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R&D News.......................... 1 Pharma Notes..................... 7 Appointments..................... 6 New Products................... 15 Calendar........................... 17 App Reviews...................... 19
A better way to build DNA scaffolds In new research published in Nature Communications, McGill University chemistry professor Hanadi Sleiman and her team at McGill say that they have devised a new technique to create much longer strands of DNA with custom-designed sequence patterns. What’s more, this new approach also produces large amounts of these longer strands in just a few hours, making the process potentially more economical and commercially viable than existing techniques. The new method involves piecing together small strands one after the other, so that they attach into a longer DNA strand with the help of an enzyme known as ligase. A second enzyme, polymerase, is then used to generate many copies of the long DNA strand, yielding larger volumes of the material. The polymerase process has the added advantage of correcting any errors that may have been introduced into the sequence, amplifying only the correctly sequenced, fulllength product. The team used these strands as a scaffold to make DNA nanotubes, demonstrating that the technique allows the length and functions of the tubes to be precisely programmed. “In the end, what we get is a long, synthetic DNA strand with exactly the sequence of bases
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Prof. Hanadi Sleiman (left) and Janane Rahbani in their lab at McGill. Photo: McGill University
that we want, and with exactly as many repeat units as we want,” explains Sleiman, who coauthored the study with Graham Hamblin, who recently completed his doctorate, and PhD student Janane Rahbani. “This work opens the door toward a new design strategy in
DNA nanotechnology,” Sleiman says. “This could provide access to designer DNA materials that are economical and can compete with cheaper, but less versatile technologies. In the future, uses could range from customized gene and protein synthesis, to applications in nanoelectronics, nano-optics, and medicine, including diagnosis and therapy.” Funding for the research was provided by the Natural Sciences and Engineering Research Council of Canada, the Fonds de recherché du Québec – Nature et technologies, the Canada Foundation for Innovation, the Canadian Institutes of Health Research, and the Centre for Self-Assembled Chemical Structures.
To see this story online visit http://www.laboratoryfocus. ca/?p=2977
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news researchers ‘un-can’ the hiv virus The human immunodeficiency virus (HIV) is a bit like a hermetically sealed tin can no one has yet been able to break open, till now. Researchers at the CHUM Research Centre, affiliated with the University of Mon-
tréal, have identified a way to use a “can opener” to force the virus open and expose its vulnerable parts, allowing the immune system cells to then kill the infected cells. This breakthrough, published in the Proceedings
of the National Academy of Sciences, opens a new path in the fight against HIV and could ultimately lead to the design of a vaccine to prevent transmission of the virus. This innovative approach could also be part of the solution for
one day eradicating the virus. Despite recent advances, 35 million people are infected with HIV-1 worldwide. “We found that people infected with the HIV-1 virus continued on page 3
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Continued from page 2 have naturally occurring antibodies that have the potential to kill the infected cells. We just have to give them a little push by adding a tiny molecule that acts as a can opener to force the viral envelope to expose regions recognized by the antibodies, which forms a bridge with some cells of the immune system, initiating the attack,” says study lead author Andrés Finzi, researcher at the CRCHUM and a professor at the University of Montréal. Outwitting HIV’s bodyguards In an earlier study, also published in 2015 (Veillette et al., 2015), the same team of researchers showed that the serum of patients infected with HIV-1 facilitated the elimination of infected cells when two proteins specific to the virus, Nef and Vpu, were deactivated by gene mutation. The experiments were conducted with serum samples from the AIDS and Infectious Diseases Network (SIDAMI) cohort of the Fonds de recherche en santé du Québec (FRSQ). In real life, however, wild-type HIV-1 virus, responsible for the vast majority of infections in the world, still contains these proteins, which act like bodyguards. So how can we outwit them? By adding a tiny molecule to the cell surfaces of infected patients
Photo: NIAID. HIV-infected T cell. CC BY 2.0. – called JP-III-48 – which imitates a protein called CD4. CD4 proteins are located at the surface of T lymphocytes and allow immune system cells to be infected by HIV. “The virus has to get rid of the CD4 proteins to protect itself,” explains Jonathan Richard, postdoctoral researcher at the CRCHUM and lead author of the study. “Adding the small
molecule forces the viral envelop to open, like a flower. The antibodies that are naturally present after the infection can then target the infected cells so they are killed by the immune system.” The JP-III-48 molecule was developed by researchers at Harvard University and the University of Pennsylvania. This is the first time it has
been successfully tested on patients infected with HIV. For decades, scientists have been trying to devise a vaccine to block HIV infection, which causes AIDS. Antiretroviral drugs can slow the spread of the virus, but it remains hidden dormant in cells and returns when the treatments cease. This is called HIV “reservoirs.” “The solution is to develop a ‘shock and kill’ therapy. We have to reactivate HIV reservoirs to force the virus out of its hiding place, then kill the infected cells with this molecule and the already present antibodies,” argues Finzi, who is also the Canada Research Chair on Retroviral Entry. The discovery by Finzi’s team could help develop a two-part vaccine to prevent HIV infection: through antibodies that are easy to generate and using this new family of molecules. Furthermore, this discovery opens the way for the development of strategies to eliminate the viral reservoirs of individuals already infected. The next step is to test the potential of this canopener molecule in monkeys. To see this story online visit http://www.laboratoryfocus. ca/?p=2981
Just hit “print”: office inkjet printer could produce simple tool to identify infectious disease, food contaminants Consumers are one step closer to benefiting from packaging that could give simple text warnings when food is contaminated with deadly pathogens like E. coli and Salmonella, and patients could soon receive real-time diagnoses of infections such as C. difficile right in their doctors’ offices, saving critical time and trips to the lab. Researchers at McMaster University have developed a new way to print paper biosensors, simplifying the diagnosis of many bacterial and respiratory infections. The new platform is the latest in a progression of paper-based screening technologies, which now enable users to generate a clear, simple answer in the form of letters and symbols that appear on the test paper to indicate the presence of infection or contamination in people, food or the environment. “The simplicity of use makes the system easy and cheap to implement in the field or in the doctor’s office,” says John Brennan, direc-
tor of McMaster’s Biointerfaces Institute, where the work was done with biochemist Yingfu Li and graduate student Carmen Carrasquilla. “Imagine being able to clearly identify contaminated meat, vegetables or fruit. For patients suspected of having infectious diseases like C. diff, this technology allows doctors to quickly and simply diagnose their illnesses, saving time and expediting what could be life-saving treatments. This method can be extended to virtually any compound, be it a small molecule, bacterial cell or virus,” he says. The research, in its formative stage, addresses a key problem facing current paper-based biosensing techniques which are labour-intensive, sometimes costly and inconvenient, and often difficult to mass produce. Using state-of-the-art methods to produce “bio-inks”, researchers can now use conventional office ink-jet printers to print man-made DNA molecules with very high molecular weight on paper, much like printing a letter in an office. The sheer size of the
John Brennan, Director of McMaster University’s Biointerfaces Institute, examines a printout of a biosensor. DNA—which produces a signal when a specific disease biomarker is present—is enough to ensure it remains immobilized and therefore stable. The paper sensor emerges from the printer ready to use, like pH paper. The implications are significant, says Brennan, since the new technology could be used in many fields where quick answers to important questions are critical.
“We could conceivably adapt this for numerous applications which would include rapid detection of cancer or monitoring toxins in the water supply,” says Brennan. “There are hundreds of possibilities.” To see this story online visit http://www.laboratoryfocus. ca/?p=2938
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Gut immune system identified as a new and effective target in treating diabetes A commonly-used drug to treat inflammatory bowel disease, such as Crohn’s disease, has been shown to lower blood sugar levels in obese mice, potentially identifying the gut immune system as a new and effective target in treating diabetes in humans. “These results are novel and important because we have identified the immune system that lives in the gut as a new player in the control of blood sugar. This opens up the entire field of bowel immunology to the study of obesity and its complications such as high blood sugar,” says Dan Winer, Scientist, Diabetes Research Group in the Toronto General Research Institute (TGRI), whose laboratory spearheaded this work, along with his twin brother Shawn Winer, and who are both co-senior authors on this paper. Their research is published in an article entitled, “Regulation of ObesityRelated Insulin Resistance with Gut Anti-inflammatory Agents,” in the prestigious journal Cell Metabolism. Being overweight, especially around the abdomen or waistline, increases the chances of developing type 2 diabetes. The question many scientists are trying to answer is: why does obesity contribute to insulin resistance? In their previous work, the Winers demonstrated that immune cells inside abdominal fat cause the release of ‘pro-inflammatory’ chemicals, which make the body less sensitive
to insulin, the hormone that regulates blood sugar levels. This is known as insulin resistance – a major trigger for type 2 diabetes. In their research, the focus shifted from the fat to the gut, where the Winers found that mice fed a high-fat, high-calorie diet had larger amounts of pro-inflammatory immune cells and less of the regulating cells which help end an immune response, than in normal mice. The researchers found this same result in 14 humans, seven of whom were obese. The high-fat diet induces inflammatory changes in the immune cells in the bowel, upsetting the immune balance, which in turn sets off a chemical cascade, damaging the bowel wall, allowing bacterial products to leak into the blood stream. This leakage is what contributes to insulin resistance, when the cells can no longer respond to and use insulin effectively to stabilize blood sugar. “If we could block the pro-inflammatory immune cells at the very beginning of this process, we could treat the disease more effectively,” reasons Shawn Winer, who is a gastrointestinal pathology fellow in the Laboratory Medicine Program at University Health Network (UHN). “By refocusing on the bowel, we open up many more therapeutic options as we already have a number of approved drugs available to treat an inflamed bowel.” The researchers then targeted the bowel inflammation found in the obese mice with 5-ASA, or mesala-
Dr. Shawn Winer, gastrointestinal pathology fellow in the Laboratory Medicine Program, UHN, and twin Dr. Dan Winer, Scientist, Diabetes Research Group in the Toronto General Research Institute (TGRI). Photo: UHN mine, a commonly used drug to treat inflammatory bowel disease. They found that the drug reversed insulin resistance and lowered blood sugar significantly in the mice to near normal levels. “By using this drug, we found that we could prevent type 2 diabetes in mice,” says Dan Winer, who is also an endocrine pathologist at UHN and an assistant professor in Laboratory Medicine and Pathobiology at the University of Toronto. “If this works in humans, it could change the whole field of diabetes prevention and treatment.” Other researchers involved in the study include co-first authors Helen Luck, who is a graduate student in Immunology at the University of Toronto, and Sue Tsai, who is postdoctoral researcher and winner of the esteemed
McEwen Centre researchers unlock ability to create human articular cartilage
A team of scientists, led by Drs. Gordon Keller and April Craft from the McEwen Centre for Regenerative Medicine in Toronto has been able to generate articular chondrocytes and cartilage tissue from human pluripotent stem cells in a Petri dish.
