AU700666B2 - Isoenzyme calibrator/control products - Google Patents

Description

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,1.d -I PI DATE 04/09/95 APPLN. ID 18448/95 11 11 AOJP DATE 19/10/95 PCT NUMBER PCT/US95/01927 AU9518448

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(51) International Patent Classification 6: (11) International Publication Number: WO 95/22602 C12N 9/00, 9/96 Al (43) International Publication Date: 24 August 1995 (24,08.95) (21) International Application Number: PCT/US95/01927 (81) Designated States: AU, CA, JP, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, (22) International Filing Date: 17 February 1995 (17.02.95) SE).

Priority Data: Published 08/198,919 18 February 1994 (18.02.94) US With international search report.

(71) Applicant: GENZYME CORPORATION [US/US]; One Kendall Square, Cambridge, MA 02139 (US).

(72) Inventors: MAGNESON, Gerald, 550 Great Plains Avenue, Needham, MA 02192 NAG, Sumatra; 54 West Wood Street, Burlington, MA 01803 (US).

(74) Agents: GOSZ, William, G. et al.; Genzyme Corporation, One Mountain Road, Framingham, MA 01701-9322 (US).

(54) Title: ISOENZYME CALIBRATOR/CONTROL PRODUCTS (57) Abstract The invention relates to buffers used to stabilize isoenzymes with formulations which upon 4 drying and reconstitution recover essentially all of the isoenzyme activity and immunological mass for long q) 3 term shelf-life storage without experiencing dissociation- C and rearrangement of its 52 subunits. The buffer used for the long term dry storage of 0 an isoenzyme is a serum-free and a salt-free aqueous 1 buffer which comprises: 1) a glass-forming sugar; 2) an antioxidant mixture; 3) a pH buffer, and 4) a non-ionic 0 surfactant and/or a synthetic polymer and/or a gelatin.

The buffer used for the reconstitution of a serum-free and salt-free isoenzyme 3.2 3.3 3.4 3.5 3.6 3.7 sample comprises an aqueous1000 TEMPERATURE buffer containing a salt. In addition, a serum-isoenzyme 0 SERUM-FREE MATRIX: 13.1 YEARS TO FAIL AT 5 C is iscosed bffhich cm) a sa; SERUM-BASED MATRIX: 7.6 YEARS TO FAIL AT 5 C an antioxidant mixture; and 4) a non-ionic surfactant and/or a synthetic polymer andlor a gelatin. The figure shows the accelerated stabilir isoenzyme mass in a serum-based and a serum-free aqueous buffer as determined by an arrnenius plot of the days to reach stability failure against the reciprocal of temperature, extrapolating a storage shelf-life at 5 *C.

WO 95/22602 PCT/US95/01927 ISOENZYME CALIBRATOR\CONTROL PRODUCTS Background of the Invention Isoenzymes, different molecular forms of a given enzyme, play an important role in clinical chemistry and immunochemistry in the evaluation of the pathological factors in certain diseases that can lead to the alteration of normal isoenzyme concentration in blood serum. Since different molecular forms of enzymes are formed in different tissues, the serum levels of a particular isoenzyme may be increased or decreased due to the effect that a disease state may have on the rate of release of an isoenzyme into the bloodstream as well as its production and deposition in the tissue. The degree to which the serum level of an isoenzyme is preferentially influenced by a particular disease determines its clinical importance.

It is essential that an isoenzyme present in serum can be readily identified by standard physical chemical means. These physical chemical means include, for example, determination by gel electrophoresis, and inactivation of enzymatic activity in the presence of enzyme inhibitors, or heat treatment. A more sensitive and specific means of detection involves the recognition of specific isoenzyme epitopes that exist due to differences in amino acid composition and/or subunit-subunit interactions using immunochemical procedures.

Furthermore, to enhance its worth as a diagnostic analyte, it is important that sufficient quantities of such an isoenzyme are commercially available in a highly purified, stable format for use as reference material. It is particularly helpful for worldwide standardization of a diagnostic assay to have freeze dry lyophilized primary reference material that can be distributed for storage at 2- 8.C.

