WO1998056900A1 - Stabilized compositions of cardiac markers - Google Patents

Abstract

Stabilized compositions for use in clinical assays for cardiac markers are disclosed. The compositions contain myoglobin and may contain troponin in the form of troponin T or troponin I, CK-MB, myosin, and CA-3.

Description

STABILIZED COMPOSITIONS OF CARDIAC MARKERS

Field of Invention

The present invention relates generally to clinical chemistry. In particular, it relates to a stable liquid clinical chemistry control matrix for cardiac markers.

Backσround of the Invention

The determination of the presence cr amount cf certain constituents or analytes is useful in the diagnosis of disease and physical well-being. Compositions which behave similarly to how constituents present in human bodily fluids (e.g. blood, blood serum, plasma, spinal fluid or urine) behave are used in clinical laboratories. These compositions assist in the determination of whether the clinical instrumentation and procedures used by the laboratory to measure the constituents are accurate. These compositions are also used to calibrate the clinical devices which measure the amount or presence of constituent in a sample. These compositions will be referred to hereinafter as control compositions or controls.

It is clear that the analytes' concentration in a control composition should not change substantially over time (e.g. - be stable).

Predictive models of stability are often used to assess long term stability. For instance, in the Arrhenius Model, reagents are stored at different temperatures. A failure rate is defined (e.g. 90% of the originally recovered value) . The concentration of reagent at each temperature is repeatedly determined over time to assess when the reagent "fails". For each failure, temperature is plotted against log time and a best curve is drawn through the points to determine predicted stability at any temperature. In addition, it is important that the analyte or analyte analog present m the control composition behave similarly to the correspoⁿding analyte to be tested for m a patient' s bodil fluid --that is, the control composition must mimic the patient sample. Often lyophilization or "freeze-drying" processes assist in stabilizing analytes. U.S. 3,466,249 and U.S. 3,629,142 both discuss freeze- dried blood products for use as reference standards (control compositions). The lyophilization process provides stability to the analytes and the use of serum in the matrix or base material allows the control to mimic the patient sample. Improved lyophilized serum-based compositions and methods have been disclosed including using alkylene polyols to reconstitute the product (U.S. 4,121,905 and 4,189,401), or addition of amine chlorides (U.S. 4,324,685). However, the reconstitution process leads to irreproducibility . Thus, liquid products are preferred and are easier to use than lyophilized products . Analytes m control compositions must mimic the shape of a concentration versus response curve using analytes from normal human serum or urine. This is important to ensure that results read off of a standard curve generated with analytes contained in the matrix used to formulate the control composition are accurate when comparing the results to the actual biological milieu. Many previous control compositions include human or animal serum or urine to mimic the patient sample, however these additions provide very little stability to the composition and the preparation of the control composition is often accompanied by cumbersome procedures. For instance U.S. 3,682,835 discloses a liquid blood serum control sample. The blood serum must be treated with a strong acid cation exchange resin to reduce certain cation levels. Then the resin treated serum is dried under vacuum. Next, fat solvents such as Freon are used to extract lipoprotems . Further, it is disclosed that such control standard is only stable for up to three months at aoout 4° C. U.S. 4,324,687 discloses a blood control comprising aqueous red blood cells which have been stabilized by treatment with aldehyde and saline.

In addition, the use of serum and urine from serum and urine pools add an inconsistency or irreproducibility to the product. Moreover, use of normal or processed human serum or urine presents health issues to both clinicians and manufacturers. Thus, a matrix which lowers health risks is also highly desirable.

Further, the use of serum or urine m a control adds turbidity to the product. Thus, many methods have been devised to overcome this disadvantage. For instance, U.S. 4,438,202 provides a method for improving optical quality of serum using acid treatment, U.S. 4,626,511 discloses adding lipase, non-ionic surfactants and cylodextπns to blood, serum or plasma and U.S. 4,716,119 discloses adding sucn chemicals as methanol, sodium acetate, triethanolammonium acetate and other chemicals to the serum to overcome problems caused by turbidity.

In formulating a control material it is desirable that the control material function in the wide variety of methods and on the wide variety of instrumentation that are used in the laboratory to determine the concentration of an analyte. Thus, increasing the number of instruments or procedures with which the control composition can be used increases the value and versatility of the control, but it makes the design more complex. In addition, it is desirable that the control have a common base matrix that stabilizes and accommodates different analytes, even in the liquid state.

In a diagnostic assay, non-specific binding of the analyte to the test surface (e.g. solid support such as test tubes, paper, slides, etc.) must be minimized in order to keep calibration accurate and eliminate any risks of "discrepant" results. Thus, the non-specific binding of the analyte in a matrix must be minimized and must be the same as the non-specific binding of samples. It is also important that during storage, the analytes do not appreciably bind to the storage container .

Controls that contain analytes indicative of cardiac function are important. These analytes are often termed cardiac markers. Some of the known cardiac markers include CK-MB, troponin, myoglobin, myosin, and D-dimer.

Stabilized solutions for cardiac markers nave oeen disclosed. U.S. 5,583,200 and Bodor et al . , Developmen t of Monoclonal An tibodies for an Assay of Cardia c Troponin -I and Preliminary Resu± ts in Suspected Cases of Myocardial Infarction _. Clinical Chemistry Vol. 38, No. 11, pp. 2203-2214 at 2204 (1992) disclose stabilized troponin T and/or troponin I using troponin C and calcium ion. U.S. 5,583,200 discloses that serum may be added.

Troponin exists as a complex. The troponin complex consists of three sub-units: troponin T, the tropomyosm binding sub-unit; troponin C, the calcium binding sub-unit; and Troponin I, which inhibits the actmomyosin magnesium ATPase. Troponin I exists in three isoforms and the ammo acid structure is known. One isoform is cardiac specific (cTnl)and has an additional 31 am o acid residues at its N-termmus compared witr. the other isoforms.

