Genetic Engineering & Biotechnology News

MAY15 2017

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20 | MAY 15, 2017 | GENengnews.com | Genetic Engineering & Biotechnology News but more abundant peptides can still conceal low concentrations of modified peptides that might contaminate the sample. Many meth- ods exist to enrich samples for the target of interest before mass spectrometry analysis, but the post-translational modification of interest ultimately dictates the techniques available. Additional Enrichment Strategies Antibody technology can offer research- ers more options for enrichment strategies beyond conventional affinity columns and techniques that use radioactive isotopes. For example, BioPlex Pro™ magnetic cell signaling assays, a platform developed by Bio-Rad, uses magnetic beads tethered to phosphorylation state-specific antibodies to isolate phosphorylated proteins of interest. Following immunoprecipitation, researchers can further separate proteins using a West- ern blot and analyze each resulting band by mass spectrometry to identify the exact modification site on the protein. The availability of both off-the-shelf and custom antibodies from companies such as Bio-Rad and Rockland Immunochemicals has increased the number of post-transla- tional modifications that researchers can target using immunoassays. Custom anti- body collections, such as Bio-Rad's HuCAL ® (human combinatorial antibody library), are the product of recombinant protein technol- ogy and in vitro screening methods, such as phage display. In addition to antibodies that can de- tect phosphorylation, Rockland Immuno- chemicals' catalog includes antibodies that can detect glycosylation, hydroxylation, methylation, acetylation, and ubiquitina- tion. Antibody technology, given its relative cost-effectiveness, ease of use, and platform- independence, has come to be tightly em- braced by the research community, remarks Carl Ascoli, Ph.D., Rockland's chief science officer. Dr. Ascoli foresees a future in protein characterization that has "a good blend of both antibody technology and mass spec technology." Multiple Attribute Monitoring While the advent of PCR has driven prog- ress at the nucleic acid level, "mass spec- trometry in the biopharmaceutical or biotech realm has really been the driver from an analytical standpoint," according to Glenn Petrie, Ph.D., senior scientific advisor at EAG Laboratories. EAG Laboratories is a contract research or- ganization (CRO) that works with Biopharma and Biotech clients to provide comprehensive analytical characterization and GMP testing of biotherapeutic proteins (including monoclo- nal antibodies and antibody-drug conjugates). "[To characterize] PTMs in the old days, you would have had to use multiple methods," ex- plains Rowel Tobias, Ph.D., principal scientist and group lead, EAG Laboratories. "With high-resolution mass spectrometry, you can combine all of this into one MAM (multiple attribute monitoring) method." EAG Laboratories uses quadrupole time-of-flight (q-TOF) and QTRAP mass spectrometers to perform their characteriza- tions. These high-end instruments have high resolution and mass accuracy and are able to detect post-translational modifications present at low levels in the sample. These ad- vanced capabilities come with a high price tag, though. "These instruments start at a half a mil- lion [dollars] and then go up," admits Dr. Petrie. "It's a big leap in both cost and tech- nology, as well as the expertise that's re- quired to run these sorts of analyses." However, for Biopharma companies look- ing to produce biosimilars, the trade-off be- tween providing comprehensive comparisons between a biosimilar and an innovator mole- cule and having to perform additional clinical trials makes the additional cost for analyses a worthwhile exchange. It's one reason the emerging biosimilar market has driven the field of protein analytics tremendously. Nonetheless, as technology becomes more advanced and robust, the FDA continues to increase its expectations. "They want you to use the current and most powerful tools to PCR just got easier … and BETTER PCR just got easier … and BETTER Visit us at www.thermagenix.com - Use checkout promo code: "best PCR" to receive a special offer for trying Therma Stop and Therma Go You deserve the BEST results in your PCR assays - We are here to help! Good results are no longer enough to satisfy modern PCR product applications. Simply add Therma Stop ™ and Therma Go ™ to your reaction and make your results GREAT. BETTER specificity throughout PCR Therma Go eliminates unwanted PCR errors, improves the production of correct products, AND increases assay reproducibility for end-point genotyping applications. FAM (dR) FAM (dR) 1150 750 350 50 750 500 250 0 Cycles Cycles 20 25 30 35 40 20 25 30 35 40 No Therma Go Therma Go BETTER RT-PCR Therma Stop -for-RT improves sensitivity AND yield of one-step or two-step RT-PCR using gene-specific primers. Therma Stop outperforms Hot Start Polymerases AND eliminates undesired product after cool down. BETTER PCR Hot Start HS denotes a hot-start enzyme 1% Agarose : 1kb Ladder Non-HS Taq's Non-HS Taq's HotStart Taq's HotStart Taq's Therma Stop Proteins Continued from page 18 Bioprocessing Hyper-reaction monitoring (HRM) is a technology based on high-resolution mass spectrometry that can be used to quantify post-translational modifications. This image, provided by Biognosys, highlights portions of the HRM workflow (building a spectral library, acquiring data in DIA mode, and analyzing data) that can be supported by proteomics technology, namely, Biognosys' sample preparation kit, indexed retention time (iRT) kit, and Spectronaut software.

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