GB1570971A - Stabilised liquid enzyme and coenzyme compositions and method of preparing same - Google Patents

Abstract

Labile coenzymes such as nicotinamide adenine dinucleotide (NAD) and/or enzymes are stabilised by treatment with an organic solvent in aqueous medium while the pH is adjusted within a range of 6.0 to 8.5. An azide may be added to the solution, not only as bacteriostatic agent, but also as stabilising agent. A second coenzyme, such as adenosine triphosphate, may also be stabilised in the same solution. One or more enzymes, such as hexokinase or glucose-6-phosphate dehydrogenase, may also be stabilised against denaturation in the same solution. The process yields a very stable composition which can be used in assays for biological diagnosis.

Description

(54) STABILISED LIQUID ENZYME AND COENZYME COMPOSITIONS AND METHOD OF PREPARING SAME (71) I, IVAN ENDRE MODROVICH, a citizen of the United States of America, of 1043 Mesa Drive, Camarillo, California 93010, United States of America, do hereby declare the invention for which I pray that a patent may be granted to me, and the method by which it is to be performed, to be particularly described in and by the following statement:- This invention relates to stabilised liquid enzyme, coenzyme or enzyme and coenzyme compositions and to methods of stabilising such compositions.

The present commercial state of the art used for stabilising the reactive ability of enzymes or coenzymes is by locking them into a solid matrix, either by freeze drying, dry blending such as used for tableting dried powders, primarily in the pharmaceutical diagnostic and related industries and immobilisation by locking the chemical structure of the enzyme into a solid matrix. Contrary to the sophistication these terms imply, these approaches are neither practical nor desirable and are also expensive. The manufacturer is forced to remove the water and supply a partial product, thus relinquishing part of the quality control cycle in the dilution and use of the final product. Laboratories are forced to pay the high cost of packaging, reagent waste, freeze drying and dry blending, and usefulness of the produce is further limited by packaging modes and sizes.

Furthermore, good product uniformity is difficult to achieve. This condition is exemplified by the fact that most commercial freeze dried control sera (reference serum) list the acceptable bottle-to-bottle variation of enzyme constituents at +100/, of the mean.

Labile enzymes and coenzymes which are treated according to the invention have long-term stability without the enzymatic or coenzymatic reactivity or photometric absorptivity being affected. Providing enzyme and coenzyme reagents in a stable liquid form enhances the colorimetric applicability of present day NAD/NADH coupled methodologies, as well as other methodologies, primarily because the separation of ingredients is easily accomplished. Stable liquid reagents are especially advantageous where NADH and other coenzyme consumption is the basis of measurement and the colour reagent must be separated from NADH and the reaction main. In the ultraviolet mode, the liquid system offers better reagent homogeneity and packaging, as well as flexibility in usage, in contrast to the freezedried or dry media preparations.

The liquid media which is designed to provide for stabilisation of enzymes and coenzymes as hereinafter described may be formulated so that one or more coenzymes may be stabilised in the media. Otherwise, one or more enzymes may be stabilised in the liquid media. Moreover, both coenzymes and enzymes may be stabilised in the same liquid media in a single container.

Stabilisation of the enzymes and/or coenzymes may be accomplished by dissolving a polymer, such as gelatin, in distilled water. The gelatin is preferably dissolved on a 0. 1% w/w basis. Thereafter, the solubilised gelatin in water is heated to about 300 to fully dissolve the gelatin. In some cases, an azide compound may be used, which not only serves as a bacteriostat, but as a stabiliser as well.

Thereafter, this solution is cooled down essentially to room temperature, or about 20"C.

In one case, the coenzyme, nicotinamide-adenine dinucleotide (NAD), is added to the solution, along with a buffering agent, such as tris(hydroxymethyl)aminomethane, for purposes of adjusting the pH. In this case, the pH is adjusted approximately between about 6.0 to about 8.5 with a preferred pH of 7.5. After the addition of the coenzyme, a polyol, such as glycerol, is added on about a 30% v/v basis. After addition of the polyol, the pH may again be adjusted to about 7.5.

In accordance with the present invention, more than one coenzyme may be stabilized in the above mentioned solution. In this case, the other of the coenzymes could be added prior to or after the addition of the NAD. For example, in one embodiment of the present invention, adenosin triphosphate (ATP) may be added as the other coenzyme.

After the addition of the coenzymes and the adjustment of the pH of the liquid, an enzyme, such as hexokinase (HK), may also be added. Typically, the hexokinase would be added from a suspension, such as a glycerol suspension, or an ammonium sulfate suspension. Another enzyme may also be added, as for example, glucose - 6 - phosphate dehydrogenase.

After the liquid stabilized enzyme, coenzyme or enzyme and coenzyme solution is prepared, it is then preferably dispensed into amber-glass bottles and which are sealed in an air-tight condition. Moreover, these bottles are typically stored under refrigeration. The projected shelf life of the stabilized enzymes, coenzymes or enzyme and coenzyme compositions is up to four years under these conditions without appreciable degradation.

