WO1991016913A1

WO1991016913A1 - Stabilized heparin solution

Info

Publication number: WO1991016913A1
Application number: PCT/US1991/002174
Authority: WO
Inventors: Jack Debrauwere; Jean-Marie Mathias; Jean-Marc Payrat; Michel Baes
Original assignee: Baxter International Inc.
Priority date: 1990-05-07
Filing date: 1991-04-01
Publication date: 1991-11-14
Also published as: CA2062807A1; JPH04507108A

Abstract

A heat-sterilizable, stable aqueous solution typically includes in the solution from 5 to 1000 USP units of heparin per ml. and from 10 to 250 millimoles of calcium lactate per liter of said solution. This solution stabilizes the heparin present and permits its pasteurization or sterilization without significant loss of heparin activity. The solution may be used with collected blood or preferably blood plasma for improvements in the extraction of factor VIII activity therefrom.

Description

STABILIZED HEPARIN SOLUTION

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of U.S. Application Serial No. 157,547 filed February 18, 1988.

TECHNICAL FIELD

In Rock U.S. Patent No. A,359,463 a method is described in which initial factor VIII activity normally present in blood collected into a calcium- chelating anticoagulant (for example, CPD or ACD) may be maintained by mixing freshly collected blood or plasma with a calcium-heparin solution in sufficient quantities to restore calcium to substantially normal physiological levels. Typically, the solution used contains calcium chloride and heparin.

Heparin containing solutions for storage in collapsible plastic containers are typically buffered with a phosphate, for example monosodium phosphate. Because of this, difficulties are encountered when calcium ions are added to such heparin solutions in an attempt to provide a stable calcium-heparin solution. Calcium phosphate can easily precipitate from the solution in that circumstance.

The heparin must, however, be buffered in order to be stabilized in solution. Particularly, the heparin must be buffered to withstand a heat sterilization and pasteurization cycle without undue loss of heparin activity. Such is of course needed as a prior step to provide an aseptic heparin solution which may be safely administered to collected blood or plasma. Other references which discuss the effect of heparin and calcium on factor VIII activity in blood or plasma include articles by Morgenthaler et al. "Influence of Heparin and Calcium Chloride on Assay, Stability and Recovery of Factor VIII" Vox Sang. 48, 8-17, (1985) and the article by Rock et al . entitled "Stability of VIII: C in Plasma: The Dependence on Protease Activity and Calcium". Thrombosis Research 29, 521-535, (1983).

DESCRIPTION OF THE INVENTION

By this invention, a heat-sterilizable, stable, aqueous solution of calcium and heparin may be provided, for use in improving the yields and other process parameters of the collection of factor VIII from blood. The solution typically comprises from 5 to 1000 USP units heparin, preferably sodium heparin, per ml. of solution present, and from 10 to 250 milli- moles of calcium lactate per liter of such solution. These solutes are preferably completely dissolved in the solution, sufficient heparin being present to retard clotting when calcium ions are added to blood or plasma.

The calcium lactate present has been found to exhibit a double function. First, it is an improved stabilizer for heparin, so that such a solution can be heat sterilized under conventional sterilization cycles and stored with less loss of heparin activity. Specifically, the solution can be stored for a period in excess of six months, or elevated temperatures a period in excess of three months, without major heparin loss. The solution is nontoxic, and useful for providing improved yields and other improvements in known, conventional processes for isolating factor VIII in a therapeutic dosage form from blood. The preparation of such therapeutic dosage forms is a present day commercial activity, and is a well-known process. While concentrations of only about 50 millimoles or less of calcium lactate per liter provide good stabilizing characteristics to the heparin, it has been found that the best factor VIII:C yield improvement is obtained when the calcium-heparin solution used in processes for isolating factor VIII and converting it into a therapeutic dosage form contain preferably from 100-250 millimoles per liter of calcium lactate, but typically no more than about 200 millimoles per liter.

