Title:  Stabilization of enzymes during freezing

United States Patent:  6,579,707

Issued:  June 17, 2003

Inventors:  Fitzgerald; Stephen Peter (Co. Antrim, GB); Campbell; John (Co. Antrim, GB); Lamont; John Victor (Co. Antrim, GB); King; Marlene (Co. Antrim, GB)

Assignee:  Randox Laboratories Ltd. (Northern Ireland, GB)

Appl. No.:  949757

Filed:  September 12, 2001

A method for stabilizing an enzyme during freezing, wherein the enzyme is in a zwitterionic buffer solution. The zwitterionic buffer is able to maintain the activity of the enzyme during freezing and thawing.

DESCRIPTION OF THE INVENTION

The present invention relies on the use of zwitterionic buffers to prepare the liquid reagent formulations. Zwitterionic buffers are sometimes referred to as "Goods" buffers (Good et al, Biochemistry, 1966; 5:467) and are commercially available. The buffers are generally zwitterionic aliphatic amines, with the majority being either substituted glycines or taurines. The buffers are distinct from the phosphate buffers used in the prior art to stabilise glucose oxidase reagents.

Suitable buffers which may be used in the present invention include Mops (3-[N-Morpholino]propanesulphonic acid), Mopso (3-[N-Morpholino]-2-hydroxypropanesulphonic acid) and Hepes (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulphonic acid]). Each of these buffers is available from commercial sources. Alternative zwitterionic buffers will be apparent to the skilled person.

The preparation of the buffer with the enzyme will be apparent to the skilled person, and the buffer will typically be prepared at a concentration of 20-250 mmol/l. Preferably, the buffer solution will be prepared with a pH of 7.

Although the buffers will prevent inactivation of the enzymes on freezing, it is preferred that the solutions are stored at 4^oC.

The buffers may be used to stabilise any enzyme against the effects of freezing. In a preferred embodiment, the enzyme is glucose oxidase or horse-radish peroxidase. The enzymes may be the only active molecule present in the buffer solution. For example, the solution will not contain triglycerides or cholesterol which are sometimes present in some diagnostic kits which require peroxidase. The solutions may also be free from polysaccharides or oligosaccharides, i.e. sugars.

The following Example illustrates the invention.

EXAMPLE

Four different formulations, containing either Mops, phosphate buffer or a combination of both were prepared according to the formulations described below. Glucose oxidase (EC 1.1.3.4) from Aspergillus niger and horse-radish peroxidase (EC 1.11.1.7) were used. The other components of each formulation were included to promote the colourimetric glucose reaction or to help stabilise the reagents.

Formulation A

50 mmol/l Mops buffer

20 KU/l glucose oxidase

1.6 KU/l Peroxidase

11.0 mmol/l Phenol

0.05% sodium azide

0.03% polyvinylpyrrolidine

0.77 mmol/L 4-amino antipyrine

The pH is adjusted to 7.0 with sodium hydroxide and/or hydrochloric acid.

Formulation B

150 mmol/l Mops buffer

20 KU/l glucose oxidase

1.6 KU/l Peroxidase

11.0 mmol/l Phenol

0.05% sodium azide

0.03% polyvinylpyrrolidine

0.77 mmol/L 4-amino antipyrine

The pH is adjusted to 7.0 with sodium hydroxide and/or hydrochloric acid.

Formulation C

50 mmol/l Mops buffer

19.5 mmol/l sodium dihydrogen phosphate

30.5 mmol/l disodium hydrogen orthophosphate

19.20 KU/l glucose oxidase

1.6 KU/l Peroxidase

11.0 mmol/l Phenol

0.04% sodium azide

0.03% polyvinylpyrrolidine

0.77 mmol/L 4-amino antipyrine

The pH is adjusted to 7.0 with sodium hydroxide and/or hydrochloric acid.

Formulation D (Control without Mops Buffer)

39 mmol/l sodium dihydrogen phosphate

61 mmol/l disodium hydrogen orthophosphate

20 KU/l glucose oxidase

1.6 KU/l Peroxidase

11.0 mmol/l Phenol

0.04% sodium azide

0.03% polyvinylpyrrolidine

0.77 mmol/L 4-amino antipyrine

The pH was adjusted to 7.0 with sodium hydroxide and/or hydrochloric acid.

All of the formulations were frozen at -20^oC. for 2 weeks and the activity of each enzyme measured after thawing. Peroxidase activity was measured using the method of Theorell, Acta Chem. Scand., 1950; 4:22. Glucose oxidase activity was measured using the method of Bergmeyer et al, Methods of Enzymatic Analysis, 1974; 1: 457 (Academic Press).

The results were compared to those obtained with the same reagent stored at 4^oC. for 2 weeks. The absorbance of the reagent was measured at 500 nm and a factor determined from the absorbance, obtained after reacting the reagent with a 100 mmol/l glucose standard.

The formulations were also subjected to conditions of freezing and elevated temperature to mimic the conditions that could result on shipping of the reagent.

The reagents were subjected to two sets of test conditions; storage at either -20^oC. or +37^oC. Each test cycle consisted of 3 days at either -20^oC. or 37^oC., followed by 3 days at the normal storage temperature of 4^oC. The reagents were subjected to 3 cycles for each test condition.

After completion, the stability of each reagent was determined by assessing the linearity and the ability to obtain the correct value for a range of quality control materials.

All glucose and enzyme assays were performed using an automated analyser.

Results

1) Enzyme activity

The concentrations of both glucose oxidase and horse-radish peroxidase were measured in all reagents after 2 weeks at -20^oC. The results are shown in Table 1. Reagent D which did not contain Mops buffer had no detectable glucose oxidase activity present and a decreased level of peroxidase after freezing and thawing. The other 3 reagents containing either solely Mops buffer at 2 different concentrations or Mops and phosphate buffers combined retained their glucose oxidase and peroxidase activity after freezing and thawing.

 

2) Linearity (Tables 2, 3 4)

All formulations when stored at +4^oC. showed linearity to 22 mmol/l of glucose. All formulations containing Mops buffer maintained their linearity of 22 mmol/l after 3 cycles at -20^oC. Formulation D, without Mops buffer, was inactivated after 3 cycles and the glucose concentration could not be determined. All formulations after 3 cycles at +37^oC. maintained their linearity.

 

3) Recovery of glucose in quality control serum (Tables 5, 6 and 7)

The formulations were tested against control serum samples. In the Tables, Precie-Path and Precie-Norm are available from Roche Diagnostics, and the multisera samples are available from Randox Laboratories.

All formulations gave acceptable values when stored at +4^oC. After 3 cycles at -20^oC., all Mops containing formulations gave values within ranges and deviations of less than 3% from the control reagent stored at 4^oC. After 3 cycles at 37^oC., all formulations gave acceptable values for each control serum sample.

 

In summary, the use of Mops buffer as a sole buffer, or in combination with phosphate buffer, maintains the function of the enzymes after freezing and thawing.

Claim 1 of 8 Claims

What is claimed is:

1. A method for stabilising the activity of an enzyme selected from the group consisting of glucose oxidase and horseradish peroxidase during storage, said method comprising storing the enzyme in a zwitterionic buffer solution wherein the enzyme is stored in said zwitterionic buffer solution for at least three days.
 

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