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“The industry has more than 20 years of manufacturingexperience with these types of molecules, and we’re seeingproducts that are extremely pure and really well characterized.”Another option for identifying proteinsis peptide mapping, which involves usinga protease enzyme to cleave an antibodyinto characteristic segments that can thenbe separated by gel electrophoresis or columnchromatography to yield a particularfingerprint.ON THE QUALITY front, tests such asion-exchange chromatography, isoelectric<strong>focus</strong>ing, or capillary electrophoresis willpick up variations in charge within theprotein structure that might indicate oxidation,deamidation, or protein clipping.In ion-exchange chromatography, howproteins interact with a charged stationaryphase determines how quickly they elute.Isoelectric <strong>focus</strong>ing takes advantage of thefact that protein charges are a function ofpH and separates proteins using a gel witha pH gradient. Capillary electrophoresisseparates proteins by applying an electricfield to a capillary tube and can be doneusing either free solution (capillary zoneelectrophoresis) or a gel (capillary gelelectrophoresis).For glycosylation, manufacturers oftencreate a glycan map similar to a proteinmap. The antibody is enzymatically orchemically treated to remove the sugars,which are then given a fluorescent tag andseparated using high-performance or ultraperformanceliquid chromatography. Theglycans’ identities determine when theyelute from the column. Sialic acids, however,need a different approach. Because theyare destroyed by the standard acid treatmentto remove sugars, they are insteadremoved using mild acid and then analyzedby high-pH anion-exchange chromatographywith electrochemical detection.To further pin down the specifics ofchemical modifications or changes inglycosylation patterns, liquid chromatography-massspectrometry (LC/MS) has becomethe standard approach. An antibodyor its sugars are digested and separated asin other tests, but the resulting fragmentsare analyzed using mass spectrometry.LC/MS provides exact mass identificationof the fragments but requires a highlyskilled operator, so it’s mostly used in developmentor problem solving, says TaegenClary, marketing manager for biopharmaceuticalsat Agilent .Nevertheless, LC/MS data have becomea necessary part of drug application packagesto demonstrate that manufacturersunderstand what they’re seeing in theirquality-control suite of assays. “I believethat if you don’t supply some LC/MS datain a package, you’ll be asked for it because itis so common now,” Covance’s Kaiser says.To pick up aggregation, one approach issimply to look at a protein solution. If theaggregates are big enough, the solution willappear hazy rather than clear. To check forsmaller particles, size-exclusion chromatography,also known as gel-filtration chromatography,is the standard method, withdetection by either ultraviolet light absorptionor multiangle laser light scattering.Other techniques to look for aggregationinclude light obscuration and microflowimaging. Microflow imaging is amicroscopy technique that allows scientiststo distinguish, for example, whether aparticle is composed of protein or of siliconoil, which is used in drug formulations as alubricant for syringes. Those techniques aswell as analytical ultracentrifugation mightbe used in development to help understandantibody aggregation or in production todiagnose a problem, Kaiser says, but theprocess workhorse is high-throughputsize-exclusion chromatography.ASIDE FROM THE QUALITY of the IgGmolecule, manufacturers must also keepan eye on the purity of the drug solution.Common impurities that manufacturerswatch for include residual DNA or otherproteins from the host cells, plus anyprotein A that might have carried throughpurification. Quantitative real-time polymerasechain reaction (Q-PCR) methodsare used to amplify and quantify DNA,while ELISA targets protein A and host-cellproteins. In early development, manufacturersmight use cell-line-specific ELISAkits that contain antibodies to commoncellular proteins. In late-stage development,however, companies often developprocess-specific assays by taking cells thatdon’t produce the therapeutic through thepurification process and using the outputto develop antibodies to whatever proteinsremain, Kaiser says.One additional area of MAb analysis isassays for potency. Potency tests, however,cannot readily be standardized across theclass. Different clinical actions by necessityrequire different potency tests, which areoften cell-based assays in which adding thedrug product elicits some kind of measurableresponse.What’s being looked at is also a consideration,whether it’s IgG proteins by themselves;antibody-drug conjugates, whichtether a small-molecule drug to an IgG; orantibody fusion proteins, which typicallycombine the IgG Fc component with anotherprotein. For antibody-drug conjugates,most assays would be similar to those forunconjugated IgGs, but scientists might paymore attention to a peptide mapping assayto make sure they know where and how successfullythe small molecule attached, Kaisersays. For fusion proteins, manufacturerswould probably turn to the same analyticaltools as for intact IgGs, but they would haveto do more development work to optimizeassays and understand what the data show.Many of the analytical methods generallyused on MAbs, such as ELISA, chargeseparation procedures, or size-exclusionchromatography, are time-honored biochemicaltechniques, says Martina Bielefeld-Sevigny, vice president and generalmanager of drug discovery and researchreagents at PerkinElmer . The push now isto make those methods faster and easier,with better precision, accuracy, reproducibility,and sensitivity. In many cases, thatmeans going to microfluidic, lab-on-a-chipplatforms that use smaller amounts of materials,provide faster response times, andenable high-throughput analysis.“I think that the monoclonal industry isa trailblazer,” USP’s Morris says. Pointingas an example to common use of capillarytechniques instead of plain gel electrophoresis,she adds, “I think they use fairly sophisticatedanalytical techniques comparedto plasma or peptide industry people.”That’s something that consumers canappreciate, for the extra assurance that adrug will work as advertised. ◾Reprinted from C&EN, Jan. 16, 2012WWW.CEN-ONLINE.ORG 18 MARCH 2012