ABSTRACT
@#Objective To develop and verify a whole-column image capillary isoelectric focusing(iCIEF) electrophoresis method to analyze the charge heterogeneity of recombinant human growth hormone Fc fusion protein(Fc-rhGH).Methods The iCIEF analysis method of Fc-rhGH was developed by optimizing the target protein concentration,cosolvent(urea)concentration and focusing time.The target protein was simultaneously analyzed by this method and traditional flat plate isoelectric focusing(IEF) electrophoresis,and the results were compared;The specificity,accuracy,precision,limit of quantitation(LOQ) and durability of the developed method were verified.Results The optimized method was using the mixed solution of 8 mol/L urea,0.35% methyl cellulose(MC),4% amphoteric electrolyte and 0.5% isoelectric point marker as the sample buffer,and the focusing condition was 1 500 V 1 min,3 000 V 5.5 min.IEF was not suitable for analyzing the charge heterogeneity of Fc-rhGH solution.Using the optimized iCIEF for analysis,the target protein was significantly different from the unrelated protein,and the baseline of blank reagent was stable;The recovery rate of accuracy verification was within 90%～110%,and the linear range was 0.25～0.75 mg/mL(50%～150% of the target loading volume);The RSD of each isomer pI in the repeatability verification was less than 0.3%,and the RSD of peak area percentage was less than 5%;The LOQ was 0.04 mg/ml.The sample storage time durability,amphoteric electrolyte pharmalyte 3-10 durability and MC durability of this method were good.Using this method to analyze the charge heterogeneity of Fc-rhGH physicochemical reference substance,eight charge heterogeneities of the reference substance were effectively separated,and the pI ranged from 5.9 to 6.4.Conclusion The developed iCIEF method had good specificity,accuracy,precision and durability,and was more suitable for efficient analysis of charge heterogeneity of Fc-rhGH than traditional flat plate IEF,which was of great significance for the quality control of Fc-rhGH and other Fc fusion proteins.
ABSTRACT
OBJECTIVE: To develop a heterogeneity analysis method of an antibody-drug conjugate (anti-CD30-vc-MMAE). METHODS: The size and charge heterogeneity of the antibody-drug conjugate (anti CD30-vc-MMAE) was analyzed by SEC-HPLC, CE-SDS, and iCIEF. RESULTS: The main peak purity was (95.69±0.01)% as shown by SEC-HPLC. The total peak purity of the heavy and light chains determined by reduced CE-SDS was (98.38±0.25%)%. At non-reduced CE-SDS level, there were six main peaks, and the total purity of which was (97.82±0.44)%. The acid modification, base modification, and main proportion could be separated effectively by iCIEF, and the peak area percentage of the main peak was (43.52±2.03)%. CONCLUSION: A heterogenecity analysis method of the antibody-drug conjugate (anti-CD30-vc-MMAE) has been preliminarily established and can provide reference for the quality control of this product.