An example of a clinically important isoenzyme is creatine kinase a dimeric enzyme consisting of either M or B type subunits which associate to form the major isoenzymes: CK-MM, CK- MB and CK-BB. The MB isoenzyme, which is predominantly produced in the myocardial tissue in the heart and is present in human serum, is a

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WO 95/22602 PCT/US95/01927 useful indicator for the diagnosis of acute myocardial infarction (AMI). Antigen grade CK-MB, which denotes the highly purified isoenzyme formulated in the presence of essentially nonimmunogenic stabilizing substance(s), may be obtained from human heart or recombinant DNA-derived sources. Since antigen grade CK- MB is readily inactivated by air oxidation at 2-8°C and its subunits tend to dissociate and rearrange to form significant amounts of MM and BB isoenzyme on long term storage or after a freeze/thaw event, the prior method for stabilizing human CK-MB was to store it in the presence of 50% glycerol at -70°C (Landt et al., Clin. Chem., 35:985- 989, 1989). Limitations of this formulation include the difficulty in maintaining active enzyme for long term storage and its impracticality for purposes of distribution and storage. Furthermore, glycerol should be removed from CK-MB prior to its use in preparing liquid calibrators in order to reduce interferences in certain clinical chemistry assays or immunoassays, resulting in a partial loss of isoenzyme activity and mass. Additionally, since glycerol does not freeze, its presence would interfere with the freeze dry lyophilization of calibrators or controls supplemented with a glycerol-based CK-MB formulation.

It has been previously reported that isoenzymes formulated in serum-based calibrator\control material can be stabilized in solution and in lyophilized form in the presence of a stabilizing agent (Hoskins, U.S. Patent No. 4,684,615 and 4,883,762, and Li Mutti et al., U.S. Patent No. 4,127,502). However, serum-based isoenzyme reference materials would present various problems due to the many components in serum, including: assay interference by extraneous components; variation between lots; immunogenicity problems; and biohazardous risks viral contamination).

In general, a need exists for a serum-free buffer formulation with a composition that can stabilize isoenzymes for long term storage in both a liquid and/or a dry format. It is a desirable objective that such a formulation maintains the enzymatic activity and protein structure of an isoenzyme during long term dry storage at 2-8°C, as well as preventing the dissociation and rearrangement of its subunits. Furthermore, it also is a desirable objective to develop a serum-free aqueous buffer that stabilizes isoenzymes during long term liquid storage at 2-8°C, while eliminating the biohazards, assay 1 artifacts, and batch-to-batch variability associated with serumbased calibrator\control materials.

Summary of the Invention The invention relates to buffers used to stabilize isoenzymes with formulations which upon drying and reconstitution recover essentially all of the isoenzyme activity .nd immunological mass for long term shelf-life storage without experiencing dissociation and rearrangement of its subunits. The buffer used for the long term dry storage of an isoenzyme is a serum-free and a salt-free aqueous buffer which comprises: 1) a glass-forming sugar; 2) an antioxidant mixture; 3) a pH buffer; and 4) a non-ionic surfactant and/or a synthetic polymer and/or a 'gelatin. The buffer used for the reconstitution of a serum-free and salt-free isoenzyme sample comprises an aqueous buffer containing a salt. In addition, a serum- .free aqueous stabilizing buffer for long term liquid storage of an isoenzyme is disclosed which comprises: 1) a pH buffer; 2) a salt; 3) an antioxidant mixture; and 4) a non-ionic surfactant and/or a synthetic polymer and/or a gelatin.

":5099 The method of stabilizing an isoenzyme for dry storage consists of: 1) desalting an aqueous solution containing the isoenzyme; 2) adding to the desalted solution: a glass-forming sugar, an antioxidant mixture, a pH buffer and a non-ionic surfactant and/or a synthetic polymer and/or a gelatin; and 3) removing essentially all of the water component of the product of step 2).

'The invention further relates to methods using the reconstitution buffer mentioned above.

oo. The method of stabilizing an isoenzyme for long term liquid comprising: a pH buffer, a salt, an antioxidant mixture, and a nonionic surfactant and/or a synthetic polymer and/or a gelatin.

