EP0751989A1 - Isoenzyme calibrator/control products - Google Patents
Isoenzyme calibrator/control productsInfo
- Publication number
- EP0751989A1 EP0751989A1 EP95910271A EP95910271A EP0751989A1 EP 0751989 A1 EP0751989 A1 EP 0751989A1 EP 95910271 A EP95910271 A EP 95910271A EP 95910271 A EP95910271 A EP 95910271A EP 0751989 A1 EP0751989 A1 EP 0751989A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- buffer
- isoenzyme
- free
- serum
- salt
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
Definitions
- 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.
- 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.
- CK creatine kinase
- 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 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 non- immunogenic stabilizing substance(s), may be obtained from human heart or recombinant DNA-derived sources.
- 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.
- 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.
- 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.
- a serum- free aqueous stabilizing buffer for long term liquid storage of an isoenzyme 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.
- 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 wate component of the product of step 2).
- the invention further relates to methods using the reconstitution buffer mentioned above.
- the method of stabilizing an isoenzyme for long term liquid storage consists of adding an isoenzyme to an aqueous solution comprising: a pH buffer, a salt, an antioxidant mixture, and a non- ionic surfactant and/or a synthetic polymer and/or a gelatin.
- 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 stability failure against the reciprocal of temperature, extrapolating a storage shelf-life at 5°C.
- 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 5°C.
- 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 5°C.
- This invention relates to in one aspect a serum-free and salt- free 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 (i.e., > 5 months shelf-life at about 2-8°C, 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.
- 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 (i.e., salt- free) 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.
- 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 (i.e., salt- free) 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.
- 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.
- a glass-forming sugar such as a reducing monosaccharide or disaccharide sugar, or a nonreducing monosaccharide or disaccharide sugar.
- glass-forming sugar is intended to include the sugars mentioned above, or a mixture thereof.
- the group of reducing and non-reducing monosaccharide sugars include arabinose, xylose, glucose, fructose, galactose, and mannose at a concentration of 1 to 30% (w/v).
- the group of reducing and non-reducing disaccharides include lactose, maltose, cellobiose, raffinose, sucrose, and trehalos at a concentration of 1 to 30% (w/v).
- 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 % (w/v).
- 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.
- 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 poiy(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 th concentration of 0.01 % (w/v).
- Synthetic polymers include agents suc as polyvinyl sulfate, polyvinylpyrrolidone (molecular weight range from 10,000 to 360,000), and hydroxyethylstarch at concentrations between 0.01 and 10% (w/v).
- Gelatins include agents (or mixtures thereof) such as those derived from mammalian and fish skin and vegetable gelatins in the range of 0.01 to 10% (w/v).
- 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.
- this buffer may contain an antioxidant and/or a non-ionic surfactant as defined above.
- Figure 1 shows an Arrhenius plot of the time to reach stability failure (i.e., loss of enzymatic activity and protein immunological mass) for recombinant human CK-MB (derived from a cloned E. coli cell line) which was lyophilized in a buffer containing Tween 20. Vials were stressed for different time intervals at temperatures of 45, 37, and 30°C, 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 5°C, about 3.9 and 41 .1 years, respectively.
- Figure 2 shows a dashed line estimating the time to reach stability failure for CK-MB mass and enzymatic activity at 5°C, about 0.5 and 30.7 years, respectively, for a buffer formulation containing gelatin and Tween 20.
- 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 2-8°C as compared to 32 and 68 days, respectively, for a glycerol-containing formulation (see Tables 1 and 2 of Examples 3 and 4). 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 serum- free and salt-free aqueous stabilizing buffer as explained above.
- 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.
- standard dialysis or diafiltration procedures known to those of skill in the art are suitable in the method of the present invention.
- 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.
- standard freeze dry lyophilization procedures known to those of skill in the art are suitable in the method of the present invention.
- lyophilization procedures include any freeze drying method.
- the present invention further relates to a serum-free aqueous stabilizing buffer for stabilizing an isoenzyme for long term liquid storage at 2-8°C, 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 above, where the preferred embodiment is 50 mM HEPES, pH 7.2, 10 mM N-acetyl-cysteine, 0.2 mM HEDTA, 0.01 % (v/v) Tween 20, and 3% (w/v) 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.
- preservatives include NaN ⁇ , oxaban A, and ProClin 300 in a range of 0.05 to 5% (w/v or v/v), where the preferred embodiment is 0.1 % NaN ⁇ (w/v).
- FIG. 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 serum- free buffer to be 13.1 years as compared to 7.6 years for a serum- based buffer (see Example 5).
