Magnetic immunoprecipitation assay for the isolation of adalimumab and degradation products in preclinical biological samples.

Evaluation of pharmacokinetics is a cornerstone in the developability assessment of therapeutic monoclonal antibodies (mAbs). For accurate evaluation of mAbs' concentration-time profile, administered mAbs should be isolated from the interfering host proteins in serum samples. While there are multiple approaches to purify and quantify the administered mAb, they are limited by drawbacks such as low specificity, relatively-large plasma volume requirements, and tedious procedures for sample preparation and analysis. Here, we present an immunoprecipitation assay that uses a highly specific anti-human IgG1 antibody coupled with magnetic beads (R10-coupled Dynabeads) for the purification of adalimumab and forced-oxidized variants. The specificity and binding capacity of R10-coupled Dynabeads were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. To demonstrate the wide applicability of this method, two different stressed variants involving mixtures with rat proteins were examined which are chemical oxidation stress and physical agitation stress. The results showed comparable recovery of native adalimumab and oxidized variants from in vitro and in vivo rat proteins mixture. R10-coupled Dynabeads also recovered aggregated species, but the method itself induced more aggregate formation as evident from analysis by dynamic light scattering and background membrane imaging. Pharmacokinetic evaluation showed a typical concentration-time profile for native adalimumab which is broadly consistent with reported profiles. This method can provide an efficient and highly-specific recovery of human IgG1 and degradation products from preclinical biological samples, provided that the epitope recognized by the used anti-human IgG antibody is retained and accessible. Furthermore, the integration of R10-coupled Dynabeads purification, Trypsin/Lys-C digestion, and QToF LC-MS/MS analysis enabled efficient capture of the oxidized species generated by AAPH and H2O2 (% sequence coverage ≥ 90%). Accordingly, this combined workflow can be applied to study in vivo modifications of therapeutic proteins (e.g. oxidation, deamidation) after exposure to an anatomical environment.