Pluripotent stem cells have the potential to make most cell types in the body. In this study, the McEwen Center team identified for the first time the combination of factors that direct the human stem cells to specifically produce articular chondrocytes. Articular chondrocytes are cells that make the cartilage that lines our joints. They also showed that these chondrocytes can make cartilage tissue in the Petri dish. With these advances, it is now possible to produce an unlimited supply of chondrocytes and cartilage tissue for studying how osteoarthritis develops and for creating new regenerative medicine-based therapies for treating patients with cartilage damage. The findings are reported in the co-authored paper, “Generation of articular chondrocytes from human pluripotent stem cells”, published online in Nature Biotechnology. “Articular chondrocytes are found on
the surface of the bones within the joints and provide the cushioning that deteriorates in osteoarthritis,” explains Dr. Craft, assistant professor of Orthopaedic Surgery at Boston Children’s Hospital & Harvard Medical School. “If we can grow and use these chondrocytes to generate and maintain stable cartilage tissue, we have a tremendous opportunity to study the early events that lead to arthritis, to screen for new drugs to treat this disease and to investigate how to use this cartilage to repair damaged joints.” Osteoarthritis (OA) is the most common type of arthritis and affects one in 10 Canadian adults. There is no cure for OA and the only treatment for advanced OA is joint replacement surgery. Cartilage is an essential part of the joint; it absorbs the impact of movement and enables the joint to move smoothly. Osteoarthritic cartilage progressively deteriorates and
Banting Fellowship from Canadian Institutes of Health Research. Additional collaborators include Jason Chung, Xavier Clemente-Casares, Magar Ghazarian, Xavier Revelo, Helena Lei, Cynthia Luk, Sally Yu Shi, Anuradha Surendra, Julia Copeland, Jennifer Ahn, David Prescott, Brittany Rasmussen, Melissa Hiu Yen Chng, Edgar Engleman, Stephen Girardin, Tony Lam, Kenneth Croitoru, Shannon Dunn, Dana Philpott, David Guttman, and Minna Woo. The work was funded by the Canadian Institutes of Health Research, the Canadian Diabetes Association, and the Banting and Best Diabetes Centre at the University of Toronto. To see this story online visit http://www.laboratoryfocus. ca/?p=2988 eventually causes pain, stiffness and swelling as a result of bone-on-bone movement in the affected joint. “This is an exciting and encouraging first step in producing functional tissue for joint repair,” says Dr. Keller, director of the McEwen Centre for Regenerative Medicine. “Working with our partners at Mount Sinai Hospital, the Arthritis Program at Toronto Western Hospital, and the University of Guelph, we are proceeding to transplant the stem cell-derived tissue into the joints of animal models to test its ability to repair damaged cartilage.” Funding was provided to the project by generous donors including Rob and Cheryl McEwen, the McEwen Centre for Regenerative Medicine, the Canadian Institutes of Health Research (CIHR), Krembil Foundation, and Michael and Yetta Bregman in collaboration with the Campaign to Cure Arthritis, Toronto General and Western Hospital Foundation. To see this story online visit http://www.laboratoryfocus. ca/?p=2991
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tacklinG transfusion with a universal blood type Researchers have developed a technique to turn nearly any blood into a universal type resembling O-type blood, a development which could transform blood transfusion and human health. Stephen Withers and David Kwan, University of British Columbia researchers, used a technique called directed evolution along with synchrotron-based structural imaging to develop an enzyme that removes A and B type antigens from blood. The resulting red
blood cells, without antigens, are very similar to the O-type blood cells which are the only blood types that can be transfused into anyone without adverse reactions. “The idea of changing blood type by removing antigens is certainly not ours, it’s been around for a long time,” says Withers. “But when they first came up with this idea, only a very limited repertoire of enzymes was available, and these had very limited activity.”
These first A and B type antigen-removing proteins, known as enzymes, came from green coffee beans, and while they worked, it was only when used in massive doses. Withers’ lab harnessed a variety of modern techniques to build a better enzyme, one that would work in smaller quantities and with more efficiency. Based on Canadian Light Source synchrotron imaging of the structures active in removing antigens, Withers
and his collaborators created a library of enzyme “mutants” by introducing targeted variations into the protein structure and selecting the ones that gave the best results in removing antigens. After five rounds of this process where the best enzyme from each round was used as the basis for another series of mutations, their enzyme was 170 times more effective than it had been setting out. The resulting enzyme works nearly perfectly con-
verting B-type blood to O, but there is work left to be done on A-type, which has several subtypes. “That’s going to be our challenge, pushing it to 100 per cent,” he says.“ This paper shows that conceptually, it’s possible. There’s going to be a lot of work to do, but it’s doable.” To see this story online visit http://www.laboratoryfocus. ca/?p=2993
arctic beetles may be ideal marker of climate chanGe
Where you live is what you eat The team of researchers, led by Prof. Chris Buddle and Dr. Crystal Ernst of McGill’s Dept.
of Natural Resource Sciences, were able to identify more than 460 different species of Arctic beetles in locations ranging from the edge of
who is the first author on the study published in PLOS ONE. “In the far north, there are generally very high numbers of predators and far fewer beetles which eat plants, while further south the reverse is generally true.” Beetles are sensitive types The discovery that Arctic beetles may be especially sensitive to temperature has implications for future climate change monitoring. “As temperatures in northern regions rise or become more variable, there is a strong possibility that the
beetle communities will undergo significant changes in response,” says Buddle, the lead researcher. “Whether these changes will have positive or negative effects on Arctic ecosystems and the other animals and plants living there remains to be seen, but it is clear that beetles’ sensitivity to climate make them ideal targets for longterm biodiversity monitoring in the far north.” To see this story online visit http://www.laboratoryfocus. ca/?p=2996
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Want to know about climate change? Ask a beetle. Scientists have been logging changes in weather patterns and temperatures in the Arctic for some time. Now they need to find ways to measure how these changes in climate are affecting biodiversity. One of the best places to look may be down at our feet, at beetles. That`s because, as a McGill research team discovered after doing the first large-scale survey of Arctic beetles, these sixlegged critters are not only abundant in number but also diverse in feeding habits and what they eat is closely linked to the latitude in which they are found. As a result, McGill researchers believe that Arctic beetles may prove to be ideal markers of climate change, since any changes in climate that affect the soil, plants and animals on which the beetles depend, are likely to be quickly reflected in changes in the beetle communities.
