NO145563B - PROCEDURE FOR PREPARING A PURIFIED CONCENTRATE OF FACTOR-VIII. - Google Patents

Description

The present invention relates to a method

for large-scale production of a purified concentrate of fac-

Thu VIII. Among the characteristic features of the invention, one can mention a selective precipitation of excess quantities of fibrinogen and denaturation and degradation products, elimination of other unwanted proteins, such as isohemagglutinin

ner, and concentration in one step of factor VIII by cold precipitation using polyol, without unwanted loss of the AHF (antihemophilic factor) activity. Another characteristic feature is the surprisingly high yield of factor VIII, between 20 and 40% of the theoretical amount of plasma used.

Furthermore and despite the high yield of AHF, the protein content turns out to be reduced in a significant way and especially the content of fibrinogen, compared to other products. This is illustrated in Table I below. The need for a

high yield of concentrated lyophilized AHF with high purity is generally recognized and has met with great international attention. Reference may be made to "Recent Advances in Hemophilia", Ann.N.Y. Acad.Sci. 240, 1975 (Reference 11); pool,

J. G. "Recent Chapters in the Factor VIII saga: Perils of

a protein". West J. of Med. 122:406, 1975 (reference 12) and it is generally accepted that commercial concentrations are generally given as less than 20% of AHF theoretically present in the plasma and that the yield in the most highly purified products is even lower (reference 11, pages 156, 208; reference 5 and 8).

In the table I below, the values are, apart from

from those relating to the result according to the present invention

see, taken from an article by Robert Breckenridge "Recent Advances in Hemophilia", Ann.N.Y. Acad. of Science, 1975.

Several methods have been described for preparing the anti-haemophilia factor (AHF or factor VIII) for therapeutic use, e.g. selective precipitation, fractional absorption and elution, extraction in a weak ionic environment and chromatography. The chemical products most used for precipitation include alcohol, tannic acid, ammonium sulphate, gly-

cin and polyethylene glycol. Although the purification of factor VIII entails the elimination of a certain number of other plasma proteins, among these proteins fibrinogen is the most prominent and the most troublesome, especially since it is denatured by the methods, such as precipitation with alcohol, freezing and melting. This denatured fibrinogen damages the filtration of AHF, leads to loss of AHF during the purification step and reduces the solubility of the lyophilized product in the reconstitution fluid. Therefore, all purification methods for AHF require the removal of significant amounts of fibrinogen. The techniques

which include selective precipitation are geared towards this, but all have the disadvantage of having to denature both fibrinogen and AHF or of not being able to tolerate insignificant losses of AHF.

The methods that use simple precipitation

during cooling without chemical products (cryoprecipitation) is limited to the production of small amounts of factor VIII usually in blood banks, and leads to high levels of fibrinogen in the blood, which is considered undesirable by some experts for therapeutic use.

The methods that use extraction of factor VIII based on cryoprecipitation in buffers with weak ionic strength, which to some extent reduces the content of fibrinogen in the final product, still give an undesired high protein level and require special equipment and procedures for centrifugation and are limited with regard to the total amount of AHF that can be extracted from the cryoprecipitation, without damaging the purification.

Other common problems associated with large-scale production of AHF are, in particular, contamination of the final product with various pyrogenic substances, isohemagglutinins and hepatitis-causing viruses (hepatitis-associated antigen (HAA)). Certain chemical precipitants favor the presence of unwanted pollutants. Polyethylene glycol is now widely used in the production of factor VIII with high purity, but these methods use such high concentrations of the polymer that final steps consisting of washing and reprecipitation are necessary to eliminate excess ethylene glycol from the final product. These methods thus lead to new unwanted losses of AHF. In this connection, reference should be made to Wickerhauser, M. "Large-scale fractionation of Factor VIII - Concentrate from cryo-ethanol precipitate (reference 1); Shanbrom, E. & Fekete, L. "Production of stable high-potency human AHF using polyethylene glycol and glycine to fractionate a cryoprecipitate of AHF concentrate", U.S. patent no. 3,631,018 (reference 2); Sgouris, J.T. and Wickerhauser, M "Use of frozen cryoprecipitate for the preparation of clinical Factor VIII concentrate: Transfusion", 13:399, 1973 (reference 3); Newman, Johnson, A.J., Karpatkin, M.H. and Puszkin, S "Methods for the production of clinically effective intermediate and high-purity Factor VIII concentrates", Brit.J.Haemat, 1971, 211 (Reference 4); Fekete, L.F. and Holst, S.L. "Stabilization of AHF using Heparin", U.S. Patent No. 3,803,115 (Reference 5).

