Assuntos
Leite/química , Ácido N-Acetilneuramínico/análise , Ácidos Neuramínicos/análise , Animais , Bovinos , Cromatografia Líquida de Alta Pressão/economia , Cromatografia Líquida de Alta Pressão/métodos , Limite de Detecção , Modelos Lineares , Ácido N-Acetilneuramínico/química , Ácidos Neuramínicos/química , Reprodutibilidade dos TestesRESUMO
AIM: Recombinant glycoprotein produced in nonhuman mammalian cell lines can be modified with the immunogenic nonhuman sialic N-glycolylneuraminic acid (Neu5Gc). We describe here a validated method for detection of antidrug antibodies against both protein and Neu5Gc-containing glycan epitopes. RESULTS: An electrochemiluminescent method was established with drug conjugates as capture and detection reagents. Rabbit antidrug polyclonal antibodies were used as the positive control for protein moiety-specific antibodies, while chicken anti-Neu5Gc polyclonal antibodies were used as the positive control for antibodies against Neu5Gc glycan epitope. Specificity to Neu5Gc was verified by signal inhibition with bovine γ-globulin that contains Neu5Gc. CONCLUSION: The assay illustrated here discerns the immunogenicity of the protein backbone and the sialic acid Neu5Gc glycan moiety of a recombinant protein containing Neu5Gc.
Assuntos
Glicoproteínas/química , Glicoproteínas/imunologia , Imunoensaio/métodos , Ácido N-Acetilneuramínico/química , Ácidos Neuramínicos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Animais , Especificidade de Anticorpos , HumanosRESUMO
In this study, we demonstrate the utility of ultra-performance liquid chromatography coupled to mass spectrometry (MS) and ion-mobility spectrometry (IMS) to characterize and compare reference and biosimilar monoclonal antibodies (mAbs) at an advanced level. Specifically, we focus on infliximab and compared the glycan profiles, higher order structures, and their host cell proteins (HCPs) of the reference and biosimilar products, which have the brand names Remicade® and Inflectra®, respectively. Overall, the biosimilar attributes mirrored those of the reference product to a very high degree. The glycan profiling analysis demonstrated a high degree of similarity, especially among the higher abundance glycans. Some differences were observed for the lower abundance glycans. Glycans terminated with N-glycolylneuraminic acid were generally observed to be at higher normalized abundance levels on the biosimilar mAb, while those possessing α-linked galactose pairs were more often expressed at higher levels on the reference molecule. Hydrogen deuterium exchange (HDX) analyses further confirmed the higher-order similarity of the 2 molecules. These results demonstrated only very slight differences between the 2 products, which, interestingly, seemed to be in the area where the N-linked glycans reside. The HCP analysis by a 2D-UPLC IMS-MS approach revealed that the same 2 HCPs were present in both mAb samples. Our ability to perform these types of analyses and acquire insightful data for biosimilarity assessment is based upon our highly sensitive UPLC MS and IMS methods.