Improvement of liquid chromatography for analysis and purification of proteoforms via rational protein purification parameter screening and sample displacement chromatography

Abstract

In contrast to small molecule drugs, therapeutic proteins are characterised by their composition of many very similar but not identical species (proteoforms) regarding their composition of atoms. This heterogeneity may arise due to alternatively spliced RNA transcripts, genetic variations, or post translational modifications (PTMs), thus there are many proteoforms encoded by a single gene. The presence of some proteoforms may reduce efficacy of therapeutic proteins and may even induce harmful side effects. Therefore, the analysis and purification of proteoforms are essential. However, the fractionation and purification of proteoforms is highly challenging because many proteoforms are physically and chemically very similar and often present in a very low abundance. Sample displacement chromatography (SDC) in previous studies was described as more efficient compared to other chromatography modes. This thesis aims to answer the question whether SDC is suitable for separating proteoforms of the same protein and to develop a strategy for the purification of proteoforms using sample displacement batch chromatography (SDBC). Ovalbumin from chicken egg was used as a model sample. Protein purification parameter screening (PPS) was conducted to obtain the most optimal parameters for SDBC, yielding a two-fold increase in the number of detected proteoforms in comparison to the original sample. A significant enrichment of low-abundant basic ovalbumin proteoforms was obtained which gives a proof that SDBC is effective even for the separation of closely related proteoforms. SDBC was also able to separate ovalbumin proteoforms into two categories: highly enriched proteoforms in the SDBC early fractions and highly enriched proteoforms in the later fractions. Low-abundant basic proteoforms were well enriched in the early fractions, while in the later fractions, less basic proteoforms were present. In addition, with SDBC a complete removal of non-ovalbumin proteins such as ovomucoid, ovatransferin, and other ovalbumin related proteins, being impurities in the original sample, was achieved. The application of SDBC for separation of proteoforms of the recombinant monoclonal antibody adalimumab showed comparably well results. In conclusion, the results obtained in this study show the utility and possibility of SDBC for simple and effective separation of proteoforms, which in the future can be used for removal of harmful proteoforms and tool for enrichment of low abundant proteoforms for further analysis.