Preparation,Performance And Application Of High Performance Protein A Affinity Chromatography Medium

Staphylococcus aureus protein A（SPA）has the ability to bind specifically to immunoglobulins,so SPA is used as an affinity ligand to prepare affinity chromatography media,which is widely used for the purification of monoclonal antibodies.However,the high price of commonly used commodity media has limited its further development.In order to obtain high-performance domestic SPA affinity chromatography media,this study used recombinant strains to obtain SPA by induced expression,and obtained affinity chromatography media by Ni²⁺purification and coupling with substrates.The adsorption capacity,alkali resistance and reusability of the media were tested in comparison,and the purification effect of the media was evaluated in practical applications.The main studies are as follows:（1）Acquisition of recombinant alkali-resistant SPA.The B structural domain of SPA was modified,and the main substitutions carried out were N23T,N3A,N6D and G29A,which enhanced the basic resistance of SPA;single-factor optimization of the fermentation conditions of recombinant bacteria was carried out in five aspects:different inoculum levels,bacterial concentration(OD₆₀₀),IPTG concentration,induction time and induction temperature,and the optimal conditions for SPA expression were determined;the obtained proteins were purified using Ni-NTA affinity chromatography medium,and the target proteins were eluted using imidazole buffer containing 500 mmol·L^-1,and the purity of the obtained proteins reached more than 90%with good purification results.（2）Preparation of high performance SPA affinity chromatography media.Optimization of the coupling reaction during the preparation of media in terms of four aspects:reaction time,reaction temperature,p H of the reaction system and protein dosage,and clarification of the optimal conditions for the coupling reaction;under the optimal reaction conditions,the static saturation adsorption capacity of the prepared affinity chromatography media could reach115.54 mg·m L^-1 media,which is 17.18%higher compared to the commercial media Cytiva Mab Select Su Re LX under the same conditions.（3）Performance testing and application study of self-made affinity chromatography media.Determination of the dynamic loading of the prepared media of 58.6 mg·m L^-1 media is higher than that of 55.2 mg·m L^-1 media of Cytiva Mab Select Su Re LX under the same conditions;after soaking in 200 mmol·L^-1 Na OH for 72 h,the load of the self-made filler could still maintain about 81%of the initial load;after 100 simulated reuse cycles,the adsorption load of the self-made media was still about 88%of the initial load,and the alkaline resistance and reuse performance of the self-made media were good and similar to those of the commercial media;the self-made medium was applied to purify monoclonal antibody samples,simulating industrial purification of monoclonal antibodies,and three-step and two-step purification tests were performed,and the purity of antibodies obtained by one-step affinity purification was around 95%,with a yield of not less than 90%;the amount of SPA shed,host protein and exogenous DNA residue in the final product is comparable or better than the purification of commercial media.