parameters, including water quality, alarms and events, water usage, and consumables.

Web: http://www.emdmillipore.com/CA/

Bio-Rad Laboratories’ release Liquichek Tumour marker controls Bio-Rad Laboratories’ has released a serum tumour marker to aid in the detection of cancerous cells. Cancer is one of the leading causes of death worldwide and early discovery of malignant cells and biomarkers is key to survival. The Liquichek Tumour Marker Controls offer a wide-ranging collection of tumour marker analytes designed to cover a variety of cancer disease conditions, and is now featuring four new ones; including HE4 and HER-2/neu. These autonomous human-based controls mimic patient samples for reliable monitoring on most major testing platforms used in cancer screening. The controls have a two-year shelf-life if frozen between -20˚ and -70˚, and have a thirty-day stability if kept between 2˚ and 8˚ for most analytes. They include low levels of PSA for high sensitivity screenings and elevated levels of cancer antigens and Ferritin.

Web: http://www.bio-rad.com/

E-Gel Agarose system for routine NA electrophoresis Thermo Fisher Scientific has released its Invitrogen E-Gel Power Snap Electrophoresis system for rapid, real-time nucleic acid analysis, and high-resolution image capture. The Power Snap Electrophoresis system takes up minimal space in the laboratory and offers a range of high-quality functions to make valuable time spent more efficiently. The product can analyze the E-gel precast agarose gels faster to eliminate tedious work, and go from

New Products sample loading to image capture in as little as fifteen minutes. The electrophoresis has also been developed with a large touchscreen and intuitive operating system to make it much simpler to use. The power snap system and E-gel precast cassettes take away the need to handle hazardous chemicals, making it a safer environment for those in the vicinity.

Web: https://www.thermofisher.com/

HyPerforma five-in-one single-use bioreactor The HyPerforma single-use bioreactor by Thermo Fisher Scientific is water-jacketed with AC motor and load cells, and the stainlesssteel bioreactor principles ensure optimal cell culture performance. The bioreactor vessel is singleuse and has sterile contact surfaces for mixing, venting, sparging and temperature sensing. The complete line of single-use bioreactors has a wide-range of sizes from 50 to 1,000 litre capability. The instrument features a unique propriety mixing system that allows conventional overhead mixing while maintain sterility and integrity of the single-use bioreactor bioprocess container. It is customizable due to the large qualified standard component library and has a minimal vessel footprint, allowing more space in and around the laboratory. This single-use bioreactor offers use with AC or DC motors for precise agitation control and has the option of being a stand-alone or a complete turnkey system when used in conjunction with control systems for bioproduction. This fivein-one system mixes turn-down ratio in combination with cross-flow sparger technology to create gas above liquid’s surface, facilitates efficient mass transfer of CO2 stripping and reduces sparges and foam damage to cells. All of this combined improves productivity in the lab and reduces costs.

Web: https://www.thermofisher.com/ca/en/

Sartorius compact system for laboratories requiring low volumes of ultrapure water This newly developed system by Sartorius, a laboratory and pharmaceutical equipment provider, offers laboratories and larger research facilities a compact, low volume water purifier with exceptional quality. The arium mini has a specifically developed system with a flow rate of one litre per minute and is designed for ultrapure water requirements of less than ten litres a day. This compact solution easily integrates into nearly any lab environment and is a width of only 28 centimetres. The feed water that is needed to create the ultrapure water is provided by the five-litre bag that has been integrated on the side of the system. Since the original feed water bag was designed for the pharmaceutical industry, it provides optimal consistent and long-term water quality. The closed bagtank system prevents secondary contamination while ensuring strong purified water. The uncomplicated nature of exchanging the bag simplifies the upkeep of the system and considerably reduces maintenance time compared with conventional tank systems. As a result, the bagtank does not require any hazardous cleaning chemicals, to ensure safer handling. Aside from the bagtank, the arium mini sports a high-resolution, touch-activated display with easy-to-use icons to save time and energy within the laboratories. The instrument automatically stores the volume of water last dispensed for more consistency, reliability, and efficiency.

