Genetic Engineering & Biotechnology News

OCT15 2017

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20 | OCTOBER 15, 2017 | GENengnews.com | Genetic Engineering & Biotechnology News See Antibody Characterization on page 22 Antibodies are critical research reagents, says Nicolas Schrantz, Ph.D., senior manager for antibody development at Thermo Fisher Scientific. They can lead to research suc- cess, but only if they perform as desired—which is to say, as advertised. "Developing an antibody that recognizes the correct target using a specific application is an art that re- quires scientific knowledge and technical expertise," notes Dr. Schrantz. "Unfortunately, many antibodies on the mar- ket that claim to have been validated for a specific applica- tion simply do not recognize the target they are supposed to, or worse, recognize the wrong target. The existence of unreliable antibodies wastes researchers' time and money." Two-Step Validation Thermo Fisher Scientific has been working with indi- vidual researchers and the International Working Group for Antibody Validation (IWGAV) to define and implement stan- dards for antibody testing. The company has already adopt- ed validation standards for its Invitrogen antibody portfolio that adhere to IWGAV recommendations. Thermo Fisher Scientific uses a two-step validation ap- proach. The first step is target specificity verification. The second step is functional application verification. Target specificity verification assures that antibodies bind to the correct target. Validating an antibody's specificity also ensures the absence of nonspecific binding. To accomplish target specificity validation, Thermo Fisher uses at least one standard method from a collection of stan- dard methods. These methods span several categories: im- munoprecipitation/mass spectrometry; genetic modification (knockout and knockdown testing); independent antibody verification (a testing approach that uses two differentially raised antibodies that recognize the same protein target); and biological verification (cell treatment, relative expression, neutralization, peptide array, and orthogonal approaches). Thermo Fisher employs several knockdown and knock- out methods to test antibody performance against genetically modified samples. They include mouse knockout models, dominant negative mutants, morpholinos, short interfering RNA, and most recently, gene editing. CRISPR/Cas9 gene editing allows creation of knockout cell models for use as controls for validating antibody specificity. In independent antibody verification, antibodies should target non-overlapping epitopes on an antigen. "Obtaining comparable [affinity] results increases confidence that these antibodies are specific and suitable for the detection of their intended target," Dr. Schrantz tells GEN. With the orthogonal approach, the idea is to correlate two methods, an antibody-dependent method and an anti- body-independent method. For example, Western blot could be correlated with quantitative RT-PCR, or flow cytometry could be correlated with Thermo Fisher's own PrimeFlow ™ RNA assay kit. The second level of validation involves determining how well antibodies work in applications such as Western blot- ting, immunofluorescent imaging, flow cytometery, chroma- tin immunoprecipitation, and immunohistochemistry. "We test our antibodies using at least one of these methods," states Dr. Schrantz."And yes, we even use antibodies or kits from other vendors for this purpose." Middle-Up Approach Monoclonal antibodies (mAbs) are inherently heteroge- neous because they are produced in living cells and can un- dergo unanticipated modifications during the biomanufac- turing process. Living cells are capable of variability at every stage of protein expression, from the generation of amino acid chains during the formation of protein backbones, to the introduction of post-translational modifications (PTMs), some of which occur enzymatically (glycosylation), and some of which occur non-enzymatically (oxidation and deamida- tion). Additional modifications may occur during purifica- tion and storage. GEN readers are familiar with chromatographic and compound analytical methods, as well as the importance of using multiple, sometimes orthogonal methods, for example, size-exclusion or ion-exchange chromatography and peptide mapping. Peptide mapping followed by liquid chromatogra- phy–tandem mass spectrometry (LC–MS/MS)—the method of choice for understanding site-specific PTMs—involves se- rious sample preparation and lengthy chromatography runs. Researchers refer to proteolysis-based mapping as bottom- up proteomics, and those based on intact proteins as top- down methods. Peptide mapping is a multistep process generally involv- ing antibody denaturing, reduction, and alkylation; diges- tion with a protease (usually trypsin); high-performance liq- uid chromatography (HPLC) on a octadecyl carbon chain (C18)-bonded silica column; and finally, mass spectrometry "online" with HPLC to identify the separated peptides. To- gether, the steps in peptide mapping take about one full day, including overnight digestion. Antibody Characterization Balances Rigor and Reason Bioprocessing Feature Figure. Major glycoforms and minor isoforms alike are clearly seen with the Agilent 6545XT AdvanceBio LC/Q-TOF system. The data was deconvoluted using the maximum entropy algorithm in the Agilent MassHunter BioConfirm software. This algorithm carefully preserves low-level peaks so that the heterogeneity of a molecules may be fully characterized. Continued from page 1 MS Deconvolution of Intact NIST mAb (0.5-μg Injection).

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