JPS596845B2 - Method for improving yield of antihemophilic factor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for concentrating antihemophilic factor (AHF, factor Ⅰ).

In particular, the present invention provides AHF obtained from plasma and plasma fractions.
This invention relates to a method for improving the yield of.

The coagulation mechanism is a complex biochemical process that involves the interaction of several substances that are normally present in the body.

It is known that certain factors related to the clotting mechanism are absent or highly deficient in some individuals.

Therefore, it is known that classical hemophilia (type A hemophilia) is a deficiency disease caused by a lack of AHF (factor II).

In individuals suffering from congenital hemophilia, known as hemophilia type B, the blood is deficient in plasma thromboplastin forming factor (PTC, factor II).

Some other factors important in the coagulation mechanism are
, factor (■), and factor X.

Until recently, the treatment for hemophilia was to transfuse the patient with whole blood or plasma.

However, in reality, it is said that an excellent treatment method is to administer only the deficient blood component to the patient, if possible.

Since the total amount of therapeutic blood is in short supply, fractionating blood into its various components is also advantageous in that these components can be used for patient treatment as needed.

Various methods are known for fractionating or concentrating blood and plasma into their individual components.

In the development of alcohol fractionation methods...E at Bard College
The work of Dwin Cohn and his collaborators is particularly noteworthy.

Specifically regarding AHF generation, recent U.S. Pat.
No. 18 and No. 3,652,530 disclose improved methods for obtaining high purity concentrates of the factor.

Wagner, Thelin, Brinkhouse
The existence of prothrombin (factor Ⅰ) and its associated factor group complex has been shown to be of paramount importance in the research activities of and other people interested in AHF production, both in the long term and in the short term. It was found that the stability of the factor was impaired.

Typical treatments to solve this problem include aluminum hydroxide, magnesium hydroxide, barium carbonate, barium sulfate, ribanol (6,9-diamino-2-ethoxyacridine lactate), IRC-50 ion exchange resin. (
The prothrombin complex was removed with various reagents such as XE-6 (4-rivanol), glycine ethyl ester.

Although the reagents described above are generally effective, it has become apparent that there are various serious drawbacks to their use in clinical applications.

Factor II is actually stabilized by the use of these reagents since no thrombin is generated by removal of prothrombin.

Kisker, “Thromb.
emorrhagicaJ Volume 17, Page 381 (19
1967) and Penick and Brinkh.
ous, “Amer. J. Med, S.
Sciences, J Vol. 232, No. 434 (19
As reported in 1956, thrombin is the main factor responsible for the degradation of factor II.

Therefore, in the preparation of factor Ⅰ with aliphatic amino acids, w
In the seminal work of Agner et al., hydroxylation was performed to (1) reduce the degradation of factor II during the concentration procedure and (2) increase its long-term stability even after freeze-drying and reduction. Aluminum was used.

"Hemophilia", K, M. Edited by Brinkhous, International Symposium, Washington, DC. C. ,
See University of North Carolina Press, p. 81 (1964).

However, the presence of trace amounts of aluminum was considered clinically dangerous.

cryoprecipitation method
) and the one-step preparation of relatively high strength concentrates apparently made it unnecessary to use any means to remove the prothrombin complex.

See Pool et al., Nature J, vol. 203, p. 312 (1964).

However, in practice, it has been found that there is a high variability in the yield of AHF, up to and sometimes exceeding 50% of the reported average value.

Therefore, one object of the present invention is antihemophilic factor (AHF),
An object of the present invention is to provide an improved method for concentrating factor (I).

Another object of the present invention is to provide a method for improving the yield of AHF obtained from plasma and plasma fractions.

Other objects and benefits of the invention will be apparent to those skilled in the art after reading the following description of the invention.

In summary, the present invention involves adding heparin to an aqueous medium containing plasma or plasma fractions and then fractionating the AHF
Obtain concentrate.

The invention may be better understood with reference to the following figures.

This figure is a schematic diagram showing the coagulation mechanism.

Roman numerals represent several clotting factors.

However, it will be understood that the schematic represents only a working hypothesis based on current knowledge, and that the inventors are not limited or bound by this hypothesis.

As mentioned above, high variability in AHF yield was observed in the actual preparation of AHF.

The longer the concentration step, the lower the AHF yield.

Yield losses are reduced to some extent by careful blood cell removal.

These results can be explained by reference to the blood coagulation schematic.

Of particular note is the feedback mechanism represented by the dashed line, which shows the effect of factor A (thrombin) on factor V (gloaccelelin and factor II (AHF)).

The action consists of two parts.

That is, the effects of factor Ⅰ and factor V (1) first increase due to the tautomeric tertiary structure, and then (2) decrease to suppress excessive blood coagulation that causes circulation difficulties.

According to the blood coagulation diagram, if factors ①, ②, It is clear that

AHF prepared by concentration using cell-free plasma
Our observation that the yield of can be increased can also be explained by the coagulation scheme, in which case thromboplastin is not produced and therefore clotting does not begin.

