WO2001007921A2

WO2001007921A2 - Stabilized coagulation control reagent

Info

Publication number: WO2001007921A2
Application number: PCT/US2000/040453
Authority: WO
Inventors: Rebecca J. Hunt; Lori J. Elder; Seyyed Mohsen FAKHARI
Original assignee: Medical Analysis Systems, Inc.
Priority date: 1999-07-23
Filing date: 2000-07-21
Publication date: 2001-02-01
Also published as: WO2001007921A3; AU7137900A

Abstract

This invention relates generally to the preparation of coagulation control compositions suitable for measuring human coagulation characteristics. The coagulation controls provided herein include novel combinations of reagents and novel processing which provide superior performance characteristics including long term stability as a liquid formulation at 4 °C after freeze thaw or lyophilization and reconstitution. The control is based on mammalian plasma, of which the majority is primate plasma and a minority is non-primate plasma as well as a variety of other stabilizing materials such as a buffer, a fibrinolysis inhibitor, a coagulation factor stabilizer, a carbohydrate for stabilizing proteins upon freeze-thaw and a preservative to maintain sterility. The invention also includes a new method to prepare coagulation controls with abnormal clotting times by adding coagulation control inhibitors to the plasma.

Description

STABILIZED COAGULATION CONTROL REAGENT AND METHOD OF

MAKING SAME

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to United States provisional application serial no. 60/145,454, filed on July 23.1999.

FIELD OF THE INVENTION

This invention relates generally to blood coagulation and, more particularly, to coagulation controls for clinical coagulation analysis which have superior stability and performance characteristics. BACKGROUND OF THE INVENTION

Hemostasis is the process by which the body regulates coagulation of blood and involves the interplay of two biochemical pathways, which are controlled by various protein and other substances known as coagulation factors. Blood coagulation as it is presently understood involves a multi-step cascade of activation steps of the coagulation factors that culminates in fibrin formation and clotting.

There are two pathways for activating protein coagulation factors; An intrinsic pathway involving only blood factors and an extrinsic pathway that is understood to require the participation of a tissue lipoprotein (tissue factor). The operation of both pathways appears to be necessary for effective hemostasis.

It is important to accurately measure the functioning of both coagulation pathways because deficiencies in either pathway can be life threatening, particularly when large blood loss is involved as in surgery. In addition, there is a need to carefully monitor hemostasis in patients undergoing coagulation therapy in thromobo-embolytic conditions.

An overdose of anti-coagulation drugs can lead to serious complications, including peptic ulcer bleeding and gastrointestinal disorders, while an underdose can lead to life threatening or debilitating stroke.

A variety of tests have been developed to measure the coagulation pathways. The most common tests are the prothrombin time (PT), which measures the extrinsic pathway and the activated partial thromboplastin time (APTT), which measures the intrinsic pathway. These tests are routinely performed in the clinical laboratory using automated instrumentation. The accuracy of such tests depends on the quality of commercially prepared plasma controls representing normal plasma and particular abnormal plasma states.

Although there is a large body of literature relating to the preparation of plasma as coagulation controls, some of which are commercially available, such products do not have reliable stability characteristics as a liquid formulation. Thus, there is increased cost associated with discarding controls after only one use. In addition, the methods to prepare abnormal plasma controls require time consuming and expensive steps to absorb critical protein factors from the plasma to obtain the abnormal clotting properties. SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a superior stable coagulation control for monitoring coagulation capability of a human patient, the composition having a predetermined clotting time ranging from normal to abnormal. The coagulation control of the present invention is stable for at least seven days at 4°C, following storage in a liquid frozen or lyophilized state.

It is also an object of the present invention to provide a method of preparing abnormal coagulation controls with abnormal coagulation times which avoid the laborious step of physically removing clotting factors and other materials from plasma.

To accomplish these and other objectives, there has been provided, in accordance with one aspect of the present invention, a stable coagulation control composition having a predetermined clotting time ranging from normal to abnormal comprising mammalian plasma devoid of red cells, white cells and platelets, wherein the mammalian plasma comprises a majority of primate plasma and a minority of non-primate plasma for the normal clotting time.

According to one embodiment of the present invention, the non-primate plasma represents between 2% to 15% of the total plasma.

According to another embodiment of the present invention, the primate plasma is human plasma and the non-primate plasma is selected from leporine, canine, porcine, and feline plasma.

