Methodologies for Biotherapeutic Biotransformation Studies

The importance of biotransformation for engineered protein and peptide drug has been well recognized in the recent years. The biotransformation for protein and peptides may impact the pharmacology, further impacting the efficacy or safety of the molecule. To date, there have been various methods applied to the study of the biotransformation of therapeutic protein and peptides. Biotransformation for proteins and peptides are unique compared to the well-established route of small molecule drug candidates in the following aspects: 1) the similarity of the biotherapeutic and endogenous molecules. 2) the biotransformation impact on the protein backbone structure or chemical modifications may impact the 3-dimentional structure of the biotherapeutic, 3) biotherapeutic and therapeutic peptides often have an engineered branched structure that further complicates the identification of the biotransformation species. Combinations of sample preparation methods, chromatographic separation as well as mass spectrometry methods can help comprehensively characterize the biotransformation biotherapeutics. Here, the advanced methods used are summarized and their applications are discussed with case studies.
1) Advancements in the sample preparation methods

Biotherapeutics and their structurally similar biotransformed products can be enriched from the matrix. Afterwards, they can be dissected to smaller pieces to allow further characterization of the structure. The enrichment step can utilize various capture reagents or solid-phase extraction techniques. With selective enrichment, it is possible to retain all the biotransformed product(s) with a certain key feature.

Among many methods to dissect the enriched biotransformation species, enzymatic digestion and selective reduction can be employed. Enzymatic digestion for biotherapeutics can selectively cleave the protein to a smaller size, that may be easier to ionize and to fragment. On the other-hand, inter-chain disulfide bonds are important features for the secondary and tertiary structure of proteins, comparing the data with and without reduction of the disulfide bond can help provide more insight of the protein structure.
2) LC-MS methods
Various chromatographic and mass spectrometric methods can be used for the characterization of biotransformation. Native separation and ionization, alternative fragmentation mechanism, middle down fragmentation are advanced techniques that may be applied to the characterization of biotransformations.

Native separation and ionization techniques can further separate the biotransformation species based on the native structure. However, the native separation often suffers from low separation power or compatibility with mass spectrometry.
While most structural confirmation on the sequence level still utilize a bottom-up approach with enzymatic digestion, it is possible to confirm the sequence for certain subunits (terminal amino acid truncations) with middle-down fragmentation. In addition to this, alternative fragmentation mechanism can also bring additional information for side-chain structures, which is of particular interest for lipidated peptide biotransformation analysis.

3) In vitro incubation systems:
While there is substantial difference behind the species and the abundance of biotransformation products observed from in vitro system incubation and in vivo collected samples, in vitro systems can complement the in vivo analysis as they provide a range of potential biotransformation products that may be specific to organ/tissue of interest, at easy-to-monitor concentrations that can be difficult to achieve with in vivo study samples.