Qualification of a Quantitative Method for Monitoring Aspartate Isomerization of a Monoclonal Antibody by Focused Peptide Mapping.
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 of a monoclonal antibody may affect the target binding and thus a sufficiently robust quality control 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 complementarity-determining regions of a therapeutic monoclonal antibody. To quantitate the site-specific Asp isomerization of the monoclonal antibody, 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, sufficiently robust quality control method for routine site-specific monitoring and quantitation to ensure product quality. This approach first identifies and locates a product-related impurity (a critical quality attribute) caused by isomerization, deamidation, oxidation, or other post-translational modifications, and then utilizes synthetic peptides and MS to assist the development of a LC-UV-based chromatographic method that 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 a good manufacturing practice environment for lot release and stability testing.
Aspartate isomerization is a common post-translational modification of recombinant proteins during manufacture process and storage. Isomerization in the complementarity-determining regions (CDRs) of a monoclonal antibody A (mAb-A) has been detected and has been shown to have impact on the binding affinity to the antigen. In this work, we utilized a mass spectrometry-based peptide mapping approach to detect and quantitate the Asp isomerization in the CDRs of mAb-A. To routinely monitor the CDR isomerization of mAb-A, a focused peptide mapping method utilizing reversed phase chromatographic separation and UV detection has been developed and qualified. This approach is generally applicable to monitor isomerization and other post-translational modifications of proteins in a specific and high-throughput mode to ensure product quality.
Analytical Biotechnology Department, MedImmune Inc, Gaithersburg, MD.
Mingyan
M
Cao
Analytical Biotechnology Department, MedImmune Inc, Gaithersburg, MD.
Wenjun David
WD
Mo
Analytical Biotechnology Department, MedImmune Inc, Gaithersburg, MD.
Anthony
A
Shannon
Analytical Biotechnology Department, MedImmune Inc, Gaithersburg, MD.
Ziping
Z
Wei
Analytical Biotechnology Department, MedImmune Inc, Gaithersburg, MD michael.washabaugh@theraproteins.com Patriciacash8@gmail.com.
Michael
M
Washabaugh
Analytical Biotechnology Department, MedImmune Inc, Gaithersburg, MD michael.washabaugh@theraproteins.com Patriciacash8@gmail.com.
Patricia
P
Cash
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001224:Aspartic Acid; D002851:Chromatography, High Pressure Liquid; D022801:Complementarity Determining Regions; D007536:Isomerism; D010449:Peptide Mapping; D011994:Recombinant Proteins; D053719:Tandem Mass Spectrometry
D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D011994:Recombinant Proteins; D001224:Aspartic Acid
Aspartic acid isomerization; Complementarity-determining regions (CDRs); Focused peptide mapping; Method qualification; Monoclonal antibody (mAb)