According to yet another embodiment of the present invention, the coagulation control further comprises one or more of a buffer, a fibrinolysis inhibitor, a coagulation factor stabilizer, a carbohydrate and a preservative. In another aspect of the present invention, there is provided a method of making a coagulation control composition for monitoring coagulation capability of a human patient, the composition having a predetermined clotting time ranging from normal to abnormal, the method comprising the steps of:

(a) obtaining a source of primate plasma from which red cells, white cells and platelets have been removed; and

(b) adding non-primate plasma to the primate plasma, wherein the amount of primate plasma is in the majority and wherein the non-primate plasma is added in an amount sufficient to achieve a normal clotting time, wherein the abnormal clotting times are achieved by physically removing coagulation factors from the plasma or by adding coagulation inhibitors to the plasma.

According to one embodiment of the present invention, the method further includes the step adding a stabilizing solution to the plasma. In accordance with this step, the stabilizing solution comprises one or more of a buffer, a fibrinolysis inhibitor, a coagulation factor stabilizer, a carbohydrate and a preservative.

According to another embodiment of the present invention, the step of obtaining the plasma comprises the step of plasmaphoresis.

According to yet another embodiment of the present invention, the method further comprises the step of freezing the control composition prior to thawing and use as a control or lyophilizing the control composition prior to reconstituting and use as a control.

In another aspect of the present invention, there is provided a method of making a coagulation control composition, the composition having a predetermined clotting time, the method comprising obtaining a source of plasma suitable for use as a control and adding one or more coagulation inhibitors to achieve the predetermined clotting time.

According to one embodiment of the present invention, the coagulation inhibitor is a serine protease inhibitor.

According to another embodiment of the present invention, the serine protease inhibitor is benzamidine. According to yet another embodiment of the present invention, the composition further comprises a stabilizing compound such as heparin to stabilize the clotting times.

Other objects, features and advantages of the present invention will become apparent from the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improved coagulation controls for measuring human coagulation characteristics. The coagulation controls provided herein include novel combinations of reagents and novel processing that results in superior performance characteristics including long term stability as a liquid formulation at 4° Centigrade ("C") after freeze thaw or lyophilization and reconstitution.

The control is based on mammalian plasma, of which the majority is primate plasma and a minority is non-primate plasma. Non-primate plasma from particular species of animal are particularly suited for achieving the coagulation performance characteristics of the coagulation control while maintaining a stable composition. A variety of other stabilizing materials can be included in the coagulation control such as a buffer, a fibrinolysis inhibitor, a coagulation factor stabilizer, a carbohydrate for stabilizing proteins upon freeze-thaw and a preservative to maintain sterility. Each of these components is discussed in detail below.

Definitions:

Stable coagulation control: A coagulation control composition that exhibits less than 13% difference in the PT or APPT clotting test when the control composition is incubated for several days at 4°C. The degree of stability varies with the components included in the control composition. A stability between 2 to 5 days of incubation is useful in some circumstances, however, stability for 6 to 7 days is more preferred, and stability exceeding seven days, including up to 15 days incubation is most preferred. Predetermined clotting time: Refers to a performance characteristic of a coagulation control which has been chosen for a particular clotting test time. For example, the predetermined clotting time of a normal (i.e., "level 1") human coagulation control is generally between 10 to 13 seconds for the PT test and 22 to 32 seconds in the APTT test. Predetermined clotting times for a abnormal plasma include, for example, a PT of 17 to 22 seconds (i.e., "level 2") or 23 to 35 seconds (i.e., "level 3"), and an APTT of 42 to 65 seconds (level 2) or 65 to 90 seconds (level 3).

Devoid: As used in reference to plasma from which red cells, white cells and platelets means that the plasma contains 2% or less of the starting amount of such cells. Fibrinolysis inhibitor: A substance that inhibits one or more components of the fibrinolytic system in plasma. Such inhibitors can act at any part of the pathway leading to fibrinolysis, including, for example, inhibiting the formation of a plasminogen activator, or inhibiting the activity of plasmin. Fibrinolysis inhibitors include those compounds which inhibit tissue plasminogen activator, anistreplase, urokinase or streptokinase. The term anistreplase refers to anisoylated plasminogen streptokinase activator complex, as described, for example, in European Patent Application No. 028,489. Epsilon aminocaproic acid ("EACA"), which can inhibit the activity of plasmin also is an example of a fibrinolysis inhibitor. Coagulation factor: Any of the factors that are involved in effecting coagulation or fibrinolysis. Coagulation factor as used herein includes factors of both the intrinsic and the extrinsic coagulation pathways, such as Factor I to V and VII to XIII (based on the international nomenclature used for clotting factors). Coagulation factor stabilizer: A substance that stabilizes one or more coagulation factors. The stabilizer reduces loss of activity of the factor over time. Glycine, which increases the stability of clotting factor VIII, is an example of a coagulation factor stabilizer.

