Identification of critical chemical modifications by size exclusion chromatography of stressed antibody-target complexes with competitive binding

Abstract

Chemical modifications (attributes) in the binding regions of stressed therapeutic proteins may affect binding to target and efficacy of therapeutic proteins. The method presented here describes the criticality assessment of therapeutic antibody modifications by size-exclusion chromatography (SEC) of competitive binding between a stressed antibody and its target, human epidermal growth factor receptor-2 (HER2), followed by SEC fractionation and peptide mapping characterization of bound and unbound antibodies. When stressed antibody and its target were mixed at a stoichiometric molar ratio of 1:2, only antibody-receptor complex eluted from SEC, indicating that binding was not decreased to break the complex. When a smaller amount of the receptor was provided (1:1), the antibody species with modifications reducing binding eluted as unbound from SEC, while the antibody-receptor complex eluted as the bound fraction. Peptide mapping revealed ratios of modifications between unbound and bound fractions. Statistical analysis after triplicate measurements (n = 3) indicated that heavy chain (HC) D102 isomerization and light chain (LC) N30 deamidation were four-fold higher in unbound fraction with high statistical significance. Although HC N55 deamidation and M107 oxidation were also abundant, they were not statistically different between unbound and bound. Our findings agree with previously published potency measurements of collected CEX fractions and the crystal structure of antibody and HER2. Overall, competitive SEC of stressed antibody-receptor mixture followed by peptide mapping is a useful tool in revealing critical residues and modifications involved in the antibody-target binding, even if they elute as a complex from SEC when mixed at 1:2 stoichiometric ratio.

Multiattribute Monitoring of Aggregates and Charge Variants of Monoclonal Antibody through Native 2D-SEC-MS-WCX-MS

“…Moreover, the acidic peaks of thermal stressed sample exhibited an increase in masses in comparison to the control mAb A, likely due to the deamidation of asparagine. Other researchers have observed that the elevated temperature results in deamidation of the asparagine, which increased the acidic species in the sample, by enhancing deamidation, N-terminal glutamine cyclization, and oxidation-based modifications . In the present study, deamidated acidic species were observed according to their deconvoluted masses.…”

“…Other researchers have observed that the elevated temperature results in deamidation of the asparagine, which increased the acidic species in the sample, 27 by enhancing deamidation, N-terminal glutamine cyclization, and oxidation-based modifications. 24 In the present study, deamidated acidic species were observed according to their deconvoluted masses. Additionally, two basic peaks were observed in the thermal stressed sample, peak B1 with a ∼16 Da and peak B2 with a ∼30 Da additional mass, likely due to 1X and 2X oxidation of the stressed sample (Table 3).…”

“…Peaks B1 and B2 elute after the main peak due to higher pI values with mass shifts of ∼+16 and +36 Da compared to the deconvoluted mass of the main peak, respectively. Oxidation of methionine in the Fc region of the mAb contributes toward the elution of the peak as a basic variant and the addition of the mass of ∼16 Da . The analysis of peak B1 showed a mass difference equal to 1X oxidation, whereas peak B2 showed 2X oxidation with ∼3–4 Da additional mass, which could be due to possible deamidation of asparagine (Std ±0.25).…”

“…Oxidation of methionine in the Fc region of the mAb contributes toward the elution of the peak as a basic variant and the addition of the mass of ∼16 Da. 24 The analysis of peak B1 showed a mass difference equal to 1X oxidation, whereas peak B2 showed 2X oxidation with ∼3−4 Da additional mass, which could be due to possible deamidation of asparagine (Std ±0.25). Methionine-mediated conformational changes can increase the level of deamidation for several residues located in the CH2 domain.…”

A competitive binding-mass spectrometry strategy for high-throughput evaluation of potential critical quality attributes of therapeutic monoclonal antibodies

“…Subsequent bottom-up MS analysis of these SEC fractions could identify attributes that were enriched in the unbound fraction due to impaired antigen binding. 23 Shi et al 24 further incorporated a competitive binding step to this workflow and showed improved method sensitivity in identifying attributes with less significant effects on target binding. This SEC fractionation and bottom-up MS-based approach significantly improved the method throughput in pCQA evaluation.…”