Preparation method and application of high-purity plant-derived recombinant human serum albumin

A method for preparing high-purity recombinant human serum albumin from Oryza sativa (OsrHSA), comprising the following steps of:

• (a) preparing a crude or unpurified extract of OsrHSA;
• (b) subjecting the crude extract of OsrHSA to cation exchange chromatography (A) comprising a cation exchange column, whereby OsrHSA binds to the cation exchange column,
• (c) providing:
• a first washing buffer comprising between about 10-20% absolute isopropanol by volume,
• an equilibration buffer I comprising between about 0-10% absolute isopropanol by volume, and
• an equilibration buffer II comprising between about 5-15% absolute isopropanol by volume,
• (d) washing the cation exchange column with the first washing buffer, then washing the column with equilibration buffer I, then washing the column with equilibration buffer II,
• (e) eluting OsrHSA from the cation exchange chromatography, thereby obtaining a primary product I;
• wherein the cation exchange chromatography comprises a cationic/hydrophobic composite resin;
• (f) subjecting the primary product I to anion exchange chromatography B comprising an anion exchange column, to obtain an intermediate product II,
• and the parameters of the anion exchange chromatography B comprise:
• (i) adding or loading onto to the anion exchange column a loading sample under conditions comprising:
• pH 8.0, conductivity between about 1 to 5 mS/cm;
• (ii) adding or loading onto to the anion exchange column a washing buffer under conditions comprising:
• pH 8.0, conductivity between about 9 to 9.5 mS/cm;
• (iii) adding or loading onto to the anion exchange column an elution buffer comprising:
• pH 8.0, conductivity between about 30 to 50 mS/cm;
• thereby eluting off the anion exchange column the intermediate product II,
• wherein the anion exchange chromatography B comprising an anion exchange column comprises use of the following buffers:
• an equilibration buffer comprising:
• between about 10 to 20 mM phosphate buffer (PB), between about 7.9 to 8.1 pH, conductivity between about 1 to 3 mS/cm;
• a re-equilibration buffer comprising:
• the same as the equilibration buffer;
• a second washing buffer comprising:
• between about 10 to 20 mM PB, between about 150 to 170 mM NaCl, between about 7.9 to 8.1, between about 9 to 9.5 mS/cm conductivity;
• an elution buffer comprising:
• between about 10 to 20 mM PB, between about 420 to 460 mM NaCl, between about pH 7.9 to 8.1, between about 41 to 43 mS/cm conductivity;
• and
• (g) subjecting the intermediate product II to hydrophobic chromatography (C) comprising use of a hydrophobic column to obtain the high-purity OsrHSA, comprising:
• loading the intermediate product II onto the hydrophobic column in a buffer comprising between about 10 to 20 mM PB, between about 500 to 600 mM (NH4)2SO4, between about 1 and 3 g/L, or about 2 g/L sodium octanoate, between about pH 6.5 to 6.6,
• washing the hydrophobic column by loading the second washing buffer onto the hydrophobic column,
• and eluting high-purity OsrHSA from the hydrophobic column by loading an eluting buffer onto the hydrophobic column, thereby generating the high-purity OsrHSA,
• and the hydrophobic chromatography uses a hydrophobic resin.