Typically, the solution of this invention contains about 15 to 50 millimoles of calcium lactate per liter if stabilization of heparin is the main concern. Otherwise more than about 50 millimoles per liter of calcium lactate should be present to increase factor VIII:C yield. Also, the solution typically contains from about 25 to 200 USP units of heparin per ml. The heparin concentration in accordance with this invention may be assayed in accordance with the USP assay for heparin sodium as generally described in the USP Official Monographs, Volume 21, pages 481 and 482. This assay method may be modified by measuring the clotting time in an available clot timing apparatus rather than by visually measuring the extent of clotting, for greater accuracy. The data herein was collected by such a modified method. For use of the solution of this invention, one may add the solution to freshly collected blood or preferably freshly separated blood plasma (separated by any conventional means), for example by means of plasmapheresis using an Autopheresis-C unit sold by the Fenwal division of Baxter Travenol Laboratories, Inc. One may add from 20 to 50 ml. of the solution of this invention per unit of blood or plasma, a unit of plasma being about 600 ml. in volume. Typically, the addition step may take place immediately after separation of the plasma into a separate unit, the plasma containing an anticoagulant such as ACD or CPD from the original blood collection process. The addition of the solution of this invention after a substantial amount of plasma has entered the separate plasma container prevents the initial aliquots of plasma from sensing an excessive concentration of solution of this invention, which can sometimes initiate an amount of clotting. After the plasma has been mixed with the desired amount ofsolution (for example 40 ml. of solution per unit of plasma) the resulting plasma-solution mixture may be processed by a conventional cryoprecipitation process such as described in U.S. Patent Nos. 3,652,530, or 3,631,018, as described in Rock U.S.

Patent No. 4,203,891. The solution of this invention not only exhibits significant advantages in the stabilization of heparin in solution, but it also provides improved yields of clinically useable antihemophilic factor (factor VIII:C) in many conventional processes for the isolation of antihemophilic factor for therapeutic use.

While lactate ion is used in some peritoneal dialysis solutions and intravenous solutions (for example lactated Ringer solution), and while calcium lactate is known as a stabilizing agent for certain prostaglandin compositions (British Patent No.. 1,582,162), calcium lactate has apparently never been used as a stabilizer for heparin in solution. The heparin solutions of the prior art have been less stable in heat resistance and/or shelf life than the solution of this application, so that the advantages of this invention have not previously been readily obtainable. To illustrate the invention of this application, the following examples are disclosed.

Example 1

To 5 liters of water there is added 2.07 g. of heparin sodium (1641 USP units per 30 ml.) and 45 g. of sodium chloride. Three 1 liter portions of this solution were then separated for further processing. To a first one liter portion (solution 1) there was added 2.94 g. of calcium chloride dihydrate plus sufficient sodium hydroxide solution to adjust the pH to 8.5. To a second 1 liter portion (solution 2) there was added 8.97 g. of calcium gluconate monohydrate. To the third one liter solution portion (solution 3) was added 6.17 g. of calcium lactate pentahydrate . Each of solutions 1-3 thus contained essentially 20 mh per liter of calcium.

From the supply of solutions 1-3, primary blood collection bags (Fenwal division of Baxter International, Inc.) were each filled with 30 ml. of one of the solutions to provide a large plurality of such bags. Some of the bags were sterilized in a conventional sterilization cycle (115-120 degrees C for 50-75 minutes). The bags were then pouched for storage in aluminum foil bags and pasteurized at 96 degrees C for 170 minutes, prior to assay for heparin in accordance with the assay method referred to above.

The original, bulk, 5 liter heparin solution before sterilization exhibited about 61.4 USP units of heparin per ml.

Referring to solution 1, the nonsterile solution had a pH of 6.40, while the sterilized solution had a pH of 4.44 and assayed for 58.6 USP units of heparin per ml. With respect to solution 2, the nonsterile solution had a pH of 6.42, while the sterilized solution had a pII of 4.44, and assayed at about 51.5USP units of heparin per ml.

