Formulation of Stable Recombinant Alpha-Fetoprotein Conjugated with Anti-Tumor Substance in Target-Delivery System for Treatment of Cancer and Autoimmune Disease

摘要

Disclosed is a stable formulation based on rAFP conjugated with drug delivery system of various classes of pharmacological agents such as dactinomycin, cardionolide, and bufadinolide, among others. In another embodiment, the invention discloses a method of producing rAFP in a strain of methylotrophic Pichia pastoris yeast, allowing the yeast to replicate, and extracting the newly created rAFP. The method provides a nonpyrogenic, highly stable, recombinant, fully refolded rAFP and its fragments that are free from stable folding intermediates caused by non-native disulfide bridges and irreversible aggregates. The newly created rAFP is purified using a two-step chromatographic purification process in the presence of 0.5% polysorbate-20 and 10 mM inosine that prevents aggregation of the monomers of the target protein, increasing the yield of biologically active rAFP. The invented formulation of rAFP is conjugated with active pharmacological substances or target drug delivery system for prevention or treatment of oncological and autoimmune-diseases.

主权项

1) A method for increasing production and purification of rAFP, comprising the steps of:
introducing rAFP into a methylotrophic strain of Pichia pastoris yeast; allowing said yeast to replicate; extracting newly created rAFP; precipitating said newly created rAFP using an ammonium sulfate solution to form a precipitate; purifying said precipitate using a chromatographic purification process in the presence of an inosine solution and a polysorbate-20 solution; conducting gel filtration in a first ammonium acetate buffer containing said inosine solution and said polysorbate-20 solution; conducting ion-exchange chromatography on DEAE-C; conducting elution of the target protein by gradient of concentration of a sodium chloride solution in said first ammonium sulfate buffer containing said inosine solution and said polysorbate-20 solution.