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@#Objective Establish quality control methods for critical quality attribute of bispecific antibody against programmed cell death protein 1(PD-1)/cytotoxic T-lymphocyte-associated protein 4(CTLA-4).Methods The biological activity of PD-1 target was determined by reporter gene assay,and the competitive binding activity of CTLA-4 target was determined by flow cytometry;The antibody molecular size variants were controlled by reducing/non-reducing capillary electrophoresis-sodium dodecyl sulfonate(CE-SDS) and size exclusion chromatography-high performance liquid chromatography(SEC-HPLC);Charge heterogeneity was determined by imaging capillary isoelectric focusing electrophoresis(iCIEF);Bispecific anti-PD-1/CTLA-4 antibody was identified by peptide map analysis;Glycosylation was analysed by high performance liquid chromatography(HPLC)Results The concentration for 50% of maximal effect(EC_(50)) of PD-1target was(6.91±0.78) nmol/L,and the relative biological potency to the reference was(103.50±13.08)% with the RSD of 12.64%;The EC_(50) of CTLA-4 target activity was(0.35±0.28) nmol/L,and the relative biological potency was(99.30±9.15)% with the RSD of 8.32%.The percentage of peak area of light chain and heavy chain of reducing CE-SDS was(98.86±0.02)%.The main peak area percentage of non-reducing CE-SDS was(93.07±0.13)%,fragment percentage was(4.44±0.13)%,and polymer percentage was(2.49±0.15)%.The peak area percentage of SEC-HPLC monomer and polymer were(97.20±0.01)% and(2.68±0.01)%,respectively.The area percentage of peak A group,peak B group,peak C group and peak D group were(38.43±0.54)%,(43.26±0.32)%,(11.31±0.14)% and(7.00±0.17)%,respectively.Peptide mapping showed the specific spectrum of the bispecific anti-PD-1/CTLA-4 antibody,which could be adopted for identification test.The highest proportion of glycotype was GOF,with a content of(41.06±0.11)%,There were three types of glycan containing sialic acid,namely G2F+G1F-NANA,G2F-NANA and G2F-2NANA,with the content of(12.44±0.12)%,(12.00±0.05)% and(5.37±0.05)%,respectively.The total content of glycan containing sialic acid was(29.80±0.20)%.Conclusion The critical quality attributes of bispecific anti-PD-1/CTLA-4 antibody were studied and the corresponding quality control methods were established to ensure its safety,effectiveness and quality control,which provides a reference for the quality control methods and strategies of this type of monoclonal antibody products.
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@#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.
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OBJECTIVE: To establish a capillary zone electrophoresis(CZE) -based method for charge heterogeneity analysis of IgG2 monoclonal antibody. METHODS: The concentrations of TETA and EACA, pH of separation buffer and temperature of capillary were optimized, to obtain a method which can efficiently separate the charge variants of IgG2 mabs. The reproducibility and repeatability of the optimized CZE method were validated and a battery of mabs were evaluated. RESULTS: The separation effect was significantly influenced by TETA/EACA concentration, pH and separation temperature. Through a series of optimization, a CZE method which showed good resolution and precision(RSD of area percentage was below 3.0% was established and the method parameters were as below: 400 mmol•L-1 EACA/0.05% TETA, pH 6.2, separation under 30 kV voltage at 35℃. This method was also suitable for IgG1 and IgG4 mabscharge heterogeneity analysis. Based on the specific electrophorogram and migration time, it could also be used as an identification method for monoclonal antibodies. CONCLUSION: The optimized CZE method can efficiently separate the charge variants of IgG2 mabs and shows good precision and specificity, which can be used to analyze the charge heterogeneity and identification of monoclonal antibodies.
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Objective To analyze the differences of size and charge heterogeneities between origi-nal humanized anti-TNF-αantibody and four similar biotherapeutic products ( SBP ) .Methods The size exclusion chromatography ( SEC-HPLC ) and weak cation exchange chromatography ( WCX-HPLC ) were used to analyze the size and charge heterogeneities , respectively.Carboxypeptidase B (CpB) treatment was employed to analyze the source of charge heterogeneity of the antibody products .Results Four SBPs showed the same pattern with the originator in SEC-HPLC, and no significant difference with the percentage of mono-mer was observed .The percentages of the aggregates of SBP-3 and SBP-4 were a little higher than those of the originator .The charge distribution of SBPs was significantly different from the originator ′s, especially in the basic region .The results from the samples treated with CpB indicated that the difference of charge distri -bution in the basic region might be caused by the C-terminal lysine variants .Conclusion Four SBPs showed similar size heterogeneity with the originator , but significant differences with charge heterogeneity were observed among them .The study suggested that more attention should be paid to the charge heterogene -ity analysis of the biosimilar products .
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Semliki Forest, Sindbis and Chikungunya viruses were grown and radiolabeled with [3H]-amino acids in Vero cells. Analysis of virus infected cell lysates by two dimensional polyacrylamide gel electrophoresis resulted in detection of polypeptides of molecular, weights corresponding to those of E1, P62, ns60, ns70/72 for Semliki Forest virus, the C, E1, 6K, 14Κ, ΡΕ2, P97, ns60, ns82 for Sindbis virus and E1. P62, P97, ns70/72 for Chikungunya virus. Charge and molecular weight heterogeneity in the precursor polypeptide P62 of Semliki Forest virus was detected. Structural polypeptides e.g. E1 and E2 of Semliki Forest virus and C, E1, E2 of Sindbis virus and E1 of Chikungunya virus were detected when purified radiolabeled virus preparations were analyzed by two dimensional polyacrylamide gel-electrophoresis. Membrane glycoprotein Ε1 and E2 of Semliki Forest and Ε1 of Sindbis and Chikungunya viruses exhibited charge heterogeneity. In contrast to the marked difference in isoelectric points of E1 and E2 of Sindbis virus; E1 and E2 of Semliki Forest virus had almost identical isoelectric points.