Free and total biotherapeutic evaluation in chromatographic assays: interference from targets and immunogenicity

Measurement of drug concentrations is critical during drug development, supporting evaluation of safety and efficacy in the context of pharmacokinetics. Protein-based therapeutics have been historically measured by immunoassay methods. Technological advances provide new opportunities to measure these biotherapeutics using previously incompatible chromatographic techniques, such as MS. These advances are breaking down the barriers between 'large-molecule' and 'small-molecule' bioanalysis, and pushing scientists outside their comfort zones. One challenge in measuring biotherapeutic concentration is potential impact from other matrix components, such as therapeutic target or antidrug antibodies. Depending on the specific assay development objective, target interference could be either desired (favoring free measurement) or undesired (favoring total measurement). Orthogonal techniques provide additional tools to meet this challenge. The goal of this review is to introduce both small- and large-molecule bioanalytical scientists to the opportunities and challenges to consider while evaluating orthogonal methods for biotherapeutic bioanalysis.