The method described here largely eliminates all of the aforementioned problems and has none of the disadvantages associated with the chemical precipitation techniques described above, and is based on a simple selective precipitation under cooling of fibrinogen and its denaturation and degradation products and for selective concentration of factor VIII with small amounts of polyol, e.g. polyethylene glycol or polyol ("Pluronic"). As explained below, the elimination of fibrinogen also relates to the elimination of its denaturation and breakdown products.

Selective elimination of fibrinogen without associated losses in factor VIII has previously not been carried out as... a practical method for producing a pure concentrate of AHF on a large scale. Wickerhauser emphasizes the importance of limiting the duration and temperature when extracting AHF: "Slightly higher yields of AHF are provided by long-term extraction with Tris buffer (based on tris(hydroxyethyl)aminomethane) at 30° C and 60 minutes, but the extract is far me: contaminated by fibrinogen aggregates, which makes the final concentrate difficult to filter."

A method using selective precipitation of fibrinogen at low temperature for the production of concentrate of factor VIII with high yield and with high purity on a large scale is the object of US-PS 4,188,318 entitled: "A simple method for the production of high- yield, high-purity Factor VIII", (reference 7). It is stated that preparations . can be further refined and concentrated using a polyethylene glycol (PEG) precipitation under cooling.

Until recently, all processes using PEG for the production of concentrates of AHF required an additional step consisting of washing and/or reprecipitation with glycine or alcohol since the polymer was used in high concentrations (10 - 12%) . (See the aforementioned references). The idea has recently been applied by cold precipitation with PEG, see Vermeer, C, Soute, B.A.M. and Brummelhuis, H.G.J., "Improvement of the preparation of Factor VIII Concentrates", Proe. of Int. Soc. Haemostasis and Thrombosis, Paris, July 1975, (reference 8). In order to precipitate fibrinogen, the concentration of PEG used in this case is only 2.5%, which leads to a less pure product. Furthermore, these authors have not considered the concentration of proteins to be important, they have used mandelate (salt of mandelic acid) to produce an irreversible precipitation of fibrinogen and have consequently obtained a product which is considerably less pure and gives a lower yield. In a similar way, the method of Shanbrom and Fekete (cited above) uses a concentration of the protein that is relatively small during the precipitation step with PEG, i.e. 1 volume part precipitation per 10 volume parts buffer and this leads to a very small yield with limited purity. These authors and likewise Johnson et al (reference 4) and Johnson et al "Antihemophilic factor prepared from blood plasma using polyethylene glyeol", US-PS No. 3,652,530, (reference 6) have used 10 - 12% PEG for the final concentration of AHF, which makes it necessary with a further dissolution and precipitation step with glycine or ethanol during cooling to reduce the content of PEG in the final product.

Another proof that the concentration of proteins and precipitation with PEG during cooling are important and lead to an original product lies in the fact that "incomplete" isohemagglutinins (antibodies) are largely eliminated, which reduces the risk of hemolytic reactions that could act since massive injections of the relevant concentrates which are available in the trade are necessary. (See: Seeler, R.A., Telischi, M, Langehennig£ P.L, Ashenhurst, J.B.: "Anti-A and Anti-B titers in coagulation concentrates", Abst. of Int. Soc. of Blood Transf., Helsinki, 1975'( reference 9).

