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SCIENCE & TECHNOLOGYCOVANCEDRUG DILIGENCEAnalysts p erformbioassays in cell-culturehoods at Covance’s NorthAmerica BiotechnologyServices facility.ASSAYING ANTIBODIESDrug manufacturers pinpoint TECHNIQUES TO ANALYZEgrowing and diverse class of therapeuticsJYLLIAN KE MSLEY , C&EN WEST C OAST NE WS B UREAUMONOCLONAL-ANTIBODY-based drugsare a large and growing segment of pharmaceuticals.Since muromonab-CD3(Orthoclone OKT3) was first approved bythe Food & Drug Administration in 1986 asan antirejection drug for organ transplantpatients, more than 30 antibody-baseddrugs have entered the market, and manymore are in the pipeline. In 2011 alone,FDA approved belimumab (Benlysta) forlupus, ipilimumab (Yervoy) for metastaticmelanoma, and brentuximab vedotin (Adcetris)for two types of lymphoma. Marketresearch company Datamonitor estimatesthat sales of antibody therapeutics willgrow by 8.2% from 2010 to 2016, the fastestof any therapeutic class, with sales expectedto surpass $65 billion by 2016.Although drugs based on monoclonalantibodies—MAbs—have widely variedtherapeutic action, they are all based on thesame protein, immunoglobulin G (IgG).But this shared attribute does not meanthat these drugs are simple to assay. MAbsare composed of more than 1,000 aminoacids in four peptide chains, and they bearsugars and other chemical modifications.As the MAb class grows, scientists aresettling on some standard critical proteinqualities and analytical testing approachesfor drug identity, quality, and purity forthese large, complex molecules.Along those lines, the U.S. PharmacopeialConvention (USP), the pharmaceuticalstandard-setting organizationin the U.S., is in the midst of developing“best practice” guidelines and regulatorystandards for MAb drug developers. “Theindustry has more than 20 years of manufacturingexperience with these types ofmolecules, and we’re seeing products thatare extremely pure and really well characterized,”says Tina S. Morris , USP’s vicepresident for biologics and biotechnology.“Every antibody has slightly differentpurification and analytical characteristics,but you’re never starting from zero with acompletely unknown protein.”One set of standards, to be known asChapter 1260, will <strong>focus</strong> on how to develop,define quality attributes for, and manufactureMAbs.These standards will not be legallybinding on MAb manufacturers but insteadare intended to provide worldwidebest practice guidance, says AnthonyMire-Sluis, corporate vice president forproduct and device quality at Amgen andchair of the USP Recombinant TherapeuticMonoclonal Antibodies Expert Panel,which is developing the standards. Theexpert panel includes 13 industry members,as well as seven representatives fromregulatory and other standard-settingagencies.The other set of standards, Chapter 129,will be legally binding on MAb manufacturersand outlines the minimal quality attributescommon to all antibodies and thecorresponding recommended analyticaltests for MAb therapeutics. Several companieswill validate the proposed methodsWWW.CEN-ONLINE.ORG 16 MARCH 2012
to ensure that they work, and the standardswill also include limits derived frommanufacturer data on actual products. But,Mire-Sluis notes, companies will be ableto justify using different tests or limits aspart of their drug application package ifthey think their product merits somethingatypical.STRUCTURALLY, an antibody,or immunoglobulin, is a large,Y-shaped protein composedof two “heavy” and two “light”peptide chains. The heavychains come together at thebottom half of the Y, also calledthe crystallizable fragment, orFc. Disulfide bonds and noncovalentinteractions hold thechains together. This part ofthe antibody has a conservedamino acid sequence and bindsto receptors to activate partsof the immune system. Differentantibody classes—IgA,IgD, IgE, IgG, and IgM—havedifferent heavy chains that determinespecific immunologicalroles. IgG provides most of thebody’s immunity to invadingpathogens.At the top half of the antibodyY, the heavy chains branchout and each associates witha light chain, also through disulfidebonds and noncovalentinteractions. This region of the antibodyis called the antigen-binding fragment,or Fab, and it is here that the amino acidsequence varies to bind to assorted foreignmolecules: Both arms of a specific antibodyhave the same amino acid