In the clinical dignostic field, the commercial application of the present invention is represented by, but not limited to, the diagnostic reagents used to determine substrate concentration, as for example, glucose concentrations in biological fluids, and the like. Nevertheless, compositions prepared in accordance with the present invention can be used to determine and estimate other biological constituents, as for example, the following constituents in biological fluids: 1. Glutamic-oxalacetic transaminase (SGOT) 2. Glutamic-pyruvic transaminase (SGPT) 3. Lactic dehydrogenase (LDH-P) 4. Lactic dehydrogenase (LDH-L) 5. Creatine Phosphokinase (CPK) 6. a-hydroxybutyric dehydrogenase (cr-HBD) 7. Glucose (via hexokinase-G-6-PDH).

These above-identified reagents often react similarly, contain some common labile ingredients, and some of the chemical reactions involved are common. The following chemical reaction scheme is presented as a model to illustrate the general nature of the reactions involved: Reaction Scheme 1-General Model

Enzyme I (1) Substrate (S) ' Product Product (S) pH Enzyme 2 (2) Product/Substrate+NAD-NADH2 NADH7NAD+Product pH Catalyst (3) NADH2+Chromogen ) Chromogen+NAD (oxidized) ( (reduced) All enzymatic reactions listed above, in accordance with this invention, will follow this general scheme, where reaction (2) is usually referred to as the coupling reaction, reactions (2) or (3) are the measuring reactions, and reaction (1) may be characterized as the primary reaction. It is understood, however, that not all three reactions are required for measurement; in fact, they may be limited to two, or one.

In the case of the ultraviolet measurement of lactic dehydrogenase (LD) activity, only reaction (2) is involved, as follows: Reaction Scheme 2-LDH

LDH Lactate+NAD < NADH2+Pyruvate Conversely, more than the three reactions listed may be involved, as in the case of Creatine phosphokinase (CPK): Reaction Scheme 3-CPK

CPK (1) GP+ADP . ATP+Creatine HK (2) ATP+Glucose = ~~~~~ Glucose-6-Phos.+ADP G-6-PDH (3) Glucose-6-Phos.+NAD ~~~~~~~~~ ( -PDH2 NADH2 PMS (4) NADH2+INT z INT+NAD (ox) (red) Symbols: CP=Creatine phosphate CPK=Creatine phosphokinase ADP=Adenosine-5'-diphosphate AM=Adenosine monophosphate ATP=Adenosine triphosphate HK=Hexokinase NAD=Nicotinamide-adenine dinucleotide NADP=Nicotinamide-adenine dinucleotide phosphate NADH2=Nicotinamide-adenine dinucleotide, reduced GLDH=Glutamate dehydrogenase G-6-PDH=Glucose-6-phosphate dehydrogenase G-6-P=Glucose-6-phosphate INT=Tetrazolium salt PEP=Phosphoenol pyruvate PMS=Pherazine methosulfate PK=Pyruvate kinase.

In this case, reactions (2) and (3) may be considered the coupling reactions, reactions (3) or (4) the measuring reactions, and reaction (1) the primary reaction.

Referring to Reaction Scheme 1-General Model, it becomes obvious and is general knowledge that the use of the reaction sequence permits the analytical estimation of either the reaction substrates/products or the catalyzing enzymes.

The estimation of these constituents in biological fluids is a well accepted and widely used diagnostic tool in diagnosis and treatment of human and animal disease states.

Enzymes are large molecular weight, complex protein molecules, usually of unknown chemical structure. They are presently classified by their catalytic activity and extreme substrate specificity. Enzymes may be redefined as biological catalysts, capable of catalyzing a reaction of a single substrate, or a reaction of a similar group of substrates.

Coenzymes are lower molecular weight organic chemicals of well-defined structure, whose reactions or interactions are necessary for specific enzyme assay or reaction. The coenzymes are catalvzed resulting in a reversible change in the coenzyme's structure and/or atomic composition. Coenzymes are very useful in clinical assay procedure. Some have strong absorbance, their reactions are stoichiometric with the substrate and, therefore, the creation or disappearance of the absorbing form can be followed photometrically. Nicotinamide-adenine dinucleotide (NAD) and its reduced form (NADH2) are used in many important clinical assays such as the S.G.O.T., S/P.G.T. and LDH assays previously described. NAD and NADH2 have a molecular weight of about 700 and are very complex organic molecules. NADH, absorbs strongly at 340 nm, whereas NAD does not absorb at this wavelength.

Substrates are organic chemicals of known structure, whose reactions or interactions are catalyzed by enzymes resulting in a change in the compound's structure, atomic composition, or stereo-chemical rotation. In general, substrates are prone to microbiological degradation as they serve as food for bacteria, fungi, and other microorganisms. Otherwise, these compounds remain stable in aqueous media at or near neutral pH (i.e., pH range of 4--10). Typical substrates are glucose, lactate or lactic acid, and gluconate.

The following reactions illustrate the determination of glucose by utilization of the coenzymes ATP and NAD.

HK Glucose+ATP = G-6-P+ADP G-6-PDH G-6-P+NAD -- NADH+6-Phosphogluconic acid The enzyme which causes the primary reaction is hexokinase, and the enzyme which causes the coupling and measuring reaction is G-6-PDH. In the above reaction, the glucose is determined by measuring the NADH which is formed in the measuring reaction. In essence, the reaction is permitted to go to completion, and the amount of the coenzyme NADH formed is essentially measured.

