Dana Motabar

AstraZeneca (United States)

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Mingyan Cao

AstraZeneca (United States)

Wai Keen Chung

Biogen (United States)

Dengfeng Liu

Central South University

Jinyang Li

Wenzhou Medical University

Gregory F. Payne

University of Maryland, College Park

Chen Yu Tsao

University of Maryland, College Park

William E. Bentley

University of Maryland, College Park

Sally Wang

Institute for Bioscience and Biotechnology Research

Alan K. Hunter

AstraZeneca (United States)

Chunlei Wang

AstraZeneca (United States)

Cooperative Institutions

AstraZeneca (United States)

Advanced Bioscience Laboratories (United States)

University of Maryland, College Park

Institute for Bioscience and Biotechnology Research

Wenzhou Medical University

University of Virginia

Harvard University

Research Institute for Bioscience and Biotechnology

Bioscience Research

University of Maryland, Baltimore

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Impact of enzymatic reduction on bivalent bispecific antibody fragmentation and loss of product purity upon reoxidation

Biotechnology and Bioengineering (2020)

Nicole Swope Wai Keen Chung Mingyan Cao Dana Motabar Dengfeng Liu

Antibody disulfide bond (DSB) reduction during manufacturing processes is a widely observed phenomenon attributed to host cell reductases present in harvest cell culture fluid. Enzyme-induced antibody reduction leads to product fragments and aggregates that increase the impurity burden on the purification process. The impact of reduction on bivalent bispecific antibodies (BisAbs), which are increasingly entering the clinic, has yet to be investigated. We focused on the reduction and reoxidation properties of a homologous library of bivalent BisAb formats that possess additional single-chain Fv (scFv) fragments with engineered DSBs. Despite all BisAbs having similar susceptibilities to enzymatic reduction, fragmentation pathways were dependent on the scFv-fusion site. Reduced molecules were allowed to reoxidize with and without low pH viral inactivation treatment. Both reoxidation studies demonstrated that multiple, complex BisAb species formed as a result of DSB mispairing. Furthermore, aggregate levels increased for all molecules when no low pH treatment was applied. Combined, our results show that complex DSB mispairing occurs during downstream processes while aggregate formation is dependent on sample treatment. These results are applicable to other novel monoclonal antibody-like formats containing engineered DSBs, thus highlighting the need to prevent reduction of novel protein therapeutics to avoid diminished product quality during manufacturing.

Bivalent (engine)

Fragmentation

10.1002/bit.27264

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Citations (14)

Simple, rapidly assembled saccharide-based sensor interface that enables interrogation of antibody glycosylation and titer for improved process analytical technologies

Dana Motabar Jinyang Li Sally Wang Chen Yu Tsao Gregory F. Payne

Interrogation

Interface (matter)

10.1021/scimeetings.0c01197

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Characterization and analysis of scFv-IgG bispecific antibody size variants

mAbs (2018)

Mingyan Cao Chunlei Wang Wai Keen Chung Dana Motabar Jihong Wang

Bispecific antibodies are an emergent class of biologics that is of increasing interest for therapeutic applications. In one bispecific antibody format, single-chain variable fragments (scFv) are linked to or inserted in different locations of an intact immunoglobulin G (IgG) molecule to confer dual epitope binding. To improve biochemical stability, cysteine residues are often engineered on the heavy- and light-chain regions of the scFv to form an intrachain disulfide bond. Although this disulfide bond often improves stability, it can also introduce unexpected challenges to manufacturing or development. We report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, we developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.

Bispecific antibody

Immunoglobulin Fab Fragments

10.1080/19420862.2018.1505398

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Citations (25)

Simple, rapidly assembled saccharide-based sensor interface that enables interrogation of antibody glycosylation and titer for improved process analytical technologies

Dana Motabar Jinyang Li Sally Wang Chen Yu Tsao Gregory F. Payne

Interrogation

Interface (matter)

10.1021/scimeetings.0c01196

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