Removal of Residual DNA and Host Cell Proteins for the Purification of Recombinant Staphylokinase Expressed in Escherichia coli

ABSTRACT The elimination of residual host cell DNA (HCD) and proteins (HCPs) is a pivotal step in the purification process for biological products such as monoclonal antibodies, recombinant proteins, vaccines, gene therapy vectors, and cell‐based therapies. During the preparation of recombinant staphylokinase (r‐SAK), a potential therapeutic protein for thrombotic disorders expressed in Escherichia coli cells, an efficient chromatography purification process, incorporating anion exchange, cation exchange, and gel filtration techniques, was developed to effectively eliminate HCPs and residual DNA. This multistep chromatography approach yielded r‐SAK with a residual HCD concentration below 1 ng/mL, a residual HCP concentration below 0.01%, and purity exceeding 98%. Comparative analysis revealed that modified cellulose‐based matrix resins exhibited superior efficiency compared to dextran and agarose matrix resins for eliminating residual HCPs and HCD under identical conditions. Based on the different properties of the matrix, deductions were made regarding the reasons for the differentiation in separation efficiency. The physical strength of the cellulose‐based matrix ensures the structural stability of macroporous resin and can guarantee efficient separation under conditions of high flow and heavy load. This study suggests that maintaining the structural stability of macropores in bioseparation materials is crucial for improving the efficiency of separating biological products.