Another characteristic feature of the product produced according to the invention is the high degree of purity (i.e. the low protein content), especially when measurable amounts of fibrinogen are involved. The high degree of purity results in a very soluble product, so soluble in fact that no undissolved material is found in the container after reconstitution. This eliminates the need for the use of a special filter or a needle filter to remove undissolved protein before the injection into the patient. Contamination of the Factor VIII concentrate with metal particles is therefore avoided (Couper, I.A., McAdam, J.H., MacKenzie, M.S. and Davidson, J.F.: "Contamina-tion of Factor VIII concentrates with metal particles", Lancet, December 21, 1974, page 1515 (reference 10).

The described method is also applicable for "retreatment" of pyrogenic amounts of concentrates with factor VIII produced by other methods, whereby expensive but pyrogenic factor VIII, which is not usable, is converted into a therapeutically usable agent of great value. The reprocessed product is reduced with respect to pyrogenic material either in a single step by precipitation during cooling with PEG of 6%, or in two steps by the method

are described in more detail below.

The greatly improved achieved results described stand in contrast to previously achieved results and suggestions in prior art techniques.

The present invention aims to compensate for the shortcomings of the known technique and thus to provide

a method for producing a purified concentrate of factor VIII from an impure solution of factor VIII by precipitation using a polyol and this method is characterized according to the invention by adding polyol to the impure solution of factor VIII to a concentration of approx. . 6%, and that the temperature is kept at 0 - 5°C until precipitation of factor VIII occurs, which is worked up into a concentrate of factor VIII which is lyophilized.

The method according to the invention is preferably carried out in that the precipitation of factor VIII is redissolved in a buffer, the resulting solution is kept at a temperature of 0.5 - 5°C for a period of 12 - 24 hours and the insoluble products that are secreted during this period is removed.

In the preferred embodiments, the method according to the invention has the following characteristic features: the source of factor VIII is a cryoprecipitate dissolved in 2-4 times its volume of buffer;

the volume ratio of cryoprecipitation to buffer is 1-3;

the method consists of subsequent steps in which the precipitated factor VIII is dissolved in a buffer and the resulting solution is kept at a temperature between 0.5 and

5°C for a period of between 12 and 24 hours after which the particulate mass formed is separated from said solution to provide a factor VIII substantially free of fibrinogen, fragments of fibrinogen and their complexes;

the buffers are citrate buffers;

the polyols used include polyethylene glycols and particularly polyethylene glycol 4000, and block copolymers of the type α-hydroxy-ft-hydroxy-poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene). Among these block copolymers one can

in particular mention the commercially available "Pluronic" products, and especially "Pluronic F68" which is a copolymer having the following characteristics: Molecular weight of the poly(oxypropylene) group: 1750; % by weight poly(oxyethylene): 80%.

The precipitation of factor VIII in the presence of 6% polyol is preferably carried out at pH 6.8 - 7.2.

The preceding precipitation of fibrinogen in the presence

of 4% polyol is preferably carried out at pH 6.0 - 6.5 and at a temperature of between 20 and 30°C.

The method according to the invention allows, in particular, to eliminate pyrogenic substances from a solution of factor VIII; this method is characterized by the fact that you add approx. 6% polyol to the aforementioned solution and keeping the solution at a temperature between 0 and 5°C, to precipitate factor VIII and recover a solution of pyrogenic material. This method can be carried out in the following preferred way: Addition of polyol to buffer solutions of factor VIII takes place by adding approx. 4% polyol, the precipitate is separated from the solution, 2% polyol is added and the solution is kept at a temperature of between 0 and 5°C, until a precipitate of factor VIII is obtained.

The solution to be purified preferably contains 1-3% protein, and more precisely 2-3%.

The method comprises subsequent steps in which the factor VIII precipitate is dissolved in a citrate buffer, the resulting solution is maintained at a temperature of between 0.5 and 5°C for an operating period of between 12 and 24 hours, after which the particulate matter which has formed in the solution , is separated from this to provide a factor VIII which is practically free of fibrinogen, fragments of fibrinogen and complexes thereof.

The invention specifically aims at a method for concentrating factor VIII, which consists in keeping a solution of approx. 1 part of a blood fraction containing factor VIII obtained by cryolite precipitation of plasma, for approx. 3 parts citrate buffer containing approx. 6% polyol at a temperature of between 0 and 5°C to obtain a precipitation of said factor VIII in the solution, after which said

te factor VIII is dissolved in the buffer to provide a solution of factor VIII of high purity.

