单克隆抗体中分析表征检测方案

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21Sponsored Featurewww.tosoh.com The addition of polysaccharidechains to therapeutic monoclonalantibodies can have a dramaticimpact on their safety andefficacy - and that's whyglycosylation is a critical qualityattrihute Here we sneak withTomasz Walski， Project GroupLeader in the R&D department atMabion S.A.， to find out how thebiosimilar developer is keepingglycan analysis short and sweet. What exactly does your team do-and what analytical technology do yourely on? In short， my team develops chromatographicmethods to analyze the quality attributesof the therapeutic antibodies we develop.Our workhorse analytical tools are U/HPLC and LC-MS/MS systems. What keeps you motivated? As a company， our priority is creatingsafe， effective and accessible drugs forthe patients who need them. The safetyof a biological drug relies on the qualityattributes of the antibody， so havingsensitive and accurate methods toOanalyze them is of outmost importanceduring the drug development phase. It isincredibly rewarding to feel that our workis contributing directly to patient safety. My favorite part of the job is whenwe are able to improve a method to waste， be moreanalyst-friendly andless time-consuming-all while still generatinghigh-quality results.Ultimately，that enables us to developprojects faster and deliver safe drugsto patients at an affordable price. Weare constantly looking for technologicalinnovations that help us to achieve that. Why is N-glycosylation so important inbiopharma analysis? N-linked glycosylation describes theattachment of oligosaccharides (sugars)to a protein， via an asparagine residue. Wepay very closeattention to N-glycosylationof biologics -thecomposition of the sugarchains attached to the drug moleculeis one the key quality attributes fortherapeutic antibodies. N-glycosylation strongly affects theway the drug interacts with its target(for example， cancer cells). It determineshow efficient it is in its therapeutic action，how long it remains active and how long itcirculates in the bloodstream. In addition.making sure that the N-glycosylation staysthe same during development， throughclinical trials and onto the market helpsto minimize the possibility of any adversereaction. It is crucial to carefully monitorthe N-glycosylation throughout projectdevelopment and then in the productionprocess to ensure that patients alwaysreceive safe， high-quality medicine. What challenges does glycan variantanalysis typically present? There are a number of powerful， gold-standard methods for the accurateanalysis of antibodyN-glycosylation.These methods typically require samplepurification followed by multi-steppreparation of some sort； for instance， proteolytic digestion or glycan separationand labeling， several clean-up steps， andfinally UPLC or LC-MS analysis. All ofthese steps can be automated， of course，but it still takes significant time. The idealapproach would keep the reliability andhigh quality of results while simplifying andaccelerating the workflow. You've used a number of different LCcolumns over the years. How is the FcRcolumn different? By using an FcR HPLC column， we canmake the whole process much simpler.For a significant number of therapeuticantibodies， interaction with the Fcyreceptor is crucial for their activity. Thisinteraction is strongly dependent on the "The accuracy ofthe data reallysurprised us. Wecould predict thecontent ofgalactosylatedglycans with lessthan five percenterror in most cases. type ofN-glycosylation. By making gooduse of the unique characteristics of theTSKgel FcR-IIIA-NPR column， whichuses the Fcy receptor as the ligand，we are able to get information on theN-glycosylation of the antibody and itsbiological activity. The resulting patternof chromatographic peaks stronglycorrelates with the N-glycosylationcomposition. The accuracy of the data really cases， compared with the orthogonalmethod- HILIC-UPLC using releasedand labeled N-glycans. The method was sensitive enough touse low microgram amounts of sampleand， on top of that， we got virtuallythe same results regardless of whetherwe used highly purified sample or theantibody still in the cell medium. And thatmeans we can perform high-throughput “| am certain thatthe FcR column willbe extensively usedin our current andprospectivebiosimilardevelopment projects. and highly informative screening of clones，cell culture conditions and lead moleculeselection with minimal sample handling.Not to mention that the lack of samplepreparation makes this approach cost-efficient and environmentally friendly. Given these characteristics，l am certainthat the FcR column will be extensivelyused in our current and prospectivebiosimilar development projects. How else could the column be used? As well as targeted screening for activityand glycosylation， I believe that thecolumn has potential in a number ofareas. Estimation of binding affinity onnon-purified samples is one thing thatcomes to mind. The fact that the Fcregion of the antibody interacts withFcR can be used to detect antibodyfragmentation or changes in thequaternary structure. FcR could makea great ligand for antibody purification，so a preparative-scale FcR-IIIA column issomething I'd be interested in exploring.l imagine that our colleagues in the Quality Control department mightfind some new uses for the columntoo； for example， in one of thebatch-release methods. 抗体糖链结构直接关系到抗体生物活性以及在血液循环中的稳定性，是抗体药物的一个重要关键质量属性。从抗体的开发阶段到临床评价阶段，需要确认糖链结构有无变化，并最大程度降低发生副作用的可能性，以确保供应安全、高品质的药物。目前为止，有许多标准分析方法可以准确解析抗体药物的糖链结构。一般来说，分解纯化抗体、将糖链酶切下来并衍生化，然后经多步骤纯化处理后进行UHPLC或HPLC-MS分析是最常用的方法。虽然这些步骤可实现自动化处理，但操作仍需要耗费相当长的时间。因此，分析人员需要一种更简单、快捷并且更可靠、准确的分析方法。此篇资料是对欧洲抗体生物类似药研发公司Mabion S.A. 研发部项目负责人的采访，阐述了该公司在抗体聚糖分析中面临的挑战与应对策略。‍