An Automated and Qualified Platform Method for Site-Specific Succinimide and Deamidation Quantitation Using Low-pH...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmaceutical Science

Background:

• Monoclonal antibodies (mAbs) undergo post-translational modifications like succinimide formation.
• Succinimide can impact mAb biological activity, necessitating its detection and quantification.
• Existing methods for succinimide monitoring are often product-specific or lack robustness.

Purpose of the Study:

• To develop a platform method for stable succinimide detection and quantification in mAbs.
• To assess succinimide stability and half-life in biological matrices.
• To establish a reliable assay for site-specific deamidation and succinimide intermediate measurement.

Main Methods:

• Development of a low-pH peptide-mapping method using specialized Lys-C and modified trypsin.
• Utilizing low-pH conditions to maintain succinimide stability and prevent assay artifacts.
• Automation of the digestion and data processing workflow coupled with mass detection.

Main Results:

• The developed method efficiently digests mAbs under low-pH conditions, preserving succinimide integrity.
• In vitro studies accurately assessed succinimide stability in serum, determining a half-life of 1.5 days.
• The method was fully qualified, demonstrating accuracy, precision, linearity, and robustness for GMP environments.

Conclusions:

• A novel, robust platform method for succinimide analysis in mAbs has been established.
• This method enables accurate assessment of succinimide stability and site-specific deamidation.
• The automated and qualified assay is suitable for pharmaceutical development and quality control.