# US 6,294,365 B1 · Method and formulation for stabilization of enzymes

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- Number: US 6,294,365 B1
- Document ID: US6294365B1
- Status: Granted
- Kind: B1
- Application number: 09/164,060
- Filed: 1998-09-30
- Granted: 2001-09-25
- Earliest claimed: 1996-07-03
- Assignees: Molecular Biology Resources, Inc.
- Inventors: Therese A. De Rosier; Norberto B. de la Cruz; Richard K. Wilkosz
- Web page: https://patentgrep.com/patent/US6294365B1

## CPC

- C12N 9/127: {RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase}
- C12N 9/1276: {RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase}
- C12N 9/16: acting on ester bonds (3.1)
- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention is directed to a method and a formulation for the stabilization of an enzyme for storage at temperatures above freezing. Specifically, the invention involves the use of formulated excipients that when added to an enzyme or mixture of enzymes and subsequently lyophilized renders stability. Upon reconstitution, the stabilized materials are useful in assays, diagnostic or molecular biology research kits, and other biological applications.

## Claims

### Claim 1

A method for stabilizing one or more enzymes, or enzymatically active fragments thereof, for storage in the dry phase above freezing temperature, the method comprising:
(i) combining said one or more enzymes or enzymatically active fragment thereof with an aqueous stabilizing solution comprising in combination a carrier protein, from about 3% to about 15% w/v of one or more disaccharides, from about 1% to about 10% w/v of one or more disaccharide derivatives, from about 0% to about 10% w/v of one or more sugar polymers, and a compatible buffer thereby producing a mixture;
(ii) freezing the mixture obtained in step (i); and
(iii) lyophilizing the frozen mixture obtained in step (ii) until substantially water-free, whereby a stabilized enzyme formulation is obtained.

### Claim 2 (depends on 1)

The method of claim 1 wherein the carrier protein is bovine serum albumin.

### Claim 3 (depends on 2)

The method of claim 2 wherein the concentration of bovine serum albumin is about 3 mg/ml.

### Claim 4 (depends on 1)

The method of claim 1 wherein the disaccharide derivative is maltitol.

### Claim 5 (depends on 1)

The method of claim 1 wherein the disaccharide is trehalose.

### Claim 6 (depends on 5)

The method of claim 5 wherein trehalose is present at from about 6% to about 10% (w/v).

### Claim 7 (depends on 1)

The method of claim 1 wherein the disaccharide derivative is selected from the group consisting of maltitol, mannitol, corn syrup solids, sorbitol and branched polymers of sucrose.

### Claim 8 (depends on 7)

The method of claim 7 wherein the disaccharide derivative is maltitol.

### Claim 9 (depends on 8)

The method of claim 8 wherein maltitol is present at from about 3% to about 10% (w/v).

### Claim 10 (depends on 1)

The method of claim 1 wherein the sugar polymer is dextran.

### Claim 11 (depends on 10)

The method of claim 10 wherein dextran is present at from about 2% to about 10% (w/v).

### Claim 12 (depends on 1)

The method of claim 1 wherein the buffer is selected from the group consisting of Tris buffers, HEPES buffers, phosphate buffers and combinations thereof.

### Claim 13 (depends on 12)

The method of claim 12 wherein the buffer consists essentially of 2.92 mM potassium phosphate pH 7.2, 4.14 mM potassium phosphate pH 7.5, 1.28 mM Tris-HCl pH 7.5, 20.7 mM NaCl, 19.2 mM KCl, 0.51 mM dithiothreitol, 0.66 mM EDTA, 1.28 mM Mg-Acetate, and 0.03% of a non-ionic surfactant.

### Claim 14

A mixture for use in producing an aqueous formulation suitable for lyophilization, comprising:
(i) a carrier protein;
(ii) from about 3% to about 15% (w/v) of one or more disaccharides;
(iii) from about 1% to about 10% (w/v) of one or more disaccharide derivatives;
(iv) from about 0% to about 10% (w/v) of a sugar polymer;
(v) a compatible buffer; and
(vi) one or more enzymes.

### Claim 15 (depends on 14)

The mixture of claim 14 wherein the carrier protein is bovine serum albumin.

### Claim 16 (depends on 15)

The mixture of claim 15 wherein the concentration of bovine serum albumin is about 3 mg/ml.

### Claim 17 (depends on 14)

The mixture of claim 14 wherein the disaccharide is selected from the group consisting of trehalose and sucrose.

### Claim 18 (depends on 17)

The mixture of claim 17 wherein the disaccharide is trehalose.

### Claim 19 (depends on 18)

The mixture of claim 18 wherein trehalose is present at from about 6% to about 10% (w/v).

