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. LAY ABSTRACT: 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.

Improved detection of host cell proteins (HCPs) in a mammalian cell-derived antibody drug using liquid chromatography/mass spectrometry in conjunction with an HCP-enrichment strategy.

RATIONALE: Host cell proteins (HCPs), which are process-related impurities typically present at low levels in recombinant biopharmaceutical products, are often measured using an immunological technique, such as an enzyme-linked immunosorbent assay (ELISA). In contrast to ELISA which only provides the total amount of HCP, liquid chromatography/mass spectrometry (LC/MS) can provide both qualitative and quantitative information about the major HCP species. In this study, an HCP-enrichment step was optimized and combined with LC/MS to identify and determine the relative abundance of HCPs present in a monoclonal antibody (mAb) drug product. METHODS: An NS0 (mouse myeloma) cell-derived mAb drug product, whose total HCP level was less than 100 ng/mg of protein, was subjected to analysis by LC/MS. One-dimensional and two-dimensional chromatography options, together with the off-line HCP enrichment strategy based on Protein A chromatography, were evaluated for optimal HCP detection. RESULTS: With this approach, nineteen HCPs were detected from a therapeutic mAb, an improvement over the detection of only one HCP without depletion. CONCLUSIONS: Compared with other published HCP studies with LC/MS, the HCP-enrichment step in our method enables a more practical and relevant application to approved protein therapeutics, which are mostly mammalian cell-derived products with HCPs present at very low levels.