Abstract
Aspartate (Asp) isomerization is a common post-translational modification of recombinant therapeutic proteins that can occur during manufacturing, storage, or administration. Asp-isomerization in the complementarity-determining regions (CDRs) of a monoclonal antibody (mAb) may impact the target binding and thus a QC friendly method for routine monitoring is desirable. In this work, we utilized a liquid chromatography mass spectrometry (LC/MS)-based approach to identify the Asp isomerization in the CDRs of a therapeutic mAb. To quantitate the site-specific Asp-isomerization of the mAb, a UV detection-based quantitation assay utilizing the same LC platform was developed. The assay was qualified, and implemented for routine monitoring of this product-specific modification. Compared with existing methods, this analytical paradigm is applicable to identify Asp-isomerization (or other modifications), and subsequently develop a rapid, QC friendly test for routine site specific monitoring and quantitation to ensure product quality. This approach first identifies and locates a product- related impurity (a CQA) caused by isomerization, deamidation, oxidation or other post-translational modifications, and then utilizes synthetic peptides and mass spectrometry to assist the development of a LC-UV based chromatographic method which separates and quantifies the product related-impurities by UV peaks. The established LC-UV method has acceptable peak specificity, precision, linearity and accuracy; it can be validated and used in GMP environment for lot release and stability testing.
- Aspartic acid Isomerization
- Complementarity-determining regions (CDRs)
- Focused peptide mapping
- Method qualification
- Monoclonal antibody (mAb)
- Received October 21, 2015.
- Accepted March 31, 2016.
- Copyright © 2016, Parenteral Drug Association
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