	US 12,215,139 B2
	Preparation method and application of high-purity plant-derived recombinant human serum albumin
Daichang Yang, Wuhan (CN); Quan Zhan, Wuhan (CN); Jiquan Ou, Wuhan (CN); Wenhui Yu, Wuhan (CN); and Zhijie Qin, Wuhan (CN)
Assigned to WUHAN HEALTHGEN BIOTECHNOLOGY CORP, Wuhan (CN)
Filed by WUHAN HEALTHGEN BIOTECHNOLOGY CORP., Wuhan (CN)
Filed on Nov. 22, 2023, as Appl. No. 18/518,227.
Prior Publication US 2024/0262887 A1, Aug. 8, 2024
Int. Cl. C07K 14/765 (2006.01); A61K 38/00 (2006.01); A61K 45/06 (2006.01); A61K 47/64 (2017.01); A61P 3/00 (2006.01); C07K 1/18 (2006.01); C07K 1/20 (2006.01)

CPC C07K 14/765 (2013.01) [A61K 45/06 (2013.01); A61K 47/643 (2017.08); A61P 3/00 (2018.01); C07K 1/18 (2013.01); C07K 1/20 (2013.01); A61K 38/00 (2013.01)]

14 Claims

1. 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,

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,

wherein the anion exchange chromatography B comprises a composite resin-comprising a matrix having a pore size of about 60 μm and a flow rate of between about 150-750 cm/h;

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 loading the intermediate product II in a chromatography buffer onto a hydrophobic column,

washing the hydrophobic column by loading the second washing buffer onto the hydrophobic column, and

eluting the high-purity OsrHSA by loading an eluting buffer onto the hydrophobic column, thereby generating the high-purity OsrHSA,

and the hydrophobic chromatography comprises use of a hydrophobic resin.