Tuesday, April 26, 2016

Battling Zika: A Biotechnological Approach

By Glenn Petrie, Ph.D.
Senior Scientific Advisor
ABC Laboratories

Zika is a disease caused by the Zika virus which is spread to humans primarily through the bite of an infected Aedes species mosquito. The disease can also be by transmitted through sex with an infected partner. The symptoms of Zika are fever, rash, joint pain, and conjunctivitis. The illness is usually mild with symptoms lasting for several days to a week and rarely requires hospitalization. There is no vaccine or specific treatment for Zika virus infection. Treatment is symptomatic and supportive, including rest and the use of acetaminophen to relieve fever.

Federal health officials recently confirmed that the Zika virus causes a rare birth defect and other severe fetal abnormalities, typically, abnormally small heads and severe brain defects. There is also limited evidence of Guillian-Barre’ syndrome and multiple sclerosis being related to Zika infection. The greatest risk is to pregnant women; however the Center for Disease control has recommended that men and women should wait a minimum of 8 weeks after possible exposure before trying to conceive. This period may increase based on the latest findings regarding the latency of the virus.

In a recent article in The New England Journal of Medicine it was noted that Zika is the fourth mosquito-borne illness to infiltrate the Western Hemisphere over the past 20 years, following dengue, West Nile, and chikungunya. There is currently no available vaccines for any of these diseases. According to the World Health Organization (WHO), more than 60 research institutes and companies are working on products to combat the spread of the Zika virus, but a vaccine is likely to take years to develop.

The disease can alternatively be attacked by control of the vector, Aedes aegypti. WHO states that traditional insecticide spraying methods have had no significant impact in decreasing the spread of dengue, implying that these classical methods for battling mosquito borne disease may be equally ineffective for Zika.

There is another possibility for eradicating the Aedes species mosquito over large areas. This involves modification of the mosquitos themselves. This approach has been used successfully in the past for eradication of the screwworm, an insect that was a major cause for livestock deaths in the mid- 20th century. The screwworms were irradiated, sterilizing the male flies. Only the male flies were released, typically sprayed from small planes. The sterile males could not produce progeny, gradually eradicating the species. The technique was an unmitigated success.  The last cases of screwworm in the United States were reported in 1982, followed by eradication in Mexico and Central America.

Now, the world is focused not only on the Zika virus, but dengue, chikungunya and yellow fever, all transmitted by Aedes aegypti .  A British company, Oxitec, has approached the problem utilizing genetic engineering and inserting a gene that kills the insect. Male mosquitoes containing the gene are released to mate with wild females. The released male mosquitoes have no effect on people because males don’t bite. Offspring die before they reach adulthood, in that way suppressing the population of wild mosquitoes. Small-scale tests conducted in the Cayman Islands, Panama and Brazil since 2009 reduced local mosquito populations by as much as 99 percent.

Last month the federal government tentatively approved a field test (Florida Keys) of genetically engineered mosquitoes that might help slow the spread of the Zika virus. A final approval for the trial will not be made until the FDA receives and assesses comments from the public, which is likely to take months and the plan has faced fierce opposition from some residents in the state.

Information for this article was obtained from The Washington Post, The New York Times, The New England Journal of Medicine, CDC website and WHO website.

Monday, April 11, 2016

Biosimilars: They May Look the Same, but One is a Real Stinker

Biosimilars by nature look much like their comparator products, but they sometimes behave quite differently. That’s because the two products often originate from different cell lines or expression systems. And no matter how hard you strive for parity in the way they’re cultured, minor differences in manufacturing can lead to sticky variations in primary amino acid sequence, glycosylation chemical modifications and protein folding issues. The FDA understands biosimilar products sometimes behave differently than their name-brand counterparts. But, they do expect you to understand why those differences occur, and to demonstrate that they have no clinical significance. That’s where ABC Laboratories comes in.

Choosing the Right Analytical Partner May Be the Most Economically Important Decision You Make

FDA guidance calls for a “complete and thorough” CMC section that begins with extensive comparative characterization of the biosimilar and... click here for more.

Tuesday, March 22, 2016

Advances in Electronic Notebook Technology

By Harley Everett Wilcox, MBA
Senior Scientific Advisor
ABC Laboratories

I started my career in the pharmaceutical industry in the early 1980’s and recall how we documented experiments in laboratory notebooks. As part of the synthesis research group, most experiments were synthesis of new molecules or purification of existing compounds. Almost every data entry or summary was manual and certain data were creatively documented. For example thin layer chromatography would be described by one of three techniques, a table listing retentions based on distance, a hand drawing showing relative spot size and location on the plates, or a photocopy once color copiers were available. NMR spectra existed most as a hard copy. Early notebooks were a single copy where a user copy was provided back to the user copy once the notebook was completed. Advances in notebook technology included two sheets for experiments and several sheets of carbon paper for an immediate copy.  For higher compliance such analytical efforts, an exhibit books containing plots and chromatographic printouts accompanied the actual notebook.   Peer review signatures and some QA review were incorporated. Eventually electronic scanning allowed for the first electronic storage.

