CHAPTER 1.4. Analysis of Disulfide Bond Formation in Therapeutic Proteins

Weinfurtner, Daniel

doi:10.1039/9781788013253-00081

Cited by 10 publications

(12 citation statements)

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“…For both modifications, conventional analytical methods include NMR [ 155 , 156 ], X-ray crystallography [ 157 ], cryoEM, CD for structure analysis, Edman degradation [ 154 ] and mass spectrometry (MS) for sequencing analysis, and electrophoresis for protein migration mobility analysis [ 153 , 160 ]. Gel shift assay by electrophoresis is a readily accessible and effective analytical method widely employed to study both modifications.…”

Section: Common Methodsmentioning

confidence: 99%

Hidden Relationships between N-Glycosylation and Disulfide Bonds in Individual Proteins

Bakshi

Pham

Kaur

et al. 2022

IJMS

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N-Glycosylation (NG) and disulfide bonds (DBs) are two prevalent co/post-translational modifications (PTMs) that are often conserved and coexist in membrane and secreted proteins involved in a large number of diseases. Both in the past and in recent times, the enzymes and chaperones regulating these PTMs have been constantly discovered to directly interact with each other or colocalize in the ER. However, beyond a few model proteins, how such cooperation affects N-glycan modification and disulfide bonding at selective sites in individual proteins is largely unknown. Here, we reviewed the literature to discover the current status in understanding the relationships between NG and DBs in individual proteins. Our results showed that more than 2700 human proteins carry both PTMs, and fewer than 2% of them have been investigated in the associations between NG and DBs. We summarized both these proteins with the reported relationships in the two PTMs and the tools used to discover the relationships. We hope that, by exposing this largely understudied field, more investigations can be encouraged to unveil the hidden relationships of NG and DBs in the majority of membranes and secreted proteins for pathophysiological understanding and biotherapeutic development.

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Section: Common Methodsmentioning

confidence: 99%

Hidden Relationships between N-Glycosylation and Disulfide Bonds in Individual Proteins

Bakshi

Pham

Kaur

et al. 2022

IJMS

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“…In this section, we summarize the analytical methods that are commonly utilized to monitor and analyze antibody disulfide bond reduction. The analytical methods listed in this section could be used for both interchain and intrachain disulfide bond reduction characterization (Weinfurtner, 2018). However, interchain disulfide bond is less stable and interchain disulfide bond reduction happened more frequently in downstream processing.…”

Section: Analytical Methods For Disulfide Bond Reduction Monitoring A...mentioning

confidence: 99%

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

Ren

Tan

Ehamparanathan

et al. 2021

Biotech & Bioengineering

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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.

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“…Interchain bonds are more susceptible to reduction and, therefore, are more susceptible to an incomplete formation of bonds and shuffling than intrachain bonds ( Cordoba et al, 2005 ; Liu et al, 2010 ; Ouellette et al, 2010 ; Liu and May, 2012 ; Hmiel et al, 2015 ; Lakbub et al, 2018 ; Weinfurtner, 2018 ; Nie et al, 2022 ). For example, the larger number and hinge region arrangement of disulfide bonds an IgG2 increases its potential for covalent dimerization, which leads to an increased binding avidity ( Moritz and Stracke, 2017 ; Weinfurtner, 2018 ). Similarly, antibody-drug conjugates (ADCs) that are conjugated via thiol-maleimide chemistry are dependent upon the partial reduction of disulfide bonds.…”

Section: Introductionmentioning

confidence: 99%

“…Usually disulfide bond shuffling occurs as a protein is exposed to stressors such as heat, oxygen radicals, high pH and agitation ( Sung et al, 2016 ; Moritz and Stracke, 2017 ; Resemann et al, 2018 ; Dong et al, 2021 ). Disulfide bond shuffling can negatively impact a therapeutic protein’s safety and functionality by increasing its aggregation and degradation, modifying its folding, and/or reducing its target binding ( Zhang et al, 2011 ; Sung et al, 2016 ; Weinfurtner, 2018 ). In addition to disulfide bond shuffling, a rare modification called a trisulfide bond can occur in IgGs.…”

Section: Introductionmentioning

confidence: 99%

Streamlining the Characterization of Disulfide Bond Shuffling and Protein Degradation in IgG1 Biopharmaceuticals Under Native and Stressed Conditions

Benet

Kumaran

et al. 2022

Front. Bioeng. Biotechnol.

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Post translational modifications (PTMs) have been shown to negatively impact protein efficacy and safety by altering its native conformation, stability, target binding and/or pharmacokinetics. One PTM in particular, shuffled disulfide bonds, has been linked to decreased potency and increased immunogenicity of protein therapeutics. In an effort to gain more insights into the effects of shuffled disulfide bonds on protein therapeutics’ safety and efficacy, we designed and further optimized a semi-automated LC-MS/MS method for disulfide bond characterization on two IgG1 protein therapeutics—rituximab and bevacizumab. We also compared originator vs. biosimilar versions of the two therapeutics to determine if there were notable variations in the disulfide shuffling and overall degradation between originator and biosimilar drug products. From our resulting data, we noticed differences in how the two proteins degraded. Bevacizumab had a general upward trend in shuffled disulfide bond levels over the course of a 4-week incubation (0.58 ± 0.08% to 1.46 ± 1.10% for originator) whereas rituximab maintained similar levels throughout the incubation (0.24 ± 0.21% to 0.51 ± 0.11% for originator). When we measured degradation by SEC and SDS-PAGE, we observed trends that correlated with the LC-MS/MS data. Across all methods, we observed that the originator and biosimilar drugs performed similarly. The results from this study will help provide groundwork for comparative disulfide shuffling analysis by LC-MS/MS and standard analytical methodology implementation for the development and regulatory approval of biosimilars.

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CHAPTER 1.4. Analysis of Disulfide Bond Formation in Therapeutic Proteins

Cited by 10 publications

References 0 publications

Hidden Relationships between N-Glycosylation and Disulfide Bonds in Individual Proteins

Hidden Relationships between N-Glycosylation and Disulfide Bonds in Individual Proteins

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

Streamlining the Characterization of Disulfide Bond Shuffling and Protein Degradation in IgG1 Biopharmaceuticals Under Native and Stressed Conditions

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