Effects of antibody disulfide bond reduction on purification process performance and final drug substance stability. Issue 6 (6th March 2017)

• Record Type: Journal Article
• Authors: Chung, Wai Keen; Russell, Brian; Yang, Yanhong; Handlogten, Michael; Hudak, Suzanne; Cao, Mingyan; Wang, Jihong; Robbins, David; Ahuja, Sanjeev; Zhu, Min
• Is Part Of: Biotechnology and bioengineering. Volume 114:Issue 6(2017)
• Page Start: 1264
• Page End: 1274
• Publication Date: 2017-03-06
• Subjects: purification -- stability -- antibody disulfide bond reduction -- aggregate; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.26265
• Languages: English
• ISSNs: 0006-3592

Abstract

ABSTRACT: Antibody disulfide bond reduction during monoclonal antibody (mAb) production is a phenomenon that has been attributed to the reducing enzymes from CHO cells acting on the mAb during the harvest process. However, the impact of antibody reduction on the downstream purification process has not been studied. During the production of an IgG2 mAb, antibody reduction was observed in the harvested cell culture fluid (HCCF), resulting in high fragment levels. In addition, aggregate levels increased during the low pH treatment step in the purification process. A correlation between the level of free thiol in the HCCF (as a result of antibody reduction) and aggregation during the low pH step was established, wherein higher levels of free thiol in the starting sample resulted in increased levels of aggregates during low pH treatment. The elevated levels of free thiol were not reduced over the course of purification, resulting in carry‐over of high free thiol content into the formulated drug substance. When the drug substance with high free thiols was monitored for product degradation at room temperature and 2–8°C, faster rates of aggregation were observed compared to the drug substance generated from HCCF that was purified immediately after harvest. Further, when antibody reduction mitigations (e.g., chilling, aeration, and addition of cystine) were applied, HCCF could be held for an extended period of time while providing the same product quality/stability as material that had been purified immediately after harvest. Biotechnol. Bioeng. 2017;114: 1264–1274. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc. Abstract : Antibody disulfide bond reduction during harvested cell culture fluid (HCCF) holds generated fragments and increased aggregation when the product is subjected to low pH treatment during purification. The increase in impurity levels also affected purification performance, resulting in lower process yields. However, when antibody disulfide bond reduction was minimized during HCCF holds, decreased aggregate formation was observed during low pH treatment. In addition, the stability of formulated drug substance was also significantly improved.