Genetic Engineering & Biotechnology News

MAY15 2017

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18 | MAY 15, 2017 | GENengnews.com | Genetic Engineering & Biotechnology News See Proteins on page 20 in various forms, depending on the local dialects, the molecu- lar statements that are proteins often acquire subtle shades of meaning—post-translational modifications, for example— that may enhance cell signaling, membrane transport, and other processes. The messages may be revised in many ways. In fact, there are over 200 different known modification types. They in- fluence protein folding, localization, activation, and other processes. Such modifications are of increasing interest to developers of biotherapeutic proteins. Unlike small molecule drugs, biotherapeutic proteins may vary in terms of safety and efficacy, depending on the messages their post-transla- tional modifications convey. Understanding these messages is a challenge that pharmaceutical companies needn't confront with small molecules. Multiple Modification Types "If you think about manufacturing a chemical small mol- ecule drug, you realize it's very simple," says Laura Moriarty, Ph.D., drug discovery and development marketing manager, Bio-Rad Laboratories. "The drug is manufactured using a chemical synthesis. When creating a biotherapeutic drug, you're looking at multiple different parameters that can af- fect the final product." Cell lines, growth conditions, purification methods, and drug formulations all have a hand in decorating the final pro- tein with glycosylations, phosphorylations, ubiquitinations, and various other modification types that affect important drug attributes including efficacy, immunogenicity, pharma- cokinetics, and degradation. "Ubiquitination, for example, is a sort of a flag," explains Poulomi Acharya, Ph.D., senior global product manager at Bio-Rad. "It says, 'Hey! Come and degrade me!'" Essen- tially, ubiquitination changes the half-life of the protein. Dr. Acharya emphasizes the importance of identifying po- tential modifications and their effect on each step of drug development, from production and purification to storage and safety. In the past, protein analysts had to perform complex, multistep procedures to characterize post-translational mod- ifications. "Many people in laboratories would say that these assays take lots of time," notes Dr. Moriarty. "They're very hands-on, and they haven't evolved much over the years." The assays may not seem to have evolved, but the inter- ests of protein analysts keep expanding. For example, pro- tein analysts are aware that the types of modifications that need to be targeted have increased, in response to a greater understanding of post-translational modifications and their role in cell signaling. Targeting is about much more than just phosphorylation. In addition, Biopharma has introduced larger, more com- plex biotherapeutic molecules that have synthetic modifica- tions, such as PEGylations or, in the case of antibody-drug conjugates, toxic small molecules, in addition to naturally occurring modifications that often increase in frequency with protein size. Meanwhile, more sophisticated mass spectrometry instrumentation and analytical software has enabled researchers to move beyond merely identifying a modification. Researchers may now map a modification's location on a protein and even quantify the abundance of modified proteins. Isotopic Labeling One quantification strategy uses a stable, isotopically la- beled peptide that behaves identically to its unlabeled twin, the target peptide, in chromatography and mass spectrometry experiments as an internal standard. Mass differences make it possible to distinguish the otherwise identical pair. To facili- tate the use of this method in monoclonal antibody charac- terization, MilliporeSigma recently introduced SILu™Mab, a product line that provides stable-isotope labeled therapeutic antibodies for mass spectrometry. Researchers can use the relative abundance of the isotopically labeled standard, which has a known initial concentration, to quantify the abundance of the target peptide. According to Jason Apter, Ph.D., head of research solu- tions, strategic marketing and innovation, MilliporeSigma, "LC-MS [liquid chromatography used in conjunction with mass spectrometry] has evolved into the predominant meth- od for understanding protein modification, although tradi- tional immunoassay-based methods still exist." Mass spectrometry has become the workhorse for pro- tein analytics, but it's not without its limitations. "The de- tection of low abundance post-translational modifications is extremely challenging," explains Dr. Apter, "but is also es- sential to ensure safety and reproducibility." Improved instrumentation has helped overcome detection limitations through increased resolution and mass accuracy, Interpreting the Language of Proteins Bioprocessing Feature Sigma-Aldrich, now a part of MilliporeSigma, provides SILuMAb and SigmaMAb antibody standards for mass spec analysis. This image shows a stable isotope- labeled immunoglobulin G monoclonal antibody. This antibody, which is expressed in a CHO cell line, uses universal heavy-labeled tryptic peptides as internal standards for the quantitation of monoclonal antibodies and Fc-fusion therapeutics. Continued from page 1

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