NAD, while being unstable in water and in dry form when exposed to humid environments, is not nearly as unstable as that reduced from NADH2. Accordingly, the NADHz must be kept free of moisture, whereas the NAD may be packaged in a container with an aqueous solution, although stabilized in accordance with the present invention. Stability is better in an acid pH, whereas in an alkaline pH, there is a tendency for the NAD to decompose. Neither the exact mechanism, nor the end products, are of significance, except that the decomposed NAD can no longer effectively function as a coenzyme, nor does it necessarily possess the coefficient at the necessary wavelength.

The compounds ADP, ATP, and AMP are chemically in the form of nucleotides. However, these compounds AMP and ADP and ATP are often referred to herein as coenzymes or cofactors even though they are classically nucleotides. Thus, the ADP, ATP and AMP will be referred to as coenzymes or cofactors herein to conform to nomenclature often used for these compounds and since they do in fact constitute an integral and important part of a coenzyme structure.

In the stabilization of coenzymes, such as NAD, it has been observed that the NAD is far more stable than the NADH. Consequently, it is not necessary to use the complex stabilization techniques necessary for NADH. Accordingly, all reagents can be packaged in one solution.

In stabilizing the enzymes and coenzymes of the present invention, a polymer, such as a gelatin, is dissolved in distilled water. The polymer is preferably present in the stabilized solution up to an amount that remains in homogeneous suspension under refrigeration without precipitation. The polymer should be present in an amount from of at least about 0.01 ,/ and normally about 0.01% to about 0.5% based on the total composition, and preferably within a range of 0.05% to about 0.25%. Any water-soluble polymers which are useful as stabilizing agents in this invention are those which do not inhibit enzymatic activity and are capable of entrapping the enzyme in the polymer matrix. The polymer may be a synthetic or organic material, such as polyvinyl pyrrolidine or dextran of biologic origin, such as gelatin which is denatured collagen.

The polymer may be dissolved in the water by heating, generally to above 30"C. The rate in which the polymer is dissolved will increase with an increase in temperature.

After the polymer has been completely dissolved in water, an azide compound, such as sodium azide, may be added, preferably in amount of about 0.1% w/w.

However, the amount of azide compound which is added can range from 0.01 Sn to about 0.5. It has been found in accordance with the present invention that the azide compound exhibits the rather surprising result of aiding in the stability of the enzymes. Previously, it was only thought that the azide compound served as a bacteriostatic agent. Nevertheless, while the complete mechanism of stabilization with the azide compound, in combination with the other ingredients, is not fully understood, it has been established that the azide compound does, nevertheless, provide increased stability. In many cases, the azide salt is not necessary and can be eliminated. Thus, in many cases, the polymer and organic solvent in the aqueous media are sufficient to provide the required stabilization of the labile components.

In some few cases, the azide salt must be eliminated inasmuch as it may have a tendency to interfere with stabilization, or otherwise materially affect a substrate, as for example, glucose.

In additon to the foregoing, other bactericidal or other fungicidal agents which do not chemically react with a substrate or inhibit the enzymatic reaction may be employed. For example, some of these agents which may be used in addition to sodium azide are benzoic acid, phenol, thymol or pentachlorophenol.

In some cases, it may be desirable to employ a metal, such as magnesium, which aids in initiation of a reaction when the stabilized composition is used.

Magnesium, in the salt form of magnesium chloride is one of the preferred agents for this purpose. This agent does not not have to be incorporated in the stabilized composition of the present invention and if used, it may be added at the time of use or incorporated during preparation. This agent which activates the coupling enzymes should be used in an amount of about 0.01% to about 1% and preferably about 0.03%.

At this point in the process, the solution may be cooled to about room temperature, such as about 20"C to about 25"C in a water bath. After the solution has been cooled, a buffering agent, such as tris(hydroxymethyl) aminomethane may be added. Typically, this buffering agent is added in an amount of about 50 millimoles to about 200 millimoles, but at least sufficient to maintain the pH within a range of 6.0 to about 8.5. Other known buffering agents and other forms of buffering agents may also be employed in the process. In some cases, buffer salts of the type hereinafter described may be used. The buffer salt is adde'd ih an amount necessary to maintain the pH between 6.0 to 8.5. Generally, the buffer is a combination of .1-1% of an alkali metal hydroxide and 0.5 to 3% of an alkali metal acid carbonate or phosphate. The total salt content also affects the amount of polymer required. At higher salt content, e.g. above 4% by weight, less polymer is required due to the electrostatic stabilization provided by the salt. However, at higher salt content, the polymer may cloud the solution or precipitate requiring warming the solution to redissolve.

After the pH of the solution has been adjusted to the desired range, the first of the coenzymes, such as the ATP or the NAD, etc., may be added. In this case, the ATP is added on a basis of about 0.3 millimoles to about 30 millimoles, based on the total composition.

As indicated previously, it is possible to form solutions of both stabilized enzymes and coenzymes. Thus, two or more coenzymes and two or more enzymes may be stabilized in the same solution. For example, the coenzyme ATP may be stabilized in the manner as described herein. On the other hand, the NAD may also be stabilized individually in the manner as described herein. Nevertheless, when stabilizing two or more coenzymes, the coenzymes may generally be added simultaneously or in any order. The NAD is preferably added in a range of about .6 millimole to about 60 millimoles, based on the total composition.

At this point in the process, the pH should again be adjusted to at least within the range of 6.5 to about 8.0 or less, and, preferably, to 7.5.

After adjustment of the pH, a suitable organic solvent, such as glycercol, may be added. In this case, it is added within the range of 25% to 40% v/v, although, in the most preferred aspect, at least 30% v/v of the organic solvent is added.

