Analysis Of Structure, Effectiveness And Stability Of Therapeutic Antibody Fusion Protein TNFR-Fc Structural Isoforms

Biologics is playing a more and more important role in the global pharmaceutical market. At present, with the trend that patents for many biologics have either expired or are going to expire, the prospect of biosimilars looks promising. Our country issued ’guiding principle of research and development, evaluation technique about biosimilars’lately. The guiding principle requires that all kinds of quality problems of biosimilars need to be analysed deeply and some related methods of quality control need to be made. There are two different peaks in the hydrophobic interaction chromatogram of biosimilar TNFR-Fc fusion protein which is produced and purified to above 99% by three classical purification steps in our laboratory, but we do not know the difference between the two peaks and their effects on effectiveness and stability. And based on the above-mentioned guiding principle, there is a need to analyse the two hydrophobic peaks. Therefore, this article will take the two peaks as research objects, fully analyse the structure characteristics of the two structural isoforms and compare, research their effectiveness and stability.First, through the optimizing of buffer, elution requirement and hydrophobic padding of preparative hydrophobic interaction chromatogram, Peak A and Peak B whose prurities were aboved 95% were prepared, which layed the foundation of subsequent analysis of structure, effectiveness and stability of TNFR-Fc fusion protein structural isoforms.Secondly, we analysed roundly the structure characteristics of the two structural isoforms Peak A and Peak B through the methods of analytical hydrophobic interaction chromatogram, technology of dynamic laser light scattering, analytical size exclusion chromatography, polyacrylamide gel electrophoresis, the HPLC analysis of N- glycosyl labeled by 2-AB, means of resorcinol, peptide mapping, far ultraviolet and near ultraviolet scanning with circular dichroism spectrum, X-ray crystallography. The main achievements were that:(1) The same points about structure of Peak A and Peak B were that:their N-glycoforms and corresponding proportions were basically the same; their amino acid sequences were the same; their proportions of secondary structures were basically the same. (2) The non-core differences in the structure of Peak A and Peak B were that:the molecular particle size and the degree of inhomogeneity of Peak B was larger than Peak A; the two structural isoforms were different in O-glycosylation. (3) It was knew that the core difference on structure was the link way of disulfides and the sites of disulfides scrambling are in the area of TNFR. (4) We got the crystal of structural isoforms Peak A and Peak B, and collected the diffraction data of Peak A preliminarily.Finally, we analysed the effectiveness and stability of structural isoforms Peak A and Peak B through the methods of bioactivity detection, the analysis of heat stability, the analysis of melting temperature, and the cold model experiment which could analyse the effect of different oxidation and reducing substances on the two structural isoforms. The main achievements were that:(1) The structure change of Peak B had led to the lose of bioactivity. (2) It seemed that Peak B was more stable on structure, but in the process of gradient increased temperature, Peak B stayed in the banlance of interchange of secondary structure. (3) In the mixed solution the structure of Peak A and Peak B stayed in the dynamic adjustment and balance when the cysteine could drive the balance towards the side of Peak A, but after Peak A was removed, the cysteine had no influence on the Peak B while after Peak B was removed, the cysteine could destroy the native conformation of Peak A, and turn it to be Peak B. (4) The culture condition of 20 mmol/L H2O2 and 10 μmol/L Cu2+ could induce the fast degradation of Peak A and Peak B while other oxidation and reducing substances had no prominent effect on the degradation of Peak A and Peak B, but under the temperature of 30 ℃, both protein could form incomplete protein and it was easy for Peak B to form aggregation. The contents of incomplete protein and aggregation both gradully increased over time.The work of this article deepened the cognizance of the relationship between the structure and function of TNFR-Fc, layed the foundation of the subsequent complete structure analysis of TNFR-Fc fusion protein, provided theoretical foundation for the latter establishment of methods of quality control, and provided reference for quality analysis of other protein drugs.