The following examples illustrate the invention.

Example 1

One melts frozen plasma, e.g. 100 - 3000 litres

at a temperature of between -5 - +2°C and places the plasma in a suitable vessel. Larger volumes can be treated, but the operation will then be more difficult. The fraction which is insoluble at this temperature (the cryoprecipitation) is collected, preferably in a centrifuge (a Sharpless centrifuge or similar device) at a temperature below 3°C.

Other collection methods can be used, but these are less effective. The cryoprecipitate is weighed, divided into small pieces,

or mixed in a Waring type blender for a few seconds to produce a paste or emulsion. These parts of the cryoprecipitate or this paste are then dissolved in 2 - 4 volumes of buffer consisting of 0.02M trisodium citrate and 0.1M glycine where the pH is adjusted to 6.9 by adding citric acid at a temperature between 20 and 30°C. After the solution is complete, the fibrinogen is selectively precipitated by adding 4% "Polyethylene glycol 4000" or "Pluronic F-68"

at pH 6.0 - 6.5 and at a temperature of 25°C The precipitate is removed by centrifugation, e.g. in a Sharpless-type apparatus and the supernatant is adjusted to pH 6.8 - 7.2.

A further amount of 2% PEG or "Pluronic F-68" added-

and the suspension is cooled again to 0 - 5°C until just a visible precipitate is obtained.

A reaction vessel with a double wall is used

all these stages of dissolution and precipitation, with conti-

equal stirring and attempts are made to avoid foam formation.

The second precipitation step is followed by centrifugation

with continuous circulation at 1 - 2°C and the supernatant liquid is removed. The precipitate is then dissolved in a glycine-citrate buffer or in a citrate buffer, the volume of which depends on the quality of the cryoprecipitation and the number of units. AHF

which is present in the original solution: The following two methods are available to calculate this volume: 1. Dissolve the final precipitate in a buffer volume corresponding to 1/100 of the original plasma volume. 2. Dissolve the final precipitate in a volume of buffer corresponding to 15 - 25 times the weight of the precipitate.

The solution is then adjusted to a pH of 6.7 - 6.9 using citric acid, clarified and then sterilized by passing the liquid through microporous membrane filters (the height of the filter 293 mm corresponding to a column) where the pores have a diameter of f .ex. 1.2 - 0.65-0.45 or 0.3 ym. The sterile solution containing 20 - 40% factor VIII in the plasma is lyophilized in the usual way for storage.

Variants of this production technique can be used, in particular by using frozen cryoprecipitation or variants of fraction I according to Cohn, during this treatment the yield of factor VIII is less and depends on the concentration of the starting material. The initial dissolution or extraction of AHF can be carried out in distilled water or in buffers of weak ionic strength, such as Tris buffer. The cooling time can also vary, be increased or carried out in sequences. Likewise, the extraction times can be increased up to 24 hours in the described processes. Likewise, the processes can be interrupted at each step and resumed another day. All these variants can lead to a certain reduction in the total yield of AKF

in the output plasma.

Example II

Pyrogenic quantities of intermediate purity factor VIII concentrate prepared by other methods such as extraction with buffers of weak ionic strength, precipitation with glycine, precipitation with PEG, can be improved according to the scheme indicated in Example I. The lyophilized contents of the bottles are reconstituted in a volume of pyrogen-free water sufficient to give a protein content of 1-3%, after which the contents of the bottles are combined in a vessel and again subjected to a treatment according to the steps indicated in Example I.

If the pyrogen levels are relatively moderate and if the product already has a high degree of purity, as a variant, only one precipitation step can be carried out. The bottles are reconstituted as described above, and 6% PEG is added at a pH of 6.8 - 7.2 at a temperature of between 0 and 5°C. The precipitate which forms is then treated as before, and it is the supernatant containing the pyrogenic material which has been dissolved and which is eliminated.

Example III

The procedure described is carried out

in Example I.