However, the amount of organic solvent should be at least 5% v/v and could range from about 5% to 70% v/v.

The organic solvent should have the following characteristics: 1. pH range of 4 to 10; 2. Liquid at room and refrigerator temperatures; 3. Does not react with NAD or ATP and the like other than forming electrostatic (i.e., hydrogen) bonds 4. Miscible with water; 5. Standard free energy of solvolysis is low (normal resonance is established).

The solvent must be miscible with water, liquid at room and refrigerator temperatures, and non-degradatively reactive with reactive sites of the enzymes and coenzymes other than formation of electrostatic bonds. Useful solvents are generally stable organic solvents such as ethers, ketones, sulfones, sulfoxides and alcohols such as methanol, ethanol, propanol, butanol, acetone, dioxane, dimethylsulphoxide, dimethylsulfone and tetrahydrofuran. However, higher activity at lower solvent concentration for the treatment step is found for liquid polyol solvents. Liquid polyols containing from 2 hydroxyl groups and 2-10 carbon atoms are preferred, such as glycerol, ethylene glycol, propylene glycol or butane diol. Glycerol, propylene glycol, 1,2 - propanediol, were found to possess all these qualities and are the solvents of choice.

When the selected organic solvent is a polyol, it is not necessary to use the azide compound, or, for that matter, other bacteriostatic agents, since the polyol effectively functions as a bacteriostatic agent. Nevertheless, while the selected solvent and the polymer provide the required stability in an aqueous solution, the azide compound is sometimes preferable, inasmuch as it appears to increase the coupling between the polymer and the enzymes.

After the glycerol or other polyol is added, the pH of the solution thus formed is readjusted. Typically, the pH may be slightly basic and, therefore, a 1 normal HCI can be added in order to adjust the pH. In like manner, if the pH is slightly acidic, then a suitable base may be added to achieve a pH of 7.5.

One of the important aspects is that the coenzyme NAD is present in excessive amounts. As indicated, the determination of glucose is accomplished by measuring the NADH which is formed from the NAD. The NADH is unstable in an acidic environment and will degrade at a pH of 6 and, even more so, will degrade extremely rapidly at a pH of 4. The pH of the solution is therefore maintained above a neutral pH of 7. While the NAD is actually more stable in the acid environment, it has been found in accordance with the present invention that it does not materially degrade in a slightly basic environment of a pH of 7.5.

Nevertheless, the NAD is added in considerable excess so that there is always sufficient undegraded NAD present, even after several years in this liquid environment.

There is typically no maximum amount of coenzyme present although the maximum amount will be limited by commercial practicalities and considerations of coenzyme inhibition of an assay.

After the coenzymes have been added to the liquid solution, the selected enzymes may be added. As with the case of the coenzymes, the enzymes may be added in any order. Again, one or more enzymes may be added to the solution. In the preferred aspect of the invention, and in accordance with the enzyme system identified above, the two enzymes are HK and G-6-PDH. The HK is also preferably added in no less than 111 I.U. per liter (pH of 7.6, 25"C). However, it is preferable to add at least 1,000 I.U. per liter of the HK.

The G-6-PDH should, preferably, be formed from the L-mesenteroides bacteria, and should be concentrated in a range of about 100 I.U. per liter to about 30,000 I.U. per liter or above. In the preferred aspect of the invention, it is normally about 3000 I.U. of the G-6-PDH of this type which is used at a pH of about 7.8 at 25"C.

The enzymes, when present, must each be present in an amount of at least 100 I.U. (International Units) per liter, although in most commercial reagents, the enzyme, as for example, the hexoinase, should be present at about a minimum of 1000 I.U. per liter. In the normal commercial packages, the enzyme is present in about 1000 to about 10,000 I.U. at a pH of 7.6 and at a temperature of about 25"C However, the maximum amount of the enzyme is unlimited, although, normally, in almost all applications the amount of enzyme will not exceed 100,000 I.U.

It is important in the process of the present invention that the enzymes are added after the final pH is adjusted. While the full mechanism for accomplishing the stabilization of the enzymes and coenzymes is not fully understood, it is believed that the selected solvent stabilizes the enzyme in the liquid media by protecting the functional group site, that is the part of the molecule where a substrate reaction may actually occur, or is otherwise catalyzed. Moreover, stabilization is believed to occur by protecting the enzymes and coenzymes from microbial contamination and thus degradation. The coenzyme NAD differs from the coenzyme NADH in that the NAD will not appreciably dissolve in the selected solvent, such as propyleneglycol. However, the NAD is more stable in water and the coenzyme does appear to be stabilized by the polyol. A pure polyol will denature the enzymes, but in the presence of an aqueous solution, such as watersolvent mixture, the enzymes do not denature. Apparently, a polar group is required in the organic solvent to maintain the active sites of the enzymes in a stable condition. Obviously, some form of physical or chemical reaction occurs in the concentrated aqueous-organic solvent media, inasmuch as the enzymes and coenzymes retain catalytic activity and do not degrade in the specified concentrations.

In addition to the above, the polymer appears to react in some fashion with the azide compound in order to form an electrostatic or covalent bond between the enzymes and the polymer. In essence, it may also appear that the polymer may stretch to somewhat encapsulate, and thereby protect, the active sites of the enzymes. In this way, enzyme denaturation or other form of degradation is inhibited or does not occur.