Coagulation factor inhibitor: A substance that inhibits a coagulation activity associated with one or more coagulation factors. Protease inhibitors include serine proteases, cysteine proteases and the like. Benzamidine is an example of a coagulation factor inhibitor.

Stable Coagulation Control The present invention provides a stable coagulation control composition for monitoring the coagulation capability of a human patient. The coagulation control of the invention have a predetermined clotting time ranging from normal to abnormal and comprise primarily a mammalian plasma devoid of red cells, white cells and platelets.

The mammalian plasma comprises a majority of primate plasma and a minority of non-primate plasma. Human plasma from normal individuals is the preferred primate plasma because of its availability and the similarity of it's performance characteristics to a normal human coagulation control.

In achieving reproducible lot to lot long term 4°C stability of a liquid formulation coagulation control, it is important to have uniform criteria for the population of individuals used as donors of human plasma. The donor population of healthy males between 18 to 40 years old who have not exercised prior to collection is preferred. Also preferred are non-lipemic plasma samples.

Plasma can be collected by any means known in the art, provided that it is devoid of red cells, white cells and platelets. However, to obtain the long term 4°C stability characteristics of the present coagulation control, it is important to collect primate plasma using an automated plasmaphoresis apparatus. Plasmaphoresis apparatuses are commercially available and include, for example, apparatuses that separate plasma from the blood by ultrafiltration or by centrifugation. An ultrafiltration-based plasmaphoresis apparatus such as manufactured by Auto C, A200 (Baxter International, Largo, Florida) is preferred for collection of plasma having the best stability characteristics because it effectively removes red cells, white cells and platelets while preserving coagulation factors. Plasma is collected with an anticoagulant as is well known in the art. Preferred anti-coagulants are those that chelate calcium such as citrate. Sodium Citrate at 0.38% (final concentration in the plasma) is the preferred anticoagulant for collecting plasma.

Mammalian plasma used in the invention comprises a majority of primate plasma and a minority of non-primate plasma. Non-primate plasma is added to primate plasma to achieve a predetermined coagulation time. Generally, the coagulation time of pooled human plasma requires the addition of some non-primate plasma with faster coagulation properties to speed up the coagulation time in such tests as the PT and APPT. The amount of non-primate plasma to be added thus depends on the performance characteristics of the starting primate plasma and the potency of the non-primate plasma for speeding up coagulation. The percentage of non-primate plasma (relative to the total plasma) in the coagulation control is generally between about 2% to 18% (v/v) and, more typically between about 4% to 8%(v/v).

Non-primate plasmas useful in the present coagulation control formulation include those non-primates that have high levels of coagulation factors, particularly factors V and VIII. Non-primate plasma with low levels of factor VII are preferred for long term stability because factor VII can become unstable upon storage in a liquid formulation at 4°C or colder. Accordingly, preferred non-primate plasma is from animals of the group leporine, canine, porcine, and feline. Animals from the group leporine which comprise the Leporidae family and include such species as the rabbit (Oryctolagus cuniculus) and hare, are the most preferred sources of non-primate plasma. However, the choice of non- primate plasma can vary depending on the other components of the control formulation. One of ordinary skill can make such determinations without resort to undue experimentation.

Although non-primate plasma is preferably used herein to modify the coagulation time of primate plasma, the predetermined clotting times can be achieved by addition of purified clotting factors in place of non-primate plasma. Useful clotting factors include those generally known in the art, with factor V and factor VIII being preferred. The source of the clotting factor is preferably from primate plasma and most preferably from human plasma.

A coagulation control of the present invention also can include a buffer to help maintain the pH at a desired range, thereby stabilizing coagulation factors. A variety of buffers are useful and include, for example, HEPES (N-[2-Hydroxyethyl]peperazine-N^τ-

[2-ethanesulfonic acid]): pKa 7.5 @ 25° range 6.8-8.2; BTP (1,3 bis[tris(hydroxymethyl)methylaminopropane): pKal 6.8 pKa2-9.0 useful range 6.3-9.5; BES (N,N-bis[2-Hydroxyethyl]2-aminoethanesulfonic acid) pKa=7.1 @25°C range 6.4- 7.8; MOPS = (3-[N-Morpholino]propanesulfonic acid): pKa 7.2 @ 25°C range 6.5-7.9; TES (N-tris[Hydroxymethyl]-methyl-2-aminoethane-sulfonic acid): pKa=7.4 range 6.8-

8.2; MES (2-[N-Morpholino]ethane-sulfonic acid): pKa = 6.1 useful range 5.5 - 6.7; and TRIS or TRIZMA (tris[Hydroxymethyl]aminomethane): pKa = 8.1 @25°C range 7-9; and PIPES (piperazine- N-N'- bis[2-ethanesulfonic acid].