Human cardiac troponin I is difficult to obtain because it must be obtained from human heart. Moreover, native human cardiac troponin I is highly subject to proteolytic degradation during purification. However, human cardiac troponin I has been expressed by recombmant techniques . U.S. Serial No .08/564 , 526, also owned bj applicants, discloses controls containing recomcmant cardiac troponin I fragments that are more stable and more immunologically active tnan full length recombmant cardiac troponin I both with and/or without complexation of troponin C. U.S. 5,217,890 discloses a method to stab_lιze the cardiac marker CK-MB using sulfnydryl modified BSA, magnesium acetate, a buffer, EDTA, anti-fungal reagents, antibiotics, and methyl paraben. U.S. 08/400,158, also c.ned by applicants, the contents of which are incorporated herein by reference, discloses a matrix for stabilizing cardiac markers, particularly troponin and/or troponin fragments comprising a buffer, reducing agent, stabilizing protein, chelatmg agent and a salt.

There are instances when an analyte analogue may oe more desirable than the actual analyte for use the control. If an analyte analogue is used instead of the analyte, the binding of the analogue must mimic the binding of the analyte. Particular care must be used when selecting analogues for proteins because the immunobindmg of the analogue must mimic that of the protein. Thus, any conformational dependence of the protein for the binding site must be maintained in the analogue. In addition, stability of the analogue should be the same or greater than that of the analyte. Again, the stability of the protein analyte may be related to its conformation. Finally, if an analogue is substituted for an analyte, it is desirable that the analogue be more readily available or be more stable than the analyte.

Analogues for cardiac markers have been disclosed. For instance, U.S. Application No. 08/564,526, the contents of which are incorporated herein by reference, discloses stable cardiac troponin I fragments generated from human recombinant cardiac troponin I that result from the cleavage of recombmant troponin I by cyanogen oromide or endoproteinase Asp-N ("EndoAsp") . U.S. 08/400,158 discloses recombinant and synthetic peptides of troponin I . A liquid control that stabilizes a cardiac marker is desirable and a liquid control that stabilizes a variety of cardiac markers is even more desirable. Summary of the Invention

The present invention discloses a composition for use in clinical assays to stabilize myoglobin comprising an aqueous solution of a means to substantially obtain and maintain anoxia and antioxidants and a stabilizing protein.

The present invention also discloses a composition for use m clinical assays to stabilize troponin I or T comprising an aqueous solution of troponin I and/or T and troponin C and calciαm ion wherein such composition is stabilized through a neat treatment process.

In addition, the present invention includes a defined base material useful as a control matrix for containing and stabilizing analytes or analyte analogs, particularly that class of analytes Known as cardiac markers, the base material comprising an aqueous solution of a stabilizing protein, a means for maintaining a low protease activity, a means for substantially obtammq and maintaining anoxia, antioxidants, a buffer to maintain the pH at 5-8, anti-microbial agents and other stabilizers including calcium

The defined base material may be utilized to prepare controls containing a wide variety of analytes including troponin or troponin fragments, CK-MB, myoglobin, myosm, and/or CA- III. The resulting solution can be stored as a liquid or frozen or also can be lyophilized if appropriate fillers are included.

Brief Description of Figures

Figure 1A, IB and 1C show the data from an ongoing stability study of myoglobin at 4° C m a control also containing troponin I and CK-MB in a base material as described in Example 4. Data was obtained using a Stratus® II Fluorometric

Analyzer, available from Dade International Inc. Figure 2 shows a Thermal Stability of Purified Troponin Complex (incubated at 37C) and shows the stability of troponin I complexed with about a 30 molar ratio of troponin C. Data was obtained using a Stratus® II Fluorometric Analyzer, available from Dade International Inc. Figure 3A and 3B show the stability of Myoglobin with and without Oxyrase® material. Data was obtained using a Stratus® II Fluorometric

Analyzer, available from Dade International Inc. Figure 3A shows Myoglobin Stability without Oxyrase (predicted stability at 4C = 100 days) . Figure 3B shows Myoglobin Stability with Oxyrase (predicted stability at 4O1000 days) .

Figure 4 shows the effect of Oxyrase® material on the recovery of CK-MB after a freeze-thaw cycle. Figure 5A shows the stability of low levels of cardiac markers (Troponin) . Figure 5B shows the stability of low levels of cardiac markers (CK- MB) . Figure 5C shows the stability of low levels of cardiac markers (Myoglobin) .

Figure 6A shows the stability of middle levels of cardiac markers (Troponin) . Figure 6B shows the stability of middle levels of cardiac markers

(CK-MB) . Figure 6C shows the stability of middle levels of cardiac markers (Myoglobin) . Figure 7A shows the stability of high levels of cardiac markers (Troponin) . Figure 7B shows the stability of high levels of cardiac markers (CK- MB) . Figure 7C shows the stability of high levels of cardiac markers (Myoglobin) . Data for Figures 5A through 7C was obtained jsing a Stratus® II Fluorometric Analyzer, available from Dade International Inc.

Figure 8 shows the stability of a recombmant Troponin I fragment (r-153 Tnl Fragment/TnC Complex) . Data was obtained using a Stratus® II Fluorometric Analyzer, available from Dade International Inc.

Detailed Description of the Invention

The present invention includes a stabilized composition of myoglobin used as a control assays for myoglobin. It has been found that myoglobin is stabilized in a substantially oxygen free environment. Thus, a means for substantially obtaining and maintaining anoxia and antioxidants are utilized in an aqueous buffered medium to stabilize a cardiac marker control including myoglobin. Roughly 8 ppm oxygen gas is saturation. The oxygen should be less than 1 ppm and preferably less than about 0.1 ppm.

Anoxia is substantially obtained and maintained by degassing the matrix, adding biocatalytic oxygen-reducing agents and/or by adding other oxygen scavengers. Preferentially the means for substantially obtaining and maintaining anoxia are achieved by degassing and by including a biocatalytic oxygen reducing agent into the solution.

