title: Monoclonal antibody disulfide reduction during manufacturing: Untangling process effects from product effects.
abstract: Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production.
authors.affiliation: Process and Product Development, Amgen Inc., Thousand Oaks, CA USA. hutterer@amgen.com
authors.forename: Katariina M
authors.initials: KM
authors.lastname: Hutterer
authors.forename: Robert W
authors.initials: RW
authors.lastname: Hong
authors.forename: Jonathon
authors.initials: J
authors.lastname: Lull
authors.forename: Xiaoyang
authors.initials: X
authors.lastname: Zhao
authors.forename: Tian
authors.initials: T
authors.lastname: Wang
authors.forename: Rex
authors.initials: R
authors.lastname: Pei
authors.forename: M Eleanor
authors.initials: ME
authors.lastname: Le
authors.forename: Oleg
authors.initials: O
authors.lastname: Borisov
authors.forename: Rob
authors.initials: R
authors.lastname: Piper
authors.forename: Yaoqing Diana
authors.initials: YD
authors.lastname: Liu
authors.forename: Krista
authors.initials: K
authors.lastname: Petty
authors.forename: Izydor
authors.initials: I
authors.lastname: Apostol
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Flynn
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000302:isolation & purification*; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000302:isolation & purification; D007146:Immunoglobulin lambda-Chains / Q000737:chemistry / Q000302:isolation & purification; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D004220:Disulfides; D007074:Immunoglobulin G; D007145:Immunoglobulin kappa-Chains; D007146:Immunoglobulin lambda-Chains; D011994:Recombinant Proteins; D010100:Oxygen; D004229:Dithiothreitol
keywords: CE-SDS; antibody disulfide reduction; capillary electrophoresis; free cysteine; harvest