Characterization and analysis of scFv-IgG bispecific antibody size variants

Self Cite

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.

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Swope

Chung

Cao

et al. 2020

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“…Many of these novel formats rely on engineered disulfide bonds to maintain the correct structure for in vivo activity. While there are examples demonstrating reduced mAbs can have similar biological activity compared with non‐reduced controls, (Wang, Liu, Cai, Huang, & Flynn, 2015) this has not been the case for novel formats where partial reduction can result in disulfide bond mis‐pairing, stable aggregates, and reduced biological activity (Cao et al, 2018). These results indicate that the consequence of reduction for therapeutics with novel structures are likely even more severe than for standard mAbs.…”

Section: Discussionmentioning

confidence: 99%

Online control of cell culture redox potential prevents antibody interchain disulfide bond reduction

Handlogten

Wang

Ahuja

2020

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The phenomenon of monoclonal antibody (mAb) interchain disulfide bond reduction during manufacturing processes continues to be a focus of the biotechnology industry due to the potential for loss of product, increased complexity of purification processes, and reduced stability of the drug product. We hypothesized that antibody reduction can be mitigated by controlling the cell culture redox potential and subsequently established a threshold redox potential above which the mAb remained intact and below which there were significant and highly variable amounts of reduced mAb. Using this knowledge, we developed three control schemes to prevent mAb reduction in the bioreactor by controlling the cell culture redox potential via an online redox probe. These control methodologies functioned by increasing the concentration of dissolved oxygen (DO), copper (II) (Cu), or both DO and Cu to maintain the redox potential above the threshold value. Using these methods, we were able to demonstrate successful control of antibody reduction. Importantly, the redox control strategies did not significantly impact the cell growth, viability, mAb production, or product quality attributes including aggregates, C‐terminal lysine, high mannose, deamidation, and glycation. Our results demonstrate that controlling the cell culture redox potential is a simple and effective method to prevent mAb reduction.

“…To do this, CHO-RS cells were stably transfected with a plasmid encoding a proprietary bispecific antibody (MEDI-X) bearing the reversible GPI-membrane anchor. MEDI-X is a symmetrically bispecific antibody consisting of an IgG with an inserted scFv in the CH3 domain (Cao et al, 2018). This bispecific was selected in part as an extensive conventional screen had been recently conducted that resulted in the isolation of cell lines capable of 1 g/L yields.…”

Section: Clonal Enrichment Of Difficult-to-express Proteinsmentioning

confidence: 99%

Amber suppression coupled with inducible surface display identifies cells with high recombinant protein productivity

Chakrabarti¹,

Li²,

Roy³

et al. 2019

Cell line development (CLD) for biotherapeutics is a time‐ and resource‐intensive process requiring the isolation and screening of large numbers of clones to identify high producers. Novel methods aimed at enhancing cell line screening efficiency using markers predictive of productivity early in the CLD process are needed to reliably generate high‐yielding cell lines. To enable efficient and selective isolation of antibody expressing Chinese hamster ovary cells by fluorescence‐activated cell sorting, we developed a strategy for the expression of antibodies containing a switchable membrane‐associated domain to anchor an antibody to the membrane of the expressing cell. The switchable nature of the membrane domain is governed by the function of an orthogonal aminoacyl transfer RNA synthetase/tRNApyl pair, which directs a nonnatural amino acid (nnAA) to an amber codon encoded between the antibody and the membrane anchor. The process is “switchable” in response to nnAA in the medium, enabling a rapid transition between the surface display and secretion. We demonstrate that the level of cell surface display correlates with productivity and provides a method for enriching phenotypically stable high‐producer cells. The strategy provides a means for selecting high‐producing cells with potential applications to multiple biotherapeutic protein formats.