The power of high-resolution LC-MS for mAb characterization.

Monoclonal antibodies (mAbs) represent one of the fastest growing classes of biopharmaceuticals today. Together with a huge therapeutic potential, mAbs are large (ca. 150 kDa) and heterogeneous proteins that come with an enormous structural complexity. Even small changes in the structure of antibody-based therapeutics can have significant impact to the safety and efficacy of the pharmaceutical end product. Structural changes in mAbs such as glycoforms, post-translational modifications (e.g. deamination, isomerization, oxidation etc.) and sequence variations can occur during manufacturing and storage.

Comparable to the development of small-molecule drugs, structural and comprehensive analytical characterization of biologics is critical and required at many points in the pipeline to ensure comparability to the original product. Liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) is a powerful and highly sensitive analytical tool that is perfectly suited to perform an in-depth analysis of therapeutic proteins such as mAbs (including antibody-drug-conjugates and host-cell protein analysis). LC-MS can be used to characterize antibody features at different levels ranging from intact molecular weight determination, over subunit examination to glycan profiling and post-translational modification analysis. Besides providing site-specific information at qualitative level, LC-HRMS can also be used to quantify the amount of structural changes in mAbs.

In this application note we describe the comprehensive characterization of adalimumab at different levels by means of the analytical LC-HRMS based platform that was developed at AnaBioTec.