sequence, whilethe variable portion differs between differentantibody molecules.But because MAbs are made by clonedcell lines with identical cells, all of the antibodiesproduced by a cell line engineeredfor producing a particular therapeuticshould be identical. Monoclonal IgG therapeuticswere originally based on mouseantibodies, but they now can be chimeric,which means a part-mouse/part-humansequence, or “fully” human. Structurally,the three types are very similar, but theymay provoke different immune responsesin patients.In addition to their basic structurallikeness, MAb therapeutics also tend tobe purified similarly, USP’s Morris notes.Purification typically starts with affinitychromatography using a solid phase thatanchors protein A, a bacterial proteinthat selectively binds to the antibodies’conserved Fc region. After that, the drug islikely treated to inactivate any contaminatingviruses and may undergo fine-tuningpurification steps. Most antibodies arealso currently produced from two popularcell lines, which means that their impurityCONFIGURATION The basic IgG structure involvestwo heavy chains and two light chains held together bydisulfide bonds and noncovalent interactions. The proteinmay be glycosylated at a conserved site (shown) orelsewhere on the structure.SSSSSSOligosaccharideHeavy chainVariable ◼Conserved ◼Light chainVariable ◼Conserved ◼SSSSSSSSprofiles are similar. The combination ofsimilar cell culture and purification, on topof targeting the same protein, further lendsanalysis of MAb therapeutics to similarprotocols, Morris says.But despite these similarities, MAbsmay show heterogeneity depending onhow they were produced and processedby their originating cells. Problems withDNA translation or transcription may leadto different protein sequences. Proteinsmay misfold or mismatch their disulfidebonds. Posttranslational modification mayproduce different glycosylation patterns.Side chains may also be subject to chemicalreactions, such as oxidation or deamidation.Proteolytic enzymes may clip theprotein, especially the lysines at the ends ofthe heavy chains in the Fc. MAbs may alsodenature or aggregate as they go throughpurification or formulation.Because of these effects, even a perfectlyprepared MAb therapeutic will includeIgG proteins with varied structuresWWW.CEN-ONLINE.ORG 17 MARCH 2012SSSSSSSand properties, says William Whitford,senior market manager for cell culture andbioprocessing at Thermo Fisher Scientific .“Unlike small-molecule drugs, biologicsshow a distribution of slightly differentmolecules,” he says. The analytical challengeis therefore to ensure that the populationdistribution doesn’t vary significantlyfrom the drug that was developed,tested clinically, and approved.SOligosaccharideSSSOME PROPERTIES are aprocess red flag. Oxidation ordeamidation, for example, byitself generally doesn’t affectan antibody’s clinical function,says Raymond Kaiser,global science leader and vicepresident for biotechnologyservices at Covance , a contractdrug development company.But variability in those categoriesis usually a sign thatthe manufacturing process isnot well controlled, and otherprotein characteristics, such asaggregation, may be affected,Kaiser says.Other modifications are aclinical concern. A misfoldedprotein or one with abnormaldisulfide bonds probably won’thave the structure needed tofunction properly. Additionally,IgGs have a conservedglycosylation site on the Fc,but variations in those sugars and glycosylationelsewhere on the protein may elicitan unwanted immunological response,such as anaphylaxis. Protein aggregatesmay also provoke an immune response.And so a suite of analytical tests is necessaryto ensure that a MAb therapeuticis within the bounds of the drug that wasoriginally prepared, tested, and approved.“You live and die by your analyticals,” Kaisersays. “If you don’t have your analyticals,you don’t know anything.”The first order of business is often toidentify whether a manufacturer has madethe correct protein. One common approachis to do an immunoassay, such asan enzyme-linked immunosorbent assay(ELISA). In these types of assays, a stationarysolid phase is loaded with a moleculethat will bind specifically to the target antibody.A drug sample is loaded, incubated,and washed, and some sort of label is usedto assay whether and how much of the targetantibody is bound.
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