the boreal forest in Northern Ontario, to Ellesmere Island in the far north. More significantly, they found that there were clear differences in what beetles are found where along this north-south gradient, and the ecological roles they fulfilled differed depending on the latitude in which they lived. “Depending on the latitude and the temperature, Arctic beetles perform a range of ecological functions such as pollinating or feeding on plants, preying on other insects, and breaking down decaying matter,” says Ernst,
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aPPointments
NEOMED has added two new members to its board of directors. Joining the board are Dr. Laurence Rulleau, principal at CTI Life Sciences, and Paul Buron, executive vice president and chief financial officer of the Business Development Bank of Canada (BDC). Prior to joining CTI, Dr. Rulleau served as a life sciences analyst covering the Canadian life sciences sector for more than 10 years. She worked at different firms including Desjardins Securities, Yorkton Securities and Blackmont Securities. She was involved in numerous mergers and acquisitions as well as IPOs. More recently she served as vicepresident business development of Univalor, the technology transfer office of Montréal’s university and affiliated schools. Under her responsibility at Univalor in the last three years, more than 40 licences with the industry were signed and eight companies started. Mr. Paul Buron has been the executive vice president and chief financial officer of the BDC since 2006 and assumed the additional title of chief risk officer in 2015. He has over 30 years of experience in finance. Before
joining BDC, he held leadership positions in major corporations, such as the Société générale de financement du Québec, Donohue Inc. and the TVA Group Inc. Verlyx Pharma Inc. has named Dr. Patrick Colin as its new chief development officer. Dr. Colin had been a consultant with Verlyx since 2011 overseeing the clinical development and pharmacovigilance activities of the company. He has spent more than 12 years at Axcan Pharma (today Aptalis Pharma), where he established and led the drug development department as vice-president, R&D. His credentials include 27 years of global drug development experience, and more than 15 market approvals in the US, EU, Canada and Australia. During his career, and especially during his Axcan years, Dr. Colin built strong ties with the international hepatology community, and played a key role in the development and approval of liver drugs such as ursodiol in the treatment of Primary Biliary Cirrhosis. Dr. Michael Toth has taken the helm of the Ontario Medical
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Dr. Michael Toth Association as its new president. Dr. Toth is a family physician from Aylmer, a small town in southwestern Ontario, who has practiced for nearly 30 years. He is also the medical director at a long-term care facility and a member of the Medical Advisory Committee at the St. Thomas Elgin General Hospital. Dr. Toth is the 134th president of the OMA; he takes over from Dr. Ved Tandan, a surgeon from Hamilton. He will serve for a one-year term. Dr. Michel Goldman, former executive director at the Innovative Medicines Initiative (IMI), is the newest member of CQDM’s board of directors. Goldman led the IMI from 2009 to 2014 and was highly successful with this major European initiative. Under his leadership, the IMI created nearly sixty publicprivate consortia to meet pressing needs in areas of great importance as antimicrobial resistance, Alzheimer’s disease, autism, diabetes, immuno-inflammatory diseases, chronic lung diseases and drug safety. A professor of immunology at the Université Libre de Bruxelles (ULB) in Belgium, he has also served as head of the department of Immunology-Hematology-Transfusion at the Hôpital Erasme in Brussels and as director of the ULB Institute for Medical Immunology. His scientific achievements have resulted in more than 400 articles in peer-reviewed journals. In 2006, he was also recognized by the Thomson Institute for Scientific Information (ISI) as one of the most cited scientists. Clinical-stage company Aptose Biosciences Inc. has added Dr. Michael Andreeff to its scientific advisory board. Dr. Andreeff joins Brian Druker, M.D., director, Oregon Health & Science University (OHSU) Knight Cancer Institute and Daniel D. Von Hoff, M.D., physician-in-chief and distinguished professor of Translational Research at Translational Genomics Research Institute (Phoenix,
10/23/2012 2:16:52 PM
AZ), as scientific advisors for Aptose. Dr. Andreeff received his M.D. and Ph.D. degrees at the University of Heidelberg, Germany, and additional training and faculty appointments at the Memorial Sloan-Kettering Cancer Center (MSKCC) in New York, NY, in the Departments of Pathology and Leukemia. Dr. Andreef is professor of medicine and holds the Paul and Mary Haas chair in Genetics at MDACC. He has received uninterrupted funding from the National Cancer Institute (NCI) for more than 30 years, serves as the principal investigator (PI) of a grant entitled “The Therapy of AML”, and participates as a PI in research grants focused on leukemia, lymphoma, ovarian and breast cancer, among others. He has published more than 450 peerreviewed papers, five books and 75 book chapters. Serge Maltais is stepping into the role of president and CEO of
Serge Maltais
Héma-Québec effective May 18. Serge Maltais brings extensive experience in operations management from his positions with major pharmaceutical companies. Before joining Héma-Québec, he was vice-president, Supply Chain, for commercial operations at Sandoz Canada Inc., the Canadian pharmaceutical subsidiary of Novartis A.G. specializing in generic products. He joined Sandoz in 2002 and was responsible for customer service, sales forecasting, purchasing and distribution for Canada. From 1985 to 2002, he held various management positions with the Canadian subsidiary of Baxter International, where he proved to be a highly successful leader in the areas of logistics and production. He holds a Bachelor of Industrial Engineering degree from the École Polytechnique de Montréal and a diploma in Business Administration from the Université de Sherbrooke.
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Pharma Notes Resverlogix Corp. (Calgary, AB) reports it has entered into an agreement with Shenzhen Hepalink Pharmaceutical Co., Ltd. (Hepalink) for an equity investment and a license of its lead product RVX-208, for all indications in China, Hong Kong, Taiwan and Macau markets. Under the terms of the transaction, Hepalink will subscribe for 13,270,000 Resverlogix common shares and 1,000,000 common share purchase warrants, for aggregate proceeds of approximately C$35 million. Hepalink will also be entitled to nominate one mutually agreed representative for election to the board of directors of the company. Aeterna Zentaris Inc. has filed an application for a patent (European Patent Office priority application: EP15000132) on a method of manufacturing zoptarelin doxorubicin. The hybrid cytotoxic molecule is the subject of a pivotal ZoptEC (Zoptarelin doxorubicin in Endometrial Cancer) Phase 3 clinical study in women with advanced, recurrent or metastatic endometrial cancer. The molecule combines a synthetic peptide carrier with doxorubicin, a well-known chemotherapy agent. The synthetic peptide carrier is a Luteinizing Hormone Releasing Hormone (LHRH) agonist, a modified natural hormone with affinity for the LHRH receptor. The design of the compound allows for the specific binding and selective uptake of the cytotoxic conjugate by LHRH receptor-positive tumours. Potential benefits of this targeted approach include a better efficacy and a more favourable safety profile with lower incidence and severity of side effects as compared to doxorubicin alone. The patent application, which is entitled “Enzymatic process for the regioselective manufacturing of NFmoc-doxorubicin-14-O-dicarboxylic acid mono esters,” may, if granted, make it difficult for generic manufacturers to produce the compound on a financially feasible basis after the company’s composition-of-matter patent on zoptarelin doxorubicin expires. Transition Therapeutics Inc. (Toronto, ON) through its whollyowned subsidiary, Transition Therapeutics Ireland Limited has exclusively licensed the worldwide rights to a novel small molecule drug candidate (TT701) from Eli Lilly and Company. The molecule, TT701, is a selective androgen receptor modulator that has been shown in a Phase 2 study to significantly increase lean
body mass and a measurement of muscle strength in male subjects. This Phase 2 study of 350 subjects also demonstrated additional beneficial effects, including significant fat mass reduction with no significant change in prostate specific antigen (PSA) levels. Transition is evaluating multiple development paths for TT701, including as a new therapeutic option for patients with androgen deficiency. Under the terms of the agreement, Lilly will receive a contingent upfront consideration of up to US$1 million. In addition, Lilly is eligible to receive up to US$100 million in commercial milestones and a mid-single digit royalty on sales of TT701 products should such products be successfully commercialized. Privately held biotechnology company Northern Biologics Inc. reports it is collaborating with Celgene Corporation. As part of the collaboration, Northern Biologics has received a US$30 million upfront cash payment. It will use the funds to discover and develop first-in-class therapeutic antibodies in oncology and fibrosis from preclinical discovery through human clinical trials. Northern Biologics will also have the right to receive additional future payments that support the advancement of its portfolio. In return, Celgene will have options to in-license drug candidates and acquire Northern Biologics upon conclusion of the collaboration. Northern Biologics was launched in June 2014 by Blueline Bioscience, a Canadian biotechnology incubator backed by venture capital firm Versant Ventures, in partnership with the University of Toronto and University Health Network’s Princess Margaret Cancer Centre. Versant committed a US$10M Series A financing round to Northern Biologics in October 2014. Cyclenium Pharma Inc. and Haplogen GmbH have signed a research agreement aimed at the discovery of novel pharmaceutical candidates in multiple disease areas. The collaboration brings together two next generation drug discovery platforms: Cyclenium’s CMRT™ Technology-derived QUEST Library™ and associated hit-tolead optimization expertise with Haplogen’s unique haploid genetics technology and expertise in host target identification, validation and screening. The companies will initially focus on certain anti-viral Haplogen targets, with targets in other therapeutic indications being
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phased-in as the research progresses. For Cyclenium, this is the fifth discovery collaboration established over the past year.
by the center, MUSC shall pay the company a royalty on net sales of all commercialized products developed from the candidate.
Cipher Pharmaceuticals Inc. (Mississauga, ON) reports it has expanded its Canadian dermatology portfolio acquiring the Canadian rights to Vaniqa® and Actikerall® from Almirall S. A., a Spanish pharmaceutical company. Both products have been approved by Health Canada. VANIQA is a prescription cream clinically proven to reduce the growth of unwanted facial hair in women. VANIQA cream is an enzyme inhibitor and works by blocking an enzyme necessary for hair to grow. The product was approved by Health Canada in May, 2001. Actikerall is indicated for the topical treatment of slightly palpable and/or moderately thick hyperkeratotic actinic keratosis, a pre-cancerous patch of thick, scaly, or crusty skin. The product was approved by Health Canada on July 31, 2014 and will be launched by Cipher in the first half of 2015. Under the terms of the agreement, Almirall will receive an upfront payment of CDN$0.45 million and is eligible for certain milestones from product sales in Canada. Almirall will supply the finished product to Cipher.
