Rapid Identification of Disulfide Bonds and Cysteine-Related Variants in an IgG1 Knob-into-Hole Bispecific Antibody Enhanced by Machine Learning

Abstract

Bispecific antibodies are regarded as the next generation of therapeutic modalities as they can simultaneously bind multiple targets, increasing the efficacy of treatments for several diseases and opening up previously unattainable treatment designs. Linking two half antibodies to form the knob-into-hole bispecific antibody requires an additional in vitro assembly step, starting with reduction of the antibodies and then reoxidization. Analysis of the disulfide bonds (DSBs) is vital to ensuring the correct assembly, stability, and higher-order structures of these important biomolecules because incorrect disulfide bond formation and/or presence of cysteine-related post-translational modifications can cause a loss of biological activity or even elicit an immune response from the host. Despite advancements in analytical methods, characterization of cysteine forms remains technically challenging and time-consuming. Herein, we report the development of an improved nonreduced peptide map method coupled with machine learning to enable rapid identification of disulfide bonds and cysteine-related variants in an IgG1 knob-into-hole bispecific antibody. The enhanced method offers a fast, consistent, and accurate workflow in mapping-out expected disulfide bonds in both half antibodies and bispecific antibodies and identifying cysteine-related modifications. Comparisons between two versions of the bispecific antibody molecule and analysis of stressed samples were also accomplished, indicating this method can be utilized to identify alterations originating from bioprocess changes and to determine the impact of assembly and postassembly stress conditions to product quality.

Rapid multi‐attribute characterization of intact bispecific antibodies by a microfluidic chip‐based integrated icIEF‐MS technology

“…The biotherapeutic studied here is a knob‐into‐hole bispecific mAb produced at Genentech [ 18 ]. The knob‐into‐hole bispecific construct substantially reduced impurities resulting from mismatched heavy and light chains.…”

“…The system included a novel microfluidic chip that enabled high‐resolution icIEF separations and quantitation of charge variant peaks by UV absorption followed by mobilization and on‐chip ESI for in‐line identification of each peak's corresponding mass proteoforms by MS. Here, we describe an improved approach, which entails applying nebulization gas at the electrospray tip of the microfluidic chip during the delivery of peaks into the MS, and optimized assay conditions to improve the peak resolution and signal intensity in the MS base peak electropherogram (BPE)—to enable a rapid assessment of multiple critical quality attributes (CQAs) of an aglycosylated IgG1 knob‐into‐hole bispecific antibody [ 18 ] in an integrated icIEF‐MS run. To challenge the icIEF‐MS system's ability to identify multiple intact posttranslational modifications (PTMs) such as deamidation, the antibody's structural complexity was enhanced by exposure to high pH and temperature conditions.…”

BiSpec Pairwise AI: guiding the selection of bispecific antibody target combinations with pairwise learning and GPT augmentation

“…Nonetheless, several researchers have begun to apply computational models, including machine learning, to BsAbs development, making significant strides. Baker et al ( 2019 ) reported on the development of an improved nonreduced peptide map method coupled with machine learning to enable rapid identification of disulfide bonds and cysteine-related variants in IgG1 knob-into-hole BsAbs. Multi-scale model calculations have also been used to simulate the spatio-temporal dynamics of three-body interactions among BsAb, CD3 and TAA to maximize drug efficacy and avoid off-target effects (Su et al 2024 ).…”

Cysteine Redox Chemistry in Peptide Self-Assembly to Modulate Hydrogelation

“…There, they are exposed to harsher conditions, and disulfide bridges can exert an important stabilizing role to preserve their function [ 10 ]. Important examples include: Hormones, such as insulin, whose function is strongly dependent on correct disulfide formation [ 11 , 12 ]; Functional proteins of the immune system, such as antigen-presenting major histocompatibility complexes (MHCs) [ 13 ] and antibodies [ 14 ]; Natural antimicrobial peptides [ 15 ], such as defensins [ 16 , 17 ]; Respiratory complexes that are key for cell metabolism, such as cytochrome c [ 18 ]; Proteins of the extracellular matrix, such as collagen [ 19 ]; Focal adhesion complexes that link integrins to the cytoskeleton in key processes, such as cell adhesion and migration [ 20 , 21 ]; Several toxins and venom peptides [ 22 , 23 , 24 ]; Ubiquitin transfer between catalytic cysteines leading to protein degradation [ 25 , 26 ]; Enzymes controlling transduction pathways, such as phospodiesterases [ 27 ]. …”