Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Stablizing an enzyme by forming a mixture – an adduct or a...
Reexamination Certificate
2001-09-12
2003-06-17
Meller, Michael V. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Stablizing an enzyme by forming a mixture, an adduct or a...
C435S190000, C435S192000, C435S183000, C424S094300, C424S094400
Reexamination Certificate
active
06579707
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the preparation of stable enzyme formulations. In particular, this invention relates to a method for stabilising an enzyme during freezing.
BACKGROUND TO THE INVENTION
The preferred form for the supply of diagnostic reagents is a liquid. Liquid reagents require no preparation by the user, and therefore there is less risk of error.
If the reagents are to be prepared in a liquid form, the maintenance of stability is a major concern as it will usually be necessary to store and transport the reagents.
Examples of reagents which are bought commercially in a liquid form are those that include the enzymes glucose oxidase and horse-radish peroxidase, required for the measurement of glucose using a calorimetric method known as the Trinder method (Barham and Trinder, Analyst, 1972; 97:142). It is necessary to stabilise both the glucose oxidase and peroxidase enzymes to ensure that the enzymes function over their shelf-life. Typically, these enzymes are formulated with phosphate or Tris (hydroxymethyl)aminomethane buffers to maintain the pH of the reagent during storage and during the reaction. One difficulty that has been encountered is that the liquid preparations are often frozen during transport or on storage, and in these circumstances the enzymes are inactivated.
It is therefore desirable to provide compositions which effectively stabilise the diagnostic reagents on storage, and which offer further protection if freezing occurs.
U.S. Pat. No. 6,071,706 discloses a composition comprising &agr;GST enzyme and a zwitterionic buffer, which may be stored at −20° C. The purpose is to retain the immuno-reactivity of &agr;GST, and there is no mention of retaining enzymic activity. Horse-radish peroxidase is mentioned, but this is only in the context of an enzyme-labelled anti-&agr;GST IgG, which is used in the immunoassay. Stabilisation of this enzyme in solution is carried out in phosphate-buffered saline.
U.S. Pat. No. 5,910,422 and U.S. Pat. No. 4,465,770 both describe the stabilisation of specific enzymes (&agr;-amylase and urease, respectively) in solution using polysaccharides or long chain oligosaccharides, e.g. sorbitol. A zwitterionic buffer may be provided to aid stabilisation of the enzymes in solution.
Although, in general, there are various methods for the stabilisation of various enzymes in solution, there is still a requirement for a useful method for stabilising enzyme activity, particularly for diagnostic reagents, during and after freezing.
SUMMARY OF THE INVENTION
According to the present invention, a method for stabilising the activity of an enzyme during freezing comprises providing the enzyme in a zwitterionic buffer solution.
The zwitterionic buffer solution has the capacity to effectively stabilise the enzyme in the liquid state and also provide protection if the end solution is frozen, either accidentally or otherwise.
In a preferred embodiment, the enzyme is glucose oxidase or horse-radish peroxidase.
According to a second aspect of the invention, a frozen solution of an enzyme is in a zwitterionic buffer.
Without wishing to be bound by theory, the ability of a zwitterionic buffer to stabilise the activity of enzymes may be a consequence of the ability of the buffers to prevent significant shifts in pH during freezing. It is known that phosphate buffers can cause large pH shifts during freezing (Rose et al Arch. Biochem. Biophys., 1959; 81:319-329), and it is believed that this may cause the denaturation of the enzymes. Typically, in a buffer composition, water will freeze first and ice crystals will grow. As the temperature approaches the eutectic points of the salts that are present, the salts crystallise out. The less soluble salts in a buffer will crystallise out of solution first, and this can cause a drastic change in the pH before the frozen solid is formed. However, a zwitterionic buffer may prevent this shift in pH, and thereby impart stability to the enzyme.
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° C.
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.
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van Os, et al., Analytical Chimica Acta, 335, 1996, 209-216.*
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K. Kobayashi et al., “Isolation of Reductase for SoxR that Governs an Oxidative Response Regulon fromEscherichia coli”, FEBS Letters, vol. 451, No. 3, May 28, 1999, pp. 227-230.
N. Eisaki et al., “Pyruvate Phosphate Dikinase from Thermophilic Actinomyces Microbispora Rosea Subsp. Aerata: Purification, Characterization and Molecular Cloning of the Gene”, Biochimica Et Biophysica Acta Protein Structure and Molecular Enzymology, vol. 1431, No. 2, May 18, 1999, pp. 363-373.
Campbell John
Fitzgerald Stephen Peter
King Marlene
Lamont John Victor
Meller Michael V.
Oliff & Berridg,e PLC
Randox Laboratories Ltd.
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