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b~ *:i P-\OERP\JH\18448.95,CLM 5/10/98 -3A- In a particular embodiment, the present invention provides a serum-free and salt-free aqueous stabilizing buffer for the long term dry storage of an isoenzyme, which is selected from creatine kinase, lactate dehydrogenase, alkaline phosphatase, acid phosphatase, aamylase, aspartate aminotransferase, 6-glutamyl transferase, leucine aminopeptidase, cholinesterase, lipase and carbonic anhydrase, comprising: a) a glass-forming sugar, which is a reducing monosaccharide or disaccharide sugar or a nonreducing monosaccharide or disaccharide sugar or a mixture thereof; b) an antioxidant mixture; 0 c) a pH buffer; and d) a non-ionic surfactant and/or a synthetic polymer and/or a gelatin.

Brief Description of the Drawings 5 Figure 1 is a graph showing the accelerated stability of isoenzyme activity and mass for a buffer formulation containing Tween 20 as determined by an Arrhenius plot of the days to reach a a a, *O a a. aD a.

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57- WO 95/22602 PCT/US95/01927 stability failure against the reciprocal of temperature, extrapolating a storage shelf-life at Figure 2 demonstrates the accelerated stability of isoenzyme activity and mass for a buffer formulation containing gelatin and Tween 20 as determined by an Arrhenius plot of the days to reach stability failure against the reciprocal of temperature, extrapolating a storage shelf-life at Figure 3 shows the accelerated stability of isoenzyme mass in a serum-based and a serum-free aqueous buffer as determined by an Arrhenius plot of the days to reach stability failure against the reciprocal of temperature, extrapolating a storage shelf-life at Detailed Des-ription of the Invention This invention relates to in one aspect a serum-free and saltfree aqueous isoenzyme stabilizing buffer which upon drying and reconstitution has a superior recovery of isoenzyme activity, mass, and/or subunit homogeneity on long term dry storage 5 months shelf-life at about 2-80C, for an isoenzyme with a residual moisture of 5% weight per volume(w/v)) relative to that of material stored in glycerol, while eliminating the contamination of purified antigen grade isoenzyme with the stabilizing presence of immunogenic protein(s) or a serum-based aqueous buffer.

More specifically, there is provided a serum-free formulation to which a native or a recombinant isoenzyme is added to and/or diafiltered against to reduce its endogenous salt content to a residual level, preferably to less than 10 milliequivalents of salt saltfree) not associated with the pH buffer, to protect isoenzyme structural integrity and/or subunit homogeneity upon freezing and subsequent drying such as the removal of essentially all of the aqueous component of the formulation during freeze dry lyophilization. It should be noted that, in order to attain maximal stability for reuse and storage at 2-80C, lyophilized human CK-MB needs to be reconstituted with a buffer containing a salt.

The serum-free and salt-free aqueous stabilizing buffer of the present invention contains a glass-forming sugar such as a reducing monosaccharide or disaccharide sugar, or a nonreducing monosaccharide or disaccharide sugar. The term, "glass-forming WO 95/22602 PCT/US95/01927 sugar", is intended to include the sugars mentioned above, or a mixture thereof. In the present invention, the group of reducing and non-reducing monosaccharide sugars include arabinose, xylose, glucose, fructose, galactose, and mannose at a concentration of 1 to 30% The group of reducing and non-reducing disaccharides include lactose, maltose, cellobiose, raffinose, sucrose, and trehalose at a concentration of 1 to 30% The serum-free and salt-free aqueous stabilizing buffer of the present invention also contains a mixture of antioxidants, which may include a reductant, a metal chelator, and/or chain terminators.

Reductants include agents such as glutathione, N-acetyl-cysteine, or thiourea at concentrations between 10 and 30 mM. Metal chelators include agents such as EDTA, HEDTA, and EGTA at a concentration between 0.1 to 1 mM. Chain terminators include agents such as ascorbityl palmitate, nordihydroguaiaretic acid, and propyl gallate at concentrations of 0.001 to 0.01% The serum-free and salt-free aqueous stabilizing buffer of the present invention additionally includes a pH buffer, which include agents (or mixtures thereof) such as PIPES, HEPES, and Tris at a concentration of 50 to 200 mM to maintain a solution pH in the range of 6 to 9 to stabilize isoenzymes. The preferred pH for stabilizing human CK-MB enzymatic activity and immunological mass is between pH 7.1 and 7.3 for both liquid and dry states, the preferred embodiment in this invention is 50 mM HEPES, pH 7.2.

