| Objective:Rituximab (common name, international generic drug name: Rituximab),produced by Roche Pharmaceuticals, is a human-mouse chimeric monoclonalantibody affected on human CD20, is the first monoclonal antibody to be approvedfor clinical treatment of non-Hodgkin’s lymphoma (NHL). CD20is mainly on thecell surface of B lymphocytes, so the products can be used to treat excessive Blymphocytes diseases, including lymphoma, leukemia, transplant rejection and someautoimmune diseases. Currently, the products have been allowed to enter the Chinesemarket (trade name: rituximab) by the Chinese Food and Drug Administration(SFDA), for:(1) treatment of aggressive (diffuse large B-cell) lymphoma incombination with CHOP programs of eight courses；(2)first-line treatment ofindolent (follicular) lymphoma in combination with CVP programs of eight courses；(3) treatment of palindromic or chemotherapy resistant indolent B-cellnon-Hodgkin’s lymphoma. In clinical practice, the monoclonal antibody showedgood efficacy and safety. However, there are also some problems in clinicalapplications, such as some patients does not produce a clinical response on rituximabtherapy；the clinical cure rate is very low when some patients used the drug alone；effective part of the patients who have taken clinical treatment will soon palindromiaor progressed after drug withdrawal, or shows treatment resistance phenomenon inre-use, and has the potential risk of human anti-mouse antibody(HAMA)response. For these phenomena, the development of a humanized anti-CD20monoclonal antibody drugs is particularly important.By repurifying the humanized anti-CD20monoclonal antibody-standard and theantibody sample liquid, a suitable concentration was prepared. Comparing andidentificating the configuration information of both,including ultraviolet absorptionspectrum, peptide mapping, molecular weight of heavy and light chain, isoelectricpoint, charge heterogeneity content;detecting and comparing the qualityinformation of both,including the purity of protein, Protein A residue (ELISA), and CHO cell protein residues (ELISA), etc. On such basis, explore the preliminarycrystallization conditions of the two antibody samples.Methods:The repurification, preparation, packaging and calibration of the antibodystandard and the antibody sample;The purity of the antibody sample and the standard detected by SEC-HPLCassay;The purity and molecular weight of light and heavy chain of the antibodysample and the standard detected by reduced SDS-PAGE assay;The charge heterogeneity content of the antibody sample and the standardanalysed by CEX-HPLC assay;The purity of the antibody sample and the standard detected bynon-reduced SDS-PAGE assay;The isoelectric point of the antibody sample and the standard determined bycIEF assay;The ultraviolet absorption spectra analysis of the antibody sample and thestandard;The peptide mapping of the antibody sample and the standard analysed byRP-HPLC assay;CHO cell’s protein residues of the antibody sample and the standard detected byELISA assay;ProteinA residues of the antibody sample and the standard detected by ELISAassay;Initial crystallization conditions exploration and X-Ray analysis of theantibody sample and the standard.Results:Through SP Sepharose FF cation chromatography, the protein contents of theantibody sample and the antibody-standard were16.1mg/ml and16.3mg/mlrespectively; each kind of antibody protein was diluted to1.0mg/ml and10mg/ml specifications after preparation;The purity of the antibody sample and the antibody standard detected bySEC-HPLC assay were99.2%and99.3%respectively.The results of the purity and molecular weight of light and heavy chain of theantibody and the standard detected by reduced SDS-PAGE showed a consistencyof them, molecular weight of light and heavy chain were58kD and27kD whichcoincide with the theoretical value;The charge heterogeneity contents results of the antibody and the standardanalysed by CEX-HPLC assay showed a good charge uniformity which havemeet the requirements of the protein crystallization;The purity results of the antibody sample and the standard detected bynon-reduced SDS-PAGE assay showed100%purity of both, molecular weightwere about135kD which shows a consistency of them;Through a more accurate determination by cIEF,the isoelectric point of theantibody sample and the standard were9.23and9.20respectively.Consideringthe system error, they showed a consistency which coincide with the theoreticalvalue;Through ultraviolet absorption spectra analysis of the antibody sample and thestandard, the results showed that the maximum UV absorption wavelengths wereboth277nm, within the scope of the theoretical value;The peptide mapping results of the antibody and the standard analyzed byRP-HPLC assay showed a consistency of them;The CHO cell’s protein residues results of the antibody sample and the standarddetected by ELISA assay showed that residual host proteins were not detectedfrom them which were suitable for protein crystallization;Protein A residues results of the antibody sample and the standard detected byELISA assay showed that residual protein A were not detected from neither ofthem, suitable for protein crystallization;The preliminary crystallization conditions exploration of the antibody and the standard showed that an initial crystallization conditions have been discovered.The crystals were all twinned crystal. X-Ray analysis showed that the crystalswere protein.The resolution of the protein was about8, which the X-Raydiffraction information could not be collected from it.Conclusions:By identifying and comparing the quality and structure of the humanizedanti-CD20monoclonal antibody-standard and the antibody sample, the resultsshowed a consistency of them and were coincide with the theoretical value; qualitytest results showed that both of them have meet the requirements of proteincrystallization; initial crystallization conditions exploration were carried out on suchbasis, and twinned crystal were obtained successfully. Those have,laid a goodfoundation for optimization of crystallization conditions as well as function andmechanism study of the antibody by structure, also opened up a new research idea forthis humanized antibody in pharmaceutical and clinical applications. |
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