The amount of buffer to add in the coagulation control generally varies with the buffer pKa, the pH range desired and the effect of other chemicals included in the coagulation control formulation. The pH of the coagulation control is preferably between about 6.8 to 7.6 at 4°C, and more preferably between about 7.1 to 7.4 at 4°C. A preferred buffer is HEPES which is present in the coagulation control at a concentration preferably between about 20 to 150 mM and more preferably at 50 mM. A coagulation control of the present invention also can include a fibrinolysin inhibitor. A fibrinolysin inhibitor can provide additional stability by inhibiting components of the fibrinolytic pathway (including plasmin) that may be present or activated during storage. Such inhibitors are well known in the art and include, for example, lysine analog inhibitors such as epsilon aminocaproic acid ("EACA") and tranexamic acid ("TA"), Aprotinin (i.e., Trasylol) and natural inactivators of plasmin such as plasminogen activator inhibitor- 1 (PAI-1) alpha- 1 antitrypsin and alpha-2 macroglobulin. EACA is a preferred fibrinolysis inhibitor and is preferably present in an amount between about 25 to 250 mM, more preferably between about 100 to 200 mM and most preferably at 150 mM. A coagulation control of the present invention also can include a coagulation factor stabilizer. A coagulation factor stabilizer can provide additional stability to the coagulation control formulation by stabilizing particular factors that become unstable upon storage and contribute to the instability of the coagulation control. Compounds that stabilize factor V and factor VIII are particularly preferred. Coagulation control stabilizers are well known in the art and include, for example, glycine, glyclglycine, glycylglycylglycine, a non-ionic surfactant such as block co-polymers, e.g. polyoxamers or polyoxyethylene (20) fatty acid esters e.g. polysorbate 20 or polysorbate 80, or a carbohydrate such as a monosaccharide, disaccharide or sugar alcohol, (see e.g., U.S. Pat.

Nos. 5,147,803 and 5,919,76 and 5,919,908). A coagulation stabilizing factor that stabilizes factor VIII is preferably included when stability of the coagulation control is desired for more than 8 hours at 4°C. A preferred coagulation factor stabilizer of the present invention is glycine. Glycine is preferably included in an amount between about 75 to 150 mM and, more preferably, at 100 mM.

A coagulation control of the present invention also can include an excipient such as a carbohydrate when the freezing before use is anticipated. Such material generally is not needed if thawing of frozen coagulation control is performed at temperatures around 37°C. Carbohydrates useful in the invention include, for example, monosaccharides, disaccharides, polysaccharides or sugar alcohols. Carbohydrate such as sorbital, trehalose, raffinose, and allobiose are preferred. Carbohydrates are generally effective when added at between 1.5 and 7% (w/v).

A coagulation control of the present invention also can include an anti-microbial agent as a preservative. Suitable such agents are well known in the art and include, for example, sodium azide or PROCLLN^® (Supleco, Supleco Park, Bellefonte, Pennsylvania), which are suitable alone or in combination. A combination of sodium azide (0.05%) and PROCLLN^® (0.05%) is preferred. If the preparation of the coagulation control is performed under sterile conditions, then there is no need for a preservative unless the product is opened and stored for later use. The coagulation controls of the present invention are stable as a liquid formulation at 4°C for at least 7 days as shown in Examples 2 and 3. Such stability is obtained regardless of whether the coagulation control sample is prepared and stored frozen or prepared and lyophilized for storage.

Method of Preparing the Coagulation Control Another aspect of the present invention is a method to prepare the stabilized coagulation control compositions described above. A first step in the method is to obtain a source of primate and non-primate plasma to be used in making the composition. Plasma can be collected by any means known in the art, provided that it is devoid of red cells, white cells and platelets. However, to obtain the long term 4°C stability characteristics of the present coagulation control, it is important to collect primate plasma using an automated plasmaphoresis apparatus. Plasmaphoresis apparatuses are commercially available and include, for example, apparatuses that separate plasma from the blood by ultrafiltration or by centrifugation. An ultrafiltration-based plasmaphoresis apparatus is preferred for collection of plasma with long term stability characteristics because it effectively removes red cells, white cells and platelets while preserving the coagulation factors. Non-primate plasma to be used in preparation of a coagulation control formulation also can be obtained by any means known in the art. A preferred means is to obtain plasma from the jugular vein of a healthy non-primate animal and separate plasma from red cells, white cells and platelets by centrifugation. Plasmaphoresis of non-primate animals is generally not needed to achieve long term stability of coagulation controls because of the relatively small amounts of such plasma used in the composition.