The serum-free and salt-free aqueous stabilizing buffer of the present invention further contains a non-ionic surfactant and/or a synthetic polymer and/or a gelatin. Non-ionic surfactants useful in the present invention include dodecylpoly(oxyethyleneglycolether)n (Brij 35), poly(oxyethylene)n-sorbitane-fatty acid derivatives such as poly(oxyethylene)n-sorbitane-monolaurate (Tween 20) and poly(oxyethylene)n-sorbitane-monooleate (Tween 80) at a concentration range between 0.005% to 0.1% volume per volume(v/v).

The preferred embodiment in the present invention is Tween 20 in the concentration of 0.01% Synthetic polymers include agents such as polyvinyl sulfate, polyvinylpyrrolidone (molecular weight range from 10,000 to 360,000), and hydroxyethylstarch at concentrations between 0.01 and 10% Gelatins include agents (or mixtures thereof) such as those derived from mammalian and fish WO 95/22602 PCT/US95/01927 skin and vegetable gelatins in the range of 0.01 to 10% This invention also relates to a buffer for reconstituting a serum-free and salt-free isoenzyme sample with a salt-containing buffer and a method of reconstituting with such a stabilizing buffer.

This buffer contains a salt, agents which include potassium and sodium salts (or mixtures thereof) such as KCI and NaCI at a concentration of 10 to 200 mM. In addition, this buffer may contain an antioxidant and/or a non-ionic surfactant as defined above.

Examples of the long term storage stability of human CK-MB in the buffers of the present invention are illustrated using an Arrhenius plot format (see Figures 1, 2, and 3 in Examples 1, 2, and Time plots were constructed to determine the first-order reaction rate constant for the loss of isoenzyme activity or mass at each incubation temperature by measuring the slope of the linear regression line as described by Anderson, G. and Scott, M. (Clin. Chem.

37:398-402, 1991). The estimated time to reach stability failure is determined as the product of the difference between 90% of the yintercept and the y-intercept of the regression line divided by the reaction rate constant for the loss of isoenzyme activity or mass y mx b or x y Thus, these stability failure time points at different temperatures are employed to construct an Arrhenius plot using the logarithm of days to reach stability failure against the reciprocal of incubation temperature. Estimates of storage shelf-life at 50C are determined by measuring the time point on the regression line that coincides with Figure 1 shows an Arrhenius plot of the time to reach stability failure loss of enzymatic activity and protein immunological mass) for recombinant human CK-MB (derived from a cloned E. coil cell line) which was lyophilized in a buffer containing Tween Vials were stressed for different time intervals at temperatures of 37, and 300C, reconstituted with a salt-containing buffer, and assayed to determine their recovery of CK-MB enzymatic activity and mass values. Figure 1 shows the dashed line estimating the time to reach product failure for CK-MB mass and enzymatic activity at about 3.9 and 41.1 years, respectively. In addition, Figure 2 shows a dashed line estimating the time to reach stability failure for CK-MB mass and enzymatic activity at 50C, about 0.5 and 30.7 years, respectively, for a buffer formulation containing gelatin and Tween -6- WO 95/22602 PCTJUS95/01927 These estimates of storage shelf-life for lyophilized human CK- MB using the buffer of the present invention demonstrate the long term stability of this isoenzyme at 50C as compared to that of the glycerol-containing CK-MB formulation stored at 2-80C, 68 days (see Table 1 in Example 3).

The liquid stability of an isoenzyme reconstituted with the salt-containing buffer of the present invention is shown in Table 2 of Example 4. These results indicate that the reconstituted CK-MB formulation containing Tween 20 recovers greater than 90% of its initial mass and enzymatic activity for at least 84 days at 2-80C as compared to 32 and 68 days, respectively, for a glycerol-containing formulation (see Tables 1 and 2 of Examples 3 and Thus, the present invention stabilizes a reconstituted isoenzyme for use in preparing calibrator\control materials, for example, for up to three months at 2-8°C.