STEMCELL Technologies Inc. has signed a license agreement with the Salk Institute for Biological Sciences for the rights to commercialize BrainPhys™ Neuronal Medium. Invented by Cedric Bardy in the lab of neuroscientist Fred H. Gage, this formulation promotes optimal neuronal and synaptic activity while supporting long-term culture of both human pluripotent stem cell (hPSC)derived neurons and primary tissue-derived neurons. BrainPhys™ Neuronal Medium will complement STEMCELL’s portfolio of products for neural cell culture applications, including the NeuroCult™ product line for tissue-derived neural cells, and the STEMdiff™ Neural System for hPSC-derived neural cells.
Aeterna Zentaris Inc. (Quebec City, QC) announces that it has agreed to transfer its discovery library of roughly 100,000 unique compounds to the South Carolina Center for Therapeutic Discovery & Development pursuant to a just concluded Material Transfer Agreement. The library will be used to discover drug development candidates for the company in the areas of oncology, neurology, endocrinology and women’s health. The center has agreed to conduct screening and pre-clinical activities with respect to the library and will submit to the company at least one development candidate in its areas of therapeutic interest per year during a ten-year period beginning in 2018. The company will receive the right of first refusal to license the development candidates. Should the company decide to further develop a candidate submitted by the center, MUSC will license the compound to the company, and be entitled to a royalty on the net sales of all commercialized products developed from the development candidate. However, should the company decide not to further develop the development candidate submitted
OncoGenex Pharmaceuticals, Inc. announced that its wholly owned subsidiary, OncoGenex Technologies Inc., has executed a termination agreement with Teva Pharmaceuticals Ltd. under which OncoGenex will regain rights to custirsen, an investigational compound currently in Phase 3 clinical development as a treatment for prostate and lung cancers. This transfer of rights occurs in connection with the termination of the 2009 collaboration agreement between OncoGenex and Teva. The agreement between the two parties to terminate the collaboration includes a $23.2 million payment from Teva. The Fight Against Cancer Innovation Trust and its partners announce the formation of Turnstone Biologics Inc., a biotechnology company focussed on developing treatments for cancer that harness the patient’s own immune system. The company represents a unique collaboration between the Children’s Hospital of Eastern Ontario (CHEO) Research Institute, McMaster University, the Ontario Institute for Cancer Research (OICR), the Ottawa Hospital Research Institute, the University of Ottawa and FACIT. The new company hopes to accelerate clinical translation and commercialization of oncolytic vaccine immunotherapies for the treatment of cancers. Turnstone’s lead technology platform, Marabex™, combines the benefits of oncolytic viral therapy with a tumour-targeted vaccine into a single treatment.
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May/June 2015 Laboratory Focus www.laboratoryfocus.ca
feature
b y k at he r ine Gillis, J ulie cl or , ka ma l a tya G a ra J a n
Plate-based drug and toxin screening using multiplexed benchtop flow cytometry
D
rug-based screening, identification of positive ‘hit’ compounds, and evaluation of mode of action require an understanding of how the drug modulates critical cellular parameters and activities which eventually lead to cytotoxicity or alter cell proliferation.1 This is particularly important in oncology research, where combinatorial strategies using drug mixtures can have additive/synergistic effects on prolif-
eration and apoptosis. Given the increased need for assays with statistically-rich informational content and mechanism-based data, drug researchers and toxicologists have turned to flow cytometry assays. With its potential for precise and quantifiable comparisons between cell populations, flow cytometry enhances dose-response calculations with the statistical power afforded by large sample size.2 The guava easyCyte™ benchtop
flow cytometry systems (EMD Millipore) enable plate-based compound screening because they require significantly fewer cells per well, and because its powerful InCyte™ software enables multiparametric comparisons of concurrent responses. The addition of a violet laser in the easyCyte™12 instrument permits the simultaneous measurement of up to 12 parameters, offering flexibility and more powerful analysis in multiparametric screening studies. This article demonstrates the power of the guava® system and accompanying software to provide enriched data during drug screening processes. Using just two fundamental assays, plate-based screening was performed for various cytotoxic compounds and identified key ‘hits’. The same assays were used to further evaluate select compounds in greater detail with dose-response and time-response studies. These assays also provided information on the mechanism of action of the compounds gambogic acid, celastrol and thimerosal, and their modulation of apoptotic and proliferative responses.
Multiparameter analysis The ability to obtain multiparametric information during compound screening is invaluable to the drug
discovery process. Screening multiple compounds while multiplexing cell health markers leads to many data points and often makes ‘hit’ identification complex. The guava easyCyte™ benchtop flow cytometry systems simplify this process by analyzing up to six parameters simultaneously, whether a single parameter from up to six plates, or six parameters from each sample in a plate, as demonstrated here. The relationship between changes in mitochondrial membrane potential (MMP) and cell health permits the use of a powerful assay for apoptosis. Combining EMD Millipore’s MitoSense Red for assessment of MMP with Caspase 3/7 FAM detection reagent, Annexin V Brilliant Violet™ 421 (BioLegend) and 7-AAD enables multiparametric assessment of apoptosis with simultaneous discrimination of necrotic cells in a single, simplified assay. Upon treatment with gambogic acid, Jurkat cells showed a decrease in MMP, increased caspase activity, increased Annexin V staining and increased cell death, as expected. Combining multiple cell health markers in the manner described permits discrimination of the stage of apoptosis. For example, not all cells that were positive for Annex-
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in V were also found to be simultaneously positive for Caspase 3/7.
Monitoring proliferation Assessment of proliferation is critical to cell analysis, and is of particular importance when screening for bioactivity or cytotoxicity. Because its expression is required for progression through the cell cycle, measuring expression of the nuclear protein Ki67 has become the prototypical assay for cell proliferation. A robust Ki67 signal was observed in untreated Jurkat cells. In contrast, a subpopulation of cells treated with the anti-inflammatory compound celastrol demonstrated the diminished Ki67 expression that is indicative of a reduction in proliferation. Celastrol has been shown to inhibit proliferation in numerous tumor cell types via downregulation of cyclins D1 and E3.3
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Laboratory Focus May/June 2015
with negative controls were eliminated to focus on compounds that showed cytoactivity at either concentration. Twenty compounds induced cell response at 10 μM while one additional compound induced response at 40 μM only. Some compounds, such as gambogic acid, induced a large change in MMP, with a
lower percentage of cells being Annexin V positive and even fewer showing Caspase 3/7 activity. These results were consistent with published reports that gambogic acid induces apoptosis specifically in tumor cells while having less impact on normal cells.4-6 Other compounds, such
as the anti-tumor agent juglone,7 induced a change in mitochondrial depolarization and an almost identical degree of caspase response at 10 μM, with changes in Annexin V binding only occurring at the 40 μM concentration. Several compounds showed large impacts on MMP changes with a low-
feature er percentage for the other markers, while others demonstrated impact on all assays in parallel. From these screening results, a subset of compounds was selected for dose and time-response analysis.
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Plate-based screening The assays described above were next applied to the platebased screening of cytotoxic compounds. A panel of 80 cytotoxic, immunosuppressive, anti-proliferative and anti-inflammatory compounds was obtained from Microsource Discovery Systems, Inc. at a concentration of 10 mM in dimethyl sulfoxide (DMSO). Compounds were diluted in complete growth medium to either 10 μM or 40 μM. Plate setup included negative controls containing 0.2 per cent DMSO, and positive controls treated with 1 μM staurosporine, a known protein kinase inhibitor. The population percentage altered for each parameter was compared in heat map format using the InCyte™ software. The software allows for quick identification of ‘hit’ compounds and comparison of all parameters simultaneously, as shown in the pie graphs and population percentages in Figure 1. While the number of hits between the 10 μM (1A) and 40 μM (1B) plates look similar, the percentage of cells showing higher percentages of effects varied, as shown by the darker shades of blue in hit wells. From the results shown in Figure 1, autofluorescent compounds and those treatments causing less than 20 per cent change compared
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feature heat map results output from incyte™ software showing four-parameter data for cell health impact of 80 compounds on Jurkat cells.
figure 1
each well is mapped to a four-quadrant circle with color intensity reflecting the percentages of cell population positive for loss of mitochondrial membrane potential (top left quadrant), annexin v staining (top right quadrant), caspase activation (bottom left quadrant) and cell death (bottom right quadrant). drug concentrations used were 10 µm (panel a) and 40 µm (panel b).
IR provides more universal accuracy in protein quantification.
Dose and time-response experiments
dose response of Jurkat cells to celastrol treatment.
figure 2
Percent of Population
100 80 60 40 20 0 0.05
5
0.5
µM Inducer MMP Loss
Caspase 3/7
Annexin V
7-AAD
Proliferation Loss
samples were stained with a combination of cell health markers as well as a ki67-based proliferation assay reagent to characterize the mechanism by which celastrol affected Jurkat cell health. Jurkat cells were incubated with various concentrations of celastrol (0.08-10 µm) for 24 hours.
