Studies On Preparation And Critical Factors Of Recombinant Anti-HER2 Antibody-Drug Conjugate

Antibody-drug conjugates(ADCs)are biological drugs that conjugated by specific targeted monoclonal antibodies and cytotoxins via linkers.ADCs drugs can specifically recognize target and guide drug internalization,then release cytotoxins in target cells or target organs,achieving the effect of killing related cancer cells inside.It can significantly reduce the non-specific systemic side-effects of common drugs in chemotherapy and improve the safety and effectiveness of tumor treatment.And it may also reduce the resistance of antibody drugs.The ADC drug development mainly depends on complete antibody development technology,chemical conjugating technology and effective selection of small-molecule toxin.The recombinant anti-HER2 antibody drug-conjugate(HER2-ADC)studied in this paper was a biosimilar of Kadcyla that developed by Roche,Switzerland.Kadcyla was an antibody drug-conjugate(ADC)formed by trastuzumab and maytansine(DM1)through a thioether linker.In this paper,the preparing of the biosimilars of Kadcyla was studied from four aspects of construction and evaluation of cell strain,cell culture process,antibody purification process,and ADC chemical conjugating process and pharmacodynamic evaluation.The main findings were as follows:(1)Construction and evaluation of cell strain.Referring to the amino acid sequence published by trastuzumab,the corresponding DNA sequence was synthesized and cloned on the pUC57 plasmid.Using primer design,PCR amplification,restriction enzyme digestion and ligation technology,the light and heavy chain gene sequences from variable region of the antibody were cloned on an expression vector for mammalian cells,Freedom pCHO1.0,and transfected into commercial CHO DG44 cells.After multi-level systematic clonal screening,candidate cell strains were selected and further evaluated for protein titer and quality attribute.Finally,an engineered cell strain was obtained for this study.The trastuzumab analogue(HER2mAb)expressed by this cell strain had a typical antibody symmetrical structure consisting of two heavy-and two light-polypeptide chains with a molecular weight of 150 kDa.Using the 300F/300S medium combination,process parameters were optimized and metal ion concentrations were regulated.At the same time,the charge heterogeneity and expression potential of the antibody was also evaluated.The result showed that the addition of 80 μM Zn2+into the broth in the fed-batch culture of 14 days in a 3 L bioreactor significantly affected the distribution of charge isomers.Compared with 50%main peak and 43%acid peak in the charge heterogeneity of the control,the titer was increased by approx.15%,the main peak was increased to approx.62%and the acid peak was declined to approx.30%after using the new strategy,which significantly improved the medicinal quality of HER2-mAb.2)Study on cell culture process.The types and components of basal and feed medium were screened and optimized,as well as cell culture process parameters,to form a scalable fedbatch culture strategy.Experimental studies found that the addition of some components such as glutamine,insulin-like growth factor(IGF),manganese chloride,uridine and galactose in the basic and feed medium,and the optimization of key control parameters such as pH and temperature during cell culture significantly affected cell culture performance.It was found that in the shake-flask culture,the antbody expression reached the highest peak after adding 6 mM glutamine by two stages and 75 μg/L IGF and declining the culture temperature.After adding the same amount of glycoform regulator containing 4 mM galactose,0.1 mM uridine and 1.2μM manganese chloride into the broth on the 2nd,4th,6th,8th and 10th day,respectively,the sum of the proportions of GIF,G1’F,and G2F was up to 39%in the 300 L bioreactor from 7.52%in the 5 L bioreactor,similar to 37%of trastuzumab.After the process was scaled up to a 300 L bioreactor,the highest viable cell density and expression was about 18×106 cells/mL and 2.5 g/L,respectively;the purity of SEC-HPLC was about 96%;the main peak of IEX-HPLC was about 60%;and the purity of reduced CE was about 98%.These met the filing requirements for drug-substance of trastuzumab biosimilar.3)Study on antibody purification process.The combination of purification methods such as affinity,cation and anion exchange chromatography was applied to purify HER2-mAb and design of experiment(DoE)strategy was adopted to optimize the key operating parameters.After optimizing the eluent pH,arginine concentration and load of affinity chromatography,the purity of SEC-HPLC and yield was both up to 99%;after optimizing the eluent pH,tris concentration and load of cation exchange chromatography,the product purity and yield was up to 99.5%and 95%,respectively;after optimizing the eluent pH,PB concentration and load of anion exchange chromatography,the product purity and yield reached about 100%and 91%,respectively.The above processes were applied in combination at a protein sample of 600 g to verify scale-purity,the purity of SEC-HPLC of the recombinant protein was about 99%,the main peak of IEX-HPLC was about 60%,the purity of reduced CE was about 98%,and the total yield was about 75%,meeting the quality requirements of the recombinant protein for drug registration and the drug-substance requirements for preparing ADC drugs.The degree of pigment removal was an important indicator for sensory testing of antibody drugs.Studies found that Fe3+ was the main cause of the formation of yellow-brown pigment in the recombinant protein stock.The method to remove the pigment in Capto adhere ImpRes with phosphoric acid solution increased the column load after regeneration by 22%compared to the conventional method,up to 76 mg/mL.4)Study on ADC chemical conjugating process and pharmacodynamic evaluation.A twostep conjugating method using HER2-mAb,DM1 and 4 N-Succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate(SMCC)as raw materials was applied to prepare HER2-ADC.In the first step,SMCC reacted with HER2-mAb to generate HER2-mAb-MCC,its main reaction conditions were determined:the ratio of SMCC to HER2-mAb,7.5;protein concentration,10 g/L;reaction time,75 min;DMA ratio,15%;pH,7.0,which was confirmed on the 15 g-and 150 g-scale antibody experiment.The second step was to conjugate HER2-mAb-MCC with DM1,its main reaction conditions were determined:the ratio of DM1 to HER2-mAb,6;protein concentration,10 g/L;reaction time,210 min;DMA ratio,10%;pH,5.2,which was confirmed on the 150 g-scale HER2-mAb-MCC experiment.The product yield after two-step conjugating reaction and ultrafiltration was 92%.Followed by size exclusion chromatography and ultrafiltration replacement,HER2-ADC stock solution was obtained.The concentration of two batches of stock solution was both about 20 g/L,the DAR was about 3.4,the purity of SECHPLC was about 99%,the purity of reduced CE was about 90%.HER2-ADC stock solution remained the biological activity that specifically recognized and bound HER2,and the total yield was about 70%.The residues met the standard requirement for drug registration.The prepared HER2-ADC was used to conduct pharmacological,necessary acute and long-term toxicity studies in the transplanted tumor models from three kinds of nude mouse to confirm the effectiveness and low toxicity of the Kadcyla biosimilar.The ADC process developed in this paper is reproduced.And the quality of the product after scaled up was similar to commercially available product Kadcyla.We also improved the distribution of charged isomers,glycosylation characteristics and remining residues to comply with requirements of biosimilar drugs.Meanwhile,the issue of Capto adhere ImpRes resin polluted by Fe3+ was found and solved,the load of the resin was restored,and its life was effectively prolonged.