This invention further relates to a method of using a serumfree and salt-free aqueous stabilizing buffer as explained above. In the method of the present invention, any standard desalting procedure for removing substantially all of the salt component from a liquid isoenzyme sample would be suitable for the method of the present invention. For example, standard dialysis or diafiltration procedures known to those of skill in the art are suitable in the method of the present invention.

In addition, any standard drying procedure for removing substantially all of the water component from a liquid isoenzyme sample would be suitable for the method of the present invention. For example, standard freeze dry lyophilization procedures known to those of skill in the art are suitable in the method of the present invention. Generally, lyophilization procedures include any freeze drying method.

j In another aspect, the present invention further relates to a serum-free aqueous stabilizing buffer for stabilizing an isoenzyme for long term liquid storage at 2-80C, while eliminating the various problems associated with serum-based aqueous buffers: assay artifacts, variation between lots, immunogenicity problems, and biohazardous risks. The serum-free aqueous stabilizing buffer contains a pH buffer, an antioxidant mixture, and a non-ionic surfactant and/or a synthetic polymer and/or a gelatin as defined n clrr _1IIII(I srr WO 95/22602 PCT/US95/01927 above, where the preferred embodiment is 50 mM HEPES, pH 7.2, mM N-acetyl-cysteine, 0.2 mM HEDTA, 0.01% Tween 20, and 3% polyvinylpyrrolidone (molecular weight of 10,000).

Furthermore, this aqueous stabilizing buffer contains a salt as defined above in the range of 10 to 200 mM, where the preferred embodiment is 140 mM KCI.

It should be noted that long term liquid storage typically requires the inclusion of preservatives and/or sterile procedures to prevent contamination by microbial growth. Typical preservatives include NaN 3 oxaban A, and ProClin 300 in a range of 0.05 to 5% (w/v or where the preferred embodiment is 0.1% NaN 3 A comparison of the thermal stability of an isoenzyme in the serum-free aqueous stabilizing buffer of the present invention is shown in Figure 3. The data wherein represents the Arrhenius plot of the time to reach stability failure for CK-MB mass against the reciprocal of temperature for both a serum-free and a serum-based buffer. Vials were stressed for different time intervals at temperatures of 37, 30, and 25°C and assayed to determine their recovery of CK-MB mass values. Figure 3 shows a dashed line estimating the storage shelf-life of CK-MB mass at 5°C in a serumfree buffer to be 13.1 years as compared to 7.6 years for a serumbased buffer (see Example EXAMPLE 1 To remove salt from the purified protein, human recombinant CK-MB (1 mg/mL) was diafiltered against a buffer containing 50 mM HEPES, pH 7.2, 6% maltose, 0.2 mM HEDTA and 10 mM N-acetylcysteine at 5°C by using an Amicon ultrafiltration cell (Amicon, Beverly, MA) with a 10,000 molecular weight cutoff filter. After diafiltration, an aliquot of the above sample was added into an aqueous solution containing 50 mM HEPES, pH 7.2, 6% maltose, 0.2 mM HEDTA, 10 mM N-acetyl-cysteine and 0.01% Tween 20 to give a final concentration of 100 gg/mL human recombinant CK-MB. The formulated CK-MB was then sterile filtered and freeze dry lyophilized using a FTS Tray Dryer (Model No. TDS-00065-A). The lyophilized vials were reconstituted using an aliquot, 1 mL, of aqueous buffer WO 95/22602 PCT/US95/01927 c_-taining 50 mM HEPES, pH 7.2, and 120 mM KCI. CK-MB activity was measured using the Roche CK-NAC assay reagent on a COBAS FARA II autoanalyzer (Roche Diagnostics). CK-MB immunological mass i concentration was determined using Abbott Human CK-MB reagent on an Abbott IMx immunoanalyzer. Time plot data for the recovery of CK-MB activity and mass was evaluated by linear regression analysis as explained above the time to reach stability failure). Vials of CK-MB were stressed for different time intervals at 45, 37 and 300C in order to estimate the storage shelf-life at 50C. Figure 1 shows an Arrhenius plot of the days to reach stability failure against the reciprocal of temperature. This plot indicates that the estimated storage shelf-lifr at 50C for CK-MB mass and activity is about 3.9 and 41.1 years, respectively.