As indicated above it is possible to stabilize at least two or more encoenzymes or at least two or more enzymes in the same solution. Moreover, and more importantly, it is possible to stabilize both enzymes and coenzymes in the same solution. It is believed that the aqueous solution of the organic solvent is the primary factor in stabilizing the coenzyme although the polymer does appear to provide some stabilizing effect. In stabilization of the enzyme, the organic solvent and the polymer appear to be the primary factors resulting in stabilization. In addition, and in many cases the azide salt aids in increasing stabilization. In either case, it can be observed that both enzymes and coetizymes are still in fhe same single solution.

Some of the additional reactions which have been performed with the stabilized enzyme and coenzyme compositions are set forth below. The reaction involving the phospholation of creatine is:

CPK Creatine+ATP ' CP+ADP [pH 891 The remaining reactions are all self explanatory with reference to the list of symbols set forth above. For an NADP reaction,

G-6-PDH G-6-P+NADP ' NADPH+6-Phosphogluconic acid for an ADP reaction.

CPK CP+ADP , Creatine+ATP [pH 6-7] For the following reaction the starting reaction of creatine+ATP would be employed to provide the ADP. Thereafter,

PK ADP+PEP = ATP+Pyruvate LDH Pyruvate+NADH Lactate+NAD The following reactions show the use of urease and GLDH enzymes in the stabilized liquid compositions.

Urease Urea ~~~~~~~~~~ ( 2NH 4+CO2 2NH+4+CO2 [pH 6-8] GLDH NH4+cx-Ketoglutarate+NAD ( Glutamate+NADH The invention is further illustrated by, but not limited to, the following Examples: EXAMPLE I About 0.7 grams of a gelatin polymer is added to about 700 milliliters of water.

This solution is then heated above 30"C in order to dissolve the gelatin polymer.

After the addition of the polymer the solution is inserted in a water bath in order to reduce the temperature to about 220 C.

The pH is then adjusted within the range of 6.5 to 8.0. After the temperature has been reduced and pH adjusted, about 2.0 grams of ATP is added to the solution, which is in turn followed by 4.0 grams of NAD. Three grams of a magnesium chloride salt is added along with the NAD. Three hundered ml glycerol is then added.

After the addition of the glycerol, the pH is adjusted to about 7.5 by the addition of 1 normal hydrochloric acid.

After complete solution is attained, the solution is added to a plastic or glass container, which is then closed. The containers are sealed and stored under refrigeration. It has been found that a stabilized coenzyme composition in this manner provides a storage stability of up to four years without significant degradation.

EXAMPLE II The sample produced in accordance with Example I is provided with the enzyme hexokinase prior to sealing in the glass container. The same shelf life is obtained without significant degradation.

EXAMPLE III The sample of Example II is also provided with the enzyme G-6-PDH prior to sealing in the glass container and the same long shelf life without significant degradation is obtained.

EXAMPLE IV About 1.0 grams of a dextran polymer is added to about 700 milliliters of water. This solution is then heated above 30"C in order to dissolve the polymer.

The sol

EXAMPLE VI Stabilized ADP, AMP, NAD, HK and G-6-PDH About 300 I.U. to about 15,000 I.U. per liter of G-6-PDH and about 100 I.U. to about 10,000 I.U. per liter of HK is added to the solution of Example V prior to packaging thereof.

EXAMPLE VII 1.5 grams of NAD Dissolve in 5 ml water Add 5 ml of glycerol Adjust pH to less than 5.

EXAMPLE VIII Stabilization of NAD and HK 1.5 grams of NAD Dissolve in 10 ml of pH 7 buffer of 0.1 molar PIPES* buffer Adjust pH to 6 to 7 Add 10 ml of glycerol Readjust pH Add and dissolve 10 mg HK of activity of 150 I.U. per milligram.

*PIPES=Piperazine[bis] ethane sulfonic acid.

EXAMPLE IX Stabilization of creatine, ATP and PEP 1000 ml of water Add 12.1 grams of tris(hydroxymethyl) aminomethane Add 1.0 grams gelatin Dissolve with heat above 30"C Cool to room temperature Add 2.0 grams ATP Add 2 grams PEP Add 10.0 grams creatine Dissolve and adjust pH to 9.

EXAMPLE X To the stabilized solution of Example IX, 100 I.U./liter to 10,000 I.U./liter of LDH was added and 100 I.U./liter to 10,000 I.U./liter of PK was added, prior to packaging.

Each of the compositions of Examples V through X have the same long shelf life without any substantial degradation. Moreover, each of the above examples are based on samples actually prepared and tested in accordance with the present invention.