Plasma is collected with an anticoagulant as is well known in the art. Preferred anti-coagulants are those that chelate calcium such as citrate. Sodium citrate at 3.8% (final concentration in the plasma about 0.38%) is the preferred anticoagulant for collecting plasma. To prepare coagulation controls that are stable for at least 7 days at 4°C, the plasma should include one or more chemicals (i.e. base chemicals). The stabilizing chemicals can be added directly to the plasma, but it is preferred to first dissolve the chemicals as a concentrate and then add the concentrate to the plasma. For example, the base chemicals used for stabilization can be compounded as a 1 OX concentration so that when 1 part of the 10X concentrate is mixed with 9 parts of plasma, the base chemicals will be at the correct concentration in the final coagulation control product (i.e., IX). As discussed above, the 10X base chemical solution can contain one or more of a buffer, fibrinolysis inhibitor, coagulation factor stabilizer, carbohydrate and a preservative. To assist in dissolution of the chemicals, it is preferred to stir the base chemical mixture at approximately 40-45°C. In the case where a specific buffer is included in the chemical mix, it is preferred to adjust the pH of the base chemical solution (after all the chemcials are dissolved) to about 7, more preferably 6.94. A preferred approach to stabilizing the plasma is to add the chemical base to the plasma within about 1-2 hours after collection if the plasma is not frozen earlier. It is preferred to add the chemical base immediately after plasma collection but not before any samples are removed for testing such as to meet FDA requirements. The volume of plasma to receive base chemical solution is measured and, in the case of the 10X concentrate, 1 part of concentrate is added to 9 parts of plasma.

It is preferred to add the base chemicals slowly to the plasma with gentle mixing to facilitate retardation of coagulation factor activation. After addition of the base chemical, the plasma can be stored at 2-8°C. Alternatively, plasma can be frozen and stored at about -60°C or colder. It is preferred not to freeze human plasma but to maintain it at about 4°C for up to 48 hours before further processing.

Primate plasma from separate donors can be pooled by gentle mixing in a plastic container. It is important to use plastic rather than glass to avoid activation of coagulation factors. It is preferred to expose the container to a temperature of 37°C such as provided in a waterbath or other heat source. All splashing and foaming should be kept to a minimum to prevent surface activation. Once the temperature of the pooled plasma reaches about 22-24°C (i.e., room temperature), the plasma can be removed from the heat source and further processed at room temperature (i.e., 22-24°C).

It is generally preferred to make abnormal coagulation controls (e.g., level 2 and level 3 type controls) from the same batch of plasma used to make the normal coagulation control (i.e„ referred to as level 1 control). The preferred strategy is to first prepare the entire batch of plasma including addition of base chemicals and then divide the plasma into the number of levels needed. Each level is then prepared accordingly.

As discussed above, appropriate non-primate plasma is added to primate plasma preferably by gentle mixing to achieve a predetermined coagulation time for level 1.

Generally, the coagulation time of pooled human plasma requires addition of some non- primate to speed up the coagulation time in such tests as the PT and APPT. The amount of non-primate plasma to be added thus depends on the performance characteristics of the starting primate plasma and the potency of the non-primate plasma for speeding up coagulation. The percentage of non-primate plasma added (relative to the total plasma) in the coagulation control is generally between about 2% to 18% (v/v) and, more typically between about 4% to 8%(v/v). Although non-primate plasma is preferably used herein to modify the coagulation time of primate plasma, the predetermined clotting time can be achieved by addition of purified clotting factors rather than by addition of non-primate plasma. Useful clotting factors include those generally known in the art, with factor V and factor VIII being preferred. The source of the clotting factor is preferably from primate plasma, most preferably from human plasma.

A coagulation control of the present invention also can be filtered to improve clarity and reduce microbial burden (if any). Filtering is preferably performed using a mixed cellulose esters filter with a 0.2μ to 0.45μ diameter pore. The pressure imposed during filtration should be kept to a minimum to avoid shearing of protein molecules.

