FAPP: Filter-Assisted Protein Precipitation sample pre-treatment to enhance bottom-up proteomics

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In the current study, we have developed a sample pre-processing protocol for protein digestion, and the outcome has significant implications for several areas of the biopharmaceutical/ biotherapeutic domains.

Of late, the biopharmaceutical market has been growing in interest with the development of biotherapeutics, in particular monoclonal antibodies (mAbs), which are used to treat, diagnose, or prevent a wide range of illnesses and medical disorders. This has also had a great impact on the foundation of the development of biosimilars by establishing the analytical and functional comparability of highly similar copies of licenced biologic medications to gain the potential cost reduction on healthcare systems. As a result, this could provide more access to biologic medications through the biopharmaceutical industry.

Bottom-up proteomics/protein digestion is one of the most crucial areas of research to achieve this goal because it aids in sequence confirmation, post-translational modification identification, contaminant detection, batch-to-batch consistency assurance, mapping epitopes, PK/PD research, and comparative analyses. Which all work together to advance the development, characterization, and quality control of biotherapeutics, ultimately improving their safety, efficacy, and therapeutic effectiveness. 

It could be challenging to prepare a bottom-up proteomic sample, digest it effectively, and then extract peptides for subsequent mass spectrometric analysis. The sample preparation for traditional protein digestion techniques requires highly trained professionals and still carries the risk of allowing chemicals that are required for extraction but likely to obstruct digestion, resulting in complex chromatographic profiles due of semi-cleavages, insufficient peptide cleavages, and other unwanted events. Additionally, peptide clean-up using frequently used immobilised C-18 pipette tips may cause significant peptide loss and individual peptide yield variability, resulting in artefacts of various product-related alterations. Therefore, we proposed an easy-to use, highly reproducible, appropriate and robust enzymatic protein digestion protocol for less trained professionals, that includes multiple filtration steps in between digestion processes, by various molecular weight filters, alongside protein precipitation, in order to minimise interference of necessary digestion chemicals. This efficiently overcomes the shortcomings of conventional digesting technology, boosts peptide yield, and removes the need for peptide clean-up. 

The proposed method, which we termed “filter-assisted protein precipitation (FAPP),” allowed us to minimise digestion-related alterations and locate nearly all possible peptides with the site-specific modifications that retain great importance for biosimilarity assessment. Additionally, the thorough coverage of the CDR peptides greatly boosted confidence in the FAPP method’s capacity to confirm the primary structure of the under-researched biosimilars.