Xiaojun Lu, R. Paul Nobrega, Heather Lynaugh, Tushar Jain, Kyle Barlow, Todd Boland, Arvind Sivasubramanian, Maximiliano Vásquez, Yingda Xu

mAbs, 11(1), 45–57. DOI: 10.1080/19420862.2018.1548233

December 10, 2018

Chemical stability is a critical factor in the development of therapeutic monoclonal antibodies (mAbs), influencing their formulation, efficacy, safety, and shelf-life. However, this aspect is often overlooked in the early stages of antibody development. Adimab researchers published the results of a comprehensive study investigating the susceptibility of existing clinical-stage mAbs to deamidation and isomerization—two common post-translational modifications that can adversely affect antibody drug production and chemical stability.

Key hypotheses and objectives

Our scientists’ central hypothesis was that a comprehensive analysis of chemical liabilities in a large panel of clinical-stage mAbs would reveal amino acid sequence patterns and inform better developability assessments. The goal was to create a transparent dataset linking specific sequence motifs to their susceptibility to deamidation and isomerization. This information would support the early selection of highly developable, drug-like antibodies and streamline formulation activities later in development.

Approach and techniques

1. Antibody production: Samples were produced for a total of 131 IgG₁ isotype matched mAbs, each with variable regions matching published sequences of clinical-stage antibodies.
2. Experimental assessment: These mAbs were subjected to accelerated degradation conditions, specifically low pH (5.5) and high pH (8.5) at elevated temperatures (40°C) for varying durations. These conditions preferentially induce isomerization and deamidation, respectively. Tryptic peptide mapping was used to identify and quantify modifications at specific amino acid residues, providing a detailed picture of where and how degradation was occurring.
3. High-resolution site-specific analysis: The focus of this analysis was CDR-based degradation events (due to the low frequency of framework region isomerization and deamidation modifications).  Sequence liabilities, as well as hot-spots (frequently modified sequence positions) and cold-spots (frequently unmodified sequence positions) were described in the context of “canonical” and “non-canonical” motifs.

Major findings and impact

The study revealed key insights:

• Deamidation motifs and hotspots: Asparagine deamidation was frequently observed in complementarity-determining regions (CDRs), specifically the CDRH2 (H2) and CDRL1 (L1) regions, particularly under alkaline pH conditions. These post-translational modifications can potentially alter antigen-binding affinity and specificity. The resulting data is in agreement with previous studies which concluded that deamidation of a canonical motif within mAb CDRs is likely dependent on local structure or conformation. 
• Isomerization vulnerabilities: CDRs H3, H2, and L1 also showed higher frequencies of aspartate isomerization in canonical motifs than all the other CDRs, predominantly at acidic pH, with the observed labile motifs being generally consistent with those described previously. Three locations were identified that were frequently susceptible to deamidation and/or isomerization modifications under the stress conditions implemented in this study: H54, H98 and a stretch of amino acids in positions L30 through L30F.
• Implications for discovery workflow: We identified at least one deamidation and/or isomerization site (in the variable region) in 56 of 131 mAbs, with a wide range of observed degradation. Similar approaches are usually applied during late-stage discovery of mAbs for lead selection to identify candidates with the best chemical stability profiles or to identify degradation sites from the leading candidates for re-engineering to de-risk downstream process development.
• Implications for developability: This diverse dataset provides a valuable resource for improving in silico predictive models for chemical stability, enabling more accurate identification of potential liabilities early in the antibody development pipeline.

This extensive analysis underscores the necessity of evaluating chemical stability early in the antibody discovery process to mitigate risks and streamline formulation development. By leveraging our findings from extensive scientific research into antibody engineering and developability optimization, Adimab empowers our partners to develop more robust, stable, and ultimately, more successful antibody therapeutics.

For more details, read the full article in mAbs.