The impact of standard accelerated stability conditions on antibody higher order structure as assessed by mass spectrometry

Abstract

Protein therapeutic higher order structure (HOS) is a quality attribute that can be assessed to help predict shelf life. To model product shelf-life values, possible sample-dependent pathways of degradation that may affect drug efficacy or safety need to be evaluated. As changes in drug thermal stability over time can be correlated with an increased risk of HOS perturbations, the effect of long-term storage on the product should be measured as a function of temperature. Here, complementary high-resolution mass spectrometry methods for HOS analysis were used to identify storage-dependent changes of biotherapeutics (bevacizumab (Avastin), trastuzumab (Herceptin), rituximab (Rituxan), and the NIST reference material 8671 (NISTmAb)) under accelerated or manufacturer-recommended storage conditions. Collision-induced unfolding ion mobility-mass spectrometry data showed changes in monoclonal antibody folded stability profiles that were consistent with the appearance of a characteristic unfolded population. Orthogonal hydrogendeuterium exchange-mass spectrometry data revealed that the observed changes in unfolding occurred in parallel to changes in HOS localized to the periphery of the hinge region. Using intact reverse-phase liquid chromatography-mass spectrometry, we identified several mass species indicative of peptide backbone hydrolysis, located between the variable and constant domains of the heavy chain of bevacizumab. Taken together, our data highlighted the capability of these approaches to identify age-or temperature-dependent changes in biotherapeutic HOS.

Denaturing and Native Mass Spectrometric Analytics for Biotherapeutic Drug Discovery Research: Historical, Current, and Future Personal Perspectives

“…Sulfation, acetylation, N-terminal pyroglutamination, and C-terminal lysine variants were also identified (Figure 4b). Kerr 155 also described how rpLC−MS was used to map clipping in mAbs as a result long-term storage at six months 25 °C and 60% relative humidity, and was key in identifying quality attributes and therefore assessing molecule shelf life. Denaturing rpLC−MS has also been used to determine drug-to-antibody ratios (DAR) values.…”

High-Resolution Ion-Mobility-Enabled Peptide Mapping for High-Throughput Critical Quality Attribute Monitoring

“…Figure demonstrates the identification of an Fc oxidation CQA that has been shown to be a marker for oxidative stress of the NIST-mAb and other therapeutic IgGs (M255 on the peptide HC 252–258). , Both a control and a chemically oxidized sample were analyzed in this study for identification purposes. The native peptide and its modified counterpart are resolved chromatographically using the shortened gradient, as shown by Figure A.…”

Comparison of Three Complementary Analytical Techniques for the Evaluation of the Biosimilar Comparability of a Monoclonal Antibody and an Fc-Fusion Protein

“…Ion mobility spectrometry would also be an interesting additional technique to provide information on the three-dimensional conformation of adalimumab and etanercept. Moreover, collision-induced unfolding analysis could be applied to study how the molecules unfold when submitted to an accelerating electric field ( Kerr et al, 2019 ).…”