| Adeno-associated virus（AAV）is widely used as a vector for gene therapy because of its good safety,wide host range（mitotic and non-mitotic cells）,stable physical properties,long-term stable expression of foreign genes,and non-physiological or pathological changes after infection.AAV-related vectors show great hope for the treatment of various diseases.To obtain adequate AAV-product for clinical evaluation,large-scale production are required instead of laboratory-scale.Cultivation process may produce many impurities that are difficult to remove,resulting in complex purification process,low recovery rate and poor product quality.Therefore,it is necessary to develop a downstream purification method suitable for large-scale production of high-quality AAV.In this study,a preliminary process for downstream purification of recombinant adeno-associated virus serotype 5（rAAV5）was developed,and the process was optimized according to the preliminary process results.The process include four section:crude purification,affinity chromatography,purification（ultracentrifugation）and preparation of stock solution.（1）The crude purification:the cell precipitation and supernatant were collected respectively after centrifugation.Harvesting cells According to the established preliminary process,the optimization of lysate salt concentration and cell concentration,and optimization of cell lysate salt concentration and p H are carried out.The harvest supernatant was purified through clarification,filtration,concentration,buffer-exchange using tangential flow filtration system（TFF1）,frozen-thawing,enzymolysis,regulation of conductance and filtration.Subsequently,the parameters of enzymatic process were optimized and the deep filtration process and the filter were investigated.（2）Affinity chromatography:using Capto AVB column,the buffer formula was optimized and the volume of loading sample was determined according to the results of the preliminary process.（3）Ultracentrifugation:the concentration of the sample was 2-8E12vg/m L.The samples were collected via the bottom collection method,and the 3.5m L product was collected after discarding 2m L.（4）Preparation of stock solution:PES syringe filter was used in fermentation scale below 50L,wherease,PES capsule filter was used in larger scale.Results:the downstream purification process of rAAV5 was established.The process include（1）crude purification:the harvested cells were concentrated 10 times with the lysate（20 m M Tris,2 m M Mg Cl2,p H8.0）,and the harvested supernatant was clarified and filtered,followed by 20 times concentration of TFF1,and buffer-exchanged for 8-15 times.After partial thawing,the cells were hydrolyzed by enzyme（400 U/m L,40 rpm,120 min）,regulated conductance,clarified by centrifugation,deep filtration,and 0.8+0.2μm membrane.The supernatant was hydrolyzed by enzyme（400 U/m L,40 rpm,120 min）,adjusted conductance and filtered by 0.8+0.2μm after partial thawing;（2）Affinity chromatography:the optimized buffer for AVB equilibrium solution contain 20 m M Tris-HCl,100 m M sodium citrate,2 Mm Mg Cl2,p H8.0;AVB wash solution:100 m M sodium citrate,2 m M Mg Cl2,p H2.5;AVB eluent:100 m M sodium citrate,100 m M phosphoric acid,2 m M Mg Cl2,p H1.7;AVB PAB buffer:120 m M phosphoric acid,167m M acetic acid,2.2%v/v benzyl alcohol.（3）Ultra-centrifugation:the sample containing2-8E12vg/m L was ultracentrifuged and the product was collected by the bottom collection method（the 3.5m L samples was obtained after discarding 2m L）.（4）preparation of stock solution:syringe filter was used in fermentation scale below 50L,wherease PES capsule filter was used in larger scale. |