The mechanism of celastrol, a known antioxidant and anti-inflammatory compound, was investigated by evaluating its dose-dependent impact on Jurkat cells. Population percentages for cell health markers at various concentrations are plotted in Figure 2. The degree of MMP decrease and Annexin V staining was similar, with a lower percentage of cells showing caspase activity across the concentration range. The loss of cell proliferation increased with increased celastrol concentration. Proliferation recovery was observed at the two highest concentrations, but this can likely be attributed to the high percentage of cells exhibiting cell death at the same concentrations. The study demonstrated that treatment with celastrol caused mitochondrial depolarization, apoptosis and proliferation loss at lower concentrations than those causing any significant cell death. To assess the effect of increasing compound exposure time on cell health, Jurkat cells were treated with gambogic acid, an antitumour compound, and thimerosal, an antifungal and antiseptic compound, for
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Laboratory Focus May/June 2015
feature time course for Jurkat cell response to gambogic acid and thimerosal.
figure 3a and figure 3b
80
80
Percent of Population
100
Percent of Population
100
60 40 20 0
60 40 20 0
0hr
3hr
6hr
16hr
24hr
0hr
3hr
Time
6hr
16hr
24hr
Time MMP Loss
Annexin V
Caspase 3/7
7-AAD
Proliferation Loss
Jurkat cells were incubated with 10 µm gambogic acid (3a) or 2.5 µm thimerosal (3b) for 0, 3, 6, 16 or 24 hours and either stained with a cocktail of cell health markers or evaluated using a ki67-based proliferation assay.
These studies showed that plate-based screening using the guava easyCyte™ 12 benchtop flow cytometer enabled uncomplicated comparison of mechanisms of action of diverse compounds while also providing the sample-conserving benefits of microcapillary flow cytometry. various durations. Time-dependent responses to gambogic acid (Figure 3A) demonstrated mitochondrial and apoptotic impacts. Results obtained with gambogic acid demonstrate that a rapid and significant loss of MMP occurred at earlier time points when compared to other cell health markers tested. Annexin V- and Caspase 3/7-positive population percentages showed a steady increase at three and six hours with maximal percentages at 16 hours. Significant cell death and proliferation loss were not seen until 16 hours. Time-dependent responses to thimerosal underscored different cell health impacts (Figure 3B). Even with short incubation times, thimerosal caused almost complete depolarization of the mitochondrial membrane by three hours. At three hours, this was also accompanied by significant changes in Annexin V binding, with a continued increase at six, 16 and 24 hours. A high percentage of cells showing caspase activity and cellular death was not seen until the six-hour time point, with maximal caspase activity attained
at 16 hours. Low levels of proliferation inhibition were seen starting at six hours, with a gradual decrease in proliferation over time. While equivalent cell death and proliferation loss responses were seen with gambogic acid, an increased percentage of cells displayed death responses to thimerosal.
Conclusion Multiparametric analysis at a singlecell level provides comprehensive mechanistic and cell health information, which greatly facilitate assessment of cytoactive compounds. These studies showed that platebased screening using the guava easyCyte™ 12 benchtop flow cytometer enabled uncomplicated comparison of mechanisms of action of diverse compounds while also providing the sample-conserving benefits of microcapillary flow cytometry. Specifically, gambogic acid induced significant and rapid mitochondrial depolarization, celastrol acted by inhibiting proliferation and inducing apoptosis, and thimerosal triggered rapid cell death. The ease of these
multiplexed assays, coupled with plate-based microcapillary cytometry and the heat mapping functionality in the InCyte™ software, enables researchers to obtain insights into how compounds modulate apoptotic and proliferation processes and their relationship to mitochondrial dysfunction and cell death pathways.
References 1. Wesierska-Gadek, J., Gueorguieva, M., Ranftler, C., ZerzaSchnitzhofer, G. (2005, Sep.) A new multiplex assay allowing simultaneous detection of the inhibition of cell proliferation and induction of cell death. J Cell Biochem,1;96(1):1-7. 2. Lombardo, T., Anaya, L., Kornblihtt, L., Blanco, G. (2012) Median effect dose and combination index analysis of cytotoxic drugs using flow cytometry. In Flow Cytometry - Recent Perspectives, M.Sc. Ingrid Schmid (Ed.) ISBN: 978-953-51-0626-5, InTech, DOI: 10.5772/38214. 3. Kannaiyan, R., Manu, K.A., Chen, L., Li, F., Rajendran, P., Subramaniam, A., Lam, P., Kumar, A.P., Sethi, G. (2011, Oct.) Celastrol inhibits tumor cell proliferation and promotes apoptosis through the activation of c-Jun N-terminal kinase and suppression of PI3 K/ Akt signaling pathways. Apoptosis, 16(10), 1028-41. 4. Shi, X., Chen, X., Li, X., Lan, X., Zhao, C., Liu, S., Huang, H., Liu, N., Liao, S., Song, W., Zhou, P., Wang, S., Xu, L., Wang, X., Dou, Q.P., Liu, J. (2010, Jan. 1) Gambogic acid induces apoptosis in imatinib-resistant chronic my-
eloid leukemia cells via inducing proteasome inhibition and caspase-dependent Bcr-Abl down regulation. Clin Cancer Res., 20(1):151-63. 5. Zhao, L., Guo, Q.L., You, Q.D., Wu, Z.Q., Gu, H.Y. (2004) Gambogic acid induces apoptosis and regulates expressions of Bax and Bcl-2 protein in human gastric carcinoma MGC-803 cells. Biol. Pharm. Bull., 27, 998–1003. 6. Li, X., Liu, S., Huang, H., Liu, N., Zhao, C., Liao, S., et al. (2013) Gambogic acid is a tissue-specific proteasome inhibitor in vitro and in vivo. Cell Rep., 3(1), 211–22. 7. Xu, H.L., Yu, X.F., Qu, S.C., Zhang, R., Qu, X.R., Chen, Y.P., Ma, X.Y., Sui, D.Y. (2010, Oct. 25) Anti-proliferative effect of Juglone from Juglans mandshurica Maxim on human leukemia cell HL-60 by inducing apoptosis through the mitochondria-dependent pathway. Eur. J. Pharmacol., 645(1-3), 14-22.
Katherine Gillis, Applications Scientist, katherine.gillis@emdmillipore.com; Julie Clor, Applications Scientist, julie.clor@emdmillipore. com; Kamala Tyagarajan, R&D Manager, kamala.tyagarajan@ emdmillipore.com
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feature by ken cook,frank steiner,mauro de Pra
Development of Ultra-fast pH-Gradient Ion Exchange Chromatography for the Separation
of Monoclonal Antibody Charge Variants Introduction The pre-formulated buffers for pH gradient, introduced by Thermo Fisher Scientific, have greatly simplified the development of ion exchange chromatography (IEX) of monoclonal antibodies (mAbs). Three features make this simplification possible. The first feature is that the buffers can cover a pH range from 5.6 to 10.2; this pH window enables the characterization of mAbs with a wide range of isoelectric points, providing a global pH gradient ion exchange screening method that will accommodate the majority of therapeutic mAbs. The second feature is that, if a lin-
figure 1
ear solvent gradient is programmed in the pump, the actual pH gradient produced in the column will be linear as well. Although this sounds trivial, scientists involved in pH gradient studies know how difficult it is to develop buffer formulations capable of fulfilling this requirement. The advantage of a genuine linear pH gradient is that the method can be confidently finetuned merely by narrowing down the pH range around the mAb and its variants, thus allowing for the adjustment of the gradient time according the resolution requirements. The third feature is the mobile phase preparation: the pre-formulated pH buffers only needs to be dilut-
Separation of 5 mAbs with a generic 0−>100 %B in 10 minutes at 0.45 mL/min (method #1 Table 1). Samples are as follows: A) bevacizumab, B) cetuximab, C) infliximab, D) trastuzumab, E) mAb A.
ed by a factor of 10 in deionized water, and the mobile phase is ready to use. Compared to a salt gradient ion exchange chromatography method, where no generic screening can be easily designed, and where method development goes through the rather tedious preparation of several buffers at different pH values, the time and effort invested in method development are substantially reduced. High resolution pH gradient separations are obtained with 30 minute gradients and relatively long columns, such as the Thermo Scientific™ MAbPac™ SCX-10 column, 10 μm, 4 × 250 mm.1 Fast separation of mAb variants were demonstrated by using the MAbPac SCX-10 column, 5 μm, 4 × 50 mm with no significant loss in resolution2 on a Thermo Scientific™ UltiMate™ 3000 BioRS system. In this case, the pH gradient from 5.6 to 10.2 was completed in 7.5 minutes, and the resolution between variants was still satisfactory, despite the short analysis time. In this work, we show how to push the throughput of pH gradient IEX even further. To achieve this, a MAbPac SCX-10 RS column, 5 μm, 2.1 × 50 mm was operated on a new Thermo Scientific™ Vanquish™ UHPLC system. The Vanquish UHPLC is a fully biocompatible system, suitable for the analysis of intact proteins. The combination of low gradient delay volume and high precision gradient formation makes it the ideal system for high throughput analysis with gradient elution. Here the system was used with the default configuration and a total system gradient delay volume of 175 μL. Fast charge variant separations of 5 mAbs are shown.
