Genetic Engineering & Biotechnology News

OCT15 2017

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22 | OCTOBER 15, 2017 | | Genetic Engineering & Biotechnology News Peptide map data analysis is complex by virtue of the sheer number of peptides gen- erated, all of which require identification and quantitation. Analysis gets even trickier when full digestion does not occur, or when products of nonspecific digestion are present. Investigators involved in mAb character- ization are therefore interested in alterna- tives to full peptide mapping. Many of these methods, including middle-up or middle- down, employ proteolytic enzymes that are somewhat more selective than trypsin. Among these is the immunoglobulin- degrading enzyme from Streptococcus py- rogenes (IdeS), which was first reported by Swedish researchers in 2002, 2 and is now pro- vided in commercialized in kit form by several vendors. IdeS reagents and kits are sold by Genovis, MilliporeSigma, Creative Enzymes, and Promega. "IdeS is effective due to its high cleavage specificity and simple operation," ex- plains Chris Hosfield, Ph.D., a senior research scientist at Promega. A related enzyme, IdeZ (from S. equi subspecies zooepidemicus), has identical specificity but cleaves mouse IgG2a more efficiently than IdeS. IdeS cleaves the IgG heavy chain below the hinge region. Post-digestion addition of a reducing agent yields a sample containing three fragments of about 25 kDa in size. This is the starting point for subsequent analy- sis—hence the "middle-up" designation. The approach identifies domain-specific oxi- dation, charge profiling, and N-glycan pro- files. In a study published in MAbs, 3 domain separation was achieved with a 30-minute HPLC gradient, and oxidations were quanti- fied through ultraviolet detection. IdeS is faster than peptide mapping, tak- ing up to about one hour instead overnight. The separation stage is faster because only three fragments are involved. "HPLC gradi- ents are in the range of 30 minutes instead of 3 hours," asserts Dr. Hosfield. Data analysis is simpler for the same reasons. "For these reasons, many companies use IdeS-based analysis as a platform method, for example, in quality control lot-release settings." The downside is that information on modifications is not site-specific. For exam- ple, one could detect an oxidation occurring in the Fc region, but not determine defini- tively which methionine within that region was affected. A more recent study by a group at Genentech described an improved IdeS in which separation of the IgG domains and variants took just 10 minutes. 4 Mea- sured oxidation levels were comparable to those achieved by more complex and time- consuming peptide mapping. Putting Validation to Use The level at which manufacturers vali- date antibodies depends on the antibodies' intended use. An example of how validation level may complement antibody use is pro- vided by Abbiotec, which manufactures both polyclonal antibodies (pAbs) and mAbs for research purposes. The company character- izes its products for target reactivity, but ac- cording to CEO Hervé Le Calvez, Ph.D., it doesn't assay products for physicochemical properties, outside of purity determinations by ELISA or SDS-PAGE, which specifically test for contamination by other antibodies. "Our approach is similar between pAbs and mAbs, especially when we use peptides as antigens," Dr. Le Calvez says. "We main- ly produce GLP-grade antibodies, so we make sure screening, titering, and isotyp- ing, if applicable, are done correctly before moving on with purification and testing." Purification methods for pAbs vary from standard Protein A or Protein G to antigen- affinity chromatography and fractionated precipitation. "Because of the difficulties in character- izing pAbs from each bleed or animal," ex- plains Dr. Le Calvez, "we offer the option to clone the genes coding for the antibodies and produce recombinant antibodies when the end goal is the diagnostics or therapeu- tics market." One issue entering the validation/char- acterization equation is the antibody tar- get. "It's one thing to validate an antibody against a well-known target such as TNFα [tumor necrosis factor alpha] or NF-ᴋB [nu- clear factor kappa B], but quite another if the target is the latest new protein identified by sequencing," insists Dr. Le Calvez. "Our products lean towards the latter." There is much to learn, he adds, about the validation of antibodies that target new molecules that have not themselves been fully characterized. Abbiotec releases such products as a service to research groups that do not fully disclose their intentions. "In this respect," reveals Dr. Le Calvez, "we provide numerous antibodies for niches that are still undeveloped." Lest We Forget... In the characterization of intact, thera- peutic-grade mAbs, conventional analysis tools are being deployed more systemati- cally. As mass spectrometry (MS) becomes more user-friendly and generally accessible, mAb developers routinely adopt this meth- od to obtain a product's accurate molecular weight and heterogeneity (for example, with respect to PTMs). For development-stage antibody-drug conjugates, MS provides further insights into conjugation number, mAb sequence variations, and degradation products. "Confidence in the information obtained from analyzing intact mAbs depends on measurement reliability," explains David Wong, Ph.D., a senior applications scientist at Agilent Technologies. "Accurate mass de- termination, separation of protein isoforms, and detection of major and minor heteroge- neities provides reliable answers about the protein and growth conditions directly from intact protein analysis." Due to its high resolution in high mass ranges, quadrupole time-of-flight (Q-TOF) LC-MS is the mode of choice for analyzing intact proteins. Top vendors have "system- atized" these instruments toward specific purposes. For example, the Agilent 6545XT AdvanceBio LC/Q-TOF system includes hardware and software features for charac- terization of biomolecules up to 30,000 m/z. "Q-TOFs have the flexibility to analyze not just intact proteins, but also to perform peptide sequence mapping and PTM identifi- cation and localization at the peptide level," Dr. Wong adds. In a recent application note, 5 Agilent described a typical workflow involving the Agilent 1290 Infinity II UHPLC system (at the front end of separation), the AdvanceBio LC/Q-TOF, and the company's MassHunter BioConfirm software (for automatic data processing). The analyte was a NIST [Na- tional Institute of Standards and Technol- ogy] mAb standard. LC-MS analysis showed mass resolu- tion of all species falling between 2,000 and 5,000 m/z. Moreover, zoom-in spectra of each charge state showed the six major glycoforms of the NIST mAb. In addition to these major features, the analysis identified minor glycosylation heterogenicities, such as loss of N-acetylglucosamine (Figure). Agilent analysis software includes a max- imum entropy deconvolution algorithm, which preserves fine details of the intact pro- tein. Typically, a mAb can have 30–70 posi- tive charges under LC-MS conditions. Con- sequently, a mAb with a nominal mass close to 150,000 amu is generally detected in the range of 2,000–5,000 m/z. According to Dr. Wong, the maximum entropy algorithm has been widely used for deconvolving the multiple charge state enve- lope that occurs when a protein is analyzed by LC-MS: "The method transforms the raw m/z spectrum of one or more intact proteins into the actual molecular weight of the pro- tein, making it easier to directly compare [it] to theoretical sequence information or po- tential mass shifts caused by PTMs." References available online. Antibody Characterization Continued from page 20 Bioprocessing THINK BEYOND CHO, THINK ESETEC ® COST SAVING WITH ESETEC ® – 3X LOWER COGS ESETEC ® CHO $/g CREATING TOMORROW'S SOLUTIONS What if there were an innovative secretion system for production of your non-glycosylated biopharmaceutical that made your processes faster and more cost-effi cient? That would be great – and it is possible. 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