JPS5910645B2 - Method for concentrating and purifying factor 8 - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION For the production of antihemophilic factor (A}fF or factor VIII) for therapeutic use, several different methods such as selective precipitation, batch adsorption and Elution methods, low ionic medium extraction methods, chromatography methods, etc. have been disclosed so far.

The chemicals most frequently used in precipitation include alcohol, tannic acid, ammonium sulfate, chrysin, and polyethylene glycol.

Although purification of factor VIII involves the removal of various other plasma proteins, fibrinogen is the most important and troublesome of these proteins, such as when denatured by processes such as alcohol precipitation, freezing and thawing. Especially so.

This denatured fibrinogen interferes with the P filtration of M2, resulting in a significant loss of AHF during the purification process and reducing the solubility of the lyophilized product in the freshly prepared liquid.

Therefore, all satisfactory AHF purification methods require the removal of significant amounts of fibrinogen.

Although the selective precipitation methods described above have been designed for this purpose, they all have drawbacks such as further denaturation of fibrinogen and AHF and undesirable loss of AHF.

Simple cooling precipitation without using chemicals [cryoprecipitation]
) is usually limited to small-scale production in blood banks and, when used therapeutically, results in high levels of blood fibrinogen, which experts in the field In some cases, it is considered unfavorable.

Although operations involving extraction of factor VIII from cryoprecipitate in low ionic strength buffers somewhat reduce the fibrinogen content of the final product, it still has an unreasonably high protein content. This method requires special equipment and operations for centrifugation, and limits the total amount of AHF' that can be extracted from the chilled precipitate without interfering with the purification operation.

Other problems typically associated with large-scale production of AHFO are pyrogens and viruses that cause hepatitis (hepatitis-associated antigen, H
AA) contamination of the final product.

These undesirable contaminations can be further increased by the use of chemical precipitants.

The method described herein effectively overcomes these problems, eliminating the drawbacks associated with the use of chemical precipitants and selectively removing fipurinogen, its denaturation and degradation products. cold precipitate
It relies on simple operations such as:

(In the following, the removal of fibrinogen includes the removal of fibrinogen, its denaturation, and decomposition products).

Selective precipitation of fibrinogen without loss of factor VIII has not yet been perfected as a practical method for large-scale production of purified AHF concentrate.

In fact, Wickerhauser
) (see reference 1 at the end) emphasizes the importance of limiting the temperature and time when extracting AHF.

“A somewhat higher yield of AHF is due to prolonged Tris extraction at 30°C for more than 60 min.
), but the extract became increasingly contaminated by aggregated fibrinogen, which reduced the filtration of the final concentrate.

Conversely, with short or low temperature extractions, low and variable AHF yields were achieved.

” The operations described herein eliminate all these problems.

This is because all excess fibrinogen contaminants are removed during cooling, while no AHF is lost.

The effect of cooling has already been reported by Hershgold et al. (cited reference 2 at the end).
However, achieving rapid and selective precipitation of fibrinogen and allohemagglutinin at temperatures as low as 0-2°C requires 18-30 hours at 4°C. I couldn't even suggest it.

[In fact, in a later study, Hirschgold et al. omitted the cooling step altogether in an attempt to obtain a very pure AHF'' (see cited reference 3 at the end).

They found it impossible to produce therapeutic concentrates that could be filtered aseptically and had to resort to alcohol or glycine precipitation steps.

The significant improvement of the present invention was an unexpected surprise in light of the disappointing report of Schugold et al.

James and Bitskerhauser
Wickerhauser) (see reference 5 at the end) 1 Furthermore, this method must be carried out at room temperature to avoid precipitation of fibrinogen.

” commented.

However, they were unable to develop any method of producing concentrates of Factor VIII using the principles of the present invention.

Their final products were also very low in yield and purity compared to those described in the present invention.

Further treatment with polyethylene glycol to remove fibrinogen and increase purity resulted in a more significant loss of factor VIII (see references 1 and 6 below).

Thus, other investigators have concluded from their experience that the results expected from using very slightly lower temperatures in the precipitation and removal of fibrinogen after very short precipitation times may require particular improvements. It is clear that we have long reasoned or concluded that there is no such thing.

The unexpected and widely improved results discovered and reported herein are contrary to the teachings and suggestions of the prior art and were initially achieved by the inventors by chance rather than by design. It is something.

The present invention is a special method for large-scale production of factor VIII concentrates.

One of the unique features of the present invention is the selective cryoprecipitation of excess fibrinogen, its denatured products and decomposition products in low ionic strength solutions, without the addition of chemical reagents. 《And more than necessary AH
It does not impair F activity.

Yet another notable feature is that the theoretical amount in plasma of approximately 25
Factor VIII is obtained in surprisingly high yields of ~40%.

Additionally, despite the high recovery of AHF, the protein content was reduced by 50-75% compared to other products.

This is shown in Table 1.

The need for high-yield, high-purity lyophilized AHF concentrates has received international understanding and attention (see citations 7 and 8), and commercially available concentrates are typically present in plasma. M to do
It provides less than 20% of the buried potential of 廿゛ (see cited documents 7, 8, and 9 listed at the end).

