Method for purifying protein

Patent metadata

Field	Value
Publication number	US-9428544-B2
Application number	US-201214347903-A
Country	US
Kind code	B2
Filing date	Sep 28, 2012
Priority date	Sep 30, 2011
Publication date	Aug 30, 2016
Grant date	Aug 30, 2016

Abstract

It is an object of the present invention to provide a method for eluting an adsorbed protein that suppresses a decrease in protein adsorption ability, in a method for purifying a protein using a protein-adsorbing porous membrane.
The present invention provides a method for purifying a protein, comprising an adsorption step and an elution step, wherein in the elution step, at least one eluent is passed in the opposite direction with respect to the direction of the passage of a stock solution containing an adsorption target protein, in the adsorption step.

First claim

The invention claimed is:

1. A method for purifying a protein by a porous membrane having a substrate surface coated with a polymer having protein adsorption ability, comprising:
an adsorption step of passing a stock solution containing an adsorption target protein through a pore direction from one surface to another surface of the porous membrane to allow the polymer to adsorb the adsorption target protein; and
an elution step of passing an eluent through the porous membrane to allow the adsorption target protein that is adsorbed on the polymer to elute in the eluent,
wherein in the elution step, at least one eluent is passed in an opposite direction with respect to the pore direction of passage of the stock solution in the adsorption step;
wherein a degree of multilayering of the porous membrane is 1.1 or more, and the degree of multilayering is a value obtained by the following formula (3)
degree of multilayering=(equilibrium adsorption capacity)/(theoretical single-layer adsorption capacity)  (3)

where the equilibrium adsorption capacity is obtained by the following formula (4), when the stock solution comprising the adsorption target protein is passed through the porous membrane

equilibrium
⁢

⁢
adsorption
⁢

⁢

capacity
⁢

[

⁢

g
-

amount
⁢

⁢
of
⁢

⁢
adsorption
⁢

/
g

-
membrane

]

=

∫
0

Q
e

⁢

(

C
0

-
C

)

⁢

ⅆ
Q

W

(
4
)

where C 0 : concentration of protein in stock solution [g/L];
C: protein concentration in passed solution of stock solution [g/L];
Q: cumulative amount of passed solution of stock solution [L];
Q e : amount of passed solution of stock solution when adsorption equilibrium is reached [L];
W: weight of protein-adsorbing porous membrane [g]; and
the theoretical single-layer adsorption capacity is obtained by the following formula (5)
theoretical single-layer adsorption capacity[g−amount of single-layer adsorption/g−membrane]=( S M /S P )( M r /N A )  (5)

where S M : specific surface area of protein-adsorbing porous membrane [m 2 /g];
S P : area occupied by one protein molecule [m 2 ];
M r : molecular weight of BSA [g/mol]; and
N A : Avogadro's number [/mol].

2. The method for purifying a protein according to claim 1 , wherein the eluent is selected from the group consisting of an aqueous solution comprising a salt, an aqueous solution whose pH is adjusted, water, an organic solvent, and a mixed solution thereof.

3. The method for purifying a protein according to claim 1 , wherein in the elution step, the eluent is passed in a forward direction and an opposite direction with respect to the direction of the passage of the stock solution in the adsorption step.

4. The method for purifying a protein according to claim 1 , wherein the polymer is grafted onto the substrate surface, and a degree of grafting of the polymer is 5% or more and 200% or less.

5. The method for purifying a protein according to claim 4 , wherein the degree of grafting of the polymer is 30% or more and 90% or less.

6. The method for purifying a protein according to claim 1 , wherein the porous membrane is an ion exchange membrane, and the eluent comprises an aqueous solution comprising a salt or an aqueous solution whose pH is adjusted.

7. The method for purifying a protein according to claim 6 , wherein the porous membrane is a weakly basic anion exchange membrane or a weakly acidic cation exchange membrane,
the elution step comprises the steps of:
passing an aqueous solution whose pH is adjusted to other than between an isoelectric point of the adsorption target protein and an isoelectric point of the porous membrane, and
passing an aqueous solution comprising a salt,
wherein in either of the steps, the aqueous solution whose pH is adjusted or the aqueous solution comprising a salt is passed in the opposite direction with respect to the direction of the passage of the stock solution in the adsorption step.

8. The method for purifying a protein according to claim 7 , wherein in the step of passing an aqueous solution whose pH is adjusted and the step of passing an aqueous solution comprising a salt, respectively, the aqueous solution whose pH is adjusted and the aqueous solution comprising a salt are passed in the opposite direction with respect to the direction of the passage of the stock solution in the adsorption step.

9. The method for purifying a protein according to claim 6 , wherein the porous membrane is a weakly basic anion exchange membrane or a weakly acidic cation exchange membrane,
the elution step comprises
a first step of passing an aqueous solution comprising a salt,
a second step of passing an aqueous solution whose pH is adjusted to other than between an isoelectric point of the adsorption target protein and an isoelectric point of the porous membrane, and
a third step of passing an aqueous solution comprising a salt,
wherein in the first step, the aqueous solution comprising a salt is passed in the forward direction with respect to the direction of the passage of the stock solution in the

Assignees

• Asahi Kasei Chemicals Corp

Inventors

• Sato Yuta
• Shinohara Naoyuki
• Shirataki Hironobu

Classifications

• C07K1/22 Primary

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• B01J39/046

  Operations & Transport · mapped topic

• C07K1/16

  by chromatography · CPC title

• C07K1/18

  Ion-exchange chromatography · CPC title

• C07K1/14

  Extraction; Separation; Purification · CPC title