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

Abstract

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 STATEMENTUnderstanding 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.

Immunoaffinity Capillary Electrophoresis in the Era of Proteoforms, Liquid Biopsy and Preventive Medicine: A Potential Impact in the Diagnosis and Monitoring of Disease Progression

“…Recombinant DNA-derived protein therapeutic products are often interchangeably referred to as biopharmaceuticals, biotherapeutics, biologicals, or biologics. Biologics are an important class of medicines, which are administered to treat an array of different diseases such as cancers, autoimmune disorders, infectious diseases, and many others [207][208][209][210][211]. The demand for biologics has risen considerable over the past few decades, owing to increase in prevalence of chronic diseases and development of plasma-derived therapies.…”

Challenges of non-clinical safety testing for biologics: A Report of the 9th BioSafe European Annual General Membership Meeting

“…Finally, Simone Schadt (Roche) presented about the increasing relevance of biotransformation studies for complex therapeutic proteins. 69 Biotransformation studies for therapeutic proteins are conducted in an issue-driven approach for compound optimization (stability, bioavailability), PK/PD characterization and to identify biotransformation products contributing to efficacy or safety. A typical trigger to embark on biotransformation studies is a mismatch between different bioanalytical assays (e.g., total versus target-binding competent), and biotransformation products may or may not be pharmacologically active.…”

Assessment of Antibody Stability in a Novel Protein-Free Serum Model

“…Different in vitro models and analytical approaches have been developed to evaluate the in vivo stability of therapeutic proteins [ 4 , 5 , 6 , 7 ]. However, simulating physiological conditions and detecting the protein of interest in biological fluids is accompanied by challenges [ 8 , 9 ]. Purification techniques and/or fluorescence labeling have been successfully used to detect the target protein [ 10 , 11 ].…”