These oxygen reducing agents remove dissolved oxygen. Biocatalytic oxygen-reducing agents are prepared from microorganism cell membrane extracts. The crude extract contains lactate oxidase complex. Glucose oxidase and catalase were also found to produce oxygen-free solutions. Jacobson, et al., Partial Purification of an Oxygen Scavenging Cell Membrane Fraction for Use Anaerobic Biochemical Reactions,

Biotecnnology and Applied Biochemistry, 9, 368-379 (1987) .

One such biocatalytic oxygen-reducing agent, prepared from e. coli is EC Oxyrase® oxygen reducing agent available from Oxyrase, Inc. The cell extract is filtered to obtain a suspension of 0.2 microns or less. The commercially available material has an activity greater than 30 umts/mL. See also, U.S. 4,476,224; U.S. 4,996,073 and U.S. 5,240,853. It has been found that preferentially the

Oxyrase® material should be treated to remove extraneous cellular contaminants. Most preferably the Oxyrase® material is treated with gelatin using a preferential fractionation method. In this method an aqueous solution containing 0.05% to 0.15% gelatin and 5-10 umts/mL Oxyrase® material is prepared. At gelatin concentrations over 0.25%, the Oxyrase® material loses its activity. The Oxyrase® material/gelatin solution is filtered through successively smaller pore size filters, for example 8 micron, then 1 micron, then 0.45 microns and finally 0.22 microns or less. It is preferred that the filters have low protein binding so as not to bind the Oxyrase® material and/or the gelatin. In addition, a substrate for the Oxyrase® material is added to act as a hydrogen donor for the Oxyrase® material. Typical substrates are lactic acio, succmic acid, formic acid, and alpha glyceral phosphate and their salts. The L- lsomers are preferred and the most preferred are succ nates and lactates. The substrate concentration may be from 5 mM to 100 mM, although higher amounts often speed up the rate of oxygen depletion.

The antioxidants that are useful include ascorbate, phenol, glutathione, vitamin E, BHT, glucose and BHA and combinations thereof. The antioxidants are added n excess to prevent the accumulation of dissolved oxygen. The most accumulation of dissolved oxygen. The most preferred antioxidants are combination of glutathione, glucose, ascorbic acid and phenol. The aqueous solution may include a buffer and the buffer may generally be any of the Good' s buffers or TRIS that function in the pH range of 5 to 8. Of these buffers, the buffers that are preferred function are in the pH range of 6-8. The concentration of buffer is between 10 mM to 200 mM. It is preferred to keep the buffer concentration lower - in the range of 20-100 mM. The most preferred buffer is TRIS.

In addition, proteins such as albumin and/or gelatin are included in amounts from 5-15%, preferably between 9-10% and preferably substantially protease free albumin. It is preferable that any protein used be substantially protease free. Serum may be included if desired, but n the most preferred embodiments serum is omitted because of the problems associated with serurr..

Anti-microbial and anti-fungal agents may be added to prevent growth and may include those commonly found in the prior art at the concentrations found in the prior art such as gentamycin, clortrimazole, sodium azide, mycostatin, thimerasol, Kathon and/or Proclin 300.

The solution may be degassed and should be stored under an inert atmosphere prior to the addition of myoglobin. Generally, myoglobin is added in amounts from 5 ng/mL to 1000 ng/mL.

It is preferable to prepare controls having different concentration ranges of myoglobin. Generally three concentrations ranges such as low, moderate and high ranges, are sufficient for controls and two to five concentrations are sufficient for standards. Prior to filling vials, it is preferable to sterile filter each solution. It is convenient to fill small vials (e.g. 3-30 mLs) with the different concentration levels of eacn calibrator or control.

The resulting solution (or solutions) is stored at 4° C and using a predictive model is stable for greater than one year. The data used in the predictive model is shown in Table 1. Results were obtained using a Stratus® Fluorometric Immunoassay System, available from Dade International Inc.. Days to "failure" _^sing 90% of the Day 0 value of the recovered values of myoglobin were determined. Thus, the present invention allows for storage of myoglobin, even at low concentrations, at 4° C for extended periods of time. See Figure 1.

Temperature Days

Myoglobin Level I

45 C 1.5

37 C 7

25 C 35

5 C >42 Myoglobin Level II

45 C 1

37 C 14

25 C >70

5 C >70

Myoglobin Level III

45 C 1.5

37 C 10

25 C 35

5 C >42

Table I

The present invention also includes a stock solution and stabilized control composition for troponin useful m clinical assays of troponin. The troponin may be cardiac troponin I (cTnl) or troponin T (TnT) . Because of the cardiac specificity of cTnl it is preferred to use cTnl . The cTnl may oe native or recombinant. Native cTnl is difficult to obtain and difficult to purify in intact form. In addition, there is little difference m structure between human cardiac cTnl and bovine cTnl; thus, bovine cTnl can often substitute for human based material.

Further, it is preferred to use fragments of cTnl instead of full length troponin I and even more preferred to use recombinant fragments. The fragments include those cardiac troponin I protein fragments containing the sequence

AYATEPHAKKKSKISASRKLQLKTLLLQIAKQEL (SEQ ID NO: 1) or RAYATEPHAKKKSKISASRKLQLKTLLLQIAKQEL (SEQ ID

NO: 2). The fragments may be recombinant sequences such as

MADGSSDAAREPRPAPAPIRRRSSNYRAYATEPHAKKKSKISASRKLQL KTLLLQIAKQELEREAEERRGEKGRALSTRCQPLELAGLGFAELQDLCR QLHARVDKVDEERYDIEAKVTKNITEIADLTQKIFDLRGKFKRPTLRRV RISADAMMQALLGARAKESLDLRAHLKQVKKEDTEKENREVGD RKNID ALSGMEGRKKKKFEES (SEQ ID NO: 3 ) ; ADGSSDAAREPRPAPAPIRRRSSNYRAYATEPHAKKKSKISASRKLQLK TLLLQIAKQELEREAEERRGEKGRALSTRCQ (SEQ ID NO: 4); longer synthetic peptides containing SEQ ID 1; or similar human or bovine fragments. It is preferred that the fragment or fragments contain about ammo acid 20 to ammo acid 120 of troponin I. U.S. Serial No. 08/564,526 discloses the 153 ammo acid fragment generated upon cleavage of recombmant troponin I with cyanogen bromide. The 153 ammo acid fragment is ADGSSDAAREPRAPAPIRRRSSNYRAYATEPHAKKKSKISASRLQLKTL LLQIAKQELEREAEERRGEKGRALSTRC*QPLELTGLGFAELQDI_C*QL HARVDKVDEERYDIEAKVTKNITEIADLTQKIFDLRGKFKRPTLRRVRI SADAM (C*= S-carboxyamidomethylcysteme) (SEQ ID No . : 5) . SEQ ID NO: 5 has been found to be more stable and more immunologically active than rTnl alone or when it is complexed with TnC .

