Assessing the Impact of CDR Deamidation and Isomerization on mAb Target Binding Using Affinity-Resolved 2D-SEC-SCX-MS...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biopharmaceutical Development

Background:

• Post-translational modifications (PTMs) in monoclonal antibody (mAb) complementarity-determining regions (CDRs) can alter target binding, posing challenges for therapeutic development.
• Current methods for assessing PTM criticality are often low-throughput and complex.
• Subtle PTMs like asparagine (Asn) deamidation and aspartic acid (Asp) isomerization are prevalent and critical but difficult to analyze with existing techniques.

Purpose of the Study:

• To develop a highly selective and sensitive method for evaluating site-specific PTMs, particularly Asn deamidation and Asp isomerization, within mAb CDRs.
• To overcome the limitations of existing affinity-based methods for analyzing PTMs with subtle mass changes.
• To enable quantitative assessment of the impact of individual CDR PTMs on antibody-antigen binding affinity.

Main Methods:

• Development and application of a 2D-LC-MS approach combining affinity-resolved size exclusion chromatography (SEC) with online strong cation exchange chromatography (SCX).
• Utilized postcolumn denaturation (PCD)-assisted intact mass measurements for analyzing PTMs with significant mass shifts.
• Applied SCX separation for high selectivity towards mAb CDR modifications at the Fab fragment level for subtle PTM analysis.
• Demonstrated the method's utility through case studies involving an in-house mAb and trastuzumab, analyzing site-specific deamidation and isomerization.

Main Results:

• The 2D-LC-MS method successfully achieved site-specific resolution for analyzing Asn deamidation and Asp isomerization in therapeutic mAbs.
• Demonstrated effective evaluation of multiple PTMs within the CDRs of trastuzumab.
• Developed a quantitative approach to estimate the relative fold change in dissociation constant (KD) for antigen-antibody interactions affected by individual CDR modifications.
• The new approach overcomes limitations of previous affinity-resolved SEC-MS techniques for subtle PTMs.

Conclusions:

• The combined affinity-resolved SEC and online SCX-MS (2D-LC-MS) is a powerful tool for evaluating critical quality attributes related to CDR PTMs in therapeutic mAbs.
• This method provides site-specific insights into Asn deamidation and Asp isomerization, crucial for understanding their impact on antibody function.
• The quantitative assessment of binding affinity changes enables a more comprehensive understanding of PTM criticality in mAb development.