Stabilization of proteins in solution

• US 6,133,229 A
• Filed: 07/01/1996
• Issued: 10/17/2000
• Est. Priority Date: 10/08/1993
• Status: Expired due to Term

• Current Assignee: University of Leeds
• Original Assignee: University of Leeds
• Inventors: Webster, Jeanette Irene, Gibson, Timothy David, Pierce, Barry L.
• Primary Examiner(s): CELSA, BENNETT M
• Application Number:
  US08/624,585
• Time in Patent Office: 1,569 Days
• Field of Search: 514/2, 514/12, 514/21, 435/188, 525/54.1, 530/408, 530/410
• US Class Current: 435/188
• CPC Class Codes: C07K 1/00 General methods for the preparation of peptides , i.e. processes for the organic chemical preparation of peptides or proteins of any length
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Abstract

A protein stabilizer additive comprises two or more of a tris compound of the formula (1): (HOCH₂)₃ --C--R, wherein R is: C₁ -C₄ alkyl, substituted C₁ -C₄ alkyl, NH₂ ; NR¹ R² wherein R¹ and R² may be independently: H, C₁ -C₄ alkyl sulphonate, C₁ -C₄ hydroxyalkyl sulphonate; C₁ -C₄ alkyl NHC(CH₂ OH)₃, C₁ -C₄ alkyl, C₁ -C₄ hydroxyalkyl; C₁ -C₄ alkyl carboxylate; a polyelectrolyte; a buffer; and one or more additional components for example divalent metal salts.

Claims

1. A method of stabilizing an aqueous enzyme comprising the step of contacting the enzyme selected from the group consisting of peroxidase enzymes, alkaline phosphatase enzymes and antibody conjugates thereof with an aqueous solution of a protein stabilizer additive comprising:

a. a tris(hydroxymethyl)methyl compound of formula 1;

space="preserve" listing-type="equation">(HOCH.sub.2).sub.3 C--R (1)

wherein R is;

NH₂ ;

NR¹ R² wherein R¹ and R² may be independently;

H, C₁ -C₄ alkyl, C₁ -C₄ hydroxyalkyl, C₁ -C₄ alkyl sulfonate, C₁ -C₄ hydroxyalkyl sulphonate;

C₁ -C₄ alkyl NHC(CH₂ OH)₃ ;

C₁ -C₄ alkyl carboxylate;

b. a cationic polyelectrolyte selected from the group consisting of copolymers of vinyl pyrollidone and quaternary methyl methacrylate and DEAE dextran;

c. a polyol selected from the group consisting of glycerol, ethylene glycol, sorbitol and lactitol; and

d. one or more salts selected from the group consisting of salt of calcium, magnesium and cobalt.

2. A method as claimed in claim 1 wherein the tris(hydroxymethyl)) methyl compound is selected from the group consisting of:

tris(hydroxymethyl)aminomethane and salts thereof selected from the group consisting of chloride, maleate, phosphate, succinate salts;

1,3-bis{tris(hydroxymethyl)methylamino}propane;

bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane;

N-{tris(hydroxymethyl)methyl}-2-aminoethane sulfonate;

N-{tris(hydroxymethyl)methyl}-3-aminopropane sulfonate;

N-{tris(hydroxymethyl)methyl}-3-amino-2-hydroxypropane sulfonate; and

N-{tris(hydroxymethyl)methyl}-glycine.

3. A method as claimed in claim 1 wherein the cationic polyelectrolyte has a molecular weight of 150 to 5,000,000 Daltons.

4. A method as claimed in claim 1 wherein the cationic polyelectrolyte is present in an amount of 0.01 to 10% w/v.

5. A method as claimed in claim 1 wherein the polyol is present in an amount of 0.1 to 25% w/v.

6. A method as claimed in claim 1 wherein said peroxidase enzyme is selected from the group consisting of horseradish peroxidase and arthromyces peroxidase.

7. A method as claimed in claim 2, wherein the tris(hydroxymethyl) compound is selected from the group consisting of tris(hydroxymethyl)aminomethane and salts thereof selected from the group consisting of chloride, maleates, phosphate, succinate salts;

1,3-bis{tris(hydroxymethyl)methylamino}propane; and

bis(2-hydroxyethyl)aminotris(hydroxymethyl)) methane.

8. A method as claimed in claim 1, wherein said enzyme is horseradish peroxidase, said tris is 20 mM tris/HCL buffer pH 8.0, said polyol is ethylene glycol, said cationic polyelectrolyte is DEAE-dextran (MW 500K), and said salt is calcium chloride.

9. A method as claimed in claim 8, wherein at least 99% of the enzyme'"'"'s activity is retained after 33 days at 37°

C.