TNFR: Fc Fusion Protein Bio-similar Drugs Quality Comparable Preliminary Study Preliminary Inquiry & CD22 Monoclonal Antibody Effector Function

In recent years，biosimilar drug development is during their booming period dueto the fact of many "blockbuster" drug patents go expiry graduallly．The historicalopportunity promotes the transformation of international pharmaceutical market fromsmall molecules to biological macromolecules．It is particularly important to makeeffective quality control for these biosimilars．Compared with small molecule drugs，antibody and recombinant protein drugshave high molecular weight， multiple domain， and complex post-translationalmodification．Thus，to what extent a biosimilar should demonstrate similaritycompared with the Reference biotherapeutic product is currently the mostcontroversial regulatory question.For the above problems，the first section of this research focuses on therecombinant human tumor necrosis factor receptor antibody fusion protein for qualityresearch．This experiment selects the Reference biotherapeutic product(RBP) and3similar biotherapeutic products(SBPs) as experimental materials， and makecomparison between the structural characterization data(amino acid sequence，secondary structure composition and type of glycosylation) and functionaldata(neutralizing activity in vitro，binding activity，in vivo half-life)．We comparestructure and function information，in order to provide the reference for this productof generic analysis and quality evaluation．In the research of structure characterization，first，the amino acid sequences wereconfirmed．Compared with RBP，SPBs are divided into three classes: SBP1has thesame sequence with RBP; SBP2has a mutation in the67/70position; SBP3has amutation in the376/378position．In the secondary structure comparison，we foundthere was no significant difference between SBPs and RBP．In the analysis ofglycosylation，we found no significant difference between SBP2and RBP; As forSBP1, its sialic acid content is50%lower than that of RBP; G0content of SBP3ishigher than that of RBP.In the functional analysis，there is no significant difference beteeen SBP1andRBP in biological function. while，we found the neutralizing activity in vitro，TNF-αaffinity and half-life in vivo of SBP2are all significantly higher than that of RBP．Theresult not only proves the drug function is realized from the neutralization of TNF-αbut also proves that67/70mutation can promote it．For SBP3, in the Fc segment ofthe amino acid mutation, the neutralizing activity in vitro, affinity and in vivo half-lifehad no significant difference with RBP. Therefore, SBP1should be defined asbiological similar drugs, SBP2can be defined as a new drug in certain extent; SBP3，whoes amino acid sequence is different from RBP (according to the EMA standard),should be defined as a new drug. However, the experimental results show that, exceptthe amino acid sequence differences, there is no significant difference in functionbetween SBP3and RBP. Therefore, non clinical and clinical trials in china need to beundertaken to justify whether this product can be defined as a biological similar drugor not. CD22monoclonal antibody(CD22mAb) belongs to IgG1type antibody.It is usedfor the treatment of non Hodge lymphoma. The antibody internalise rapidly afterbinding to the target cells which makes it impossible to directly reflect the mechanismof antibody with traditional tumor cell proliferation inhibition method. Aiming at thisproblem，people abroad use IgM special sensitization to B cell receptor to determinethe biological activity of CD22mAb．This process does not add the complement orkiller cells. Therefore, it can’t simulate the in vivo environment actually．Based on the background above，the second section of this research use the targetcells expressing anti-CD22idiotypic antibody(artificially constructed) asmaterial． First， we use the cells to determine CDC and ADCC activity ofCD22mAb．Second，we use3methods to verify the removal effect of glycosylation.Comparing the glycosylated and deglycosylated antibody, we further validate thebinding ability and biological function of the antibody．CD20monoclonal antibody and CD147monoclonal antibody are used as positivecontrol to verify CDC and ADCC experiment can work effectively. In the CDCexperiment，the add-in order of complement and target cell types are optimized; in theADCC experiment，cell concentration and antibody dilutions were optimized．In theverification of deglycosylation，CD22mAb can be deglycosylated completely．Toexplore the effect of glycosylation on the function， two methods (Competitive flowcytometry and Biacore) were used to compare the binding ability of glycoslated anddeglycoslated antibody，and there is no significant difference between them．Then，on the basis of optimized CDC and ADCC experimental scheme，we compared theCDC and ADCC effect of glycoslated and deglycoslated antibody.The result displaysthat these two effects significantly decreased after deglycoslation．The results showthat the CD22antibody Fc fragment has CDC and ADCC effector function. Now, wedo not take CDC and ADCC evaluation of CD22monoclonal antibody intoconsideration. So this part of the research is crutial for the quality evaluation of theantibody.In this study，we made an overall evaluation of TNFR:Fc fusion protein and itsSBPs and initially carried out the quality comparability research on one hand. Weestablished the relevant analysis methods and discussed whether a product could beclassified as biological similar drugs if there were differences in non critical qualityattributes. On the other hand，this paper has initially solved the difficult to evaluatethe biological activity of CD22monoclonal antibody，and brings new lights for theestablishment of special antibody quality evaluation method．This research willprovide reference and data supports for the formulation of drug quality assessmentguidelines of antibody and other related biotherapeutics.