DK157170B - CONCENTRATE OF ANTI-HAEMOFILE FACTOR VIII AND PROCEDURES FOR PRODUCING THEREOF - Google Patents

Description

DK 157170 B

The present invention discloses a concentrate of the anti-hemophilic Factor VIII, also called AHF, which is of the kind set forth in the preamble of claim 1, and a method of preparing such concentrate.

It is known that the ability of blood to coagulate is controlled by a system of coagulation proteins, of which AHF or Factor VIII is an important component.

People with haemophilia-A or haemorrhagic disease have completely or partially lost the ability to produce AHF. For the treatment of this disease, AHF-containing preparations are injected, and it is therefore important to have preparations containing suitably high concentrations of AHF and with the lowest possible content of other proteins, such as fibrinogen and immunoglobulins.

Especially in the treatment of inhibitor patients, i.e. for patients who produce antibodies against AHF and who therefore need to raise the preparation in excess, with high doses of AHF, it is important that the specific activity (unit of AHF / mg protein) is high, as a case of "other 20 proteins "in sterile amounts can cause unwanted side effects. By 1 AHF unit, factor VIII is understood to be the activity of 1 ml of normal blood plasma. Side effects of AHF treatment caused by immunoglobulins are e.g. described by Tilsner et al., Munich. With. Wschr. 124 (1982) no.

25 22, pp. 553-557.

AHF is usually extracted by the so-called cryoprecipitate, which mainly consists of fibrinogen, albumin, IgG and IgM, while AHF accounts for less than 1¾ of the total protein. The specific activity of a cryoprecipitate 30 is typically 0.1-0.3 units. AHF / mg protein.

It is known that one can remove part of the unnecessary 2

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protein by precipitation with glycine or polyethylene glycol (PEG). Optionally, precipitation can be carried out several times, with one and then with the other precipitant.

It has long been known to trap AHF by approx.

5 2 M glycine at low temperature, cf. Webster et al., See Amer. J. Med. Sc. 250, No. 6, pp. 643-650 (1965).

Webster used a cryoprecipitate which was dissolved in water and fractionated at low temperature (<10 ° C) with increasing concentrations of glycine up to 2.3 molar. In this type of precipitation, it is utilized that AHF in the cold precipitates with fibrinogen by the addition of glycine. Thereby a specific activity of 0.3 - 0.5 unit is obtained. AHF / mg protein.

From the description file to E'P ip.ate, specification 32655, it is known that by passing the fractionation of redissolved cryoprecipitate with 2M glycine as a precipitant at higher temperatures, such as 30-45 ° C, it is known precipitated a large proportion of the other proteins, such as fibrinogen, such that AHF remains in the supernatant from which it can be recovered.

When PEG is used as a precipitant, a fractional PEG precipitation is most often performed, with the majority of the fibrino gene and some IgM removed by low PEG concentration (2-6?), After which AHF is precipitated from the 25 albumin-containing supernatant with PEG concentration (5-15? ô). Such PEG / PEG precipitates are described by e.g. Newman et al., Brit, J, Haematology 2_1, 1972, p.

1; U.S. Patent No. 3,652,530; DK patent. no.

146,895, FR Patent No. 2,460,305, DK Patent Application 30 No. 3602/81. By these methods, an AHF concentrate with a specific activity of 1-3 units / mg protein is obtained.

According to U.S. Patent No. 4,027,013 (Bick et al.), A mixture containing coagulable fibrinogen and Factor VIII is released which, from the Examples, is believed to be dissolved.

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3 cryoprecipitate for 90% of the fibrinogen content by precipitation with a block copolymer of the "Pluronic" type, while in a less preferred embodiment leading to substantially lower yields PEG can also be used.

The supernatant from the first fibrinogen precipitate is then treated with higher concentrations of "Pluronic") to precipitate factor VIII and residual fibrinogen.

The factor II-containing precipitate is redissolved in a more concentrated form and is then treated with a thrombin mimetic enzyme which reportedly specifically coagulates the total amount of residual fibrinogen but does not significantly affect the biological activity of factor VIII. However, this last is not further documented.

It is important to note that in order to stabilize factor VIII during subsequent lyophilization, Bick adds albumin, which is not the case with lyophilization of the concentrate according to the invention.

