Preparation And Quality Control Of RHSA-IFNβ

Interferon beta (IFN β) has antiviral, antitumor, antiproliferative and immunomod-ulatory effects. It’s mainly used for hepatitis B, hepatitis C, multiple sclerosis, as well as avariety of tumor treatment in clinical. Having a short half-life in vivo, so we need to developIFN β long-acting protein drugs.We constructed the gene of Human Serum Albumin and interferon β fusion protein(rhHSA-IFNβ) and then established the process of fermentation and purification in ourlaboratory. To meet the pre-clinical drug evaluation, the quality of the target protein is need tobe identified.In this study, Pichia yeast strains were induced to express rHSA-IFN β fusion protein in30L fermenter. The fermentation broth was concentrated by ultrafiltration and then bluedextran gel chromatography, G25gel filtration chromatography and SP Sepharosechromatography. Determined by HPLC,the purity of product was above95%,as well as29%for the protein recovery rate.There is no quality standards for rHSA-IFN β fusion proteins currently. The hole items inthis study were tested according to the requirements of recombinant human interferon α2b(yeast) dope test in Chinese Pharmacopoeia（Volumes Ⅲ，2010edition）.The relative molecular mass of fusion protein were89070,88669and88836byMALDI-TOF/TOF mass spectrometry determination. The SDS-PAGE electrophoresis bandsafter cutted sugar chains with glycosidase F was lower, proving that the fusion protein wereglycosylation. Edman degradation method was used for the determination of its N-terminalamino acid sequence. The N-terminal amino acids sequence of fusion protein was as follows:NH2-DAHKS,while isoelectric point was6.34by isoelectric focusing electrophoresisdetermination.The fusion protein showed the antigenicity of interferon beta and human albumin byWestern blotting. Trypsin cleavage-(RP-HPLC) was used for peptide maps analysis of thefusion protein.After trypsin digestion, rHSA-IFNβ fusion protein was cleaved into fourpeptides,which repeated three times showed the same results.Therefore this method is suitablefor peptide mapping analysis of rHSA-IFNβ, indicating the stability of product quality. Theproduct maximum absorption wavelength was278nm scaned by UV spectroscopy.The bacterial endotoxin content was less than10EU every1mg fusion protein by the gellimits of LAL test. Enzyme-linked immunosorbent assay results showed that Pichia yeastprotein residues is less than0.05%accounted for the fusion protein. Fluorescent stainingmethod determined that1mg fusion protein had exogenous DNA residues less than10ng/mL,meeting the quality requirements.The fusion protein activity was tested according to IFNα2b active detection methods inChinese Pharmacopoeia（2010edition）and IFN β activity detection methods in the UnitedStates Pharmacopoeia. Three batches of fusion protein activity were1.9×106IU/mg,1.5×106IU/mg and1.1×106IU/mg.