Experimental Vanquish UHPLC, consisting of: • Vanquish System Base (P/N VH-S01A) • Binary Pump H with Default Mixer (P/N VH-P10-A) • Split Sampler HT (P/N VH-A10-A) • Column Compartment H (P/N VH-C10-A) • Diode Array Detector HL (P/N VH-D10-A) Chromatographic Conditions Column: MAbPac SCX-10 RS, 5 μm, 2.1 × 50 mm (P/N 082675) Buffers: Thermo Scientific CX-1 pHGradient buffer A (pH 5.6) 125 mL (P/N 083273) CX-1 pH-Gradient buffer B (pH 10.2) 125 mL (P/N 083275) Mobile Phase A: CX-1 pH-Gradient buffer A (pH 5.6) diluted 10x in deionized water Mobile Phase B: CX-1 pH-Gradient buffer B (pH 10.2) diluted 10x in deionized water Column Compartment Temperature 30 °C, forced air Detector and Conditions Detector: LightPipe™ 10 mm Standard Flowcell (P/N 6083.0100) Detection Wavelength: 280 nm Data Acquisition Range: 5 Hz (for flow rate ≤ 0.5 mL/min) and 50 Hz (for flow rate ≥ 1.0 mL/min) Response Time: 2 s (for flow rates ≤ 0.5 mL/min) and 0.1 s (for flow rates ≥ 1.0 mL/min) Data Processing Software: Thermo Scientific™ Dionex™ Chromeleon™ 7.2 Chromatography Data System (CDS)
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Laboratory Focus May/June 2015
feature
Samples
mab name
concentration (mg/ml)
injection volume (µl)
bevacizumab
25
1
cetuximab
5
4
infliximab
10
4
trastuzumab
21
2
mab a
21
2
Results and Discussion One of the benefits of using CX1 buffers for the pH-gradient is the simplified method development and optimization. This is due to the fact that the pump running a linear solvent gradient will result in a linear pH gradient in the column. This is not the case for most of home-made buffer formulations, which would produce a non-linear pH gradient in response to a linear programmed gradient. A non-linear pH gradient makes method optimization difficult due the uncertainty of the actual effects of any changes in the programmed gradient. It is recommended to perform a generic screening from pH 5.6 to 10.2 when the pH at which a given mAb elutes is not known. In this work, the generic screening was run in 10 minutes at 0.45 mL/min. As it can be seen in Figure 1 in some cases, satisfactory separation of the charge variants was achieved during the first run. This was the case for cetuximab and infliximab: several charge variants could be resolved with sufficient resolution. After the generic screening, the method development efforts should be aimed at decreasing the analysis cycle time and at the same time improving resolution. To achieve this goal, two parameters were modified, namely the pH range and the gradient slope. Here we used gradient slope based on gradient volume, i.e. ∆(%B)/VG, where %B is the amount of B eluent and VG is the volume of mobile phase delivered by the pump during the gradient.
A narrower pH window allowed for a reduced run-time, whereas a shallower gradient slope provided better resolution. The gradient slope of the generic screening between pH 5.6 and 10.2 was 22.2 (%B)/mL; the improved and faster analysis were obtained with gradient slopes of 8 (%B)/mL and 10 (%B)/mL. Details of the conditions are listed in Table 1. Figures 2a, 3a, 4a, and 5a, show the results obtained by this approach. The number of resolved variants was larger than in the initial screening. Gradient time was five minutes, hence half of the time of the initial screening. The following step was used to develop fast analysis cycles compatible with high throughput. With this approach we aimed for a 2.5 minute gradient time or lower, and total analysis time of less than four minutes, including column re-equilibration. Data are shown in Figures 2b, 3b, 4b, 5b, and 6b. The purpose was to develop a method suitable for high-throughput that can run at least 300 samples a day. High flow rate was used for this purpose. A high flow rate allows running short gradients with relatively shallow gradient slopes; the shallow slope is required to preserve selectivity between charge variants. Additionally, column equilibration, which is directly dependent on the volume of mobile phase flowing through the column, is reached quicker. MAbPac SCX-10 RS column is pressure rated up to 7,000 psi (~ 480 bar), therefore
figure 2
Separation of bevacizumab with different gradient conditions. Chromatogram A condition #2 Table 1; chromatogram B condition #3 Table 1.
figure 3
Separation of cetuximab with different gradient conditions. Chromatogram condition A #4 Table 1; chromatogram condition B #5 Table 1
table 1 description conditions used for the separation of charge variants of the different monoclonal antibodies. method #
figure
Gradient range (%b)
Gradient ph range
Gradient time (min)
flow rate (ml/min)
Gradient slope* (%b/ml)
1
1
0-100
5.6-10.2
10.0
0.45
22.2
2
2a
20-40
6.5-7.4
5.0
0.50
8.0
3
2b
23-35
6.7-7.2
2.5
1.00
4.8
4
3a
10-35
6.1-7.2
5.0
0.50
10.0
5
3b
10-35
6.1-7.2
2.5
1.00
10.0
6
4a
20-40
6.5-7.4
5.0
0.50
8.0
7
4b
18-27
6.4-6.8
0.8
1.20
9.4
8
5a
35-60
7.2-8.4
5.0
0.50
10.0
9
5b
33-45
7.2-7.7
2.5
1.00
4.8
10
6b
5-30
5.8-7.0
2.5
1.00
10.0
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May/June 2015 Laboratory Focus www.laboratoryfocus.ca
feature figure 4
figure 5
Separation of trastuzumab with different gradient conditions. Top chromatogram condition #8 Table 1; bottom chromatogram condition #9 Table 1
figure 6
Separation of mAb A with different gradient conditions. Chromatogram A condition #1 Table 1; chromatogram condition B #10 Table 1.
Separation of inflixumab with different gradient conditions. Chromatogram condition A #6 Table 1; chromatogram condition B #7 Table 1.
it can be operated at high linear flow rate. In this work, we used flow rates up to 1.2 mL/min. The chromatographic pattern between methods at moderate and high flow rate was preserved. The separation capabilities of different methods were compared based on the resolution between charge variants. Since in several instances peak pairs were overlapping, and it was not always possible to measure peak width at half height or at the baseline, here we used resolution based on statistical moments. Resolution was calculated directly by Chromeleon 7.2 CDS according to the formula:
where t2/R and t1/R are the retention times of the more and less retained peak respectively, and μ2/2 and μ1/2 are the related second moment. In some cases, the ultra-fast separation approach was accompanied by some resolution loss. This is the case of trastuzumab, where average resolution loss was ~ 3 per cent. In the case of the complex variants pattern of cetuximab, the average resolution loss at high flow rate was ~ 13 per
cent. Infliximab resolution decreased by ~ 11 per cent, however separation of the five main charge variants and two minor ones was achieved in one minute. This impressive result was obtained by running the column at 1.2 mL/min with a 0.8 min gradient time. In the case of bevacizumab, the ultrafast separation approach even yielded to ~ 4 per cent improved resolution. The better resolving power can be explained by a slightly narrower pH range and a shallower gradient slope. mAb A was analyzed only with the generic screening and high flow rate (1 mL/min) method. The highthroughput method provided 19 per cent better resolution on average of five charge variants.
Conclusion The ultra-fast charged variant separations described here are achieved because of several advances in chromatography techniques. The mechanism of pH gradient chromatography lends itself to the use of shorter, faster columns. The availability of high pressure rated small particle size ion exchange columns are a perfect match to pH gradient methodology. The commercial buffer formulations used here form a linear gradient
which allows intelligent optimization of the methods. Finally, there is the use of the new Vanquish UHPLC system which has extremely low delay volumes, high precision gradient formation and a totally inert flow path. Ultra-fast separation that requires total analysis cycle in the order of two minutes, including column reequilibration and injection time, enables users to run more than 1,400 samples during 48 hours continuous operations. To allow unattended tasks with such large amount of samples, the Vanquish UHPLC system
table 2 overview of the resolution between charge variants at different conditions. resolution was calculated by chromeleon cds using statistical moments. mab bevacizumab cetuximab infliximab trastuzumab mab a
Gradient time (min)
1-2
2-3
3-4
4-5
5-6
6-7
5
0.94
0.87
1.81
-
-
-
2.5
1.00
0.89
1.89
-
-
-
5
0.79
1.14
1.00
1.13
1.28
0.98
2.5
0.53
1.05
0.94
0.93
1.20
0.91
5
0.68
1.56
1.33
0.87
-
-
0.8
0.59
1.40
1.30
0.70
-
-
5
1.25
1.14
1.70
0.87
-
-
2.5
1.27
1.11
1.63
0.79
-
-
10
0.14
1.31
0.94
1.26
-
-
5
0.32
0.96
1.22
2.02
-
-
can be extended with the Vanquish Charger Module. This can host up to 9,000 samples in a thermostatted environment, and transfer them to the Vanquish Autosampler.
