ABSTRACT
OBJECTIVE: To analyze the trend in quality control of anti-HER2 monoclonal antibody. METHODS: The biological activity of anti-HER2 monoclonal antibody (mAb) was monitored by the proliferation inhibition with CellTiter 96® AQueous Assay kit using BT474 cells as target, and the potency of anti-HER2 mAbs was calculated by comparison with the reference standard using the method of parallel analysis. Cation exchange chromatography method was used to detect the purity by calculating the area percentage of the main peak according to the area normalization method. Ultraviolet spectrophotometry was used to detect the protein content. And pH was detected by potential method. The warning limit (x±2s) and action limit (x±3s) were defined according to the determination results of several batches of anti-HERmAb by National Institutes for Food and Drug Control (NIFDC) and the quality control laboratory of manufacturer, based on which the trend graph was plotted, and the consistent and periodic trend of anti-HER2 mAb were analyzed. RESULTS: The potencies of 74 batches of anti-HER2 antibodies from a certain enterprise during 2012 to 2014 were analyzed. The products and reference standard both showed a dose-response effect in biological activity, presenting parallel straight lines on semilog coordinate paper. The potencies determined by NIFDC and the manufacturer were (1.04±0.11)×104 and (0.94±0.08)×104U·mg-1, respectively. And the protein contents were (442.15±13.59) and (442.07±6.60) mg·vial-1. The main peak areas of IEC-HPLC were (76.29±1.36)% and (73.76±1.17)%, while the pHs were (6.16±0.11) and (6.22±0.08), respectively. The consistent and periodic trend analysis of the above-mentioned results showed that the total trend was relatively steady. CONCLUSION: This is the first time to conduct trend analysis of critical quality attributes (CQA) of anti-HER2 monoclonal antibody. These results reveal the quality changes of the products, which would help a lot in evaluating the batch consistency and production stability, and also provide reference for trend analysis of critical quality attributes for other monoclonal antibodies.
ABSTRACT
OBJECTIVE: To perform a multidimensional study on the critical quality attributes of a therapeutic anti-HER2 humanized monoclonal antibody targeting HER2-positive breast cancer. METHODS: The critical quality attributes such as purity, impurities, structure, and function were evaluated using multidimensional analytical techniques including HPLC, CE-SDS, dynamic light scattering, circular dichroism, differential scanning calorimeter, LC-MS/MS, in vitro bioactivity assay and SPR binding kinetics. RESULTS: The monomer content of the antibody was more than 98% while the polymer impurities were less than 2%. The sum of heavy chain and light chain peak area was over 97% while the non-glycosylated heavy chain impurities area was less than 1% on the CE-SDS electrophoretogram. The antibody drug demonstrated comparable CD spectrum, predicted secondary structure and unfolding temperatures to the reference product. It had highly similar N-glycan profile involving glycosylation site and N-glycan types to the reference product. The analysis showed no significant difference in the functional CQAs like BT474 proliferation inhibiting bioactivity, ADCC efficacy, the binding affinity to HER2 and Fc receptors between the evaluated drug product and the reference. CONCLUSION: The characteristics of the monoclonal antibody drug such as high purity and few impurities indicate homogeneity and low-risk of safety issues. High similarity to the reference has been verified in multiple aspects like higher structure, glycosylation and function. The positive quality evaluation result is a prerequisite for clinical research. Moreover, to some extent, there can be potential correlation between CQAs and clinical safety and efficacy.
ABSTRACT
OBJECTIVE: To screen for the optimal CHO cell clone which stably expresses recombinant humanized anti-HER2 monoclonal antibody(rhHER2-mAb) and to measure its bioactivity. METHODS: Disposable orbital shaking bioreactor, Tubespin, was utilized for high-throughput screening, and then orbital shaking flask was used for scale-up. Protein A affinity chromatography was utilized to purify the fusion protein. SDS-PAGE and HPLC were utilized to detect its purity, ELISA was utilized to detect its production amount, and MTT was utilized to detect its bioactivity. RESULTS: Through the cell clone selection, it was found that #38 cell clone had the best expression performance, with the productivity in shaking flask of(152±20) mg · L-1. After scale up, enough supernatant was got for the purification. The molecular weight of the purified protein was about 150 × 103, and its purity was higher than 96%. Analysis of bioactivity and binding affinity showed that its activity and bind affinity were close to the commercialized product, Herceptin. CONCLUSION: A cell clone with high productivity was obtained by cell clone screening. The antibody protein was successfully obtained by orbital shaking scale-up cultivation and purification. Its bioactivity and binding affinity were consistent with the commercialized drug. This method has laid a foundation for large-scale fermentation of this recombinant protein.
ABSTRACT
Anti-HER2 monoclonal antibody (Sc7301)-paclitaxel (TAX) immunoconjugate was prepared and its specific binding to tumor cells was investigated in this study.Sc7301 was conjugated to TAX by the active ester method and then the TAX-Sc7301 immunoconjugate was obtained.After purification and labeling by Cyano-fluorescein isothiocyanate (FITC),the specific binding of TAX-Sc7301to HER2-positive tumor cells (SKOV3) and HER2-negative tumor cells (HepG2) was evaluated respectively.TAX-Sc7301 (20 nmol/L) showed distinct specific binding to SKOV3 cells rather than HepG2cells.And the uptake of the immunoconjugate by SKOV3 cells was increased with the TAX-Sc7301concentration (3-48 nmol/L) and the incubation time (P＜0.05).It was concluded that the TAX-Sc7301immunoconjugate is potentially applicable as a targeted agent against HER2-positive tumor cells.