Additional fragments are disclosed in U.S. Application No. NOT YET ASSIGNED, filed on May 29, 1997 incorporated herein by reference and also owned by applicants. This application discloses the use of cardiac troponin I fragments of the general sequence X-A-B-Y wherein X comprises any of amino acids 1-27 of full length cardiac troponin I, A comprises residues 28-69 of full length cardiac troponin I, B comprises amino acid residues 70-90 of full length cardiac troponin I, and Y comprises any sequential amino acid sequence of amino acid residues 91-170 of full length cardiac troponin I. Full length cardiac troponin I is known to have the following sequence ADGSSDAAREPRPAPAPIRRRSSNYRAYATEPHAKKKSKISASRKLQLK TLLLQIAKQELEREAEERRGEKGRALSTRCQPLELTGLGFAELQDLCRQ LHARVDKVDEERYDIEAKVTKNITEIADLTQKIFDLRGKFKRPTLRRVR ISADAMMQALLGARAKESLDLRAHLKVKKEDTEKENREVGD RKNIDAL SGMEGRKKKFES (SEQ ID NO: 6) (Armour, K.L. et al., (1993) Cloning and Expression in Escheria

Coli of the CDNA Encoding Human Cardiac Troponin I . Gene . 131 (2) :287-292) .

The cardiac troponin I or troponin T should be complexed with troponin C to stabilize the troponin I or troponin T. In contrast to methods to stabilize troponin I or T as disclosed in Bodor et al., (1992) Development of Monoclonal Antibodies for an Assay of Cardiac Troponin-I and Preliminary Results in Suspected Cases of Myocardial Infarction_/ Clinical Chemis try, 38,

(11) -.2203-2214 at 2204 and U.S. 5,583,200, in the present invention a troponin stock solution is prepared by combining the troponin I or T with troponin C in a mole ratio greater than 10:1 of troponin C to troponin I or T in an aqueous solution containing at least 1-2 mM calcium ion, but otherwise the concentration of calcium ion is not critical. Often the amount of calcium present with the TnC is sufficient. In addition, it is preferred that the troponin I or T/troponm C mixture be heated at temperatures between 37-55°C for 7 to 20 days. Heating in this manner will also stabilize troponin I or troponin T and troponin C prepared at 1:1 to 10:1 mole ratios of troponin C to troponin I or troponin T. It is most preferred that the ratio of troponin C to troponin I or troponin T be 10:1 or greater and most preferably 30:1 or greater. Tab_^e 2A and 2B demonstrate the effect of combining increasing amounts of troponin C with native fragmented troponin I (commercially available from SciPac Inc.) at a final spiked concentration of 60 ng/mL (Table 2A) or 15 ng/mL (Table 2B) as measured in the Stratus® troponin assay. The results were obtained using a Stratus® II Fluorometric Analyzer (available from Dace International Inc.) after the samples had incubated for 7 days at 45°C.

Concentration of Recovered Recovered Recovered Troponin C value of value of value of Tnl Lot 3 Tnl Lot 2 Tnl Lot 1

7.5 ng/mL 1.35 ng/mL 1.1 ng/mL 1.2 ng/mL 15 ng/mL 1.85 ng/mL 1.1 ng/mL 1.5 ng/mL 30 ng/mL 3.15 ng/mL 2.1 ng/mL 2.6 ng/mL 60 ng/mL 5.80 ng/mL 3.3 ng/mL 5.0 ng/mL 120 ng/mL 9.95 ng/mL 5.7 ng/mL 9.5 ng/mL 240 ng/mL 15.8 ng/mL 10.8 ng/mL 16.5 ng/mL 480 ng/mL 23.2 ng/mL A . 1 ng/mL 25.5 ng/mL 960 ng/mL 28.2 ng/mL 25.3 ng/mL 36.7 ng/mL 1920 ng/mL 42.1 ng/mL 50.0 ng/mL 50.0 ng/mL Table 2

Concentration of Recovered Recovered Recovered

Troponin C value of value of value of Tnl Lot 1 Tnl Lot 2 Tnl Lot 3

15 ng/mL 1.4 ng/mL 1.8 ng/mL 1.0 ng/mL 30 ng/mL 2.5 ng/mL 2.8 ng/mL 1.7 ng/mL 60 ng/mL 4.5 ng/mL 4.3 ng/mL 3.1 ng/mL 120 ng/mL 6.5 ng/mL 6.0 ng/mL 4.6 ng/mL 240 ng/mL 8.7 ng/mL 7.5 ng/mL 6.5 ng/mL 480 ng/mL 12.9 ng/mL 10.1 ng/mL 11.1 ng/mL

Table 2B

Higher ratios than 30:1 may also be used, but a commensurate increase in stability and/or reactivity is not seen. In the most preferred compositions, the troponin I or troponin T is combined with troponin C at ratios of about 30:1 and the resulting solution is heated at about 37- 55° Celsius for 7-20 days. Heating, and particularly heating for extended periods of time, is often thought to destabilize, destroy, or denature proteins. Thus, it is surprising that the composition, after an initial drop m recovery, is stable at 45°C. Other stabilizers for troponin I or T such as stabilizing proteins or chelatmg agents may also be included in the aqueous solution as can other general preservatives as discussed above. Generally the stock solution is prepared using between about 0.5 to 50 micrograms per milliliter of fragmented troponin I, either native fragments or recombinant fragments and an amount of troponin C equal to about thirty times the amount of troponin I. Typical amounts of troponin I m tne final control composition may be from 0.2 ng/mL to 200 ng/mL and for troponin T from about 0.1 ng/mL to 10 ng/mL. The control compositions are prepared by diluting the stock solutions.

