| Rheumatoid arthritis(RA) is a common inflammatory disease, which is characterized by joint pain, swelling and chronic systemic disease. There are about 20 million patients across the world. T cells play an important role in the pathophysiology of RA. Upon dual signal cascade transduction from major histocompatibility complex(MHC) and immune co-stimulatory molecules on antigen-presenting cells(ADCs) to active T cells, various cytokines such as tumor necrosis factor-α(TNF-α), interleukin(IL), Interferon(IFN) and so on are released, leading to the injury of tissues such as bones, joints and etc. The commonly used biotherapeutics to treat RA include Adalimumab(ADA), Etanercept(ETN), Infliximab(IFX), Rituximab(RTX), Tocilizumab(TOC) and Abatacept(ABT). Their mechanisms of action could be mainly divided into two categories: one targets end-stage cytokines in the cascade, blocking their biological functions, thus reducing the injure of tissuses such as skeletons, joints and etc., such as ADA, ETN, IFX, etc., and the other targets the initiation phase of the immune reaction, preventing the activation of T cells, thereby blocking immune reaction cascade from the very beginning, which are typically represented by ABT. In 2005,ABT was approved by FDA for the treatment of refractory RA. After 10 years of application,it has brought evangel to the patients.As the subject of this study, ABT is a fusion protein consisting of the extracellular domain of the human cytotoxic T-lymphocyte-associated antigen 4(CTLA-4) linked to a modified Fc(hinge, CH2 and CH3 domains) portion of human immunoglobulin 1(Ig G1).With its extracellular domain, ABT can compete with immune molecules(CD80/B7-1 or CD86/B7-2) on APCs as for binding to CD28 on T cells, thus blocking T cell actication,inhibiting immunune reaction cascade, and reducing the release of cytokines such as IL and TNF-α, which plays a very important role in RA progression. Therefore, it has been successufully applied in the treatment of RA.Compared with small molecule chemicals, protein drugs have obvious advantages, such as higher specificity, lower side effects, targeted distribution in the body, and multiple ways to play therapeutical roles. However, protein drugs are characterized by being expressed in mammalian cells accompanied by complex production processes, large molecular weights,complex structures, a large number of post-translational modifications(PTMs), and so on,which makes the quality study and release control a great challenge for pharmaceutical company. Many studies have shown that PTMs have an important impact on the structure andfunction of proteins, for example, the Fc N-glycosylation on antibody based drugs can not only affect the antibody dependent cell mediated cytotoxicity effect(ADCC) and complement dependent cytotoxicity(CDC), but also the antibody structure, stability and sensitivity to enzymes. As a fusion protein, CTLA4-Ig contains crystallizable fragments(Fc) and CTLA 4extracellular section, both with complicated glycosylation modification. Although approved by FDA in 2005, there are no detailed studies about its glycosylation modification, let alone its O-glycosylation modification. Furthermore, the influence of the glycosylation modification on its structure, stability and function was not documented. In the study, in order to provide reference to its quality control, and meanwhile to lay a foundation for biosimilar research of CTLA4-Ig fusion protein, we are aiming at characterizing the glycosylation modification, and unraveling the relationship between glycosylation modification and the stability or function of the fusion protein.In this study, by employing technologies such as ultra performance liquid chromatography-electrospray ionization-quadrupole-time of flight(UPLC-ESI-Q-To F),differential scanning calorimetry(DSC), size exclusion chromatography(SEC), differential scanning fluoroscopy(DSF), binding activity with Fortebio, and cell-based bioassays, we performed comprehensive characterization of CTLA4-Ig fusion protein at the glycosylation modification level. Furthermore, by resorting to various enzyme digestions, we studied the stability and function with various techniques. The research route is as followings:Section for characterization of the glycosylation modification: firstly, at intact protein level, direct mass spectrometric analysis without any processing of the sample is unable to get much useful information, as CTLA4-Ig fusion protein contains multiple N- and O-glycosylation modification; secondly, at subunit level, in order to reduce the complexity and get more detailed information, we treated the sample with a variety of enzymes(PNGase F, Endo F2, Neuraminidase, and O-Glycosidase) before detection with MS; thirdly, at peptide mapping level, the sample was digested by trypsin and detected with MS; Fourthly, at free glycan level, after releasing N-glycan by PNGase F digestion and labeling with 2-AB,fluorescence detection after UPLC separation and mass spectrometric analysis were applied to the quantitative and qualitative analysis respectively. Then we separated the fusion protein into two parts by digestion with Ides, and charaterized the N- glycosylation profile separately;Lastly, at O- glycosylation modification level, after digestion with PNGase F, Neuraminidase and trypsin, we located the O-glycosylation sites with LC-MS/MS technology.Section for the relationship between glycosylation or stability and function study: After being digested by a variety of enzymes(PNGase F, Endo F2, Neuraminidase, andO-Glycosidase), samples were subjected for subsequent analysis. As for the stability studies,firstly, we determined the purity of the samples before and after enzyme treatments for various time intervals with SEC; Secondly, the purified samples, sterilized with 0.22 um filter membrane after incubation under 45°C for various time intervals(0, 1, 3, 5, 7, 9, 11 and 12weeks), were studied with SEC for accelerated stability testing; Thirdly, the changes of overall thermodynamic parameters were tested by DSF for samples treated by various enzymes for various time intervals; Forthly, we detected the thermodynamic paramet- ers of different domains by DSC for the samples treated by various enzymes for various time intervals. As for the functional studies, firstly, we evaluated the binding activity changes to Fc receptor for samples treated by various enzymes with reporter gene assay, which also indirectly reflected the ADCC activity changes; Secondly, for CTLA4 binding activity, we applied Forte Bio technology to study the affinity of different samples, before and after enzyme treatments, to B7-1 and B7-2; Lastly, for biological potency, we employed report gene assay method to study the ability of samples, before and after enzyme treatments, to prevent Duadi cells from activating Jurkat cells, which may reflect their biological activity.This study indicates that the CTLA4-Ig fusion protein expressed by CHO system contains very complicated glycosylation modifications. For the six N-glycosylation modifications, the four located in the CTLA4 domain are relatively complex, containing large number of terminal sialic acid modifications, while the other two located in Fc domain are relatively simple, which are similar to the Fc glycosylation modifications of classical monoclonal antibody. At the same time, we further clarify the number and position of the O-glycosylation modifications. The O-glycans possibly link to serines located at position 2, 8,11 and 20 in the hinge region, while different tyes of O-glycosylation modification are preliminarily quantitively compared through their different mass spectrum intensity. By means of different glycosidase treatment, it is clearly been proved that the glycosylation modification on CTLA4-Ig fusion protein had a significant impact on the stability, but not on the binding activity and cell based activity. Through the above research, we have a more comprehensive understanding of the CTLA4-Ig protein glycosylation modifications, which lay a foundation for the drug quality control, as well as research and development of its biosimilar. |
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