References 1. Thermo Fisher Scientific. Thermo Scientific Application Note 20784, A Novel pH Gradient Separation Platform for Monoclonal Antibody (MAb) Charge Variant Analysis, AN20784_E, Sunnyvale, CA, 2013. 2. Thermo Fisher Scientific. Thermo Scientific Application Note 20946, A Fast and Robust Linear pH Gradient Separation Platform for Monoclonal Antibody (mAb) Charge Variant Analysis, AN20946_E, Sunnyvale, CA, 2014.
Ken Cook,Thermo Fisher Scientific; Frank Steiner,Hemel Hempstead, United Kingdom; Mauro De Pra,Thermo Fisher Scientific, Germering, Germany
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PCR System
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PAGE
WEBSITE
aeterna Zentaris ............................... 7 .............................. www.aezsinc.com bibby scientific ................................. 15 .................. www.bibby-scientific.com
Pumps
brady corporation ............................ 15 ......................... www.bradycorp.com caledon laboratory chemicals ......... 5 .....................www.caledonlabs.com canadian society for chemical .................................................................... technology ....................................... 6 ....................www.chem-tech.ca/cct children’smiraclenetwork.............. 17 .......... childrensmiraclenetwork.ca cipher pharmaceuticals inc................. 7 .....................www.cipherpharma.com cQdm .............................................. 6 .............................www.cqdm.org/en/ facit ................................................ 7 .................................. https://facit.ca eppendorf .................................... 18, 20 ...... www.eppendorf.ca.bioflo320 fluidigm corporation ......................... 18 .............................www.fluidigm.com fluid metering inc. ............................ 15 .....................www.fluidmetering.com harvard apparatus .........................16,18 ...........www.harvardapparatus.com héma-Québec.................................... 6 ....................www.hema-quebec.qc.ca
The STH and STQ Duplex metering pumps from Fluid Metering, Inc. (FMI) are ideal for precision mixing, diluting, and proportional metering for OEM medical, analytical, and industrial instrumentation. FMI’s OEM Duplex Metering Pumps consist of two FMI valveless pump heads directly coupled to a single variable stepper motor drive. The displacement of each pump head is independently adjustable, and a variety of pump head sizes can be used in combinations to achieve dispensing ratios from 1:1 up to 500:1, from microlitres to litres. Fluid Metering’s OEM Duplex Metering Pumps feature a CeramPump® valveless piston pumping technology and are the valveless-syringe alternative to conventional syringe pumps. The CeramPump® has only one moving part in the fluid path, a sapphire-hard ceramic piston, which accomplishes both pumping and valving functions, without valves.
web: www.fluidmetering.com
mcmaster university ......................... 3 ............................. www.mcmaster.ca meller optics inc. ............................. 18 ...................... www.melleroptics.com university health network.................. 4 ...................................... www.uhn.ca university of british columbia ............. 5 ...................................... www.ubc.ca university of montreal ....................... 2 .............................www.umontreal.ca resverlogix corp................................ 7 .........................www.resverlogix.com thermo scientific .............................. 16 ................ www.thermoscientific.com transition therapeutics inc. ............... 7 .........www.transitiontherapeutics.com verlyx pharma ................................... 6 ................................ www.verlyx.com vwr ................................................ 2 ...................................... ca.vwr.com wyvern/mandel ............................... 9 ...............................www.mandel.ca
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May/June 2015 Laboratory Focus www.laboratoryfocus.ca
New Products
Catheters
Microplate Reader
Harvard Apparatus has introduced a new line of rodent catheters. Designed with rounded tips, suture collars and additional features specific for the given species w:Layout 1 12/19/2012 10:01and AM vessel, Page 1 this complete line of catheters has something to suit all rodent infusion applications. Mouse and rat options are available for jugular vein, femoral artery/ vein, carotid artery and tail vein. Additional options for rat include catheters for portal vein, bile duct, gastric and intrathecal.
Web: www.harvardapparatus.com
Introducing a multimode microplate reader that simplifies your workflow and provides reliable results across a wide range of applications. The new Thermo Scientific Varioskan LUX multimode microplate reader with Thermo Scientific SkanIt Software delivers automated and versatile functionality to bioscience researchers performing a variety of microplate assays. The Thermo Scientific™ Varioskan LUX™ is ideal for variety of applications, including Absorbance (UV-Vis), Fluorescence Intensity, Luminescence, AlphaScreen / AlphaLISA, and Time-Resolved Fluorescence. The Varioskan LUX is equipped with smart safety controls to help keep research on track. These technologies include automated plate check, built-in shaker speed control, and automatic prime check and position sensors
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for the built-in dispensers, which are designed to identify and notify the user of potential errors that could compromise research. The Varioskan LUX’s SkanIt software monitors and captures measurement data throughout the run to reduce the risk of wasting expensive reagents, samples and time. Varioskan LUX also offers an innovative automatic dynamic range selection feature that eases protocol set-up by eliminating the need for users to manually adjust sensitivity settings for each assay. The instrument automatically selects the optimal reading range based on signal intensity in the well, using settings to obtain maximum sensitivity. This helps ensure that results are comparable from assay to assay—and day to day—even when a single assay exhibits very high and very low signals.
Web: www.thermoscientific.com
Cell Imaging
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Lonza announces the release of its CytoSMART™ System, an easy-to-use live cell imaging system that enables researchers to take time-lapse videos and images of their cell culture without needing to manually inspect their cells. The new system offers a number of benefits to researchers, including 24-hour peace of mind that their cells are safe and well, all without them ever having to step foot in the lab. It also provides a number of advanced functions, such as reporting ongoing cell confluency via a graphical readout, while automatic email alerts can be set to inform the user when milestones are reached (for example, once the cell culture has reached a certain confluency). The images taken with the CytoSMART™ device are transmitted into the cloud, enabling researchers to view their cell culture outside of the actual cell culture lab at any given time through a web browser, whether via a computer, tablet or smartphone. Everything is pulled together via one intuitive and easy to use interface, the CytoSMART™ Connect Cloud Project page, which allows users to view and download all the videos and images connected with their project.
Web: www.lonza.com
www.laboratoryfocus.ca Laboratory Focus
MAY 2015 May 22-24 Labcon 2015 Venue: Montreal, QC Tel: 905-667-8688 Email: labcon@csmls.org Web: www.csmls.org
May 26 Vaccine Innovation Conference Venue: Toronto, ON Tel: 780-492-0950 Web: www.biotech.ca/en/ what-biotech-is/vaccines.aspx
May/June 2015
Calendar
Web: www.bio.org/events/ conferences/world-congressindustrial-biotechnology
July 22-24 Advanced Biofuels Symposium Venue: Montreal, QC Tel. 514-508-2884 Fax. 514-508-4336 Email: info@biofuelnet.ca Web: www.biofuelnet.ca/ news-and-events/advancedbiofuels-symposium-2015/
SEPTEMBER 2015
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OCTOBER 2015
September 28-30
October 27-28
Sherbrook International Life Sciences Summit Twitter: @SILS_Sherbrooke Email: info@sils-sherbrooke.com Tel: +1 819 821-5577 / 1 877 211-5326 Web: http://sils-sherbrooke.com/
BioPort Atlantic Venue: Halifax, NS Email: jgillis@bionova.ca
JUNE 2015 June 2-5 Canadian Materials Science Conference Venue: Halifax, NS Email: contact@gmail.com Web: http://cmscconf.org/
June 7-8 Personalized Medicine Summit Venue: Vancouver, BC Email: summit.2015@ubc.ca Web: www. personalizedmedsummit.com
June 13-17 Canadian Chemistry Conference and Exhibition Venue: Ottawa, ON Tel: 613-232-6252 Email: acampbell@cheminst.ca Web: www.csc2015.ca
June 15-18
June 20-24 Canadian Laboratory Medicine Congress Venue: Montreal, QC Tel: 613-531-9210 Email: info@clmc.ca Web: www.clmc.ca/2015/
JULY 2015 July 5-9 International Rapeseed Congress Venue: Saskatoon, SK Twitter: @agwestbio #IRC2015 Tel: 306-975-1939 Fax: 306-975-1966 Email: irc2015@agwest.sk.ca Web: https://event-wizard.com/ irc2015/0/welcome/
CritiCal priority needs for
14
Children’s
hospitals
aCross Canada
eaCh day, there are
4,900
Children
Bio International Convention Venue: Philadelphia, PA Tel: +1.202.962.6655 Email: convention@bio.org Web: http://convention.bio.org/2015/
Children’s MiraCle network funds
who rely on the support of Children’s MiraCle network
MeMber hospitals in Canada
What is Children’s Miracle Network? Children’s Miracle Network® raises funds for 170 children’s hospitals in North America, 14 of which are in Canada. These hospitals, in turn, use the money where it’s needed the most. When a donation is given, it stays in the community, ensuring that every dollar is helping local kids. These donations have gone to support critical research and training, purchase life-saving equipment, and ensure excellence in care - all in support of our mission to save and improve the lives of children.