The aqueous solution used to prepare the stock and controls may include a buffer and the buffer may generally be any of the Good' s buffer that function in the pH range of 5 to 8. Of these buffers, the buffers that are preferred function are m the pH range of 6-8. The concentration of buffer is between 10 mM to 200 mM. It is preferred to keep the buffer concentration lower - in the range of 20-100 mM. The most preferred buffer is TRIS. Anti-microbial and anti-fungal agents may be added to prevent growth and may include those commonly found in the prior art at the concentrations found in the prior art such as gentamycin, clortrimazole, sodium azide, mycostatin, thimerosal, Kathon and/or Proclin 300.

In addition, stabilizing proteins such as albumin may be included. The concentration of stabilizing protein may be from 0 to 15% and preferably from 7 to 12%. Preferably the albumin is substantially protease free.

It is preferred that the solution have low protease activity, thus protease inhibitors such as aprotinin and "Protease Inhibitor" (Sigma) are effective. They may be added and may be used at the manufacturer's recommended concentration. Examples of other protease inhibitors include benzidine, (2S, 3R) -3-Amino-2-hydroxy-5- methylhexanoyl] -Val-Val-Asp (Amastatin-Sigma) , [2S, 3R] -3-Amino-2-hydroxy-4- [4-nitrophenyl] - butanoyl-L-leucine, Antipain, [2S, 3R] -3-Amino-2- hydroxy-5-methylhexanoyl ] -Val-Val-Asp (Epiamastatin-Sigma) , ( [2R, 3R] -3-Amino-2-hydroxy- 4-phenylbutanoyl) -L-leucine (Epibestatin-Sigma) , Foroxymithine, Acetyl-Leu-Leu-Arg-al (Leupeptin- Sigma) , 4-Amino-3-hydroxy-6-methyl-heptanoic acid, 4-Amino-3-hydroxy-6-methylheptanoic acid,

N- (α-Rhamnopyranosyloxy-hydroxyphosphinyl) -Leu- Trp and phenyl methane sulfonyl fluoride (PMSF) . It is most preferred that the means to provide a substantially protease free solution is to use substantially protease free proteins such as albumin which is substantially protease free.

Serum may be included if desired, but in the most preferred embodiments serum is omitted because of the problems associated with serum.

As discussed further herein, for simplicity of manufacture, the base material that comprises the base material for a cardiac control containing other cardiac markers such as CK-MB and myoglobin may be used to prepare the troponin stock solution. The stock solution may then be diluted in the same base material to provide stabilized troponin control compositions.

Controls prepared by this method may be lyophilized by adding those bulking agents that are known in the art, but the controls may also be liquid. In addition, the liquid controls may be frozen to further increase shelf-life. The present invention also includes a defined base material useful as a control matrix for containing and stabilizing analytes or analyte analogs, particularly cardiac markers. The base material comprises stabilizing proteins which may be general stabilizing proteins or specific stabilizing proteins, a means for maintaining a low protease activity, a means for maintaining anoxia, antioxidants, a buffer to maintain the pH at 5-8, anti-microbial agents and stabilizers .

The general stabilizing proteins may be one or a combination of albumin, globulins, ovalbumin, casein or gelatin. The source of the protein is irrelevant although recombinant or bovine proteins are preferred. Preferably the stabilizing protein is substantially protease free.

The concentration of the stabilizing protein may be anywhere from between 1% to over 12%, preferably 8-10%. If concentrations over 12% are employed salts can be added to improve the filterability of the resulting solution.

A means for maintaining a low protease activity are achieved by using substantially protease free analytes or other added components such as highly purified analytes or recombinant analytes or other added components or by employing physical or chemical means. Examples of physical means include pH treatment or heat treatment. Examples of chemical means include adding protease inhibitors or specific inhibitors such as benzidine analogs or those inhibitors discussed above. Protease activity may be measured by certain assays such as the Peptag® assay. These assays have picomolar sensitivity to proteases. It is desirable that the control composition have less than picomolar levels of protease activity. It has been found that the use of substantially protease free components is preferred. Since albumin is included in the most preferred embodiment of the present invention, it is most preferable to use substantially protease free albumin.

A means for substantially obtaining and maintaining anoxia and antioxidants are required if myoglobin is included. Preferred means for substantially obtaining and maintaining anoxia and antioxidants are the same as the preferred means discussed for a matrix for myoglobin stabilization as discussed above. That is -- anoxia is maintained by degassing the matrix, adding the Oxyrase® material and/or by adding other oxygen scavengers. Preferentially the means for maintaining anoxia are achieved by degassing and by including Oxyrase® material into the solution. More preferentially the Oxyrase® material is treated to remove extraneous cellular contaminants. Most preferably the Oxyrase® material is treated with gelatin using a preferential fractionation method. In this method an aqueous solution containing 0.05% to 0.15% gelatin and 5-10 umts/mL is prepared. At gelatin concentrations over 0.25%, the Oxyrase® material loses its activity. The Oxyrase® material/gelatin solution is filtered through successively smaller pore size filters, for example 8 micron, then 1 micron, then 0.45 microns and finally 0.22 microns or less. It is preferred that the filters have low protein binding so as not to bind the Oxyrase® material and/or the gelatin. Figure 3 demonstrates the stability of myoglobin in the base material with and without Oxyrase® material. In addition, a substrate for the Oxyrase® material is added to act as a hydrogen donor for the Oxyrase® material. Typical substrates are lactic acid, succinic acid, formic acid, and alpha glyceryl phosphate and their salts. The L- isomers are preferred and the most preferred are succinates and lactates . The substrate concentration may be from 5 mM to 100 mM, although higher amounts often only speed up the rate of oxygen depletion. The antioxidants that are useful include ascorbate, phenol, glutathione, vitamin E, BHT, glucose and BHA and combinations thereof. The concentrations of the antioxidants should be sufficient to provide an excess of antioxidant. The most preferred antioxidants are combinations of glutathione, glucose, ascorbic acid and phenol .