Web: https://www.sartorius.com/sartorius/en/EUR/home//

Laboratory Focus November/December 2017 www.laboratoryfocus.ca

UCalgary engineering, science, medicine, arts professors

bag Killam awards

Each year, the Killam Annual Professorship awards are accorded to faculty members of the University of Calgary who have demonstrated a high quality of teaching, research, publication and creative activities. The recognition of being acknowledged for the prestigious award comes with a $10,000 prize from the Killam Trust. This year, two women led the five professors in the areas of science, medicine, engineering and fine arts received the awards. Josephine Hill is a leading researcher in the area of catalysis and chemical engineering who has been teaching at the Schulich School of Engineering since 2002. She serves as the Canada Research Chair in Hydrogen and Catalysis, and leads the Laboratory for Environmental Catalytic Applications. Hill received the Engineers Canada Award for the Support of Women in the Engineering Profession in 2013, the inaugural Women in Engineering and Geoscience Champion Award from APEGA in 2012, the 2008 Minerva Mentoring Award, and the 2007 Mentor of the Millennium Award from the Alberta Women’s Science Network. She promotes awareness of the challenges that women face with Women in Science and Engineering (WISE) — a university-wide organization dedicated to supporting and encouraging women who are interested or engaged in science or engineering careers — and as faculty liaison for the Cybermentor Program for girls in grades 8-12. As a member of the Faculty of Arts, Susan Bennett focuses in the areas of Shakespeare and early modern English drama. Bennett is internationally sought after as a speaker in these areas and has presented at such venues as the Shakespeare Institute in Stratford-upon-Avon and the Globe Theatre in London, England. Bennett was inducted last year into the Royal Society of Canada as a Fellow of the Academy of the Arts and Humanities, one of Canada’s highest distinctions for scholars. Stem cells are the main focus of the research being carried out by Michael Kallos, a member of the Schulich School of Engineering and Cummings School of Medicine. One of the projects he is working on with University of Calgary colleague Jeff Biernaskie involves developing a stem cell and biomaterial solution that can one day help burn patients that need skin grafts. Earlier in his career, Kallos made a major bioengineering breakthrough by demonstrating in 2006 that it is possible to achieve large-scale growth of certain types of stem cells in bioreactors. This is important because growing cells in large numbers is needed for the development of therapies. Bernhard Mayer is one of the world’s leading experts in isotope hydrology and isotope geochemistry. His work involves the use of chemical and isotopic fingerprinting techniques to trace compounds in the environment including contaminants. Mayer, who is with the Faculty of Science, has been instrumental in the development of isotope tools and research programs that help to assess the environmental impacts of many anthropogenic activities, including the agricultural and energy sectors. His work also helps scientists in South Korea and Europe identify ways to treat contaminated groundwater. David Hodgins of the Faculty of Arts and Cummins School of Medicine is a leading authority in gambling and addiction studies. He heads the Addictive Behaviours Laboratory. Hodgins’ research has led to the development of an evidence-based treatment for gambling disorders that is currently used in several states in the U.S., and has also impacted therapies developed in Australia and several Asian countries.

app review Pocket Pharmacists

https://itunes.apple.com/us/app/pocket-pharmacist/ id387365379?mt=8 This free mobile app developed by Danike Inc. is available for iPhone, iPad and Android users. It is meant for both healthcare professionals and healthcare advocates alike. Created by a clinical pharmacist, the app provides summarized drug information on the top 1,700 plus medications in the United States. It enables users to automatically check for interactions, overlapping side effects, and precautions. The Med Box feature, allows users to organize the medical needs and alerts of family members. A chat feature lets users join forums dedicated to discussions about a specific drug. It free for now but makers will be implementing a subscription fee soon.

Medical Lab Test

https://itunes.apple.com/us/app/medical-lab-tests/ id307829594?mt=8 Whether you are a healthcare professional, nurse or a medical student the free Medical Lab Test iOS app from Medicon Apps can provide you with easy access to common lab tests. It also helps users remember lab values and differentials between values. The available categories to browse through inlcude: Red Blood Cells, White Blood Cells, Coagulation, Electrolytes and Metabolites, Arterial Blood Gas, Enzymes and Proteins, Ions and Trace Metals, Cardiac Tests, Liver and Pancreas, Lipids, Hormones, Immunology, Cancer Markers, Cerebrospinal Fluid, Drugs, Toxicology and Urine.

Promega Biomath Calculator

https://itunes.apple.com/us/app/promega-biomathcalculators/id987501449?mt=8 This free mobile app is available for iOS and Android users to perform everyday lab calculations. It provides a range of functions necessary for molecular biology experiments, including nucleic acid and protein conversions, melting temperature, temperature conversion, molarity, and dilution calculations.

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Laboratory Focus November/December 2017

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