Multistage precipitation processes in which more than one precipitant is used are also known. Eg.

20, low temperature glycine precipitation may be carried out before or after PEG / PEG precipitation, optionally. For example, glycine precipitates can be performed both before and after the PEG / PEG precipitates. This is described in U.S. Patent No. 3,682,881 and U.S. Patent No. 3,631,018. Generally, preparations having a specific activity of 1 to 3 units / mg protein are thereby obtained.

When precipitated with glycine at low temperature in this context, the AHF-containing precipitate, i.e. that for subsequent PEG precipitates on dissolved AHF-containing precipitate there is only a low concentration of 30 glycine in the solution.

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4

If glycine is used at high temperature in conjunction with PEG precipitation, a glycine precipitate is first left, leaving AHF in the supernatant where the concentration of glycine is high, and then AHF is precipitated therefrom with a PEG precipitate 5 at 'high PEG concentration. Hereby a specific activity of 1 - 3 units / mg protein can be obtained.

The concentrate obtained by this type of glycine / PEG precipitate is similar to a concentrate obtained by PEG / PEG precipitation. As shown in Table 1, the 1st precipitate supernatant contains more IgM and IgG after a warm 2 M precipitation

glycine than after a low PEG concentration (4%) · However, after the high PEG concentration (9¾), the glycine / PEG precipitate concentrate contains less IgM and IgG than the PEG / -15 PEG precipitate.

TABLE 1

Comparison of PEG / PEG and hot glycine / REG precipitation of AHF from cryoprecipitated cryoprecipitate

Fraction_% AHF_% IgM% IgG

Cryoprecipitate 100 100 100 1. "FEG, 4%, supernatant 72" 38 55 PEG / PEG, 4 / i, 9¾ concentrate 40 24 15 2M glycine supernatant 68 76 79 2M qlycine / PEG, 9%, concentrate 36 18 8 Reason for this is not previously recognized, but it is believed that the polar amino acid in the glycine supernatant acts as a salting agent, especially on basic proteins, so that IgM and IgG are not readily precipitated by PEG alone. However, the concentrate obtained by the glycine / PEG precipitate still has an undesirably high content of fibrinogen, IgG and IgM, cf. Table 2 below.

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5

The object of the present invention is to provide a shark pure, directly lyophilizable AHF concentrate having a shark solubility and specific activity that it can be administered intravenously in injection doses of 1000 units loaded in 2-5 ml injection medium and as contains factor VIII: second component of factor VIII: RP, which stabilizes on coagulation active factor V111: C-10

This is achieved by the AHF concentrate according to the invention, which is peculiar to the part characterized in claim 1 '. Such a concentrate according to the invention can be prepared as set forth in the characterizing part of claim 2.

It should be noted that there is no clear correlation between the specific activity of an AHF concentrate and its solubility, the latter being dependent on the nature of other proteins, especially fibrinogen, but that a shark specific activity would otherwise also involve a shark solubility.

20 U.S. Patent No. 4,361,509 (Zimmermann et al.) Discloses a Factor VIII: C preparation prepared from plasma or commercial Factor VIII concentrates and which is liberated from Factor VIIIIRP by direct immune adsorption on a column of agarose gel to which there are 25 monoclonal antibodies coupled showing shark affinity for factor VIII: RP, yet binding both factor V111: C and VIIIîRP, and subsequent desorption of factor VIII.-C with a calcium-containing buffer. Zimmermann's Factor V111: C concentrate exhibits a very shark specific activity (about 2300 units / mg) and a very shark solubility (545-1172 units / mg) in aqueous injection media, but is different from the concentrate as mentioned. according to the invention also exempted from factor VIIIRP.

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6

The invention is based on the surprising realization that a particularly pure AHF preparation can be obtained, containing both factor VIIIiC and VIIIiRP, but substantially free of other proteins, especially immunoglobulins, by fractionation. a cryoprecipitate with PEG in such a way that a substantial portion, preferably at least 80%, of the fibrinogen is precipitated and that in a subsequent step, more PEG is precipitated in the presence of a salting agent. The addition of the salting agent does not in itself entail precipitations, but on the basis of ten first years of the salting agent modi-ficexies serving the yellow exdex the eiffer general 'P'EG trap -ing, 3.81 ^ 4¾¾ to obtain a sharper fractionation, i.e. a renexe AHF concentrate with a shark specific activity, cf. Table 2 below. The high purity means that the composition has a solubility of 200-500 units of factor VIIIiC (AHF) per day. ml. As a normal injection dose is 1000 units, it can thus be sealed with approx. 2-5 * nl injection medium. Since the solubility of the best 20 AHFs on the market is stated to be 40-50 units per unit. According to the invention, the preparation thus provides a very significant relief for the patient.