The aqueous solution may include a buffer and the buffer may generally be any of the Good' s buffers or TRIS that function in the pH range of 5 to 8. Of these buffers, the buffers that are preferred function are in the pH range of 6-8. The concentration of buffer is between 10 mM to 200 mM. It is preferred to keep the buffer concentration lower - in the range of 20-50 mM. The most preferred buffer is TRIS. concentration lower - in the range of 20-50 mM.

The most preferred buffer is TRIS.

Anti-microbial agents and anti-fungal agents may added to prevent growth and may include those commonly found m the prior art at the concentrations found m the prior art such as gentamycm, clortπmazole, sodium azide, mycostatm, thimerosal, Kathon and/or Proclin 300. Serum may be included if desired, but in the most preferred embodiments serum is omitted because of the problems associated with serum.

The analytes to be added include troponin, myosm, carbonic anhydrase III (CA-III) , CK-MB, and/or myoglobin. The optimum conditions and amounts for troponin and myoglobin are discussed above. CK-MB is added to the base material to provide final concentrations m amounts ranging from 0-200 ng/mL. Light chain myosm may be added to provide final concentrations ranging from 0-200 ng/mL and CA-III may be added to provide final concentrations in amounts ranging from 0-1000 ng/mL.

It has been found that the addition of Oxyrase® material provides additional stability to CK-MB if the control material is stored frozen and subsequently thawed for use. See Figure 4.

In a preferred embodiment, the base material comprises myoglobin and at least troponin or CK- MB. When troponin is to be included, troponin I fragment (s) is preferred and troponin C should be included as a specific stabilizing protein.

Preferably the troponin I and troponin C should be prepared at over a 20:1 ratio of troponin C to troponin I and should be heated as discussed herein. Most preferably native troponin I fragments (commercially available) or recombinant troponin I fragments as discussed above are preferred. It is most preferable that the base material include myoglobin, CK-MB and troponin. Controls prepared by this method may be lyophilized by adding those bulking agents that are _no n in the art, but the controls may also be liquid. In addition, the liquid controls may be frozen to further increase shelf-life.

Example 1

Preparation of a myoglobin control.

About 200 milligrams each of glutathione, glucose, 50 milligrams of ascorbic acid, about 1.1 r lliliter of phenol, about 2 grams of L-

_^actate, 95 grams of protease free BSA and 1 gram of gelatin are combined in a one liter aqueous TRIS ouffered solution at about 50 mM adjusted to about pH 7.3. Preferably the solution is sterile filtered using filters of about 0.3 microns or less. The resulting solution is degassed.

One method to degas the solution is to use a Liqui-cel Membrane cartridge as per the manufacturer's instructions. The degassing should take place under an inert environment such as under nitrogen, helium or argon gas. After the solution is degassed, an amount of

Oxyrase® material sufficient to provide about 0.3 units of activity per mL is added under an inert atmosphere such as nitrogen or argon gas. The added Oxyrase® material is preferably purified to remove extraneous cellular contaminants. Most preferably the Oxyrase® material is treated with gelatin using a preferential fractionation method prior to addition to the base material. In this method a gelatin solution is prepared by dissolving about 1 gram of gelatin in 800 mLs of water by heating. After cooling, about 200 mL of Oxyrase® material at about 30 umts/mL is added. Thus, the resulting solution has about 6 umts/mL of Oxyrase activity. The

Oxyrase® material is separated from the liquid by filtration, although centπfugation or otner means of separation work as well. Then the purified Oxyrase® is added to the base material to provide about 0.3 umts/mL of activity.

The case material is stored under an inert atmosphere prior to the addition of myoglobin. Generally, myoglobin is added to provide final control concentrations in amounts from about 5 ng/mL to 1000 ng/mL.

It is preferable to prepare controls having different concentration ranges of myoglobin. Generally three concentration ranges such as low, moderate and high ranges, are sufficient. Example 2

Preparation of a defined base material for cardiac markers.

Antioxidants such as 200 milligrams of glutathione, 200 milligrams of glucose, 50 mgs of ascorbic acid, and 1.1 milliliter of phenol, about 2.7 grams of L-Lactate, about 225 milligrams of calcium chloride or other calcium salt to provide 1-3 mM calcium ion, anti- microbial and anti-fungal agents such as about 20 milligrams of clortπmazole, 35 milligrams of gentamicin, and about 1 milliliter of Proclin 300, about 95 grams of protease free BSA, and about 1 gram of gelatin are combined in an aqueous 50 mM TRIS buffered solution at about pH 7.3 also containing a salt such as sodium chloride (about 30 grams) to provide about one liter of base material.

It is best to add the gelatin in solution by dissolving the gelatin by adding 1 gram of gelatin to 100 milliliters of water and gently heating to dissolve the gelatin. Then the gelatin containing solution is added to the base material . The resulting solution is filtered using filters sufficient to remove any bacteria such as a .22 micron filter. A low protein binding filter is preferred. Example 3

Preparation of troponin I stock solution and controls .

The troponin I stock solution is prepared by adding about 0.5 mgs of fragmented troponin I obtained from human heart muscle and about 15 mgs of troponin C to about 85 milliliters of the defined base material prepared in Example 2. The solution is mixed and then heated at about 50° Celsius for about 10 days. The resulting stock solution is sterile filtered using low protein binding filters of 0.22 microns or less. The stock solution may be stored frozen at -20° C for up to two years . The stock solution may be stored at 2-8° C for more than one week.

