Study On The Structure And Function And Comparison Of Two Fermentation Process Of Anti-CD52 Monoclonal Antibody

Monoclonal antibodies have become the main research direction in the field of therapeutic biologicals due to their minor side effects,clear targets and remarkable effects.The research focus on antibodies accounted for 70%of therapeutic biologicals,and the antibodies were widely used in indications like cancer,autoimmune diseases,graft-verus-host diseases.The anti-CD52 antibody has already been approved by FDA and EMA for the treatment of B chronic lymphocytic leukaemia?B-CLL?and multiple sclerosis?MS?,however,but have not been launched on the domestic market.Fed-batch and perfusion cell cultures are the dominant modes of fermentation processes of recombinant therapeutic proteins production.In the light of high expression efficiency of cell lines,the fed-batch process has dominated the production of monoclonal antibody products for its large scale-up capability and robustness.The perfusion process offers consistent and steady culture conditions,allows rapid removal of product with replenishment of fresh culture media.Therefore,the perfusion process has benefits of high cell density,long culture duration,stable protein quality and high productivity.Unstable proteins like enzymes and recombinant blood coagulation factors are produced by perfusion process,along with few therapeutical antibodies.In this study,we investigated overall quality attributes and biological potency characterization of anti-CD52 antibody,meanwhile compared the productivity and quality of antibodies produced by perfusion and fed-batch process.Objective:To study the protein structure and biological activity of anti-CD52 antibody.Bioreactors with 15L working volume were used to produce anti-CD52 antibodies using fed-batch process and perfusion process respectively,as well as the productivity and quality attribution were compared.Method:Quality attribution of anti-CD52 antibody were investigated,including N-terminal sequencing,aggregation and purity measurement by SDS-PAGE,the molecular mass by LC-MS,disulfide bond,circular dichroism measurement,IEF for isoelectric point,CEX-HPLC for charge heterogeneity,glycosylation level and sialic acid level.Biological potency characterization were investigated,including binding capacity test by FACS,ability for complement dependent cytotoxicity?CDC?,and antibody dependent cell-mediated cytotoxicity?ADCC?.Fed-batch culture and perfusion culture were used to research cell growth kinetics and protein expression efficiency of the cell line for TH-6 cells in shake flask.Fed-batch culture and perfusion culture were used to research cell growth kinetics and protein expression efficiency of the cell line for TH-6 cells in 15L bioreactor,as well as purity,aggregate and charge heterogeneity of monoclonal antibody products were analyzed.Results and Discussion:Results of N-terminal sequencing,peptide sequencing and molecular mass confirmed the amino acid sequence consistency with the theoretic sequence.Disulfide bridges were identified to be four intra-heavy chain bonds and two intra-light chain bonds,the other three were inter chain disulfide bonds,which is consistent with the theoretic data as well.Results of circular dichroism measurements showed that there were 52.3%?-helix,45.6%turn,0%beta-pleated sheet and 2.1%random coil in secondary structure of production.Isoelectric point was mesaured by IEF,which was around 9.2.CEX-HPLC was used to test the charge heterogeneity,the proportion of acid peaks,the main peak and the basic peak were 13.57%?72.59%?13.83%respectively.Glycosylation measurements was carried out in the oligosaccharide level,and they are mostly G0,G0F,G1,G1F,and G2F types.According to the mechanism of action,glycoforms can be divided into non-fucose type and galactose type,which was 5.9%and 53.2%respectively.There were few sialylated glycans in anti-CD52 antibody.The results of 4 batches of products presented that there were 0.063 to 0.089mol/mol of NANA and 0.001 to 0.002 mol/mol of NAGA.The data of profile attributes was overall comparable with Campath.Results of biological potency characterization showed that the antibody's binding EC₅₀to MC/CAR cells is 5ug/mL,and its ADCC potency is much stronger than CDC potency,with EC₅₀ is about 30ng/mL.The CD52 expression cells were able to adapt to the culture both in shake flask and in bioreactor.In flask,fed-batch process produced proteins around 1.2 g/L,specific cell protein production reached 20-25 pg/cell/day;perfusion process produced proteins around 0.5g/L/day,specific cell protein production reached 25-36 pg/cell/day with 1 perfusion volume a day.IThe CD52 expression cells were cultured in 15-L bioreactors using fed-batch process and perfusion process for 3 batches respectively.The productivity of perfusion process was7.1-folder higher than that of fed-batch process.The study of quality profile and biological potency characterization indicated that proteins from perfusion process had fewer aggregation,fewer acid component,more galactose and higher CDC potency.Conclusion:We studied the structure and bioactivity data of anti-CD52 antibody,the structure data was identical with the theoretical data,and the bioacitity was similar to Campath.The process compare results showed that to produce anti-CD52 antibodies,fed-batch process had a higher specific productivity,but perfusion process performed a higher volumertric productivity.And then,antibodies produced by perfusion process performed a better quality and higher biological potency.