Structural Characterization And Analysis Of A Novel Anti-CD20Humanized Monoclone Antibody

PurposeNon-Hodgkin’s lymphomas (NHL) is the most common malignant tumor which originatesin lymphatic system. About85%of Non-Hodgkin’s lymphomas arise in B-cells. The CD20antigen is present on more than95%of B-cell lymphomas and is neither shed norinternalized after antibody binding, making it an effective target for immunotherapeuticremoval of malignant B cells. The mouse/human chimeric anti-CD20antibody C2B8(Rituximab) is the first therapeutic monoclonal antibody approved for the treatment ofB-cell non-Hodgkin’s lymphomas. Despite the effectiveness of C2B8, only10%ofpatients respond to treatment when given alone, most patients get relapsed or refractoryand have potential of HAMA reaction. In this study, we designed and prepared a novelanti-CD20humanized mAb to improve the potency and reduce the immunogenicity.Ultraperformance liquid chromatography electrospray ionization quadrupole time of flightmass spectrometry (LC-ESI-Q-Tof) was first used for detailed characterization of the novelanti-CD20humanized mAb. We also conducted the biological activities analysisin vitro.These studies provided information and made strong basis for the ongoing preclinical andclinical study.Methods1. SDS-PAGE and HPLC-SEC analysis for apparent mass and purity of recombinantanti-CD20humanized mAb2. LC/MS analysis at the level of intact proteins for accurate mass andpost-translational modifications of recombinant anti-CD20humanized mAb3. LC/MS analysis at the level of subunits for accurate mass and post-translationalmodifications of recombinant anti-CD20humanized mAb4. LC/MS analysis at the level of peptide mapping for amino acid sequence,glycosylation site, and post-translational modifications confirmation ofrecombinant anti-CD20humanized mAb5. LC/MS analysis at the level of released free glycans for glycosylation, anddistribution of different glycan structures of recombinant humanized anti-CD20 humanized mAb6. In vitro biological analysis of recombinant anti-CD20humanized mAb: bindingaffinity, CDC, ADCC and apoptosisResults1SDS-PAGE and HPLC-SEC analysis showed that the apparent mass wasconsistent with theoretical data. The HPLC purity can achieve to above99%byProtein A and hydrophobic chromatography, only less polymers exist.2After reduced and digested with PNGase F, the intact protein mass is145424.92Da, which is well consistent with theoretical data.3The accurate mass of heavy chain is50671.28Da and49226.75Da before andafter treatment with PNGase F. The accurate mass of light chain is the same as23489.18Da before and after treatment with PNGase F. These data indicated thatthere was glycosylation modification in the heavy chain, but not in the lightchain.4The novel recombinant anti-CD20humanized mAb also have a single N-linkedglycosylation site located in the Asn301of the heavy chain. LC-MS pepetidemapping analysis concluded that the MS coverage achieve to100%, the MS/MSEcoverage achieved to96.7%(b,y equal to3).5The NP-UPLC-MS overall data indicated the presence of32distinct glycanstructures in the novel recombinant anti-CD20humanized mAb, these structureswere confirmed by MS/MS analysis. Thenovel recombinant anti-CD20humanized mAb is mainly composed of two antenna complex type glycan, withsmall amount of high mannose type and nonfucolysed glycan. The nonfucolysedG0and fucolysed G0F glycan account to6.54%and45.96%individually.6In vitro biological analysis of recombinant anti-CD20humanized mAb revealedthat the novel recombinant anti-CD20humanized mAb exhibited better activityover rituximab in ADCC, CDC, apoptosis and binding affinity.