According to the present invention, it has been found that in production scale quantities the yield of AHF can be increased by adding heparin during fractionation.

In contrast to the prior art, which does not use heparin, the use of heparin eliminates (1) cell contamination, such as occurs during fractionation, that causes yield loss; and (2) does not occur during intravenous administration. (3) prolongation of the process time does not reduce the yield, and (4) the stability when reduced is higher than that of concentration without the addition of heharin. The inventors observed an increase of at least 30 times that in plasma.

Therefore, in the practice of the present invention, it is not necessary to remove the prothrombin complex in order to obtain the stability of concentrated factor II, and there is no need to remove the prothrombin complex due to cell and tissue contamination or prolongation of process time. This also solves the problem of easy yield reduction.

In the practice of this invention, the amount of heparin used during AHF fractionation can vary within suitable ranges.

A concentration of approximately 1 unit of heparin per 72 liters of plasma solution or plasma fraction II has been found to be optimal.

Concentrations above about 10 units/ml are unnecessary and dangerous and should be avoided.

Concentrations of about 0.01 units/TLl are also effective.

The preferred range is about 0.01 units to about 10 units/1 rL.
It is l.

One unit of heharin is IU, s. p. It means the unit (United States Pharmacopoeia unit).

Heparin IU. S. P. The unit is CaCl2 solution (1
:1 0 0 ) 0.21 nl is the amount that will not cause clotting of 1.0 ml of citrated sheep plasma for 1 hour after addition.

The term "heparin" as used herein also includes the sodium salt of heparin, which is preferred because it is water-soluble.

The invention defined herein is effective in a variety of methods for preparing concentrated AHF and can be applied to any plasma or plasma fraction containing AHF mixed with any of the prothrombin complex factors. .

Therefore, the present invention was applied to the following AHF fractionation method.

Namely, (1) AHF fractionation method from plasma using glycine precipitation, (2) AHF separation method from cryoprecipitate using glycine precipitation.
Fractionation method, (3) AHF fractionation method from cryoprecipitate by polyethylene glycol precipitation, (4) AHF fractionation method from cryoprecipitate by polyethylene glycol and glycine precipitation, (5) AHF fractionation method from cryoprecipitate, ( 6) AHF fractionation from plasma; and (7) AHF fractionation from cryoprecipitate chromatographed after polyethylene glycol and glycine precipitation [ecteoracellulose].

A preferred AHF adjustment method for application of the present invention is described in U.S. Pat.
31018, in which polyethylene glycol (PEG) and glycine are used for the purpose of fractionating a cryoprecipitate of concentrated AHF.

That is, in Example 4 of the above-mentioned patent, about 1 unit of heparin is added per ml of the solution during the step of dissolving the cryoprecipitate in glycine citrate saline.

Following precipitation with 10% polyethylene glycol, 1
To compensate for the heparin lost during the fractionation step using 0% polyethylene glycol, the precipitate solution is treated to contain about 1 unit of heparin per L1 of solution ITr.

For convenience, heparin may be added in a mixture with citrate saline or glycine citrate saline used to dissolve the respective precipitates.

It is believed that the following examples will clarify the invention, but of course the invention is not limited to these specific examples. It's not something you can do.

Example 1 A highly concentrated stable human blood AHF concentrate is prepared in high yield by the following method.

Reagent Citrate Saline-0. Add 1 part by weight of 1 M sodium citrate solution to 4 parts by weight of 0.9% saline. Glycine citrate saline - Add enough glycine to the citric acid saline prepared as above to obtain glycine 0.IM. Make a solution.

Washing buffer - 0.5M sodium citrate solution.
A volume of citric acid buffer adjusted to pH 6.88 with 5 M citric acid is added to distilled water.

100 Heparin - Uses USP grade material ("Lipo-Hepin J-Heparin Sodium Injection, Aqueous)".

Acetic acid-1. Prepare an ON aqueous solution and an 0, IN aqueous solution.

Glycine-1.3M aqueous solution and 1.8M aqueous solution are prepared.

Method Human plasma is received frozen (below 4°C) from a blood donation center.

The plasma was pooled in funder canisters and incubated at −20° to −40°.
Centrifuge the mixture continuously or in packets while maintaining the temperature at °C.

1 unit of heparin/TILl glycine citrate saline is added to the cryoprecipitate to bring the volume up to 1 volume of the starting plasma volume.

The cryoprecipitate and the glycine citrate saline solution are stirred and dissolved in a warm environment (normal room temperature, 30° C. or lower).

The cryoprecipitate solution was reduced to 0. Adjust pH to 6.5 with IN acetic acid.

Polyethylene glycol 4000 (average molecular weight approximately 40
00) is added to this solution to bring the PEG concentration to about 3.5%.

The mixture was stirred gently for 10 minutes at room temperature and then for 50 minutes.
00rpm. Centrifuge for 15 minutes.

Decant the supernatant to 0. pH with IN sodium hydroxide
Adjust to 6.88.