- 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-acetyl- cysteine at 5°C by using an Amicon ultrafiltration cell (Amicon, Beverly, MA) with a 10,000 molecular weight cutoff filter.
- CK-MB activity was measured using the Roche CK-NAC assay reagent on a COBAS FARA II autoanalyzer (Roche Diagnostics).
- CK-MB immunological mass 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 (i.e., the time to reach stability failure).
- FIG. 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-life at 5°C 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 20 component was replaced with 0.5% gelatin plus 0.01 % Tween 20. 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 5°C of about 0.5 and 30.7 years for CK-MB mass and activity, respectively.
- Table 1 Stability of Native Human CK-MB in 50% Glycerol-based Buffer at 2-8°C Days Incubated Percentage Recovery
- Examples 1 -3 demonstrate that human CK-MB lyophilized in an aqueous buffer containing either Tween 20 or gelati plus Tween 20 displays greater estimated storage stability at 5°C as compared to that of the native CK-MB present in the aqueous buffer containing 50% glycerol.
- 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 2-8°C.
- Table 2 (shown below) indicates that recombinant human CK-MB recovers at least 90 % of its initial mass and enzymatic activity at 5°C 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).
- 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 25°C.
- 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% (w/v) polyvinylpyrrolidone (molecular weight of 10,000), and 0.1 % (w/v) NaN ⁇ .
- the serum-based aqueous buffer contains human serum plus 0.2% (w/v) NaN ⁇ .
- 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 5°C 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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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Medicinal Preparation (AREA)
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US198919 | 1980-10-20 | ||
US19891994A | 1994-02-18 | 1994-02-18 | |
PCT/US1995/001927 WO1995022602A1 (en) | 1994-02-18 | 1995-02-17 | Isoenzyme calibrator/control products |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0751989A1 true EP0751989A1 (en) | 1997-01-08 |
EP0751989A4 EP0751989A4 (en) | 1999-07-14 |
Family
ID=22735444
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP95910271A Withdrawn EP0751989A4 (en) | 1994-02-18 | 1995-02-17 | Isoenzyme calibrator/control products |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0751989A4 (en) |
JP (1) | JPH09509064A (en) |
AU (1) | AU700666B2 (en) |
CA (1) | CA2183573A1 (en) |
WO (1) | WO1995022602A1 (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7074581B2 (en) * | 2002-08-09 | 2006-07-11 | Sysmex Corporation | Reagent for assaying lipid |
CN102628863B (en) * | 2012-04-19 | 2016-05-11 | 上海蓝怡科技有限公司 | Mark alkaline phosphatase antigen-antibody dilution |
EP3234564A1 (en) | 2014-12-18 | 2017-10-25 | Radiometer Medical ApS | Method for calibrating a device for measuring the concentration of creatinine |
CN107003326B (en) | 2014-12-18 | 2019-10-22 | 雷迪奥米特医学公司 | The method that equipment for measuring Concentrations is calibrated |
CN109298193A (en) * | 2018-09-25 | 2019-02-01 | 山东博科生物产业有限公司 | A kind of biochemistry detection that stability is strong compound quality-control product of liquid myocardial enzymes |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4883762A (en) * | 1983-06-06 | 1989-11-28 | Ciba Corning Diagnostics Corp. | Stabilized isoenzyme control products |
US5059341A (en) * | 1989-03-16 | 1991-10-22 | Olin Corporation | Cleaning composition comprising microbial lipase SD2, sodium dodecylbenzene sulfonate and gelatin |
-
1995
- 1995-02-17 WO PCT/US1995/001927 patent/WO1995022602A1/en not_active Application Discontinuation
- 1995-02-17 JP JP7521896A patent/JPH09509064A/en active Pending
- 1995-02-17 AU AU18448/95A patent/AU700666B2/en not_active Ceased
- 1995-02-17 CA CA 2183573 patent/CA2183573A1/en not_active Abandoned
- 1995-02-17 EP EP95910271A patent/EP0751989A4/en not_active Withdrawn
Non-Patent Citations (2)
Title |
---|
JACOBI R AND G\CKERITZ D: "Stabilit{t und Stabilisierung von Enzymen" PHARMAZIE, vol. 44, no. 10, October 1989, pages 678-685, XP002102673 * |
See also references of WO9522602A1 * |
Also Published As
Publication number | Publication date |
---|---|
JPH09509064A (en) | 1997-09-16 |
AU700666B2 (en) | 1999-01-14 |
EP0751989A4 (en) | 1999-07-14 |
AU1844895A (en) | 1995-09-04 |
CA2183573A1 (en) | 1995-08-24 |
WO1995022602A1 (en) | 1995-08-24 |
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