WHAT I CLAIM IS: 1. A stabilized liquid enzyme, coenzyme or enzyme and coenzyme composition for use in biological diagnostic determinations and with enzyme and/or coenzyme affect the reactivity of a biological constituent in a biological diagnostic determination, said enzyme and/or coenzyme being normally unstable in an aqueous media, said composition comprising: (a) an aqueous vehicle; (b) at least a sufficient amount of coenzyme to cooperate in a biological diagnostic determination with said enzyme to perform said determination when said coenzyme is present and said coenzyme being dissolved in said aqueous vehicle; (c) at least 100 I.U. of enzyme being dissolved in said aqueous vehicle when an enzyme is present and which enzyme is primarily effective in affecting reactivity of said biological constituent; (d) a non-reactive aqueous miscible organic solvent present in an amount of about 5% v/v to about 70 /" v/v in said aqueous vehicle and which solvent is liquid at least at room temperature, the organic solvent being non-degradatively reactive with the reactive sites of the enzymes when present or coenzymes when present except to form electrostatic bonds therewith, and where activity of the enzyme and/or the coenzyme remains unaffected by the presence of the organic solvent in the stabilized composition and in a determination reaction;

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (45)

**WARNING** start of CLMS field may overlap end of DESC **. EXAMPLE VI Stabilized ADP, AMP, NAD, HK and G-6-PDH About 300 I.U. to about 15,000 I.U. per liter of G-6-PDH and about 100 I.U. to about 10,000 I.U. per liter of HK is added to the solution of Example V prior to packaging thereof. EXAMPLE VII 1.5 grams of NAD Dissolve in 5 ml water Add 5 ml of glycerol Adjust pH to less than 5. EXAMPLE VIII Stabilization of NAD and HK 1.5 grams of NAD Dissolve in 10 ml of pH 7 buffer of 0.1 molar PIPES* buffer Adjust pH to 6 to 7 Add 10 ml of glycerol Readjust pH Add and dissolve 10 mg HK of activity of 150 I.U. per milligram. *PIPES=Piperazine[bis] ethane sulfonic acid. EXAMPLE IX Stabilization of creatine, ATP and PEP 1000 ml of water Add 12.1 grams of tris(hydroxymethyl) aminomethane Add 1.0 grams gelatin Dissolve with heat above 30"C Cool to room temperature Add 2.0 grams ATP Add 2 grams PEP Add 10.0 grams creatine Dissolve and adjust pH to 9. EXAMPLE X To the stabilized solution of Example IX, 100 I.U./liter to 10,000 I.U./liter of LDH was added and 100 I.U./liter to 10,000 I.U./liter of PK was added, prior to packaging. Each of the compositions of Examples V through X have the same long shelf life without any substantial degradation. Moreover, each of the above examples are based on samples actually prepared and tested in accordance with the present invention. WHAT I CLAIM IS:

1. A stabilized liquid enzyme, coenzyme or enzyme and coenzyme composition for use in biological diagnostic determinations and with enzyme and/or coenzyme affect the reactivity of a biological constituent in a biological diagnostic determination, said enzyme and/or coenzyme being normally unstable in an aqueous media, said composition comprising: (a) an aqueous vehicle; (b) at least a sufficient amount of coenzyme to cooperate in a biological diagnostic determination with said enzyme to perform said determination when said coenzyme is present and said coenzyme being dissolved in said aqueous vehicle; (c) at least 100 I.U. of enzyme being dissolved in said aqueous vehicle when an enzyme is present and which enzyme is primarily effective in affecting reactivity of said biological constituent; (d) a non-reactive aqueous miscible organic solvent present in an amount of about 5% v/v to about 70 /" v/v in said aqueous vehicle and which solvent is liquid at least at room temperature, the organic solvent being non-degradatively reactive with the reactive sites of the enzymes when present or coenzymes when present except to form electrostatic bonds therewith, and where activity of the enzyme and/or the coenzyme remains unaffected by the presence of the organic solvent in the stabilized composition and in a determination reaction;

(e) and said composition having a pH from about 6.0 to about 8.5, such that the enzyme and/or coenzyme are stabilized; (f) said enzyme being glucose-6-phosphate dehydrogenase, hexokinase, glutamate dehydrogenase, creatine phosphokinase, pyruvate kinase and/or alkaline phosphatase, and/or said coenzyme being selected from the class consisting of nicotinamide-adenine dinucleotide, adenosine triphosphate, adenosine - 5' diphosphate, nicotinamide-adenine dinucleotide phosphate, and/or adenosine monophosphate.

2. The stabilized liquid enzyme and/or coenzyme composition of Claim I for use in biological diagnostic determinations of creatine phosphokinase or glucose, said coenzyme when present being a first coenzyme of nicotinamide-adenine dinucleotide, nicotinamide-adenine dinucleotide reduced, and nicotinamideadenine dinucleotide phosphate and a second coenzyme of adenosine - 5' diphosphate, adenosine monophosphate and adenosine triphosphate to perform a determination of creatine phosphokinase of glucose, said enzyme when present being glucose - 6 - phosphate dehydrogenase and hexokinase.

3. A stabilised liquid enzyme and coenzyme composition for use in biological dignostic determinations and which enzyme and coenzyme are normally unstable in an aqueous media, said composition comprising: a) an aqueous vehicle, b) at least a sufficient amount of coenzyme to perform a determination dissolved in said aqueous vehicle, .c) at least 100 I.U. of enzyme dissolved in said aqueous vehicle and both said enzyme and coenzyme cooperating in a determination reaction, d) at least 5% v/v of a non-reactive aqueous miscible organic solvent in said aqueous vehicle and which is liquid at least at room temperature, e) and said composition having a pH from about 6.0 to about 8.5, such that the enzyme and coenzyme are stabilised, f) said enzyme being glucose - 6 - phosphate dehydrogenase, hexokinase, glutamate dehydrogenase, creatine phosphokinase, pyruvate kinase and/or alkali phosphatase, and said coenzyme being nicotinamide-adenine dinucleotide, adenosin triphosphate, adenosin - 5'- diphosphate, nicotinamide-adenine dinucleotide phosphate, and/or adenosin monophosphate.

