{"title":"Unravelling the reason for different binding behaviors exhibited by antibody aggregates towards preparative and analytical Protein A columns","authors":"Wanyuan Dong, Mengying Tian, Yifeng Li","doi":"10.1016/j.pep.2024.106449","DOIUrl":null,"url":null,"abstract":"<div><p>We previously showed that the root cause of low Protein A step yield observed for certain antibodies/Fc-fusions is the presence of non-binding aggregates in cell culture harvest. A pre-assumption for the above conclusion is that the aggregates, while do not bind to the preparative Protein A column, can bind to the analytical Protein A-high performance liquid chromatography (HPLC) column used for titer measurement. In the current work, using materials from a previous case with the low yield issue, we confirmed that non-binding aggregates in preparative Protein A flow-through can indeed bind to the analytical Protein A column. In addition, we showed that this discrepancy is mainly due to the different loading densities applied under these two circumstances. We also demonstrated that aggregate bound to the analytical Protein A column slightly stronger than the monomer, as it exhibited a longer retention time. In summary, the current study not only confirmed that non-binding aggregates detected in the preparative Protein A flow-through bind to the Protein A-HPLC column and contribute to the measured titer of culture harvest but also unravelled the reason for different binding behaviors exhibited by antibody aggregates towards preparative and analytical Protein A columns.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.4000,"publicationDate":"2024-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Protein expression and purification","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1046592824000214","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
We previously showed that the root cause of low Protein A step yield observed for certain antibodies/Fc-fusions is the presence of non-binding aggregates in cell culture harvest. A pre-assumption for the above conclusion is that the aggregates, while do not bind to the preparative Protein A column, can bind to the analytical Protein A-high performance liquid chromatography (HPLC) column used for titer measurement. In the current work, using materials from a previous case with the low yield issue, we confirmed that non-binding aggregates in preparative Protein A flow-through can indeed bind to the analytical Protein A column. In addition, we showed that this discrepancy is mainly due to the different loading densities applied under these two circumstances. We also demonstrated that aggregate bound to the analytical Protein A column slightly stronger than the monomer, as it exhibited a longer retention time. In summary, the current study not only confirmed that non-binding aggregates detected in the preparative Protein A flow-through bind to the Protein A-HPLC column and contribute to the measured titer of culture harvest but also unravelled the reason for different binding behaviors exhibited by antibody aggregates towards preparative and analytical Protein A columns.
我们以前的研究表明,某些抗体/Fc-融合体的蛋白 A 步进产量低的根本原因是细胞培养收获物中存在不结合的聚集体。上述结论的一个前提假设是,这些聚集体虽然不能与制备蛋白 A 柱结合,但能与用于滴度测量的分析蛋白 A 高效液相色谱柱结合。在目前的工作中,我们使用了之前出现低产率问题的案例中的材料,证实制备型蛋白 A 流动液中的非结合性聚集体确实可以与分析型蛋白 A 柱结合。此外,我们还证明了这种差异主要是由于在这两种情况下使用的装载密度不同造成的。我们还证明,聚合体与分析蛋白质 A 柱的结合力略强于单体,因为它的保留时间更长。总之,本研究不仅证实了在制备型蛋白 A 流出液中检测到的非结合性聚集体与蛋白 A-HPLC 柱结合,并对培养收获物的测定滴度起作用,而且还揭示了抗体聚集体与制备型和分析型蛋白 A 柱表现出不同结合行为的原因。
期刊介绍:
Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.