Optimization Of Cell Culture Of Rh-TPO Based On Feed-Batch Model And Improvement Of Protein Purification Method

Background: Recombinant human thrombopoietin（rh TPO）is a 332 amino acid recombinant glycoprotein expressed by engineered Chinese hamster ovary cells（CHO）that regulates the growth,differentiation,maturation and division of megakaryocytes by binding to its specific receptor Mpl.It is used for the treatment of severe thrombocytopenic purpura and idiopathic thrombocytopenic purpura caused by solid tumors and acute leukemia after radiotherapy,bone marrow transplantation,reblastosis and other bone marrow insufficiency,and human immunodeficiency virus（HIV）.thrombocytopenic purpura（ITP）.However,the current method is too complicated and cumbersome,with the presence of potential animal-derived viruses and hydrolyzed proteins in the culture medium,complicated culture process,low expression of target proteins,and many steps of protein purification,which fail to inactivate potential viruses,and the organic solvents used for protein purification are potentially hazardous to occupational health.Therefore,there is an urgent need to establish a simple,batch,stable culture mode and efficient protein purification method to improve the yield and increase the yield,while solving the problems of drug safety and occupational health hazards caused by the inactivation of potential viruses.Objective: To establish a novel Feed-Batch flow-plus culture mode and protein purification method.Methods: Screening of culture media from different manufacturers was performed by shake flask test,and a medium with well-defined chemical composition was established.Feedbatch culture mode was used for fermentation culture in 10 L and 100 L reactors,and key parameters for fermentation with high expression were established.rh-TPO stock solution preparation with complex process,low yield and potential virus inactivation were solved by ion exchange,hydrophobic chromatography and other protein purification steps.Results:1.The process adopts the most advanced imported medium Sigma EX-CELL Advanced^TM CHO as the basic medium,and adopts Hy Clone^TM Cell Boost^TM 7a/7b as the addition medium,which does not contain animal origin,does not contain hydrolyzed protein and has clear chemical composition,replacing the old traditional medium with potential virus hazards.2.The results of cell culture experiments showed that the culture volume of CHO-K1 cells could reach 1200 ml within 16 days with a live cell density of not less than 4.0×106 cells/ml and a cell viability of not less than 95% by using Sigma medium as the basal medium and 7a/7b medium as the flow addition medium for shaking flasks of CHO-K1 cells.The expression of target protein could reach more than 90 mg/L at harvest,which met the experimental requirements.3.The results of three batches of 10 L reactor experiments showed that the density of live cells could reach up to 6.0×10⁶ cells/ml,the cell viability maintained at more than 90% could reach 8-9 days in culture;the expression of target protein could reach up to 103 mg/L.4.The results of three batches of 100 L scale cell culture experiments showed that the highest density of live cells in all three batches was 3×10⁶ cells/ml,the cell viability was above 80%,and the protein expression was above 100 mg/L.5.The first step of protein purification in feed-batch mode was MMC Diamond with 20 mM PB 0.1 M NaCl,pH 6.5±0.2,Cond: 11¹5 ms/cm,wash condition: 1 M NaCl p H 6.5,elution condition: 50 m M Tris p H8.0 1 M NH4 Cl 2 M Urea.Elution conditions: 50 m M Tris p H 8.0 1 M NH4 Cl 2 M Urea.second step for Butyl HP,loading conditions: 20 mM PB 0.7 M（NH₄）₂SO₄ pH 7.0,washing with 20 mM PB 0.7 M（NH₄）₂SO₄ pH 7.0,elution with 10 mM PB one step elution.The third step was Mix A Mustang,and the final confirmed loading conditions were,20 mM Tris 0.1 M NaCl pH 8.0,washing conditions were 20% 20 mM Tris 1 M NaCl pH 8.0 washing,and elution conditions were,20 mM Tris 1M NaCl pH 8.0 one-step elution.6.The results of virus inactivation experiments showed that this process could not only remove a large amount of endogenous and exogenous lipid membrane viruses,but also remove non-lipid membrane viruses,while maintaining high protein activity and reducing the residual amount of Solvent/detergent（S/D）inactivator.7.Experimental results of p Harmacokinetic and p Harmacodynamic studies showed no serious adverse events or serious adverse reactions after rh-TPO administration;rh-TPO administration resulted in a wide distribution of the drug with a long half-life and a relatively stable Tmax;good correlations existed between rh-TPO dose increase and drug systemic exposure（all coefficients of determination were greater than 0.9）;The AUC0-t ratio of rh-TPO to TBio was 105% and its elimination half-life ratio was 1.1,The median Tmax of rh-TPO was stable at 9-12 h and comparable to the median Tmax of TBio at 12 h.Conclusion:1.This process establishes a medium with well-defined chemical composition,replacing the traditional complex medium with animal-derived and plant-derived hydrolyzed proteins of potential exogenous viruses.2.The process established the Feed-Batch flow-plus culture mode,which optimally enhanced the growth density of cells and improved protein expression by scaling up to 100 L perfusion culture step by step in shake flasks;simple and efficient cation exchange,hydrophobic chromatography and anion exchange chromatography methods were established based on protein properties,replacing the complex purification processes of isopropyl alcohol and acetonitrile reverse chromatography and hydroxyapatite,and the protein The purification yield was significantly improved,and a new method of Feedbatch cell culture and protein purification of recombinant human platelet growth factor was successfully established.3.The process has established measures to remove and inactivate a large number of endogenous and exogenous lipid cell membrane and non-lipid cell membrane viruses through the selection of inactivating agent and optimization of nanofiltration membrane,which has obvious safety advantages compared with similar marketed products.4.The process eliminates the reverse chromatography of isopropyl alcohol and acetonitrile in the purification step,which reduces the occupational health hazards and ensures safety and environmental protection.5.The rh-TPO purified by this process has good safety and platelet-raising effects in vivo,which lays a solid foundation for the development of subsequent clinical trials.