4. A stabilised liquid composition according to Claim 3 in which said composition comprises a first labile coenzyme and at least one second labile coenzyme which is also stabilised by at least said organic solvent.

5. A stabilised liquid composition according to Claim 3 or 4 in which said composition comprises a water soluble polymer which does not substantially inhibit enzymatic activity.

6. A stabilised liquid composition according to Claim 5 in which said composition comprises a first labile enzyme and at least one second labile enzyme which is also stabilised by at least said solvent and said polymer.

7. A stabilised liquid composition according to any of Claims 1 to 6 in which composition comprises a bacteriostat which provides stabilisation as well as providing bacteriostatic action.

8. A stabilised liquid composition according to Claim 7 in which the bacteriostat is an azide compound.

9. A stabilised liquid composition according to any of Claims 1 to 8 in which said solvent has the following characteristics: a) pH of 4 to 10; b) Liquid at room and refrigerator temperatures; c) Does not react with the coenzymes or enzymes other than forming electrostatic (i.e. hydrogen) bonds; d) Miscible with water; e) Standard free energy of solvolysis is low (normal resonance. is established).

10. A stabilised liquid composition according to any of Claims I to 9 in which said composition comprises at least two coenzymes and at least two enzymes.

11. A stabilised liquid coenzyme composition for use in biological determinations which coenzyme is normally unstable in an aqueous media, said composition comprising: a) at least 30% v/v of a non-reactive aqueous vehicle.

b) at least a sufficient amount of coenzyme to perform a determination dissolved in said aqueous vehicle and cooperating in a determination reaction, c) an aqueous miscible organic solvent in said aqueous vehicle and which is liquid at least at room temperature, and d) said composition having a pH of from about 6.0 to about 8.5, such that the coenzyme is stabilised, e) said coenzyme being nicotinamide-adenine dinucleotide, adenosin triphosphate, adenosin - 5' - diphosphate, creatine phosphokinase, nicotinamideadenine dinucleotide phosphate, and/or adenosin monophosphate.

12. A stabilised liquid coenzyme composition according to Claim 11 in which said composition comprises a water soluble polymer which does not substantially inhibit enzymatic activity and that said composition also comprises a labile second coenzyme which is also stabilised by at least said organic solvent or said polymer.

13. A stabilised liquid coenzyme composition according to Claim 11 or 12 in which said composition comprises a bacteriostat which provides stabilisation as well as providing bacteriostatic action.

14. A stabilised liquid coenzyme composition according to any of Claims 11 to 13 in which the organic solvent is non-reactive with said coenzyme and aqueous vehicle at room and refrigerator temperatures.

15. A stabilised liquid coenzyme composition accordng to any of Claims 11 to 14 in which the coenzyme is selected from the class consisting of NAD, ATP, ADP, CK, CP and NADP.

16. A stabilised liquid coenzyme composition according to any of Claims 11 to 14 in which the coenzyme is NAD with a concentration of above 1.2 grams per litre of liquid composition.

17. A stabilised liquid coenzyme composition according to any of Claims 11 to 16 in which said organic solvent has the following characteristics: a) pH between 4 to 10; b) Liquid at rrom and refrigerator temperatures; c) Does not react with coenzymes other than forming electrostatic (i.e.

hydrogen) bonds; d) Miscible with water; e) Standard free energy of solvolysis is low (normal resonance is established).

18. A stabilised liquid coenzyme composition according to Claim 17 in which the organic solvent is a polyol.

19. A stabilised liquid enzyme composition for use in biological determinations and which enzyme is unstable in an aqueous media, said composition comprising: a) an aqueous vehicle, b) at least 100 I.U. of enzyme dissolved in said aqueous vehicle, c) a non-reactive aqueous miscible organic solvent in said aqueous vehicle and which is liquid at least at room temperature, d) a water soluble polymer which does not substantially inhibit enzymatic activity, and e) a bacteriostat which provides stabilisation as well as providing bacteriostatic action, f) said enzyme being glucose-6-phosphate dehydrogenase, hexokinase, glutamate dehydrogenase, creatine phosphokinase, pyruvate kinase and/or alkali phosphatase.

20. A stabilised liquid enzyme composition according to Claim 19 in which said composition comprises a labile second enzyme which is also stabilised by at least said organic solvent or said polymer.

21. A stabilised liquid enzyme composition according to Claim 19 or 20 in which the bacteriostat is an azide compound.

22. A stabilised liquid enzyme composition according to any of Claims 19 to 21 in which said organic solvent has the following characteristics: a) pH between 4 to 10; b) Liquid at room and refrigerator temperatures; c) Does not react with enzymes other than forming electrostatic (i.e.

hydrogen) bonds; d) Miscible with water; e) Standard free energy of solvolysis is low (normal resonance is established).

23. A stabilised liquid enzyme composition according to Claim 22 in which said organic solvent is non-reactive with said enzyme and aqueous vehicle at room and refrigerator temperatures.

24. A stabilised liquid enzyme composition according to any of Claims 19 to 23 in which the enzyme is selected from the class consisting of G-6-PDH, HK, GLDH, CK, PK and PEP.

