Heterogeneityandcomplexityofbiopharmaceuticalagentsdevelopedoverthepastdecadeorsocanbedefined attwolevels.First,inthecaseofproteinsystemssuchasmonoclonalantibodies,theprimarysourceofheterogeneity arises from glycosylation post?translational modifications mainly at the conserved N?linked glycosylation sites in the constant (Fc) region of the heavy chain. The Fc glycosylation plays a role in maintaining antibody stability and also contributes to binding with Fc gamma receptor isoforms that in turn mediate effector functions such as antibody dependentcellularcytotoxicity(ADCC).Second,inthecaseofcomplexdrugssuchasheparinorlowmolecularweight heparin(LMWH)etc.themaincomponentofthedrugisaheterogeneousmixtureoflinear(inthecaseofheparin)or branchedglycanpolymerswithamolecularweightandanionicchargedistribution.Developingfollow?on(orgeneric) versionsofsuchcomplexbiopharmaceuticalmixturesisthereforeinconstantneedfornewtechnologiestodefineand characterizethesemixturesascomprehensivelyaspossiblefromthestandpointofchemicalandbiologicalequivalence. Despite these advances, there remain gaps in linking glycosylation heterogeneity with antibody efficacy and adverseeffectssuchasthoserelatedtohostimmuneresponsetoahumanantibodygeneratedinanon?humancellline. InthecaseofcomplexglycandrugssuchasheparinandLMWH,thereisalargergapinlinkingmixturepropertieswith biologicalactionofthedrug.Forexample,despitethefactthatheparinhasbeenqualitycontrolledandusedinclinicfor severaldecades,aseriousglobalhealthcrisisassociatedwithadministrationofheparinemergedinearlyMarch2008 whichwaslateridentifiedbyusandotherstobeassociatedwithanunnaturalglycosaminoglycancontaminantspecies. Wehaveextensiveexperienceincharacterizingcomplexbiopharmaceuticalmixturesintermsoftheirstructural attributes and how these attributes impinge on their function. A central component of our approach is the ability to employ a computational framework that we had built over the past decade or so in integrating diverse datasets pertaining to complex glycan mixtures. This framework permitted us to incorporate orthogonal datasets including analyticalandfunctionaldatatogettothebottomofstructure?functionrelationshipsofcomplexglycanmixtures.Inthis proposal we seek to build on this computational platform and our extensive experience to build a robust set of integrated tools and algorithms to determine the extent of characterization required for establishing equivalence for therapeuticsthatareusedintheclinic.Throughthestudiesproposedhere,webelievethatwewillmakeacontribution towards determining criteria for sufficiency in characterization of complex therapeutic agents in establishing equivalence.
Developing follow?on (or generic) versions of such complex biopharmaceutical mixtures is in constant need for new technologiestodefineandcharacterizethesemixturesascomprehensivelyaspossiblefromthestandpointofchemicaland biologicalequivalence.Buildingonourextensiveexperienceindevelopingcomputationaltools,hereinweaimtoconstruct a robust set of integrated algorithms or `tools' to determine the extent of characterization required for establishing equivalence of therapeutics used in the clinic. Through the studies proposed here, we believe that we will make a contributiontowardsdeterminingcriteriaforsufficiencyincharacterizationofcomplextherapeuticpharmaceuticalagents