Today, technology and compliance have significantly evolved.   I spent a few moments with our LIMs administrator, Kevin Cornel, for a Q&A on electronic laboratory notebooks (ELN), and although ELN’s have been available for some time, I was surprised to the extent the systems can help eliminate human errors and support automation.

With the introduction of laboratory information systems, instrument software, and electronic notebooks it would appear that the future may hold data generation with limited human input.  Users have electronic workbooks where menu’s for reagents, methods etc. are chosen and the entire experiment can use electronic inputs from balances, pH meters and HPLC’s interfaces etc.  Bar codes are scanned for samples, reference standards, and reagents to almost eliminate opportunities for errors and flag non-compliance such as expired reagents. Bar coding glass pipets, glassware are possibilities may serve to further refine experiment as well as provide more explicit data records. Electronic signature and permanent electronic profiles of all activities for a study allow for efficient auditing.

Clearly ELN’s will improve efficiency and quality but the information system requires significant up front resources. Existing software platforms must be compatible with ELN systems or interfaces will need to be created. Information services will create methods, workbook formats and all other desired fields and complete validations.  Analyst will require training for the chosen software systems for system set-up and management. Opportunities for errors then reside almost exclusively with the initial sample inventory and sample management where a sample/compound arrives and sample data is entered into the information system via barcode or like. A second reviewer of the upfront data entries may be worthwhile.

Will the introduction of robots interfaced with electronic data systems eliminate the need for a warm bodied chemist for certain types of analysis?

Thursday, February 18, 2016

Enantiomers: Exactly the Same… Only, Different!

By Jim Schmidt
Senior Scientific Advisor
ABC Laboratories

In chemistry, enantiomers are stereoisomers that are non-superimposable mirror images of each other - much like your left and right hands are the same except for being reversed along one axis.

*Image courtesy of Wikimedia Commons

Indeed, the study of enantiomers – chirality – comes from the Greek word, “Χειρ = Cheir =  Hand”!

Enantiomers have identical chemical and physical properties except for their ability to rotate plane-polarized light by equal amounts but in opposite directions.

However, in biological systems, they can have very different behavior.  Some of the most interesting – and important – differences are in drug metabolism.

For the better part of a century, the consideration of enantiomers in drug metabolism was limited to academic study and/or to natural products, owing in no small part to the limits of separations chemistry.

However, for many reasons (including improvements in analytical chemistry), chirality has been earning ever-greater importance in drug discovery and development, such that many new drugs reaching the market in the first decades of the 21st century are single enantiomers, rather than the racemic mixtures (or achiral drugs) that dominated the latter half of the 20th century.

Indeed, in the recent report, “The Year in New Drugs” (C&E News, February 1, 2016, pp. 12-17), it can be seen that more than half of the newly-approved small-molecule drugs in 2015 had specific stereochemistry (and often with more than one chiral center).

Examples of enantio-selective biotransformations include:
  • Prochiral to Chiral 
  • Chiral to Chiral 
  • Chiral to Achiral 
  • Chiral to Diastereoisomer 
  • Chiral Inversion

These metabolism pathways can have significant effects on pharmacology and drug safety. While the movement towards single enantiomers as drug candidates, noted above, mitigates safety problems that might  be associated with racemic mixtures, they do not necessarily alleviate the need to consider and study achiral-to-chiral, chiral-to-chiral, and/or chiral-to-diastereomer transformations.

Adapted from my chapter – “Metabolite Profiling” – in New Horizons in Predictive Drug Metabolism and Pharmacokinetics (edited by Alan G. E. Wilson; Royal Society of Chemistry, 2015).

Tuesday, February 9, 2016

Challenges in the Characterization of Antibody Drug Conjugates

By Glenn Petrie, Ph.D.
Senior Scientific Advisor
ABC Laboratories

Antibody Drug Conjugates (ADC) provide a unique new treatment for a variety of cancers. ADCs consist of a monoclonal antibody (mAb) targeted to the receptor of interest and linked to a highly cytotoxic payload. The mAb binds to the cell and enters the cytoplasm. Once inside the cell, the linker is cleaved and the toxin released. This provides the ability to use highly cytotoxic compounds without the serious side effects of systematic chemotherapy. It is estimated that there are 100-150 ADCs currently in preclinical development. ADCs present unique analytical challenges.