25. A method of stabilising a labile coenzyme and labile enzyme for use in biological diagnostic determinations and which enzyme and coenzyme are normally unstable in aqueous media, said method comprising: a) mixing water with an aqueous miscible non-reactive organic solvent to form an aqueous miscible organic solvent solution and which organic solvent is liquid at least at room temperature, b) dissolving a polymer in the aqueous miscible organic solvent solution, c) adding at least a sufficient amount per litre of a labile coenzyme to said solution to perform a determination and which is dissolved in said solution and cooperates in a determination reaction, d) adjusting the pH to within the range of 6.0 to 8.5, such that the coenzyme is stabilised, e) adding at least 100 I.U. per litre of a labile enzyme to said solution, and which enzyme is dissolved in said solution and cooperates in a determination reaction, and f) sealing the composition, g) said enzyme being glucose-6-phosphate dehydrogenase, hexokinase, glutamate dehydrogenase, creatine phosphokinase, pyruvate kinase and/or alkali phosphatase, and said coenzyme being nicotinamide-adenine dinucleotide, adenosin triphosphate, adenosin - 5'- diphosphate, nicotinamide-adenine dinucleotide phosphate, and/or adenosin monophosphate.

26. A method according to Claim 25 in which said method comprises adding a bacteriostatic agent which also functions as an enzyme stabilising agent.

27. A method according to Claim 26 in which said bacteriostatic agent is an azide compound. ' ~~ ''

28. A method according to any of Claims 25 to 27 in which said method also comprises adding a second coenzyme to said solution which is also stabilised therein.

29. A method according to any of Claims 25 to 27 in which said method also comprises adding a second enzyme to said solution which is also stabilised therein, after adjustment of the pH.

30. A method according to any of Claims 25 to 30 in which said solvent has the following characteristics: a) pH between 4 to 10; b) Liquid at room and refrigerator temperatures; c) Does not react with the enzymes or coenzymes other than forming electrostatic (i.e. hydrogen) bonds; d) Miscible with water; e) Standard free energy of solvolysis is low (normal resonance is established).

31. A method according to Claim 30 in which the organic solvent is a polyol which contains 2 1 hydroxyl groups and 2-10 carbon atoms.

32. A method according to any of Claims 25 to 31 in which said solvent is added so that it is present in an amount of about 25% to about 40% by volume.

33. A method according to any of Claims 25 to 32 in which said polymer is present in an amount of at least 0.01%.

34. A method of stabilising a labile coenzyme for use in biological diagnostic determinations and which coenzyme is normally unstable in an aqueous media, said 'method comprising: a) dissolving a coenzyme in an aqueous base in an amount sufficient to perform a determination and which coenzyme cooperates in a determination reaction, b) contacting said coenzyme containing aqueous base with at least 5% by volume of a non-reactive aqueous miscible organic solvent to provide a stabilised composition and which solvent is liquid at least at room temperature, c) adjusting the composition of pH to about 6.0 to about 8.5 such that the coenzyme is stabilised, d) said coenzyme being nicotinamide-adenine dinucleotide, adenosin triphosphate, adenosin - 5' - diphosphate, creatine phosphokinase, nicotinamideadenine dinucleotide phosphate, and/or adenosin monophosphate, e) sealing the composition in a container.

35. A method according to Claim 34 in which said method comprises dissolving a water soluble polymer in said composition which does not substantially inhibit enzymatic activity.

36. A method according to Claim 34 or 35 in which said method also comprises adding a second enzyme to said solution which is also stabilised therein.

37. A method according to any of Claims 34 to 36 in which said solvent has the following characteristics: a) pH between 4 to 10; b) Liquid at room and refrigerator temperatures; c) Does not react with coenzymes other than forming electrostatic (i.e.

hydrogen) bonds; d) Miscible with water; e) Standard free energy of solvolysis is low (normal resonance is established).

38. A method according to any of Claims 34 to 37 in which said solvent is added so that it is present in an amount of about 25% to about 50% by volume.

39. A method according to Claim 37 or 38 in which the solvent is a liquid polyol containing from 2-10 carbon atoms and 2--4 hydroxyl groups.

40. A method according to any of Claims 35 to 39 in which the polymer is gelatin present in said solution in an amount of at least 0.01%

41. A method of stabilising a labile enzyme for use in biological diagnostic determinations and which enzyme is normally unstable in an aqueous media, said method comprising: a) contacting water with an aqueous miscible organic solvent to form a solution thereof and which organic solvent is liquid at least at room temperature, b) adding at least 0.01% of a water soluble polymer to said solution, c) adding a bacteriostatic agent which also functions as an enzyme stabilising agent to said solution, d) dissolving at least 100 I.U. per litre of enzyme to said solution to form the composition, and which enzyme cooperates in a determination reaction, e) said enzyme being glucose-6-phosphate dehydrogenase, hexokinase, glutamate dehydrogenase, creatine phosphokinase, pyruvate kinase and/or alkali phosphatase, f) and sealing the composition.

42. A method according to Claim 41 in which said bacteriostatic agent is an azide compound.

43. A method according to Claim 41 or 42 in which said method also comprises adding a second enzyme to said solution which is also stabilised therein.

44. A stabilised liquid enzyme, coenzyme or enzyme and coenzyme composition substantially as herein described.

45. A method of stabilising a liquid enzyme coenzyme or enzyme and coenzyme composition substantially as herein described.