With respect to solution 3, containing calcium lactate, the nonsterile solution exhibited a pH of

6.85, while the sterilized solution had a pH of 5.61, and assayed for about 60.4 USP units of heparin per ml.

Thus it can be seen that the heparin content and pll of solution number 3 was preserved through the sterilization process, when compared with the results of solutions 1 and 2, which failed to prevent significant decrease in the concentration of active heparin present and the pH.

Example 2

(A) Samples of solution 3 as described in Example 1 above were stored for six months at room temperature in sealed, foil-enclosed blood bags of the type previously described. Initially, the sterilized solution assayed for about 60.4 USP units of heparin per ml. and had a pH of 5.61. At the end of six months, the solutions assayed at about 58.6 USP units of heparin per ml. and had a pH of 5.46. (B) Other containers of solution 3 prepared as in Example 1 above, were stored at 45 degrees C. for thnee months. Initially, the containers assayed at about 60.4 USP units of heparin per ml. and a pH of about 5.61. After the three months storage, the containers assayed at about 62.2 USP units of heparin per ml. (the increase being apparently due to experimental error of the assay procedure) and exhibited a pH of 5.35. It can be seen that little or no degradation of the heparin or the pH took place during storage, even at elevated temperatures.

(C) Another sterilized sample of the solution 3, prepared as in Example 1 above initially had the heparin assay and pH as stated in Example 2 (A). After one month of storage at 45 degrees C, the heparin assay was about 61.98 USP units of heparin per ml., and the solution had a pH of 5.49, showing essentially no degradation of either the heparin content or the pH. To the contrary, a similarly treated, bagged sample of solution 1 showed a heparin assay of about 61.6 USP units per ml. After sterilization, the solution exhibited a heparin assay of about 60.4 USP units per ml. After one month of storage at 45 degrees C the solution exhibited a heparin assay of about 56.4 USP units per ml. Thus it can be seen that the shelf life of solution 1 is much shorter than the shelf life of solution 3.

Example 3

One unit of plasma from a blood donor was separated from whole blood on an AUTOPHERESIS-C brand machine (Baxter International) using a sodium citrate anti-coagulant. Four different solutions were prepared as follows :

- Solution 1: Calcium chloride solution (3 mM.) Solution 2: Calcium lactate solution (20 mM.) containing 60 IU/ral. of heparin sodium. Solution 3: Calcium lactate solution (50 mM.) containing 135 IU/ml. of heparin sodium. Solution 4: Calcium lactate solution (170 mM.) containing 135 IU/ml. of heparin sodium.

Samples of each of these solutions were placed in plastic tubes and mixed with fresh, collected blood plasma in a calcium solution to plasma volume ratio equal to 1:27 for solutions 1, 3, and 4, and a similar ratio of 1:21 for solution 2. The calcium solutions were added within four hours of their collection to insure freshness, and the plasma-calcium solution samples were frozen at -80 degrees C. promptly thereafter. The plasma-calcium solution tubes were kept in such frozen condition until assayed.

After about two weeks of storage, the various samples were thawed for fifteen hours at 2 degrees C, followed by 20 minutes of centrifugation at 3500 g, and the cryoprecipitate was recovered. Also a control run of cryoprecipitate was prepared from the same plasma, without added calcium solution.

Six tubes of each type of solution were tested in the above-described manner. The mean values of factor VIII:C of cryoprecipitate recovered from each of the six different samples is recorded below, as well as the standard deviation. Each mean value represents the total factor VIII:C activity of the cryoprecipitate in each sample in IU/ml., multiplied by the weight of the cryoprecipitate in grams, and divided by the actual volume of plasma (ml.) in each x sample. The results are as follows:

Control Means Solu. 1 Solu. 2 Solu. 3 Solu. 4

Cryoprecipitate

Activity

0.360 0.346 0.376 0.419 0.502

Standard Deviation

0.020 0.022 0.012 0.016 0.013

Thus it can be seen that the presence of increased amounts of calcium lactate in Solutions 2-4 provide significantly increased yields of factor VIII:C activity, when compared with the control and solution 1 in which calcium chloride was present.