In addition to the complicated mAb, there is the added complexity of combining a cleavable linker and a cytotoxic drug. This introduces the necessity for determining drug loading, linkage sites and Drug Antibody Ratio (DAR). The critical technique for analysis on ADCs is ultra-high resolution QToF mass spectroscopy. This allows for determination of relative loading, DAR and linkage sites, as well as PTMs, disulfide linkages and related substances/degradants (deamidation/oxidation, truncations, and amino acid substitutions). Due to their high resolution, UHR-QTof instruments have the ability to sequence proteins up to 25-30 kDa. The specific protease IdeS (which cleaves just below the hinge region of IgG) under reducing conditions results in three polypeptide chains of ~ 25kDa: Fd region, Fc/2 region and the LC region. Analysis of these digests by UHR QTof MS yields complete sequencing of each polypeptide, DAR, payload and glycan distribution. If necessary, other proteases may be utilized for more detailed analysis. Additional characterization includes the following:

  • Intact mass
  • Deglycosylated mass
  • IEX
  • Imaging CIEF
  • CE-SDS
  • SEC
  • N-linked glycan analysis
  • HIC (secondary DAR analysis)
  • Binding assay
  • Bioassay
  • Higher Order Analysis (CD, AUC, DSC, etc.)

The FDA considers the mAb a drug substance so both the mAb and ADC must be fully characterized. In addition, the agency has been requesting characterization of charge variants of the mAb and ADC. This necessitates preparative IEX followed by characterization of the acidic and basic fractions. Based on all these considerations, characterization of ADCs require careful planning and attention to detail.

Thursday, February 4, 2016

Staying the Course (a Note to the Agribusiness Community)

By Jon Rhodes, M.S.
Senior Scientific Advisor
ABC Laboratories

Times are tough for the agribusiness community.  The pace of mergers and acquisitions is heating up in a big way and continually challenging businesses to grow their pipelines and accelerate development timelines to remain competitive.  Falling commodity prices and high inventories, coupled with slowing product sales volumes and fluctuating currency exchange rates are affecting the bottom line.  The growth in the number of herbicide resistant weed species has kicked into high gear with the United States leading the way.  Global weather patterns are becoming more variable and the continued availability of adequate water resources for agricultural use is in doubt.  Pollinators and endangered species are under pressure.  Regulatory pressures are expanding.  Crop yield enhancement technology continues to face regulatory and social obstacles to global acceptance.  Is agribusiness destined to become mired in a long and protracted slow-growth environment as a result?

Not likely.  The challenge facing humanity and the agribusiness community has never been greater.  Feeding a world population estimated at more than 9 billion people by 2050 will require a 70 to 100 percent rise in overall food production.  Rising wealth in developing countries is bringing changing dietary preferences and greater demand for food.  The total amount of arable land is shrinking.  These factors and others will put enormous pressure on social stability and global security without a concerted and collaborative effort.  The opportunities and consequences are clear and no sector is more prepared to address the challenges we’re facing than the global agribusiness community with a proven record of  investment in technological innovation and enhancement of crop production practices.

Tuesday, January 12, 2016

Well Characterized Biopharmaceuticals Meeting - 2016

By Glenn Petrie, Ph.D.
Senior Scientific Advisor
ABC Laboratories

The 20th annual Well Characterized Biopharmaceuticals (WBCP) meeting will take place in Washington, D.C. the week of January 24th. I attended the inaugural meeting and have only missed one in the last 20 years. This is always an excellent meeting covering both the scientific and regulatory aspects of biopharmaceutical development in equal measure. I am particularly excited about this year’s topics including Higher Order Structure, Combo Products, Biosimilars and Pre-clinical development. I am looking forward to seeing you at the ABC Labs booth to discuss some of our exciting studies. These include:
  • · Utilizing the WES system to quantitate both intact and degraded protein DP in fecal matter
  • · Supported two successful IND submissions for Antibody Drug Conjugates in 2015 with a third in progress. This included full characterization, method development, qualification, release and stability studies. These submissions had NO comments regarding the chemistry section of CMC.
  • · Supported our clients during difficult times
    • During a Phase II trial, reagent changes caused bioanalytical ELISA results to be “offset” from the previous results. We were able to troubleshoot and solve the client’s problem.
    • When a client suddenly severed a relationship with its CRO we were able to transfer, qualify and release methods in support of GMP DS and DP while meeting deadlines.
We hope to speak to you about your biopharmaceutical analytical needs during the breaks, as well as at the Exhibitor Reception, Wednesday, from 5:30-7:00 PM.