(M0930-01-01) The Impact of Oxidative Modifications on the Bioavailability of Subcutaneously Administered Therapeutic Monoclonal Antibodies

Purpose: Therapeutic monoclonal antibodies (mAbs) are often administered intravenously (IV), causing inconvenience, but reformulating them for subcutaneous (SC) injection enhances convenience and reduces healthcare resource use. However, SC injection is limited by reduced bioavailability, which is linked to the unique anatomy of the SC tissue and its direct effects on the mAb structure. Because of the probable involvement of reactive oxygen species (ROS) in the SC tissue, oxidation represents a critical class of these modifications. Here, we test the effect of oxidation on the bioavailability of therapeutic proteins. Understanding oxidation effects may facilitate the development of more convenient SC formulations while maintaining adequate bioavailability.

Methods: Different models of oxidation, mimicking those occurring in the SC space, were applied to adalimumab, then the oxidized variants were administered SC to a rat model to test whether bioavailability may be reduced. Immunoprecipitation assay employing anti-human magnetic beads was used for the purification of adalimumab and stressed variants from the rat proteins. The specificity and binding capacity of the beads were confirmed by LCMS/MS.

Results: Adalimumab was isolated from rat serum and SC tissues with high protein recovery ( >95%) and high specificity ( >95%). MS analysis showed that methionine (M) is the primary oxidation site, with M256 in the CH2 domain being the most susceptible, as indicated by its highest abundance in oxidized variants. Pharmacokinetic analysis of collected blood samples showed a significant decrease in SC bioavailability (˜20%) for oxidized variants compared to non-stressed adalimumab. In support for this observation, oxidized variants of adalimumab were found to significantly retain at the SC tissue as supported by dye-labeling adalimumab and monitoring its SC retention overtime using in vivo imaging system.

Conclusion: Anti-human magnetic beads demonstrated highly specific and consistent recovery of the oxidized variants of adalimumab from in vitro and in vivo rat protein matrices. Animal studies suggest that oxidation lowers SC bioavailability of adalimumab. A potential application of this assay is to study the in vivo oxidation that may occur to therapeutic proteins while they are resident in the SC space.

References: Varkhede, N.; Bommana, R.; Schöneich, C.; Forrest, M. L. J. of pharmaceutical sciences 2020, 109(1), 191-205. https://doi.org/10.1016/j.xphs.2019.08.005

Acknowledgements: This work was generously funded by Novartis Pharma AG


Figure 1 Determination of binding capacity of the beads as measured by (a) SDS-PAGE, (b) LCMS/MS, (c) Specificity of the beads by comparing the ratio of intensities at m/z 530.97 (signature peptide of rat serum) and m/z 713.68 (signature peptide of Adalimumab)


Figure 2 (a) Serum concentration-time profile of Adalimumab and its oxidized variants after SC injection (b) Comparison of bioavailability by measuring AUC between the first and the last time points, (c) Fluorescence signal-time profile for dye-labeled adalimumab in the subcutaneous injection site, (d) Comparison of adalimumab variants retention at the subcutaneous injection site by measuring the AUC between the first and the last time points


Table 1 List of oxidized methionine residues (bold and underlined) in the corresponding tryptic peptides and their location along adalimumab structure