Hereinafter, the present invention will be explained in more detail according to preferred embodiments.

Frozen plasma, e.g. 100-300
0 liters was thawed from about -5°C to about +2°C and collected in a suitable tank or container.

A larger amount can be handled, but it will be difficult to operate.

Cryoprecipitate was collected at 3° C. or below, preferably by continuous flow centrifugation (Sharpress or similar centrifuge).

Other collection methods may be used, but they are less efficient than this one.

The cryoprecipitate was weighed and then mixed with a small amount of pyrogen-free distilled water for a few seconds, preferably in a Warring-type blender, to form a slurry or emulsion.

Next, after heating the slurry to 20 to 30°C, the slurry was heated to a pH of about 7.
．． Extraction was carried out for 30 to 60 minutes in 2 to 3 times the amount of pyrogen-free distilled water at 0.0 °C.

Next, add aluminum hydroxide gel at 10 to 10% per liter.
It was added in an amount of 30 rLl and allowed to adsorb for 15 minutes.

For further purification, 0.5 to 2.0 weight percent tricalcium phosphate may be added, but in this case the amount of aluminum hydroxide is reduced.

This step helps remove lipids, denatured proteins and prothrombin complexes.

This entire operation was carried out in a jacketed reaction vessel with care to avoid foaming and with continuous stirring.

The container contents were then cooled to an internal temperature of about 1'C to 2C for about 1/2 to 2 hours.

The resulting heavy precipitate was removed by continuous flow centrifugation (eg Sharpless).

The obtained supernatant liquid was heated to 0.0°C at a temperature of 20 to 25°C.
It was stabilized with 2M trisodium citrate and 0.1M glycine and the pH was adjusted to 7.0 with citric acid.

The solution is then diluted with 1.2, 0.65, 0.45 or 0.3
Cleaned and sterilized through a 293u+ Millipore Membrane filter or cartridge equivalent with micron pore size.

This resulted in approximately 2 of factor VIII in the original starting plasma.
Sterile solutions containing 5-40% were lyophilized in conventional manner for storage.

The production method is particularly suitable for refrozen cryoprecipitate or Cohn Fraction I.
However, in that case, the yield of factor 8 will decrease and the yield will depend on the concentration of factor 8 in the starting material. Dew.

Extraction of AHF can also be performed in other low ionic strength buffers such as Tris buffer.

The cooling time can be varied and increased within the scope of the invention and can also be repeated with further agglomeration of Factor 8.

Similarly, the extraction time is within the range of the described method.
can be increased up to an hour.

The resulting product can be stored for long periods of time, at least one year, at +5° C. and can be regenerated with distilled water or brine.

Due to the relatively low fibrinogen content and high albumin content, the product goes into solution very quickly.

Compared to other manufacturing methods, the total processing time from opening the plasma bag is very short, thus limiting bacterial growth.

Since the extraction is carried out in distilled water, the amount of fipurinogen and gamma glopurin that goes into solution is reduced.

By cooling at 2° C., selective precipitation of excess fibrinogen, its modification and decomposition products as well as macroglobulins takes place without appreciable loss of AHF.

Furthermore, the heavy cohesive precipitate of fibrinogen traps any pyrogen present and reduces the amount of hepatitis associated antigen (HAA or hepatitis virus).

This makes it possible to obtain a product that is 30 to 60 times more pure than plasma and has very low amounts of fibrinogen and "saline" homologous hemagglutinin.

If necessary, this product can be further purified and concentrated by known or novel methods.

In a representative example of the new method, the ultrapure factor VIII preparation is made from 3 to 6% polyol (polyethylene glycol or pluronic, a trade name of Biandatte, Inc., see reference 10, supra). and then cooling again at 0~2°C for 1-2 hours.

Please refer to item number (7) in Table 1. One of the major advantages of the present invention is that ultra-high purity AHF can be produced in less than 8 hours, compared to 2-3 times longer with conventional methods.

The embodiments described are the best currently known methods for carrying out the invention.

It will be apparent, however, that the present invention may be practiced by applying the principles of the present invention to conventional processing methods and materials.

For example, starting from less than about 100 liters of plasma or cryoprecipitate from this amount of plasma.
LAlthough generally speaking it is economically unfeasible, it is clear that there are no limit values for either the upper or lower amounts indicated in the embodiment, and a change of around 50% of these values will not affect the present invention.

In the most preferred embodiments, the operation is carried out using known and readily available equipment, such as Sharpless centrifuges and Waring blenders.

However, these steps themselves and the equipment involved, which are unrelated to the principles of the invention, are not unique to the present invention, and major changes may be made within the operator's choice depending on available personnel, equipment, etc. This can be done within the range of

The adsorption of aluminum hydroxide is a known process per se and is not unique to the concept of the present invention.

When carrying out this step, it can be carried out with considerable discretion, such as by substituting another adsorbent, etc., achieving the same purpose by an equivalent step, or simply omitting the step.