The final coagulation control composition can be dispensed into plastic vials for storage if not immediately needed. Polypropylene and polyethylene are suitable for shorter stability times while polyethylene terephthalate/glycol ("PETG"), siliconized plastics or polyurethane vials are more preferred to achieve longer stability. The vials are preferably flash frozen using liquid Nitrogen. Slower freezing achieved at lower temperatures generally will not be suitable for achieving greater than 7 day stability of the coagulation control at 4°C. The product is then preferably stored at a temperature of -70°C or lower.

Coagulation control samples with prolonged coagulation times, indicated as level 2 and level 3 can be prepared by removing coagulation factors as is well known in the art, such as by absorption with aluminum hydroxide gel as described in U.S. Patent No. 5,891,843. In general, 10 ml of 2% aluminum hydrogel is typically added per liter of plasma and the precipitate removed by centrifugation at about 5000 rpm.

Abnormal coagulation controls with, for example, prolonged coagulation times also can be prepared as described herein by addition of coagulation factor inhibitors to slow the coagulation time. Preferred coagulation inhibitors include protease inhibitors such as trypsin type serine protease type inhibitors including aprotinin. Benzamidine is a preferred coagulation factor inhibitor for preparing abnormal controls and preferably includes heparin to achieve stabilization exceeding 7 days at 4°C. For example, a level two coagulation control is prepared by taking plasma and adding the protease inhibitor to achieve a PT of 17 to 22 seconds and an APTT of 42 to 65 seconds and adding heparin to 0.1 U/mL. A level three coagulation control is prepared by taking plasma and adding the protease inhibitor to achieve a PT of 23 to 35 seconds and an APTT of 65 to 90 seconds. Additionally, heparin to 0.1 U/mL can be added to the level two and level three preparations to increase the stability (see Example III, Table 5, for use of heparin to stabilize abnormal coagulation controls prepared using benzamidine).

EXAMPLES

Methods:

All coagulation assays were performed on the MLA Electra 1600C (Medical Laboratory Automation, Inc., Pleasantville, N.Y.). The ELECTRA 1600C is a computerized coagulation analyzer with automatic sample preparation. It uses photometric detection for both clotting and chromogenic assays and is an open system that can use reagents from different manufacturers.

Coagulation tests are performed using reagents and lyophilized controls from Dade-Behring (Deerefield, IL) following the manufacturer's instructions. The reagents include PT reagent - Dade Innovin Lot # TFS-654 (prepared form purified recombinant human tissue factor and synthetic phospholipids); APTT reagent - Dade Actin lot # 527109 (prepared from liquid rabbit brain cephalin with plasma activators) and CaCl₂ - Lot # 506861 A (Dade-Behring); Fibrinogen Reagent - Ortho/Hemoliance, Bovine Thrombin lot #BVT106A (prepared from bovine plasma thrombin); and Owren's Buffer (an aqueous saline solution containing a sodium barbital buffer (pH 7.35) and 0.08% 1,2

Benzisothiazolin-3-one as preservative) for diluting samples and calibrator used in the standard curve.

For PT, a sample plasma is warmed to 37°C and at a predetermined time, is vigorously mixed with 2 parts of PT reagent. The time begins simultaneously with the addition of the PT reagent and ends when clot formation is detected. Lyophilized PT reagent is reconstituted and used fresh daily.

APTT reagent obtained as a liquid formulation is mixed and pre-warmed with control plasma 1/3 and 1/3. After incubating for about 3 minutes at 37°C, pre-warmed calcium chloride solution (37°C) is added (the final 1/3) and the instrument begins timing. Again the time ends when clot formation is detected.

Fibrinogen is measured using the Clauss method developed in 1957 (Clauss, Acta Haemat. 17:237 (1957)). In this procedure excess thrombin is added to diluted plasma, resulting in a clotting time value that is inversely proportional to the plasma fibrinogen concentration. A six point calibration curve is generated using a reference plasma with known amounts of fibrinogen. The clotting times for unknown samples are calculated from the curve. Commercially available thrombin reagent provided lyophilized and reconstituted prior to use is stable for 7 days when stored at 2-8°C.

The samples were poured into samples cups and run in duplicate. The values reported are the average of these duplicates.

Example 1 Preparation of Stabilized Coagulation Controls

This example describes the preparation of a coagulation control that is stable for more than 7 days at 4°C. A plasma base is prepared that includes plasma and base chemicals to stabilize the plasma after it is collected.

