title: Effects of antibody disulfide bond reduction on purification process performance and final drug substance stability.
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 IgG2mAb, 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.
authors.affiliation: Purification Process Sciences, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland.
authors.forename: Wai Keen
authors.identifier: 0000-0001-7822-7131
authors.initials: WK
authors.lastname: Chung
authors.affiliation: Cell Culture and Fermentation Sciences, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland.
authors.forename: Brian
authors.initials: B
authors.lastname: Russell
authors.affiliation: Analytical Sciences, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland.
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Cell Culture Sciences, Macrogenics Inc, Rockville, Maryland.
authors.forename: Michael
authors.initials: M
authors.lastname: Handlogten
authors.affiliation: Formulation Sciences, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Hudak
authors.affiliation: Analytical Sciences, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland.
authors.forename: Mingyan
authors.initials: M
authors.lastname: Cao
authors.affiliation: Analytical Sciences, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland.
authors.forename: Jihong
authors.initials: J
authors.lastname: Wang
authors.affiliation: Purification Process Sciences, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland.
authors.forename: David
authors.initials: D
authors.lastname: Robbins
authors.affiliation: Cell Culture and Fermentation Sciences, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland.
authors.forename: Sanjeev
authors.identifier: 0000-0002-2869-6885
authors.initials: S
authors.lastname: Ahuja
authors.affiliation: Protein Science, Boehringer Ingelheim Fremont Inc, Fremont, California.
authors.forename: Min
authors.initials: M
authors.lastname: Zhu
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification*; D016466:CHO Cells; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004340:Drug Contamination / Q000517:prevention & control*; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction
chemical_list: D000911:Antibodies, Monoclonal; D003470:Culture Media; D004220:Disulfides
keywords: aggregate; antibody disulfide bond reduction; purification; stability