Development of a Process for the Cleavage of Mucin Fusion Protein by Enterokinase

In today’s pharmacological industry, fusion proteins are used for the production of recombinant proteins of therapeutic interest. However, to obtain the therapeutically important protein in its monomeric form, the fusion partner, generally a signal peptide for translocation, needs to be removed using either chemicals or enzymes. In the latter case, the cleavage of fusion proteins can be conducted with higher specificity and under milder reaction conditions. One of the biocatalysts used in laboratory scale is the serine protease enterokinase. In this study, enterokinase was applied in the cleavage of MUC1-IgG2a Fc for the generation of MUC1, a potential target in cancer immunotherapy. To make enterokinase an attractive candidate for industrial fusion protein cleavage, the process for biocatalyst production by recombinant $\textit{E. coli}$ was optimized with regard to fermentation conditions and used isolation and purification techniques. By the application of a newly developed batch-binding chamber, the downstream process was simplified and the process time could be reduced by half. Furthermore, the yield of isolated biocatalyst was increased 8-fold for an inducible expression system and 14-fold with constitutive protein expression. The enzymatic cleavage reaction needs to be economically feasible making an efficient utilization of the biocatalyst necessary. Therefore, different carrier materials for enzyme immobilization have been investigated, of which two – the porous Sepabeads$^{®}$ EC-HA203 and non-porous magnetic particles – gave promising results. Remaining activities for immobilized enterokinase of 60 % could be achieved with an additional stabilizing effect when using the porous material. Enterokinase immobilized on Sepabeads$^{®}$ EC-HA203 was successfully applied in fusion protein cleavage receiving the desired protein MUC1, compared to the non-porous support. Finally, enterokinase immobilized on porous support was applied in the preparative cleavage of MUC1-IgG2a Fc either in a continuous process or in repetitive utilization. According to the received process parameters, the repeated application of the enzyme-support preparation proved to be the more efficient method in fusion protein cleavage. Immobilized enterokinase was re-used 15 to 18 times for cleaving MUC1-IgG2a Fc increasing the total turnover number 419-fold compared to a single application of the biocatalyst