EXAMPLE 2 The same diafiltration procedure with recombinant human CK- MB was performed as described in Example 1, except that the Tween component was replaced with 0.5% gelatin plus 0.01% Tween Accelerated stability studies were conducted with lyophilized vials of this CK-MB formulation at 45, 37 and 30°C for different time intervals. Figure 2 shows an Arrhenius plot for this formulation, estimating a storage shelf-life at 50C of about 0.5 and 30.7 years for CK-MB mass and activity, respectively.

EXAMPLE 3 For comparison purposes, the storage stability of native human CK-MB in a formulation containing 50 mM Tris HCI, pH 7.5, 150 mM NaCI, 50% glycerol, 10 mM 1-mercaptoethanol, and 0.1% NaN 3 was performed at 2-80C using a single vial for sampling purposes. The results in Table 1 (shown below) demonstrate that CK- MB in this glycerol-base aqueous buffer fails to recover greater than of its initial mass and enzymatic activity value after about 32 and 68 days, respectively.

Table 1: Stability of Native Human CK-MB in 50% Glycerol-based Buffer at 2-8°C -7 WO 95122602 PCT/US95/01927 Days Incubated Percentage Recovery Mass Activity 0 100.0 100.0 32 88.2 100 85.4 99.1 68 74.5 71.8 120 44.3 60.1 The results in Examples 1-3 demonstrate that human CK-MB lyophilized in an aqueous buffer containing either Tween 20 or gelatin plus Tween 20 displays greater estimated storage stability at 50C as compared to that of the native CK-MB present in the aqueous buffer containing 50% glycerol.

Example 4 t To illustrate the liquid storage stability of reconstituted CK- MB in a buffer of the present invention, lyophilized human CK-MB was reconstituted with a buffer containing 50 mM HEPES, pH 7.2, and 120 mM KCI and stored for continuous sampling at 2-80C. Table 2 (shown below) indicates that recombinant human CK-MB recovers at least of its initial mass and enzymatic activity at 50C for greater than 84 days, demonstrating greater liquid storage stability as compared to that of native CK-MB in an aqueous buffer containing 50% glycerol (see Table 1 in Example 3).

WO 95122602 PCTIUS95/01927 Table 2: Reconstituted Stability of Recombinant Human CK-MB at 2- Days Incubated Percentage Activity Mass Activity 0 100.0 100.0 100.0 100.0 19 100.0 100.0 100.0 100.0 56 100.0 98.3 66 98.9 95.7 84 94.7 95.7 120 64.9 89.6 Example Lyophilized recombinant human CK-MB was reconstituted as described in Example 1, added to a final concentration of about 100 ng/mL into both a serum-free and a serum-based aqueous buffer, and stressed for different time intervals at temperatures of 37,30, and 250C. The serum-free aqueous buffer contained 50 mM HEPES, pH 7.2, 140 mM KCI, 10 mM N-acetyl-cysteine, 0.2 mM HEDTA, 0.01% (v/v) Tween 20, 3% polyvinylpyrrolidone (molecular weight of 10,000), and 0.1% NaN 3 The serum-based aqueous buffer contains human serum plus 0.2% NaN 3 An Arrhenius plot of the time to reach stability failure against the reciprocal of temperature displays a nearly two-fold greater storage shelf-life for CK-MB mass at 50C in the serum-free aqueous buffer of the present invention as compared to that in the serum-based buffer (13.1 years versus 7.6 years see Figure 3).

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Claims (6)

1. A serum-free and salt-free aqueous stabilizing buffer for the long term dry storage of an isoenzyme, which is selected from creatine kinase, lactate dehydrogenase, alkaline phosphatase, acid phosphatase, a-amylase, aspartate aminotransferase, 6-glutamyl transferase, leucine aminopeptidase, cholinesterase, lipase and carbonic anhydrase, comprising: a) a glass-forming sugar, which is a reducing monosaccharide or disaccharide sugar or a nonreducing monosaccharide or disaccharide sugar or a mixture thereof; b) an antioxidant mixture; 0 c) a pH buffer; and 0 d) a non-ionic surfactant and/or a synthetic polymer and/or a gelatin.