### Claim 20 (depends on 14)

The mixture of claim 14 wherein the disaccharide derivative is selected from the group consisting of maltitol, mannitol, corn syrup solids, sorbitol and branched polymers of sucrose.

### Claim 21 (depends on 20)

The mixture of claim 20 wherein the disaccharide derivative is maltitol.

### Claim 22 (depends on 21)

The mixture of claim 21 wherein maltitol is present at from about 3% to about 10% (w/v).

### Claim 23 (depends on 14)

The mixture of claim 14 wherein the sugar polymer is dextran.

### Claim 24 (depends on 23)

The mixture of claim 23 wherein dextran is present at from about 2% to about 10% (w/v).

### Claim 25 (depends on 14)

The mixture of claim 14 wherein the buffer is selected from the group consisting of Tris buffers, HEPES buffers, phosphate buffers and combinations thereof.

### Claim 26 (depends on 25)

The mixture of claim 25 wherein the buffer consists essentially of 2.92 mM potassium phosphate pH 7.2, 4.14 mM potassium phosphate pH 7.5, 1.28 mM Tris-HCl pH 7.5, 20.7 mM NaCl, 19.2 mM KCl, 0.51 mM dithiothreitol, 0.66 mM EDTA, 1.28 mM Mg-Acetate, and 0.03% of a non-ionic surfactant.

### Claim 27 (depends on 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)

The mixture of any one of claims 14-26 further comprising an aqueous phase.

### Claim 28

A stabilized mixture of one or more enzymes, or enzymatically active fragments thereof, produced by a method comprising:
(i) combining one or more enzymes, or enzymatically active fragments thereof, with an aqueous stabilizing solution comprising in combination a carrier protein, from about 1% to about 10% (w/v) of one or more disaccharide derivatives, from about 3% to about 15% (w/v) of one or more disaccharides, from about 0% to about 10% of one or more simple sugar polymers, and a compatible buffer, thereby producing a mixture;
(ii) freezing said mixture; and
(iii) lyophilizing said frozen mixture until substantially water-free, whereby a stabilized mixture of said one or more enzymes is obtained.

### Claim 29 (depends on 28)

The stabilized mixture of claim 28 wherein the carrier protein is bovine serum albumin.

### Claim 30 (depends on 29)

The stabilized mixture of claim 29 wherein the concentration of bovine serum albumin is about 3 mg/ml.

### Claim 31 (depends on 28)

The stabilized mixture of claim 28 wherein the disaccharide is selected from the group consisting of trehalose and sucrose.

### Claim 32 (depends on 31)

The stabilized mixture of claim 31 wherein the disaccharide is trehalose.

### Claim 33 (depends on 32)

The stabilized mixture of claim 32 wherein trehalose is present at from about 6% to about 10% (w/v).

### Claim 34 (depends on 31)

The stabilized mixture of claim 31 wherein the sugar polymer is dextran.

### Claim 35 (depends on 34)

The stabilized mixture of claim 34 wherein dextran is present at from about 2% to about 10% (w/v).

### Claim 36 (depends on 28)

The stabilized mixture of claim 28 wherein the disaccharide derivative is selected from the group consisting of maltitol, mannitol, corn syrup solids, sorbitol and branched polymers of sucrose.

### Claim 37 (depends on 36)

The stabilized mixture of claim 36 wherein the disaccharide derivative is maltitol.

### Claim 38 (depends on 37)

The stabilized mixture of claim 37 wherein maltitol is present at from about 3% to about 10% (w/v).

### Claim 39 (depends on 28)

The stabilized mixture of claim 28 wherein the buffer is selected from the group consisting of Tris buffers, HEPES buffers, phosphate buffers and combinations thereof.

### Claim 40 (depends on 39)

The stabilized mixture of claim 39 wherein the buffer consists essentially of 2.92 mM potassium phosphate pH 7.2, 4.14 mM potassium phosphate pH 7.5, 1.28 mM Tris-HCl pH 7.5, 20.7 mM NaCl, 19.2 mM KCl, 0.51 mM dithiothreitol, 0.66 mM EDTA, 1.28 mM Mg-Acetate, and 0.03% of a non-ionic surfactant.

### Claim 41

An aqueous enzyme formulation useful for stabilizing one or more enzymes, or enzymatically active fragments thereof, produced by a method comprising combining with an aqueous phase:
(i) a carrier protein;
(ii) from about 1% to about 10% (w/v) of one or more disaccharide derivatives;
(iii) from about 3% to about 15% (w/v) of one or more disaccharides;
(iv) from about 0% to about 10% of one or more simple sugar polymers;
(v) a compatible buffer; and
(vi) one or more enzymes, or enzymatically active fragments thereof.