The stock solution is used to prepare diluted solutions of troponin I in the clinical range of interest (about 0 to 200 ng/mL) by diluting the stock solution in the defined base material prepared in Example 2.

Example 4

Preparation of a cardiac control containing multiple cardiac markers. The base solution prepared in Example 2 is degassed and stored in the absence of oxygen (e.g. under helium, nitrogen or argon) until used. In addition, gelatin-treated Oxyrase® detailed in Example 1 is added in the absence of oxygen after the degassing process to provide a final activity of Oxyrase® material of about 0.3 units/ml .

Troponin I stock solution as described in Example 3, myoglobin and CK-MB are added to the base solution in amounts to provide final concentrations in the clinical level of interest. It is preferred to prepare three controls each control having myoglobin, CK-MB, and troponin I at three different levels in the clinical range, the levels representing a low, normal, and high clinical ranges. Each cardiac marker containing solution is sterile filtered using 0.22 micron filters prior to filling in vials. The stability of the controls prepared by this method are shown in Figures 5, 6, and 7.

Example 5

Preparation of Troponin I Controls.

Recombinant troponin I fragment designated as SEQ ID NO: 5 is substituted for the native fragments and covalently cross-linked to TnC using the EDC method described by Greaser and Gergely (1971). The molar amount of Tnl is generally greater than or equivalent to the amount of TnC. To 20 mM Mes buffer at pH 6 containing 0.1 M KC1, 0.2 mM CaCl , freshly prepared NHS at 5.7 mM final concentration was added followed by addition of TnC to provide 0.2 mg/mL. The mixture was incubated at room temperature for 15 minutes. The TnC was activated by addition of 5.7 mM EDC (final concentration) followed by incubation at room temperature for 30 minutes. The activation step was terminated by addition of β mercaptoethanol (20 mM final concentration) . Then the CNBr-rTnl (Tnl 153) in PTU buffer was added at a final concentration of 0.175 mg/mL. The mixture was incubated for two hours at room temperature. The complex is buffer exchanged. The activity of the crossed linked complex was measured using Stratus Tnl immunoassay.

The stock solution is used to prepare diluted solutions of troponin I in the clinical range of interest (about 0 to 200 ng/mL) by diluting tne stock solution in the defined base material prepared in Example 1 or other base material. Figure 8 shows the stability at 45 C of the recombinant troponin I fragment dilutions over time.

Example 6

Preparation of a cardiac control containing multiple markers.

Example 4 is repeated except that the stock solution described in Example 5 is used to provide the troponin I. SEQUENCE LISTING

(1 ) GENERAL INFORMATION:

(i) APPLICANT: PALMER, DENNIS

MORJANA, NIHMAT

(ii) TITLE OF INVENTION: CARDIAC CONTROL

(iii) NUMBER OF SEQUENCES: 6

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: DADE BEHRING INC.

(B) STREET: 1717 DEERFIELD ROAD (C) CITY: DEERFIELD

(D) STATE: ILLINOIS

(E) COUNTRY: USA

(F) ZIP: 60015

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/898,538

(B) FILING DATE: 22-JUL-1997

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/874,566

(B) FILING DATE: 13-JUN-1997

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: TYMESON, CYNTHIA G.

(B) REGISTRATION NUMBER: 34,745

(C) REFERENCE/DOCKET NUMBER: DA-9021 CIP

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (305) 591-5558

(B) TELEFAX: (305) 591-5568 (2) INFORMATION FOR SEQ ID NO:1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :

Ala Tyr Ala Thr Glu Pro His Ala Lys Lys Lys Ser Lys lie Ser Ala 1 5 10 15

Ser Arg Lys Leu Gin Leu Lys Thr Leu Leu Leu Gin He Ala Lys Gin 20 25 30

Glu Leu

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys Lys Ser Lys We Ser 1 5 10 15

Ala Ser Arg Lys Leu Gin Leu Lys Thr Leu Leu Leu Gin He Ala Lys 20 25 30

Gin Glu Leu 35 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 212 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Ala Asp Gly Ser Ser Asp Ala Ala Arg Glu Pro Arg Pro Ala Pro 1 5 10 15

Ala Pro We Arg Arg Arg Ser Ser Asn Tyr Arg Ala Tyr Ala Thr Glu 20 25 30

Pro His Ala Lys Lys Lys Ser Lys He Ser Ala Ser Arg Lys Leu Gin 35 40 45

Leu Lys Thr Leu Leu Leu Gin We Ala Lys Gin Glu Leu Glu Arg Glu 50 55 60

Ala Glu Glu Arg Arg Gly Glu Lys Gly Arg Ala Leu Ser Thr Arg Cys 65 70 75 80

Gin Pro Leu Glu Leu Ala Gly Leu Gly Phe Ala Glu Leu Gin Asp Leu 85 90 95

Cys Arg Gin Leu His Ala Arg Val Asp Lys Val Asp Glu Glu Arg Tyr 100 105 110

Asp He Glu Ala Lys Val Thr Lys Asn We Thr Glu He Ala Asp Leu 115 120 125

Thr Gin Lys We Phe Asp Leu Arg Gly Lys Phe Lys Arg Pro Thr Leu 130 135 140

Arg Arg Val Arg lie Ser Ala Asp Ala Met Met Gin Ala Leu Leu Gly 145 150 155 160

Ala Arg Ala Lys Glu Ser Leu Asp Leu Arg Ala His Leu Lys Gin Val 165 170 175

Lys Lys Glu Asp Thr Glu Lys Glu Asn Arg Glu Val Gly Asp Trp Arg 180 185 190

Lys Asn He Asp Ala Leu Ser Gly Met Glu Gly Arg Lys Lys Lys Lys 195 200 205

Phe Glu Glu Ser 210

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 80 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Ala Asp Gly Ser Ser Asp Ala Ala Arg Glu Pro Arg Pro Ala Pro Ala 1 5 10 15