When the factor VIII:C activities are compared in five different sets of six plastic tubes containing control and treated plasma after thawing in a 37 degree C. water bath, the following results are obtained.

Control Means Solu. 1 Solu. 2 Solu. 3 Solu. 4

Cryoprecipitate

Activity

0.715 0.732 0.813 0.794 0.853

Standard Deviation

0.008 0.032 0.039 0.046 0.051 It can be seen that the calcium lactate solutions retain their superiority over the control solution and solution 1 which contains calcium chloride. Also, solution 4, which contains 170 mM. of calcium lactate per liter, exhibits the best results of factor VIII yield.

Thus, by this invention a stable, aqueous heparin solution is provided, having sufficient calcium lactate to permit heat sterilization of the solution and six month (or more) room temperature storage thereof with no more than about 5 weight percent heparin loss. The solution of this invention also provides improved factor VIII yields when used in a process to isolate that blood fraction.

The above has been offered for illustrative purposes only, and is not intended to limit the scope of the invention of this application, which is as defined in the claims below.

Claims

THAT WHICH IS CLAIMED:

1. A heat-sterilizable, stable, aqueous solution which comprises in said solution an effective amount of heparin to retard clotting when added to blood or plasma, and from 10 to 250 millimoles of calcium lactate per liter of said solution.

2. The solution of Claim 1 in which pH is essentially 4 to 7.

3. The solution of Claim 1 which contains about 15 to 50 millimoles of calcium lactate per liter.

4. The solution of Claim 1 which contains about 50 to 200 millimoles of calcium lactate per liter.

5. The solution of Claim 1 in which about 5 to 1000 USP units of heparin per ml. are present.

6. The solution of Claim 1 in which said heparin is the sodium salt thereof.

7. A heat-sterilizable aqueous solution at a pH of essentially 4 to 7 which comprises in said solution from 5 to 1000 USP units of heparin per ml. of solution, and from 10 to 200 millimoles of calcium lactate per liter of said solution.

δ. The solution of Claim 7 in which said heparin is the sodium salt thereof.

9. The solution of Claim 8 which contains about 15 to 50 millimoles of calcium lactate per liter.

10. The solution of Claim 8 which contains about 100 to 200 millimoles of calcium lactate per liter.

11. The solution of Claim 8 in which from 25 to 200 USP units of heparin per ml. of solution are present.

12. The method of adding to collected blood or blood plasma from 20 to 50 ml., per unit of blood or plasma, of an aseptic, stable, aqueous solution which comprises in said solution an effective amount of heparin to retard clotting when added to blood or plasma, and from 10 to 250 millimoles of calcium lactate per liter of said solution, whereby improvements may be obtained in the extraction of factor VIII:C activity therefrom.

13. The method of Claim 12 in which the pH of said solution is essentially 4 to 7.

14. The method of Claim 13 in which said solution is added to freshly separated blood plasma.

15. The method of Claim 14 in which about 5 to 1000 USP units of heparin per ml. of solution are present.

16. The method of Claim 14 which contains about 15 to 50 millimoles of calcium lactate per liter of solution.

17. The method of Claim 14 which contains about 100 to 200 millimoles of calcium lactate per liter of solution.

18. The method of Claim 14 in which said heparin is the sodium salt thereof.

19. A stable, aqueous solution which comprises in said solution an effective amount of heparin to retard clotting when added to blood or plasma, and sufficient calcium lactate to permit heat sterilization of said solution and six month room temperature storage thereof with no more than about 5 percent heparin loss.