Furthermore, PEG4000 was added to this solution to form the final PEG.
Make the concentration about 10%.

The mixture was stirred gently at room temperature for 30 minutes until 500
0 rpm. Centrifuge for 1.5 hours.

Decant and discard the supernatant.

The precipitate was washed with cold water (2°C) and then incubated at -4°C for 5 minutes.
0 0 0 rpm. Spin wash for 5 minutes.

The supernatant is decanted and the precipitate is redissolved in glycine citrate saline containing 1 unit/TLl heparin.

Also, the amount of this solution is starting 1 The plasma volume is approximately -.

10 Add this precipitate solution to 0. Adjust to 1) H6.88 with IN acetic acid and reprecipitate with 1.8M aqueous glycine solution.

Sufficient glycine is added during this precipitation step to create a mixture with a glycine molar concentration of 1.8.

The mixture is gently stirred at 2°C to 10°C for 45 to 60 minutes and then centrifuged by continuous or packet mode.

The resulting precipitate is collected, gently washed with washing buffer, and redissolved in citrate saline.

Filter this solution using a 293 Millipore filter (filter membranes used = 1.2 micron, 0.45 micron, 0.3 micron).

This generated liquid is then subjected to shell freezing (-60
Store in a flash freezer (-20°C to -30°C) for at least 3 hours.

For the purpose of dissolving the initial cryoprecipitate or 10% PEG
For the purpose of dissolving the precipitate obtained by fractionation by
Alternatively, the above method is repeated without adding heparin to the glycic citric acid saline solution used during the fractionation step.

The above procedure is repeated both with and without added heparin.

The following table shows the results in these four fractionation experiments.

The table shows the final AHF yield of 17% with heparin addition.
On the other hand, it has been shown that the yield without adding heharin is on average 12.5-13.6%.

Therefore, there are two methods: adding heharin or not adding heperin.
It increases the yield by 5-35%.

Dividing the final AHF yield by the cryoprecipitated AHF yield, the efficiency of this process is 38% and 39% without heparin addition.
It can be seen that the improvement is from 49% to 52% when heparin is added.

In the above example, PEG6000 was replaced with an equal amount of PEG4
Substantially the same result can be obtained even if the value is replaced with 000.

Example 2 The method of Example 1 is repeated up to the step of resuspending the initial cryoprecipitate in heparinized and non-heparinized glycine citrate saline.

The final AHF yield of these two cryoprecipitated AHF concentrates is 34% without heparin and 41% with hehalin.

This corresponds to a 21% increase in sales.

Example 3 The process of Example 2 was repeated, but the difference was that the freeze-precipitated concentrate with heparin added and the freeze-precipitate concentrate without heparin were freeze-dried and then reduced with water. Then, leave it at room temperature (approximately 25°C) for 24 hours.

The initial AHFO power of the heparinized sample before standing was 9.
Only 72% of the original AHF strength remained in the sample without heparin, whereas 8% remained.

Example 4 Three commercially available concentrates were used to determine the ratio of active AHFO to total AHF in the final AHF after fractionation.

Here, active AHF refers to AHF that has coagulation activity, and total AHF refers to AHF that is a combination of active AHF and inactive AHF that does not have coagulation activity.

Sample ratio Cutter Laboratories, Inc.
(No heparin added) 0゜16-0゜18Traven
Ol (heparin added) 0.29 ~ 0.42Imm
uno (heparin addition) From the results of 0.28 to 0.42 or higher, it was found that the addition of heparin increased the ratio of active AHFO to total AHF in the final AHF by 160 to 260%.

Various other embodiments and modifications of the embodiments described above will be apparent to those skilled in the art after reading the foregoing specification and claims without departing from the spirit and scope of the invention.

All such additional embodiments and modifications are within the scope of the claims.

Embodiments of the present invention are summarized below.

(1) The method according to the claims, wherein the plasma fraction is cryoprecipitated concentrated antihemophilic factor.

(2) The method according to claims, wherein the plasma fraction is cryoprecipitated concentrated anti-hemophilic factor further fractionated with polyethylene glycol and glycine.

(3) The average molecular weight of the polyethylene glycol is about 4
000, the method according to the preceding clause (2).

(4) Precipitate the cryoprecipitate concentrate with 3-4% polyethylene glycol, then precipitate the supernatant with 10% polyethylene glycol, and further fractionate the supernatant with glycine. Method described.

(5) The average molecular weight of the polyethylene glycol is about 4
000, the method according to the preceding clause (4).

(6) The method described in item (4) above, wherein the glycine is about 1.8M.

(7) The average molecular weight of the polyethylene glycol is about 4
000, and the glycine is about 1.8M.

Claims (1)

[Claims] 1. Antihematogenic preparations obtained from plasma cryoprecipitates and plasma cryoprecipitate fractions, which consist of adding 0.01 to 10 units of heparin per ml of solution to the plasma cryoprecipitates or plasma cryoprecipitate fractions. A method for improving the yield of philic factors.