But after the final step of filtration has been carried out, the product is placed in a refrigerator (temperature 0.5 - 5°C) and the product is left here for 12 - 24 hours.

The white, faded material that forms in this period is eliminated by decantation, centrifugation or pre-filtration. The final product is sterilized by filtration or as previously described and subjected to the same lyophilization steps. This product is even cleaner than the aforementioned products and therefore even easier to filter. No loss of factor VIII occurs due to this extra step.

The resulting products can be stored at +5°C for a period as long as one year, and reconstituted in distilled water or physiological serum. Due to its high purity and very low fibrinogen content, the lyophilized product dissolves very quickly (1-2 minutes). The turnaround time for the treatment is very short, compared to other methods of production from the moment the plasma bag is opened, so that the development of bacteria is limited. The fact that the initial dissolution takes place in small amounts of buffer reduces the amount of gamma globulins that could enter the solution, and the optimal precipitation of excess fibrinogen occurs using a polyol concentration of 4%. Furthermore, the heavy, flocculating precipitate of fibrinogen will presumably trap pyrogenic material present and reduce hepatitis antigen (HAA or hepatitis virus). By only using 6% polyol for the precipitation during cooling, unwanted proteins and especially isohaemagglutinins which can be dissolved also in smaller volumes of buffer, will not be precipitated with AHF and will therefore be eliminated with the above liquid. You therefore obtain a product that is purified 200 - 400 times compared to the plasma, and the product contains very small amounts of fibrinogen and isohemagglutinins. Concentrates of factor VIII which are prepared with very large volumes of extraction buffer, e.g. 10 parts by volume (reference 2), does not give the same purity or the same yield of AHF (reference 1, 2, 3, 4, 8) probably due to the fact that optimal ratios between protein and polymer for co-precipitation are not provided. If this is desired, such a product can be purified and concentrated by known methods or the new methods. A major advantage of the present invention lies in the fact that large-scale production of very pure AHF can be carried out in half the time required by other methods.

The examples given in the foregoing are the optimal methods known for carrying out the invention. Although it is usually not economical to start with less than approx. 100 liters of plasma, or a cryoprecipitation from such a quantity of plasma, it is obviously possible to use larger or smaller amounts than indicated in the examples and variations of 50% or equivalent in these values will not damage the invention. The procedures in the optimal example are carried out using known equipment which is easy to use, such as Sharpless centrifuge, Waring mixer etc, but the equipment used is not necessary and a large number of variants are available to those who know the present techniques.

When one has provided a liquid containing factor VIII in large yield, this is treated in a known manner for storage and reconstitution, e.g. by preparation and sterilization through a standard microporous filter, it is lyophilized to concentrate factor VIII to a small volume that is easy to store and it is reconstituted using normal liquid, e.g. pyrogen-free, distilled water or a physiological serum.

The manufactured product has original properties that benefit the patient and that distinguish the product from other concentrates of factor VIII that are actually manufactured.

These characteristics are:

1) Very high purity and therefore a very high "specific activity". 2) Very high solubility,' which enables rapid dissolution before treatment and which makes the material ideal for emergency treatment. 3) Very little protein content, which reduces unwanted secondary reactions. 4) Very low content of immunoglobulins, which reduces allergic reactions. 5) Negligible content of isohemagglutinins, which eliminates hemolytic reactions. 6) Very low content of fibrinogen, which reduces hyperfibrinogenic complications. 7) Filter needle is not necessary since foreign particles are eliminated. 8) The elimination of pyrogens during the processes conserves the usable factor VIII.

Claims (2)

1. Process for producing a purified concentrate of factor VIII from an impure solution of factor VIII by precipitation using a polyol, characterized in that polyol is added to the impure solution of factor VIII up to a concentration of approx. 6%, and that the temperature is kept at 0 - 5°C until precipitation of factor VIII occurs, which is worked up into a concentrate of factor VIII which is lyophilized. 2. Method according to claim 1, characterized in that the precipitation of factor VIII is redissolved in a buffer, the resulting solution is kept at a temperature of 0.5 - 5°C for a period of 12 - 24 hours and the insoluble products that are secreted during this period, is removed.