2. A stabilizing buffer for the reconstitution of a serum-free and salt-free isoenzyme sample, wherein the isoenzyme is selected from creatine kinase, lactate dehydrogenase, alkaline phosphatase, acid phosphatase, a-amylase, aspart-te aminotransferase, 6-glutamyl transferase, leucine aminopeptidase, cholinesterase, lipase and carbonic anhydrase, comprising an aqueous solution containing a salt and additionally an antioxidant and/or a non-ionic surfactant.

3. A serum-free aqueous stabilizing buffer for long term liquid storage of an isoenzyme, which is selected from creatine kinase, lactate dehydrogenase, alkaline phosphatase, acid phosphatase, a-amylase, aspartate aminotransferase, 6-glutamyl transferase, leucine aminopeptidase, cholinesterase, lipase and carbonic anhydrase, comprising: a) a pH buffer; b) a salt; c) an antioxidant mixture; d) a non-ionic surfactant and/or a synthetic polymer and/or gelatin; and e) a glass-forming sugar, which is a reducing monosaccharide or disaccharide ".i PAOPERTEJH\1848-9S.CLM -5110/98 13 sugar or a nonreducing monosaccharide or disaccharide sugar or a mixture thereof.

4. A serum-free and salt-free aqueous stabilizing buffer for stabilizing a dry isoenzyme, which is selected from creatine kinase, lactate dehydrogenase, alkaline phosphatase, acid phosphatase, a-amylase, aspartate aminotransferase, 8-glutamyl transferase, leucine aminopeptidase, cholinesterase, lipase and carbonic anhydrase, against dissociation and rearrangement of subunits, comprising: a) a glass-forming sugar, which is a reducing monosaccharide or disaccharide sugar or a nonreducing monosaccharide or disaccharide sugar or a mixture thereof. b) an antioxidant mixture; c) a pH buffer; and d) a non-ionic surfactant and/or a synthetic polymer and/or a gelatin.

5. A method for stabilizing an isoenzyme, which is selected from creatine kinase, lactate dehydrogenase, alkaline phosphatase, acid phosphatase, a-amylase, aspartate "i aminotransferase, 6-glutamyl transferase, leucine aminopeptidase, cholinesterase, ~lipase and carbonic anhydrase, for long term dry storage, comprising: a) desalting an aqueous buffer containing the isoenzyme; b) adding to the desalted buffer of step a) an aqueous composition comprising: i) a non-reducing monosaccharide sugar or a non-reducing disaccharide sugar of a reducing monosaccharide sugar in a range of about 9% to weight per volume or a reducing disaccharide sugar in a range of about 9% to 30% weight per volume; ii) an antioxidant mixture; iii) a pH buffer; and v) a non-ionic surfactant and/or a synthetic polymer and/or gelatin; and c) removing essentially all of the product of step wherein when the product Sof step c) is reconstituted, a salt-containing aqueous buffer is added. p:\OPER\EJH\18448.95.CLM 5/1098

14- 6. A method for reconstituting a serum-free and salt-free dry isoenzyme sample, wherein the isoenzyme is selected from creatine kinase, lactate dehydrogenase, alkaline phosphatase, acid phosphatase, a-amylase, aspartate aminotransferase, 8-glutamyl transferase, leucine aminopeptidase, cholinesterase, lipase and carbonic anhydrase, comprising adding an aqueous buffer solution containing a salt, and additionally an antioxidant and/or a non-ionic surfactant. 7. A method for stabilizing an isoenzyme, which is selected from creatine ,kinase, lactate dehydrogenase, alkaline phosphatase, acid phosphatase, a-amylase, aspartate aminotransferase, 6-glutamyl transferase, leucine aminopeptidase, cholinesterase, lipase and carbonic anhydrase, for liquid storage comprising adding a serum-free Po. aqueous buffer composition comprising: a) a pH buffer; b) a salt; c) an antioxidant mixture; and od) a non-ionic surfactant and/or a synthetic polymer and/or a gelatin. 0 0 k 8. A serum-free and salt-free aqueous stabilizing buffer according to any one of claims 1 to 4 or a method according to any one of claims 5 to 7 substantially as hereinbefore described with reference to the Figures and/or Examples. 0 DATED this 5th day of October 1998 GENZYME CORPORATION By Davies Collison Cave Patent Attorneys for the Applicants