Establishment and verification of a bio-layer interferometry-based method for determining Fc receptor affinity of IgG4 monoclonal antibodies
10.13200/j.cnki.cjb.004724
- VernacularTitle:基于生物层干涉技术的IgG4型单克隆抗体Fc受体亲和力测定方法的建立及验证
- Author:
Shi ZENG
- Publication Type:Journal Article
- Keywords:
Bio-layer interferometry(BLI);
Monoclonal antibody;
Fc receptor affinity
- From:
Chinese Journal of Biologicals
2026;39(06):686-693
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish and verify a set of methods based on bio-layer interferometry(BLI) technology for the detection of Fc receptor affinity of IgG_4 monoclonal antibodies,and to provide reliable detection methods for the quality research and supervision of this category of pharmaceuticals.Methods According to the characteristics of the interaction between each Fc receptor and IgG_4 monoclonal antibodies,Protein A(ProA),anti-penta-HIS(HIS1K) and streptavidin(SA)biosensors were selected respectively,and the corresponding immobilization strategy,sample concentration,binding and dissociation time,and buffer system were set for different receptors.Among them,Fc gamma receptorⅠ(FcγRⅠ) adopted ProA sensor to immobilize antibodies and then analyze receptors.FcγRⅡa,FcγRⅡb/c and FcγRⅢ were analyzed using HIS1K sensor to immobilize the receptor.FcRn was detected by binding His-tag antibody to SA sensor followed by immobili-zation of the receptor.The specificity and repeatability of the method were verified,and bevacizumab was used as a positive control to confirm the detection performance of the method.The Fc receptor affinity of six batches of IgG_4-type programmed death-1(PD-1)-targeted monoclonal antibody products and working reference substances was determined by the established method.Results In each receptor detection,all the interference light wavelength shift signals of the assay buffer were < 0.003 nm.The relative standard deviations(RSDs) of IgG_4 PD-1 targeting monoclonal antibody injection with FcγRⅠ/CD64 and FcRn KD were 1.9% and 5.8%,respectively,and were all < 25% with FcγRⅡa/CD32a(H/67),FcγRⅡb/c(CD32b/c),FcγRⅢa/CD16a(V176) and other weak affinity receptors.The KDs with FcγRⅢa/CD16a(F176) and FcγRⅢb/CD16b were all >1 × 10-5 mol/L,showing no affinity.The measured results of bevacizumab were consistent with the theoretical values.The KDs of six batches of IgG_4 monoclonal antibody injection samples with FcγRⅠ/CD64 were(3.029-3.193) × 10~(-9) mol/L,and the KDs with FcRn were(1.398-1.622) × 10~(-7) mol/L,which were consistent with preclinical research data.The KDs with FcγRⅡa/CD32a(H/67),FcγRⅡb/c(CD32b/c),and FcγRⅢa/CD16a(V176) were(0.920-1.402) × 10~(-5),(4.673-5.682) ×10~(-6),and(2.236-3.994) × 10~(-5) mol/L,respectively,all showing weak affinity.The KDs with FcγRⅢa/CD16a(F176) and FcγRⅢb/CD16b were both > 1 × 10~(-5) mol/L,showing no affinity.The Fc receptor affinity determination results of each batch of products were basically consistent with those of the working reference substances.Conclusion The established affinity detection method based on BLI technology has good specificity and precision,which can be used for the quality research and supervision of monoclonal antibody drugs,and also provides reference for the affinity detection of other antibody drugs.
