Structure-Function Assessment and High-Throughput Quantification of Site-Specific Aspartate Isomerization in Monoclonal Antibody Using a Novel Analytical Tool Kit

Abstract

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 structurefunction 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.

Development of a Rapid Adeno-Associated Virus (AAV) Identity Testing Platform through Comprehensive Intact Mass Analysis of Full-Length AAV Capsid Proteins

“…To develop a QC-compliant workflow capable of both rapid serotype AAV identification and PTM characterization, it is important to understand the PTMs present on the AAV VPs. PTMs on biotherapeutic proteins are monitored as potential CQAs to inform process development, batch consistency, product stability, and more, as their presence can influence product quality, efficacy, and potentially patient safety. ,− Comprehensive PTM analysis is crucial for in-depth characterization of full-length AAV VPs, as it aids in the proper identification of different VP proteoforms present and any potential consequences their presence might cause. For PTM determination, peptide mapping and relative PTM quantitation of AAV2 using on-bead pepsin digestion and nano-LC separation were performed in parallel to full-length VP analysis, following the procedure described by Guapo et al One hundred percent sequence coverage was obtained after filtering identified peptides in BPF as described in the section describing AAV peptide mapping data processing for improved peptide mapping confidence (Figure S1).…”

Advancing humanized monoclonal antibody for counteracting fentanyl toxicity towards clinical development

“…Additionally, any alteration or heterogeneity induced by PTMs in mAb CDRs, such as asparagine deamidation and aspartate isomerization, pose a risk of loss of antibody efficacy. 64 , 65 Where such risks were present in mAb CDRs, PTM mitigating mutations were introduced. PTM mitigated humanized mAbs retained equivalent affinity to fentanyl and carfentanil by BLI ( Table 1 ), and they maintained optimal biophysical characteristics ( Table 4 ).…”

2D NMR Analysis of the Effect of Asparagine Deamidation Versus Methionine Oxidation on the Structure, Stability, Aggregation, and Function of a Therapeutic Protein

“…Deamidation rates can be predicted based on the amino acid sequence and three-dimensional structure of the protein. − In the case of IFNA2a, the extent of deamidation varies in the order Asn45 > Asn156 > Asn93 > Asn65 . In addition, the present study has not examined the effect of asparagine to isoaspartic acid mutation (Figure a) …”

“…69 In addition, the present study has not examined the effect of asparagine to isoaspartic acid mutation (Figure 1a). 80 In Summary, we used high-resolution 2D NMR methods to determine how asparagine deamidation affects the structure, stability, aggregation, and function of interferon alpha-2a, and we compared these with our recent results on the effect of methionine oxidation. 23 Identifying the local structural changes helped us in understanding how these two common chemical modifications are similar or different in terms of their effect on the aggregation and function of IFNA2a.…”