The base chemicals are compounded as an intermediate solution which is made at a 10X concentration. The 10X base chemical solution in this example contains 1.5 M

EACA, 0.5 M HEPES, 3.8% (w/v) sodium citrate, 1 M glycine, 0.5% sodium azide, 0.5% PROCLIN^® and 25% sorbital. The base chemical solution is warmed to approximately 40-45°C to achieve complete dissolution of all the chemicals. After dissolution is complete, the pH is adjusted to 6.94. Human plasma obtained by ultrafiltration plasmaphoresis, devoid of red cells, white cells and platelets, is measured for volume and samples are taken for any necessary analysis (e.g. FDA product testing). Within 1-2 hours after plasma collection, 1 part of the 10X base chemical solution is slowly added to 9 parts of human plasma. The plasma is mixed gently to facilitate retardation of coagulation factor activation. Rabbit plasma obtained by centrifugation of freshly drawn blood taken from the jugular vein of healthy rabbits is processed as described for human plasma except is stored frozen.

After release, cooled plasma from separate human donors is pooled together in a plastic container, which is maintained at 37°C in a waterbath or other heat source. The temperature of the plasma is closely monitored while gently mixing. All splashing and foaming is kept to a minimum to prevent surface activation. Once the plasma temperature reaches 22-24°C, it is removed from the heat source and handled at room temperature (i.e., 22-24°C). The same pool of human plasma is used to make all three levels of the control. It is divided at this stage into three aliquots.

Level 1 : rabbit plasma warmed to 22-24 °C is added with gentle mixing to achieve the target values for PT, APTT and Fibrinogen. Generally, rabbit plasma is added to achieve an amount equal to 7% of the total plasma added (human plus the rabbit).

Level 2: O.lU/ml of heparin and approximately 2 mM benzamidine hydrochloride is added with gentle mixing. This actual amount of benzamidine added is the amount needed to achieved target coagulation times for PT (17 to 22 seconds) and APTT (42 to 65 seconds). Level 3: O.lU/ml of heparin and approximately 5 mM benzamidine hydrochloride is added with gentle mixing. This actual amount of benzamidine added is the amount needed to achieved target coagulation times for PT (23 to 35 seconds) and APTT (65 to 90).

Once the chemicals are added the control is pH adjusted to 7.1 + 0.5 using Sodium Hydroxide solution. This is done while the product is maintained at 22-24°C temperature.

The completed coagulation controls are filtered using mixed cellulose esters filter composition with a 0.45 μ pore diameter. Filtration is accomplished at reduced pressure (about 10-15 psi) to avoid shearing of protein molecules.

The filtered product is dispensed into plastic vials (PETG: polyethylene terephthalate/glycol) and flash frozen using liquid nitrogen. The product is stored at a temperature of -70°C or lower.

The process from collection of plasma to freezing of final product is complete within about 40 working hours over a seven day period.

Example 2

Stability of Stabilized Coagulation Controls

This example shows the stability of coagulation controls prepared according to Example 1, above. Three separate lots of Levels 1-3 were prepared from separate pools of human and rabbit plasma.

Each batch was snap frozen at the end of the day that they were completed. The following Monday, all samples were removed from the freezer and thawed at 37°C for 5 minutes. A sample was used on one day only and then discarded (essentially the same stability was seen for vials that kept open and used for the entire time period) . All pilot lots were analyzed in parallel for PT (Table 1), APTT (Table 2) and fibrinogen (Table 3).

The results showed that all three levels of coagulation controls were stable for at least 7 days at 4°C, even up to day 15. In all conditions, the percent of change over time was less than 13% and more typically between about 1 to 6%.

Table 4 provides a statistical analysis showing the mean, standard deviation and coefficient of variation for selected samples prepared and treated as discussed above. The individual levels were prepared from the same lot of plasma and were tested on 9 separate days within the 15 day period (the same days as shown in Table 1 or 2). Duplicate readings taken for virtually all days. The stability of the three levels of samples over the fifteen day period is evidenced by a coefficient of variation that is less than 5%.

Table 1 : Coagulation Control Stability at 4°C - Prothrombin Time (Seconds)

% CHG refers to percent change in the value immediately left of the percent change column compared to the value at day 0. Table 2: Coagulation Control Stability at 4°C - Activated Partia Thromboplastin Time (Seconds)

% CHG refers to percent change in the value immediately left of the percent change column compared to the value at day 0.

Table 3 : Coagulation Control Stability at 4°C - Fibrinogen Concentration

% CHG refers to percent change in the value immediately left of the percent change column compared to the value at day 0. Table 4: Statistical Analysis of Coagulation Control 4°C Stability

S.D.: standard deviation.

C.V.: coefficient of variation.

