Intact Mass Spectrometry Analysis of Immuno-Isolated Human Therapeutic Antibodies from Serum.

Antibody-based therapeutics have emerged as a novel class of biopharmaceuticals over the last couple of decades with the advancements made in production and downstream processing technologies. The structural diversity of therapeutic antibodies has also evolved with the development of bispecific (and multispecific) antibodies and antibody-drug conjugates. With increased structural complexities and multi-modularity, there is a need to demonstrate that the entire structure is stable in vivo and arriving at its target site in an intact form. Proving that antibodies reach their target site unscathed is a challenging but essential step for showing effective delivery as well as showing whether failure in efficacy (if any) was related to its in vivo instability. This chapter describes a method for highly specific immuno-isolation followed by intact mass spectrometry of human Fc-containing antibody from serum of rats dosed with the antibody. The method provides an opportunity for evaluating antibody stability in the physiological environment by providing accurate validation of its molecular mass in vivo, as well as the potential to identify breakdown products.