Are Biotransformation Studies of Therapeutic Proteins Needed? Scientific Considerations and Technical Challenges.

For therapeutic proteins, the currently established standard development path generally does not foresee biotransformation studies by default because it is well known that the clearance of therapeutic proteins proceeds via degradation to small peptides and individual amino acids. In contrast to small molecules, there is no general need to identify enzymes involved in biotransformation because this information is not relevant for drug-drug interaction assessment and for understanding the clearance of a therapeutic protein. Nevertheless, there are good reasons to embark on biotransformation studies, especially for complex therapeutic proteins. Typical triggers are unexpected rapid clearance, species differences in clearance not following the typical allometric relationship, a mismatch in the pharmacokinetics/pharmacodynamics (PK/PD) relationship, and the need to understand observed differences between the results of multiple bioanalytical methods (e.g., total vs. target-binding competent antibody concentrations). Early on during compound optimization, knowledge on protein biotransformation may help to design more stable drug candidates with favorable in vivo PK properties. Understanding the biotransformation of a therapeutic protein may also support designing and understanding the bioanalytical assay and ultimately the PK/PD assessment. Especially in cases where biotransformation products are pharmacologically active, quantification and assessment of their contribution to the overall pharmacological effect can be important for establishing a PK/PD relationship and extrapolation to humans. With the increasing number of complex therapeutic protein formats, the need for understanding the biotransformation of therapeutic proteins becomes more urgent. This article provides an overview on biotransformation processes, proteases involved, strategic considerations, regulatory guidelines, literature examples for in vitro and in vivo biotransformation, and technical approaches to study protein biotransformation. SIGNIFICANCE STATEMENT: Understanding the biotransformation of complex therapeutic proteins can be crucial for establishing a pharmacokinetic/pharmacodynamic relationship. This article will highlight scientific, strategic, regulatory, and technological features of protein biotransformation.