Antibody disulfide bond reduction and recovery during biopharmaceutical process development—A review

Abstract

Antibody disulfide bond reduction has been a challenging issue in monoclonal antibody manufacturing. It could lead to a decrease of product purity and failure to meet the targeted product profile and/or specifications. More importantly, disulfide bond reduction could also impact drug safety and efficacy. Scientists across the industry have been examining the root causes and developing mitigation strategies to address the challenge. In recent years, with the development of high titer mammalian cell culture processes to meet the rapidly growing demand for antibody biopharmaceuticals, disulfide bond reduction has been observed more frequently. Thus, it is necessary to continue evolving the disulfide reduction mitigation strategies and developing novel approaches to maintain high product quality. Additionally, in recent years as more complex molecules (such as bispecific and trispecific antibodies) emerge, the molecular heterogeneity due to incomplete formation of the interchain disulfide bonds becomes a more imperative challenging issue. Given the disulfide reduction challenges that biotech industry is facing, in this review, we provide a comprehensive scientific summary of the root cause analysis of disulfide reduction during process development of antibody therapeutics, mitigation strategies and its potential remediated recovery based on published papers. First, this paper intends to highlight different aspects of the root cause for disulfide reduction. Secondly, to provide a broader understanding of the disulfide bond reduction in downstream process, this paper discusses disulfide bond reduction impact on product stability, associated analytical methods for disulfide bond reduction detection and characterization, process control strategies as well as their manufacturing implementation. In addition, brief perspectives on the development of future mitigation strategies are also reviewed, including platform alignment, mitigation strategy application for the emerging new modalities such as bispecific and trispecific antibodies as well as using machine learning to identify molecule susceptibility of disulfide bond reduction. The data in this review are originated from the published papers.

Chemical Tagging of Bioactive Amides by Cooperative Catalysis: Applications in the Syntheses of Drug Conjugates

“…However, heterogeneous products with different drug-to-antibody conjugation ratios (DAR = 2–4) are formed as the number of conjugation sites varies due to the nonselective nature of disulfide reduction. − Moreover, the loss of disulfide bonds and the retro-Michael reaction can cause stability issues in the ADC in vivo. − Thus, as a means to assemble various drug conjugates from 10f and/or 1b by sequential tagging and bioconjugation, we opted to not use the thiol-Michael addition (i.e., 11f → ADC, Table ). Instead, to achieve site-selective and irreversible bioconjugation, − we decided to merge an engineered azide-containing sfGFP-151-AzK ( 12b ), nanobody-5F7-69-AzK ( 12c ), or trastuzumab-LC-D122AzK ( 12d ), with the NAMPT inhibitor derivatives armed with DIBO ( 11h ) or DIBAC ( 11i ) by a strain-promoted azide–alkyne cycloaddition (Figures and ).…”

Functionally Masked Antibody to Uncouple Immune-Related Toxicities in Checkpoint Blockade Cancer Therapy

“…Instead, site-specific PEGylation has been proposed to circumvent loss of binding. Still, this method requires an additional chemical reduction procedure with disruption of disulfide bonds, which affects the structural stability of antibodies and has the risk of causing irAEs when administered to patients . However, with the development of drug delivery system and antibody–drug conjugate fields, research to circumvent off-tumor toxicities in cancer immunotherapy using linkers that can be cleaved by various tumor-specific stimuli has been actively conducted. − For this reason, the concept of introducing cleavable PEG into ICB antibodies recently gained revived interest, showing enhanced therapeutic efficacy and safety in a glioblastoma mouse model .…”

“…Still, this method requires an additional chemical reduction procedure with disruption of disulfide bonds, which affects the structural stability of antibodies and has the risk of causing irAEs when administered to patients. 56 However, with the development of drug delivery system and antibody−drug conjugate fields, research to circumvent off-tumor toxicities in cancer immunotherapy using linkers that can be cleaved by various tumor-specific stimuli has been actively conducted. 57−59 60 Unfortunately, because most linkers were designed to deliver cytotoxic payloads into the cell, they are cleaved by a more aggressive stimulus.…”

Crystal Structure of Human CD47 in Complex with Engineered SIRPα.D1(N80A)

“…This field is progressing rapidly, and some mAbs targeting the CD47/SIRPα pathway have reached clinical phase II and phase III [ 21 ]. Notably, despite the excellent clinical performance shown by CD47/SIRPα antibodies, the limitations of antibody drugs, including poor tumor permeability, undesirable oral bioavailability and poor stability, hinder their clinical application [ 22 , 23 , 24 , 25 ]. Targeting the CD47/SIRPα signaling pathway with a different strategy than anti-CD47 mAbs and potentially eliminating the problems caused by antibody drugs, SIRPα-Fc fusion proteins have attracted the attention of researchers and have become a promising research area.…”