July 12-14 World Congress on Industrial Biotechnology Venue: Montreal, QC Twitter: #BIOWC15 Email: info@bio.org
Learn more at: ChildrensMiracleNetwork.ca
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New Products Pumps Harvard Apparatus has introduced an addition to its Pump 11 Elite product family. The Pump 11 Pico Plus Elite is now available in a single channel configuration that is compatible with syringe sizes from 0.5 µl to 60 ml. The single channel configuration maintains the flow capabilities of the original dual channel Pump 11 Pico Plus Elite, while allowing for additional fluid volume without refilling the syringe. This infusion/withdrawal syringe pump can deliver flow rates down to 0.54 pl/min and is ideal for small volume injections such as drug studies and low flow rate applications including microdialysis and organ-on-a-chip experiments. It also comes with glucose clamp capability that sets the infusion rate based on subject weight, drug concentration and required dose rate.
Web: www.harvardapparatus.com
Single Cell Analysis Fluidigm Corporation has offered the first glimpse of its Polaris™ single-cell research system. This new product is the first to integrate cell selection, isolation, dose, culture and molecular preparation into a single workflow, thereby enabling researchers to directly correlate gene expression with environmental conditions and phenotypic information. The Polaris system will consist of new instrumentation and a new class of Fluidigm integrated fluidic circuits (IFCs). These new IFCs will include the processing of single cells for downstream molecular biology preparatory and analysis techniques. Data generated by the Polaris system will be processed using Fluidigm’s Singular™ Analysis Toolset, which includes features to filter, visualize and identify biologically relevant genes and variants. Fluidigm is planning shipments of the Polaris system in the coming months.
Web: www.fluidigm.com
Centrifuge Cell Windows Meller Optics Inc. announces the release of a new line of UV cell windows for use with Beckman centrifuges and other instruments. The cell windows are made from sapphire to resist chemicals, scratching, and high pressure and from single crystal quartz for less demanding applications. They are available wedged one-or two degrees and plano, with chamfers 0.20/0 mm x 45 degrees in two places. Featuring an arrow scribed on their edge that projects the optic access for easy alignment, Meller Sapphire Centrifuge Windows are 19.03 mm dia. ±0.3 mm by 0.5mm thick ±0.10mm and have a 30-5 scratch-dig surface finish. The quartz windows have a 10-5 finish with a central clear aperture of 16 mm and both types are suitable for OEM and field-replacement use.
Web: www.melleroptics.com
May/June 2015 Laboratory Focus www.laboratoryfocus.ca
Spectrofluorophotometer Shimadzu announces the release of its RF-6000 spectrofluorophotometer, an instrument able to handle challenging applications in chemical, environmental, pharmaceutical, foods and life sciences labs. Equipped with new LabSolutions RF control software it has greatly enhanced operability as well as a wealth of options and accessories for a wide variety of applications and analyses. The adoption of a much brighter light source allows the RF-6000 to achieve an S/N ratio of 350:1. Furthermore, the lifespan of the xenon arc lamp has been greatly extended, to approximately 2,000 hours. This, in combination with the fact that the range of the detector’s measurable wavelengths has been widened, assures stable analyses with high precision. It is also now possible to obtain fluorescence spectra up to 900 nm with a standard configuration.
Web: www.shimadzu.com
Tubes
Eppendorf has added two new conical tube sizes, 15 mL and 50 mL to its range of microcentrifuge tubes. Equipped with newly designed screw caps, the tubes provide not only optimal sealing properties, they also simultaneously ensure a secure slip-free grip. The optimized handling features further facilitate safe opening and closing of the tubes via convenient one-handed operation. The new tubes represent an optimal solution for cell culture and cell biology applications as well as for sample preparation protocols in microbiology and molecular biology laboratories.
Web: www.eppendorf.com
Liquid Chromatography Tosoh Bioscience LLC has extended its line of TSKgel Amide-80 hydrophilic interaction liquid chromatography (HILIC) columns. TSKgel Amide-80 columns are now available packed with 2 μm spherical silica particles, offering higher resolution and faster analysis with equivalent retention and selectivity as TSKgel Amide-80, 3μm columns. This allows for smooth method transfer from a 3μm to a 2μm column. In addition, TSKgel Amide-80, 2μm columns retain more hydrophilic compounds than the existing amide columns on the market. Ideal applications include analysis of peptides, oligonucleic acid, and other hydrophilic small molecules.
Web: www.tosohbioscience.com
www.laboratoryfocus.ca
T
Laboratory Focus May/June 2015
Genome Canada unveils new Genomics Innovation Network
he way genomics research is conducted in Canada is about to undergo a change, as Genome Canada says it is restructuring and creating a new Genomics Innovation Network. The new network will be comprised of 10 “nodes,” each receiving core operational funding from Genome Canada, with matching funds from various public and private sector partners. A total of $15.5 million in federal funding through Genome Canada is the initial investment being made in core operation funding for the nodes, with each receiving from $800,000 to $2 million in federal funds over two-years. Co-funding investments in the Nodes from other partners, including provincial governments, academic institutions, and the private sector at a required minimum one-to-one ratio, bring the total initial investment in the Genomics Innovation Network to approximately $31 million. The node selection process was the result of a competitive process involving peer review by an international review committee. Under the old model, Genome Canada was making its investments in five science and technology innovation centres. Often, the five centres were “working in their own silos.” For this reason, Genome Canada president and CEO Dr. Pierre Meullien says the new network model will place greater emphasis on collaboration and the sharing of expertise among the centres. He adds that creating the Genomics Innovation Network will contribute to Canadian leadership in the development of new genomic technologies and better fulfilling this mandate. “Breakthroughs across all sectors that form part of Canada’s growing bioeconomy – health, agriculture, fisheries and aquaculture, forestry, energy and mining – rely on researchers across Canada having access to leading-edge ‘omics technologies, which are rapidly evolving,” says Meullien. “Moreover, we want to build on past successes where we’ve seen new genomics technology development become the foundation for Canadian business growth.” He adds that the investment will also support important work in the area of bioinformatics and computational biology, including addressing major challenges associated with the storage and analysis of “big data.” Each node will also provide Canadian and international researchers with access to the leading-edge technologies required for research in genomics, metabolomics, proteomics and related areas. The nodes will include: The Proteomics Centre and the Sequencing Platform at the BC Cancer Agency Genome Sciences Centre, both led by Genome British Columbia; The Metabolomics Innovation Centre led by Genome Alberta; the Toronto Centre for Phenogenomics, the Network Biology Collaborative Centre, and The Centre for Applied Genomics, each led by the Ontario Genomics Institute; and the Canadian Centre for Computational Genomics, McGill University and the Génome Quebéc Innovation Centre, the Canadian Data Integration Centre, and the Centre for Advanced Proteomics Analyses, each led by Genome Quebéc.
app review
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Activ8rlives 3.0 Health and Food Diary Self-Monitoring App
By: Aseptika http://www.activ8rlives.com/software/smartphone-apps.html https://itunes.apple.com/us/app/activ8rlives-3.0-health-monitoring/ id570252701?mt=8 Made for iOS and Android, the third generation Activ8rlives Health and Food Diary tracking app is a well-being solution that enables users to quickly understand where a health tracker or vital sign should be to indicate good health. The user interface is brighter, cleaner and easier to navigate. Detailed help, instruction wizards and embedded instructions are available within the App on every screen. The App also works with a full range of consumer and medical devices. Data is stored and can be shared with family supporters or carers online. Those who wish can also share their data with their medical staff using a dedicated clinician’s portal. The restructuring of the App is in preparation for CE marking as a certified medical device (initially Class 1). Data generated by the user and stored within Activ8rlives can then be accepted by clinicians world-wide and used in partnership with their patients in support of self-management programmes.
RefME
By: RefME https://appcenter.evernote.com/app/refme/web-apps https://appcenter.evernote.com/app/refme/iphone RefME is an app and online tool that crowdsources information to make the process of referencing work fully automated. The app, which is available on iPhone, Android and the Google Chrome webstore, automatically generates academic references in more than 7,000 different formats, including Harvard, APA and MLA. RefME automates the whole referencing process for you. You can search by book/journal article title, DOI, ISBN, ISSN or simply copy and paste any website URL to create a reference in seconds. Essentially, its referencing made easy. Users only need to point their smartphone camera at the barcode of a weighty academic tome and the app will present a bibliography-ready reference. All references are stored in the cloud and can be accessed online through the website and across devices. The app also pulls its data from Google Books. You can import and export your work to Microsoft Word, Evernote, Mendeley and many more applications.
Highly Evolved The advanced control station The BioFlo® 320 is the next generation in bioprocess control. Designed as a universal platform, the BioFlo 320 is capable of meeting the ever-changing needs of scientists in the biotech and pharmaceutical industries. The BioFlo 320 has the right combination of features to get the job done.
> Universal gas control strategy for both microbial and cell culture applications > Multi-unit control of up to eight systems from a single interface > Direct integration of digital Mettler Toledo® ISM sensor connectivity > Built-in optical pH sensing technology for use with BioBLU® vessels
For more information visit: www.eppendorf.ca/bioflo320 131.A1.0132.A © 2015 Eppendorf AG. Mettler Toledo® is a registered trademark of Mettler Toledo AG, Switzerland.
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