### Claim 42 (depends on 41)

The formulation of claim 41 wherein the carrier protein is bovine serum albumin.

### Claim 43 (depends on 42)

The formulation of claim 42 wherein the concentration of bovine serum albumin is about 3 mg/ml.

### Claim 44 (depends on 41)

The formulation of claim 41 wherein the disaccharide is selected from the group consisting of trehalose and sucrose.

### Claim 45 (depends on 44)

The formulation of claim 44 wherein the disaccharide is trehalose.

### Claim 46 (depends on 45)

The formulation of claim 45 wherein trehalose is present at from about 6% to about 10% (w/v).

### Claim 47 (depends on 44)

The formulation of claim 44 wherein the disaccharide derivative is maltitol.

### Claim 48 (depends on 47)

The formulation of claim 47 wherein maltitol is present at from about 3% to about 10% (w/v).

### Claim 49 (depends on 41)

The formulation of claim 41 wherein the sugar polymer is dextran.

### Claim 50 (depends on 49)

The formulation of claim 49 wherein dextran is present at from about 2% to about 10% (w/v).

### Claim 51 (depends on 41)

The formulation of claim 41 wherein the buffer is selected from the group consisting of Tris buffers, HEPES buffers, phosphate buffers and combinations thereof.

### Claim 52 (depends on 51)

The formulation of claim 51 wherein the buffer consists essentially of 2.92 mM potassium phosphate pH 7.2, 4.14 mM potassium phosphate pH 7.5, 1.28 mM Tris-HCl pH 7.5, 20.7 mM NaCl, 19.2 mM KCl, 0.51 mM dithiothreitol, 0.66 mM EDTA, 1.28 mM Mg-Acetate, and 0.03% of a non-ionic surfactant.

### Claim 53 (depends on 41)

The aqueous formulation of claim 41 wherein the disaccharide derivative is selected from the group consisting of maltitol, mannitol, corn syrup solids, sorbitol and branched polymers of sucrose.

### Claim 54

A stabilized, substantially water-free mixture of one or more enzymes or enzymatically active fragments thereof comprising:
(i) a carrier protein;
(ii) from about 3% to about 15% (w/v) of one or more disaccharides;
(iii) from about 1% to about 10% (w/v) of one or more disaccharide derivatives;
(iv) from 0% to about 10% (w/v) of a sugar polymer;
(v) a compatible buffer; and
(vi) one or more enzymes.

### Claim 55 (depends on 54)

The stabilized mixture of claim 54 wherein the carrier protein is bovine serum albumin.

### Claim 56 (depends on 55)

The stabilized mixture of claim 55 wherein the concentration of bovine serum albumin is about 3 mg/ml.

### Claim 57 (depends on 54)

The stabilized mixture of claim 54 wherein the disaccharide is selected from the group consisting of trehalose and sucrose.

### Claim 58 (depends on 57)

The stabilized mixture of claim 57 wherein the disaccharide is trehalose.

### Claim 59 (depends on 58)

The stabilized mixture of claim 58 wherein trehalose is present at from about 6% to about 10% (w/v).

### Claim 60 (depends on 57)

The stabilized mixture of claim 57 wherein the sugar polymer is dextran.

### Claim 61 (depends on 60)

The stabilized mixture of claim 60 wherein dextran is present at from about 2% to about 10% (w/v).

### Claim 62 (depends on 54)

The stabilized mixture of claim 54 wherein the disaccharide derivative is selected from the group consisting of maltitol, mannitol, corn syrup solids, sorbitol and branched polymers of sucrose.

### Claim 63 (depends on 62)

The stabilized mixture of claim 62 wherein the disaccharide derivative is maltitol.

### Claim 64 (depends on 63)

The stabilized mixture of claim 63 wherein maltitol is present at from about 3% to about 10% (w/v).

### Claim 65 (depends on 54)

The stabilized mixture of claim 54 wherein the compatible buffer comprises a buffer selected from the group consisting of Tris buffers, HEPES buffers, phosphate buffers and combinations thereof .

### Claim 66 (depends on 65)

The stabilized mixture of claim 65 wherein the compatible buffer consists essentially of 2.92 mM potassium phosphate pH 7.2, 4.14 mM potassium phosphate pH 7.5, 1.28 mM Tris-HCl pH 7.5, 20.7 mM NaCl, 19.2 mM KCl, 0.51 mM dithiothreitol, 0.66mM EDTA, 1.28 mM Mg-Acetate, and 0.03% of a non-ionic surfactant.