SEC-HPLC analysis of column load and flow-through provides critical understanding of low Protein A step yield.

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2024
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Abstract
For a certain number of mAbs, bispecific antibodies (bsAbs) and Fc-fusion proteins that we worked on, the Protein A capture step experienced low yield (i.e., āˆ¼80%). A previous case study suggested that non-binding aggregate formed in cell culture was the root cause of low Protein A step yield. In the current work, we selected five projects with the low Protein A yield issue to further illustrate this phenomenon. In all cases, existence of non-binding aggregates was confirmed by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of Protein A load and flow-through. In addition, we demonstrated that aggregates failed to bind to Protein A resin mainly due to their large sizes, which prevented them from entering the resin beads. As the data suggested, SEC-HPLC analysis of Protein A load and flow-through, although not a standard procedure, can provide information that is critical for understanding the unexpected performance of Protein A chromatography in cases like those being presented here. Thus, SEC-HPLC analysis of Protein A load and flow-through is highly recommended for antibodies/Fc-fusions suffering from low Protein A yield.
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Authors Liu, Chen;Tian, Mengying;Dong, Wanyuan;Lu, Wenwen;Zhang, Ting;Wan, Yan;Zhang, Xudong;Li, Yifeng;
Journal Protein expression and purification
Year 2024
DOI 10.1016/j.pep.2023.106418
URL
Keywords
aggregate culture harvest fc-fusion flow-through protein a chromatography sec-hplc bsab mab

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