Pro He Arg Arg Arg Ser Ser Asn Tyr Arg Ala Tyr Ala Thr Glu Pro 20 25 30

His Ala Lys Lys Lys Ser Lys He Ser Ala Ser Arg Lys Leu Gin Leu 35 40 45

Lys Thr Leu Leu Leu Gin He Ala Lys Gin Glu Leu Glu Arg Glu Ala 50 55 60

Glu Glu Arg Arg Gly Glu Lys Gly Arg Ala Leu Ser Thr Arg Cys Gin 65 70 75 80 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 150 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 94

(D) OTHER INFORMATION: /note= "S-carboxyamidomethylcysteine"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 77

(D) OTHER INFORMATION: /note= "S-carboxyamidomethylcysteine"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Ala Asp Gly Ser Ser Asp Ala Ala Arg Glu Pro Arg Ala Pro Ala Pro 1 5 10 15

He Arg Arg Arg Ser Ser Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His 20 25 30

Ala Lys Lys Lys Ser Lys lie Ser Ala Ser Arg Leu Gin Leu Lys Thr 35 40 45

Leu Leu Leu Gin lie Ala Lys Gin Glu Leu Glu Arg Glu Ala Glu Glu 50 55 60

Arg Arg Gly Glu Lys Gly Arg Ala Leu Ser Thr Arg Cys Gin Pro Leu 65 70 75 80

Glu Leu Thr Gly Leu Gly Phe Ala Glu Leu Gin Asp Leu Cys Gin Leu 85 90 95

His Ala Arg Val Asp Lys Val Asp Glu Glu Arg Tyr Asp lie Glu Ala 100 105 110 Lys Val Thr Lys Asn He Thr Glu He Ala Asp Leu Thr Gin Lys lie 115 120 125

Phe Asp Leu Arg Gly Lys Phe Lys Arg Pro Thr Leu Arg Arg Val Arg 130 135 140

He Ser Ala Asp Ala Met 145 150

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 208 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Ala Asp Gly Ser Ser Asp Ala Ala Arg Glu Pro Arg Pro Ala Pro Ala 1 5 10 15

Pro He Arg Arg Arg Ser Ser Asn Tyr Arg Ala Tyr Ala Thr Glu Pro 20 25 30

His Ala Lys Lys Lys Ser Lys He Ser Ala Ser Arg Lys Leu Gin Leu 35 40 45

Lys Thr Leu Leu Leu Gin He Ala Lys Gin Glu Leu Glu Arg Glu Ala 50 55 60

Glu Glu Arg Arg Gly Glu Lys Gly Arg Ala Leu Ser Thr Arg Cys Gin 65 70 75 80

Pro Leu Glu Leu Thr Gly Leu Gly Phe Ala Glu Leu Gin Asp Leu Cys 85 90 95

Arg Gin Leu His Ala Arg Val Asp Lys Val Asp Glu Glu Arg Tyr Asp 100 105 110

He Glu Ala Lys Val Thr Lys Asn We Thr Glu He Ala Asp Leu Thr 115 120 125

Gin Lys He Phe Asp Leu Arg Gly Lys Phe Lys Arg Pro Thr Leu Arg 130 135 140

Arg Val Arg He Ser Ala Asp Ala Met Met Gin Ala Leu Leu Gly Ala 145 150 155 160

Arg Ala Lys Glu Ser Leu Asp Leu Arg Ala His Leu Lys Val Lys Lys 165 170 175

Glu Asp Thr Glu Lys Glu Asn Arg Glu Val Gly Asp Trp Arg Lys Asn 180 185 190

He Asp Ala Leu Ser Gly Met Glu Gly Arg Lys Lys Lys Phe Glu Ser 195 200 205

Claims

We claim:

1. A composition for use m clinical assays comprising an aqueous buffered solution of myoglobin, a means for maintaining anoxia and antioxidants .

2. The composition of claim 1 further comprising troponin T or troponin I or fragments thereof.

3. The composition of claim 1 further comprising a stabilizing protein.

4. The composition of claim 3 wherein the stabilizing protein is albumin.

5. The composition of claim 4 wherein the albumin is protease free.

6. The composition of claim 5 further comprising troponin T or Troponin I or fragments thereof.

7. The composition of claim 1 wherein the means for maintaining anoxia comprises degassing and an oxygen scavenger.

8. The composition of claim 7 wherein the oxygen scavenger is Oxyrase® material.

9. A composition for use in clinical assays for troponin comprising troponin I or troponin T, troponin C in a molar amount equal to or greater than the amount of troponin I or troponin T and calcium ion wherein the composition has been heated between 37-55┬░C for at least seven days.

10. The composition of claim 9 further comprising myoglobin.

11. A composition for use in clinical assays for troponin comprising troponin I or troponin T, troponin C in a molar amount equal to twenty or more times the amount of troponin I or troponin T and calcium ion.

12. The composition of claim 11 wherein the composition has been heated between 37-55┬░C for at least seven days.

13. The composition of claim 12 further comprising myoglobin.

14. A composition for use in the assay of cardiac markers comprising a buffered aqueous solution of myoglobin, at least one of either CK- MB or troponin, a stabilizing protein, a means for maintaining a low protease activity, a means for maintaining anoxia, antioxidants, and calcium ion.

15. The composition of claim 14 further comprising cardiac markers selected from the group consisting of CA-III and myosin.

16. The composition of claim 14 wherein the troponin is troponin I.

17. The composition of claim 14 wherein the troponin is a troponin I fragment.

18. The composition of claim 14 wherein the troponin fragment includes at least SEQ ID NO: 1.

19. The composition of claim 14 further comprising troponin C at a molar concentration at least equal to that of the troponin.

20. The composition of claim 19 wherein the troponin I and troponin C have been heated cetween 37-55┬░C for at least seven days.

21. The composition of claim 20 wherein the molar concentration of troponin C is at least ten times greater than the troponin I.

22. The composition of claim 21 wherein the troponin I is a troponin I fragment.

23. The composition of claim 22 wherein the troponin I fragment is SEQ ID NO: 5.