Structure-Function Assessment and High-Throughput Quantification of Site-Specific Aspartate Isomerization in Monoclonal Antibody Using a Novel Analytical Tool Kit
| Autor: | Tapan K. Das, Zhi Chen, Neil Hershey, Ming Zeng, Li Tao, James Bautista, Richard Ludwig, Xiang Cao, Kaimeng Zhou |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
medicine.drug_class
Computer science Drug Compounding Drug Storage Pharmaceutical Science Succinimides 02 engineering and technology Computational biology Monoclonal antibody 030226 pharmacology & pharmacy Peptide Mapping Isoaspartate 03 medical and health sciences chemistry.chemical_compound Immunoglobulin Fab Fragments Structure-Activity Relationship 0302 clinical medicine Succinimide Drug Stability Isomerism Tandem Mass Spectrometry medicine Critical to quality Amino Acid Sequence Throughput (business) Chromatography High Pressure Liquid Aspartic Acid Isoaspartic Acid Protein Stability Peptide mapping Structure function Antibodies Monoclonal 021001 nanoscience & nanotechnology Chromatography Ion Exchange Complementarity Determining Regions High-Throughput Screening Assays chemistry Immunoglobulin G 0210 nano-technology Isomerization |
| Zdroj: | Journal of pharmaceutical sciences. 109(1) |
| ISSN: | 1520-6017 |
| Popis: | Isomerization of surface-exposed aspartic acid (Asp) in the complementarity-determining regions of therapeutic proteins could potentially impact their target binding affinity because of the sensitive location, and often requires complex analytical tactics to understand its effect on structure-function and stability. Inaccurate quantitation of Asp-isomerized variants, especially the succinimide intermediate, presents major challenge in understanding Asp degradation kinetics, its stability, and consequently establishing a robust control strategy. As a practical solution to this problem, a comprehensive analytical tool kit has been developed, which provides a solution to fully characterize and accurately quantify the Asp-related product variants. The toolkit offers a combination of 2 steps, an ion-exchange chromatography method to separate and enrich the isomerized variants in the folded structure for structure-function evaluation and a novel focused peptide mapping method to quantify the individual complementarity-determining region isomerization components including the unmodified Asp, succinimide, and isoaspartate. This novel procedure allowed an accurate quantification of each Asp-related variant and a comprehensive assessment of the functional impact of Asp isomerization, which ultimately helped to establish an appropriate control strategy for this critical quality attribute. |
| Databáze: | OpenAIRE |
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