After obtaining a supernatant containing a high recovery of Factor VIII, the supernatant is processed in a conventional manner for storage and regeneration.

For example, purified and sterilized by filtration through a standard micropore filter, lyophilized to concentrate Factor VIII to a small, easy-to-storage volume, and stored in a normal container, such as pyrogen-free water or live brine, for example. It is regenerated using the liquid of

EXAMPLE 215 liters of frozen plasma was thawed maintaining the temperature below +2°C.

The insoluble fraction, cryoprecipitate, was collected in a continuous flow centrifuge with a cooling system kept at a temperature below 3°C.

The cryoprecipitate thus obtained was extracted with twice the volume of pyrogen-free distilled water at about 20° C. and pH 7 for 30 minutes.

Aluminum hydroxide gel was then added in an amount of 2 l/k9 cryoprecipitate.

Further, this was cooled at +2°C for 30 minutes, and then the precipitate was removed by centrifugation.

The supernatant was stabilized with trisodium citrate and glycine and sterilized by passing through a Millipore Membrane filter of the above pore size.

5.2 l of sterile solution was obtained, which is 12.2 units/r
It contained factor VIII of ILl.

The yield based on raw plasma was 30%.

In addition, fibrinogen content 6. 7 1 1f/l,
and total protein content 1 5.0 3 ? /l.

Although the invention is limited by the scope of the following claims,
The present invention is not limited to the description of the exemplary process presented in the specification as the best mode.

References cited in the specification■. M. Wickerhauser
r), "Large-Scale Fraction of Factor VIII - Concentrate from Cryoethanol Precipitation", Thromb, Diath
, Haemorrh, vol. 43, p. 165 (1971
year) 2. E. J. Hersh Gold
gold), J. G. Pool, A. R,
Papperhagen
"An Potent Antihemophilic Factor Concentrate Derived from the Chilled Insoluble Fraction of Human Plasma; Characterization and Additional Data on Preparation and Clinical Trials," J. Lab, C.
Iin. Med. , vol. 67, p. 23 (1966) 3. E, J, Hirshgold, A, M, Davison (
Devison), M.E., J.
hen), "Isolation and chemical properties of human factor VIII (antihemophilic factor)", J, Lab, CIin. Med,
, vol. 77, p. 185, (1971) 3. E, -7 Shanbrom,
L. Fekete, [a stable, high potency drug using polyethylene glycol and glycine to fractionate cryoprecipitate in AHF concentrates]
4. Process for Preparing AHF, U.S. Pat. No. 3,631,018 (December 28, 1871).
H.L. James, M. Binkerhauser “Development of large-scale fractionation methods”, VoxS ang,
, Vol. 23, p. 402 (1972) 4, J. T., Shouris, M. Bitsker Hauser “
"Utilization of Frozen Cryoprecipitate for the Preparation of Clinical Factor VIII Concentrates", Transfusion, 1
Volume 3, page 399 (1973) 7. "Recent Advances in Hemophilia", Ann. N.Y. Acad, Sci. ,2
40, pp. 1-426 (1975) 8. J. G. Poole, [Recent Topics in the Factor VIII Genealogy - The Risk of Certain Proteins J. West, J.; ofM
ed, vol. 122, p. 406 (99, 1975).
L. F. Huekete, S. L. Holst
), [Stabilization of AHF' using heparin], USA>
Patent No. 380311 (April 9, 1974) 10. BASF-Wyandatt
e Corp. ), "Alphahydromega-hydroxy-poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) block copolymer", The Amazing World of Pluronic (Trademark of Biandatte Co., Ltd.) Polyols (1971).

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Claims (1)

[Claims] 1. Cryoprecipitate prepared from frozen plasma,
Eighth step, comprising extraction in a low ionic strength aqueous medium at about room temperature and adsorption by an aluminum hydroxide gel.
In the method of concentrating and purifying factors, the extraction operation is carried out at pH
7 for 30-60 minutes and cooling the resulting solution by maintaining it at a temperature of 1-2 °C for 0.5-2 hours, and collecting the resulting supernatant. . A method for concentrating and purifying factor 8, which comprises freeze-drying. 2. The method according to claim 1, wherein the solution is purified by adding aluminum hydroxide gel before the cooling step and removing the gel together with the precipitate formed after cooling. Cryoprecipitate obtained from 3 iooz or more of frozen plasma is soaked in about 2 to 3 times the volume of pyrogen-free water at 25 to 30°C and pH 7 for 30 to 60 minutes. a step of adding aluminum hydroxide gel to remove lipids, denatured proteins and prothrombin complexes from the extract by adsorption; fibrinogen and its denaturation and decomposition products are removed from the resulting extract in large amounts Precipitation by cooling the extract at 1-2° C. for 1/2-2 hours without removing factor 8, approximately the factor 8 content in the cryoprecipitate used as starting material. Claim 1, characterized in that it includes the steps of separating, stabilizing, cleaning and sterilizing a supernatant containing 80% or more, and lyophilizing the stabilized supernatant. the method of.