Multi-epitope insert modulates solubility-based and chromatographic purification of human papilloma virus 16 L1-based vaccine without inhibiting virus-like particle assembly

The separation of heterogeneous protein mixtures has always been characterized by a trade-off between purity and yield. One way this issue has been addressed in the past is by recombinantly modifying protein to improve separations. Such modifications are mostly employed in the form of tags used specifically for affinity chromatography, though it is also possible to make changes to a protein that will have a sizeable impact on its hydrophobicity and charge/charge distribution. As such, it should also be possible to use protein tags to modulate phase separations and protein-resin binding kinetics when performing ion exchange chromatography. Here, we employed a three-step purification scheme on E. coli expressed, His-tagged, human papilloma virus 16 L1-based recombinant proteins (rHPV 16 L1) that consisted of an inclusion body (IB) wash step, a diethylaminoethyl (DEAE) anion exchange chromatography (AEX) step, and an immobilized metal affinity chromatography (IMAC) polishing step. Purification of the wild type rHPV 16 L1 protein (WT) was characterized by substantial losses during the IB wash but relatively high yield over the DEAE column. In contrast, purification of modified rHPV 16 L1, a chimeric version of the WT protein that had the last 34 amino acids replaced with an MHC class II multi-epitope insert derived from tetanus toxin and diphtheria toxin (WTΔC34-2TEp), was characterized by little to no losses in the IB wash but had a relatively low yield over the DEAE column. Since the fate of these proteins was to be used in vaccine formulations, it is important to note that the modifications made to the WTΔC34-2TEp protein had little to no effect on its ability to assemble into virus-like particles (VLPs). These results demonstrate that modifications of the WT protein via the recombinant insertion of immunofunctional polypeptides can modulate both phase-based separation and charge-based chromatographic processes. Additionally, incorporation of the specific, multi-epitope tag used in this study may prove to be beneficial in recombinant HPV vaccine development due to its potential to improve phase separation yield and vaccine immunogenicity without inhibiting VLP formation.