Research and development for industry: Advanced ... - CSIR

PHARMACEUTICAL AND CHEMICAL SECTORS| 38 |Dr Stoyan StoychevThe CSIR is currently buildingcapacity for large-scalerecombinant production ofbiopharmaceuticals. At present,there are a number of suchproducts at various stages in thedevelopment pipeline, includingtherapeutic peptides such asExenatide (diabetes treatment),Enfurvitide and Griffithsin (HIVinhibitors) as well as anti-rabiesmonoclonal antibodies (mAbs).Production of biologicsBiopharmaceuticals areconventionally produced insterile fermentation facilitiesusing mammalian cellcultures. A drawback of suchsystems is that they have aUsing biologics totransform the drugmarketplaceBiologic products now provide important therapeutic options for a range of serious clinical conditions.Therapeutic biologics such as genetically engineered recombinant proteins and monoclonal antibodies representa large portion of newly-approved therapies for conditions such as chronic inflammatory diseases and cancer.limited production capacity,and setting up such facilitiesrequires a huge capitalinvestment. Biologics mayalso be produced by usingrecombinant micro-organismsor plants engineered to expressa particular therapeutic peptide,protein, monoclonal antibodyas well as vaccine sub-unit.The pharmaceutical industryis now being transformed dueto the emergence of biologics(biopharmaceuticals) whichoffer many advantages overconventional small-moleculedrugs. These include higherefficacy, reduced side effects,and the potential to curedisease, compared to treatingsymptoms.The size and complexity ofbiologics, in comparison tosmall-molecule drugs, havebrought a unique set ofchallenges to manufacturers.Therapeutic peptides,proteins and mAbs have tobe scrutinised at primary aswell as higher-order (native)conformation. Close attentionmust also be paid to anymodifications induced bythe recombinant expressionsystem. In addition, productand process-related substancesand impurities can affect boththe structure and functioningof biologics. Hence, it is criticalto be able to detect dimidiated,isomerised, oxidised forms,or protein aggregates as wellas host cell protein, DNA andculture components introducedduring cloning, production andpurification.In-depth characterisationof biologicsUnder these circumstances,manufacturers have to ensurethat their products undergorigorous characterisation tofacilitate transition throughdiscovery into development,eventual commercialisation,and their ultimate intendedtherapeutic use. Achievingthis goal is dependent on theavailability of appropriateanalytical tools to provide anarray of chemical and physicalinformation. Hence, a rangeof approaches has beendeveloped, optimised andsuccessfully implementedin-house for in-depthcharacterisation of biologics witha focus on meeting regulatoryand commercial specifications inorder to shorten time-to-marketas well as lower risk duringproduct development stages.The identity of recombinantlyexpressed peptides, proteinsand mAbs is scrutinisedusing a combination ofproteolytic digestion followedby multi-dimensional liquidchromatography peptidefractionation, directly coupledto tandem mass spectrometry.This workflow yields highresolutionpeptide sequencingdata that not only permit indepthcharacterisation of primarystructure, but also allow directdetection of post-translationalmodifications product as wellas process-related impurities.In addition, in-house developedmicrospheres (ReSyn TM ) arebeing used to manufactureimmobilised protease microreactorsfor fast and efficientprotein digestion that yieldshigh coverage primarystructure data.Cutting-edge ultrahigh-pressureliquidchromatography systemsProduct purity is monitoredusing electrophoresisas as wellas chromatography-basedtechniques, with separationbased on a range of molecularproperties such as size, charge,as well as hydrophobicity. Theuse of cutting-edge, ultra-highpressureliquid chromatographysystems in combination withcolumns packed with smaller,sub 2 μm size particles, allowsfor more efficient as well as faster separation of moleculesof interest.Since native structure dictatesfunction, the understanding ofstructure and its interplay withfunction is essential for developingeffective, safe, and cost-effectiveprotein biopharmaceuticals.The native conformation of largerpeptides, proteins and mAbsis examined on a global scaleusing circular dichroism, whichmonitors changes in secondarystructure, and fluorescencespectroscopy to monitor thetertiary and quaternary structurallevels of complex biologics. Futureefforts will also centre on thedevelopment and implementationof cutting-edge techniquesfor in-depth characterisationof native conformation on alocal scale. This will furthersupplement existing capabilitiesby allowing site-specificand detailed conformationalcharacterisation, that would beuseful throughout all stages ofprotein drug development, fromoptimisation of target binding andreceptor interaction(s) in research,to formulation, stability andcomparability studies in processdevelopment, where informationon changes in conformation couldplay an important role in thedevelopment and optimisationof complex biologics.In order to demonstrate thatmethods are appropriate for usein drug developmentprogrammes, substantial timeand resources are required. Inthe year ahead, our focus willbe on method qualification andvalidation so that the assayscurrently being used are deemedcompliant with internationalexpectations, and mostimportantly, that the analyticaldata derived from these methodsretain their full scientific value.– Dr Stoyan StoychevEnquiries:Dr Stoyan Stoychevsstoychev@csir.co.zaWhat are biologics?A biologic is a prophylactic, an in vivo diagnostic or atherapeutic substance that:• Is produced only by a living system, but is not simply ametabolite;• Is stated to contain a single substance that has documentedbiological activity or activities;• Has a relatively large molecular weight with a high structuralcomplexity compared with biologically active substancesthat can be made by chemical synthesis; is inherentlyheterogeneous in the molecular species present, and has animpurity profile that depends critically upon the processesused to make and test each batch; and• Has activity that is often very sensitive to physical conditions(i.e. temperature, shear forces, phase) and enzymatic action.| 39 |