Investigating the Degradation Behaviors of a Therapeutic Monoclonal Antibody Associated with pH and Buffer Species

Abstract

Abstract. This study aimed in understanding the degradation behaviors of an IgG 1 subtype therapeutic monoclonal antibody A (mAb-A) associated with pH and buffer species. The information obtained in this study can augment conventional, stability-based screening paradigms by providing the direction necessary for efficient experimental design. Differential scanning calorimetry (DSC) was used for studying conformational stability. Dynamic light scattering (DLS) was utilized to generate B 22 *, a modified second virial coefficient for the character of protein-protein interaction. Size-exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) were employed to separate degradation products. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for determining the molecular size and liquid chromatography mass spectrometry (LC-MS) were used for identifying the sequence of the separated fragments. The results showed that both pH and buffer species played the roles in controlling the degradation behaviors of mAb-A, but the pH was more significant. In particular, pH 4.5 induced additional thermal transition peaks occurring at a low temperature compared with pH 6.5. A continual temperature-stress study illustrated that the additional thermal transition peaks related to the least stable structure and a greater fragmentation. Although mAb-A showed the comparable conformational structures and an identical amount of aggregates at time zero between the different types of buffer species at pH 6.5, the aggregation formation rate showed a buffer species-dependent discrepancy over a temperature-stress period. It was found that the levels of aggregations associated with the magnitudes of protein-protein interaction forces.

<p>Effect of 2nd and 3rd generation PAMAM dendrimers on proliferation, differentiation, and pro-inflammatory cytokines in human keratinocytes and fibroblasts</p>

“…There has been a very large increase in the interest of research teams in dendrimers as carriers of drugs in skin diseases 31,32. A good in vitro model for testing skin diseases are keratinocytes and fibroblasts.…”

Hyphenation of size exclusion chromatography to native ion mobility mass spectrometry for the analytical characterization of therapeutic antibodies and related products

“…Until recently, the direct coupling of SEC to MS failed, mainly because older generation of MS instruments were not sufficiently tolerant to high salt contents (even volatile salts like ammonium acetate) and the SEC columns were not enough inert (poor peak shapes were generally observed with volatile salts). Most publications report the off-line coupling of SEC to either classical denaturing MS (mostly via reversed-phase liquid chromatography coupled to MS, RPLC-MS) [17][18][19] or native MS [20]. In this configuration, SEC fractions are manually collected and reinjected into the MS instrument.…”

Formulation and Delivery of Macromolecules

“…In their article, Zheng et al investigate the influence of pH and buffer species in an IgG 1 subtype therapeutic monoclonal antibody A (mAb-A) (10). Through a combination of dynamic light scattering, differential scanning calorimetry, size-exclusion chromatography, hydrophobic interaction chromatography, gel electrophoresis, and liquid chromatography mass spectrometry as characterization tools, the authors systematically studied the antibody degradation pathways.…”