* All levels prepared for a single plasma mix: Batch No. 990513.

Example 3

Preparation of Stable Coagulation Controls with Abnormal Coagulation Times by

Addition of Coagulation Factor Inhibitors

This example shows the use of a serine protease inhibitor (i.e. benzamidine) to chemically slow coagulation time and the ability of heparin to stabilize the coagulation value over seven days at 4°C.

The coagulation control base material is prepared according to the preparation of a level 1 as described in Example 1. Increasing concentration of benzamidine was observed to reduce PT values. Addition of heparin at 0.6 to 0.1 Units/mL reduced the percent change in PT values over the seven day period by about a factor of four. Table 5 : Effect of Heparin on Abnormal Coagulation Controls Prepared with Benzamidine

*Formulation as level 1 as described in Example 1.

**Formulation as level 1 as described in Example 1 except 300 mM EACA and no sorbital added.

The examples set forth above are provided to give those of ordinary skill in the art with a complete disclosure and description of how to make and use the preferred embodiments of the compositions, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent or patent application were specifically and individually indicated to be incorporated herein by reference.

Claims

ClaimsWhat is claimed is:

1. A stable coagulation control composition for monitoring coagulation capability of a human patient, the composition having a predetermined clotting time ranging from normal to abnormal, the composition comprising mammalian plasma devoid of red cells, white cells and platelets.

2. The coagulation control of claim 1, wherein said mammalian plasma of the normal clotting time coagulation control comprises a majority of primate plasma and a minority of non-primate plasma.

3. The coagulation control of claim 1, wherein said non-primate plasma represents between 2% to 15% of the total plasma.

4. The coagulation control of claim 1, wherein said control is stable for at least seven days at 4°C.

5. The coagulation control of claim 1 , wherein said primate plasma is human plasma.

6. The coagulation control of claim 1, wherein said non-primate plasma is selected from group consisting of leporine, canine, porcine, and feline plasma.

7. The coagulation control of claim 6, wherein said leporine plasma is rabbit plasma.

8. The coagulation control of claim 1, further comprising one or more of a buffer, a fibrinolysis inhibitor, a coagulation factor stabilizer, a carbohydrate for stabilizing proteins upon freeze-thaw and a preservative to maintain sterility.

9. The coagulation control of claim 1, wherein said fibrinolysis inhibitor is epsilon aminocaproic acid.

10. The coagulation control of claim 1, wherein said coagulation factor stabilizer is glycine.

11. The coagulation control of claim 1 , wherein said coagulation control composition remains stable after freezing and thawing.

12. The coagulation control of claim 1, wherein said coagulation control composition remains stable after lyopbilization and reconstitution.

13. The coagulation control of claim 1, wherein said abnormal clotting times is accomplished by removing clotting factors from the plasma or by adding coagulation inhibitors to the plasma.

14. A method of making a coagulation control composition for monitoring coagulation capability of a human patient, the composition having a predetermined clotting time, the method comprising the steps of:

(b) adding non-primate plasma to the primate plasma, wherein the amount of primate plasma is in the majority and wherein the non-primate plasma is added in an amount sufficient to achieve a normal clotting time.

15. The method of claim 14, wherein said control is stabilized by the step of adding a stabilizing solution to the plasma, wherein the stabilizing solution comprises one or more of a buffer, a fibrinolysis inhibitor, a coagulation factor stabilizer, carbohydrate for stabilizing proteins upon freeze-thaw and a preservative to maintain sterility.

16. The method of claim 14, wherein said primate plasma is human plasma.

17. The method of claim 14, wherein said primate plasma is obtained by plasmaphoresis.

18. The method of claim 14, wherein said non-primate plasma is selected from group consisting of leporine, canine, porcine, and feline plasma.

19. The method of claim 18, wherein said leporine plasma is rabbit plasma.

20. The method of claim 14, further comprising the step of freezing the control composition prior to thawing and use as a control.

21. The method of claim 14, further comprising the step of lyophilizing the control composition prior to reconstituting and use as a control.

22. A method of making a coagulation control composition, the composition having a predetermined clotting time that is slower than normal plasma, the method comprising obtaining a source of plasma and adding one or more coagulation inhibitors to achieve the predetermined clotting time.

23. The method of claim 22, wherein said one or more coagulation inhibitors is a serine protease inhibitor.

24. The method of claim 22, wherein said serine protease inhibitor is benzamidine.

25. The method of claim 21, further comprising addition of a stabilizer compound to stabilize the clotting time.

26. The method of claim 25, wherein the stabilizer compound is heparin.