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7 TABLE 2

Effect of Glycine Addition during PEG / PEG Precipitation from Genoplast Cryoprecipitate

Process_AHF unit / mq% IgG% IgM% Fibr.

Cryoprecipitate 0.32 100 100 100 4% PEG / 9% PEG 2.53 15 24 1 2M glycine / 9 / ° ô PEG 2.27 8 18 2 4K PEG / 12% PEG + 2M glycine 4.36 4 13 0, 5

Thus, in the precipitation of the invention, glycine is used as a salting agent, stabilizing the unwanted proteins by holding them in solution, whereas the known methods utilize the saline effect of glycine on fibrinogen / AHF. Thus, using a salting agent during a PEG / PEG precipitation according to claim 2, a more selective precipitation of AHF is obtained.

Without being bound by any particular theory, it is assumed that the effect of the salting agent is due to the difference in surface charge between Factor VIII and the other proteins, in particular IgG.

It is important to remove most of the fibrinogen in the first stage because otherwise the AHF preparation would be highly contaminated with fibrinogen and because the presence of fibrinogen in the second step would counteract the salting effect and prevent a selective precipitation of Factor VIII.

As the salting agent of the process according to the invention, an amino acid, especially basic amino acids, preferably lysine or arginine added in the form of the hydrochloride is suitably used. Other useful amino acids are e.g. histidine, as well as polar amino acids such as serine, glutamic acid, glycine and β-aminohexanoic acid. André usable

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8 salts are carbohydrates, preferably mono- or disaccharides e.g. glucose, and sucrose, but also oligosaccharides and sugar alcohols, e.g. sorbitol and glycerol.

5 An overview is given in Table 3.

TABLE 3

Effect of Salts on PEG / PEG Precipitation of AHF from Genoplated Cryoprecipitate

Desiccant Spec. activity% yield of _enh. / mq_AHF from plasma

Nothing 2.53 18 2M glycine 4.36 17 0.5M lysine, HCl 8.4 18 0.5M arginine, HCl 7.0 14 l, OH £. -aminohexanoic acid 5.0 10 1.5M glucose 3.85 18 1.0M sucrose 5.35 20

The preferred salting agent is lysine HCl. In the drawing, the influence of lysine on PEG / PEG precipitation of AHF is expressed as unit, respectively. AHF / mg protein (fig.

1) and ή yield relative to the cryoprecipitate at different lysine concentrations (Fig. 2). Lysine is added advantageously at levels of Q, L - 0.9, especially 0, 3 - 0.8, most preferably 0.5 - 0.7 tnol / l.

As can be seen from FIG. 1, there is proportionality between the added lysine amount and specific activity of the final product, but even small amounts of lysine have a clear effect. However, as seen in Fig. 2, the yield of AHF decreases at the PEG concentration used if more than 0.75M lysine is used, as this amount of lysine also acts as salts on AHF.

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9

Another preferred salting agent is arginine HCl, with some influence on the PEG / PEG precipitation of AHF as shown in Table 4 below, from which it is seen that satisfactory yields are obtained at concentrations of 0.2 5 - 0.8. Considering the yield, the preferred amount is 0.3 - 0.5, especially 0.4 mol / l.

TABLE 4

The effect of arginine on PEG / PEG precipitation of AHF Mol arginine / 1% yield from plasma unit. AHF / mq protein 0 20 2.3 0.2 20 4.9 0.4 17 6.4 0.6 14 6.1 0.8 4 4.6

The concentrations of PEG and the salting agent used, as well as the pH during the precipitation, depend in particular on the salting agent used, as it can generally be said that the more loaded salting agents can be used in smaller concentrations. The chosen saline concentration is compromised, because at high concentrations the IgG amount can be almost completely reduced, but at the same time part of factor VIII is also salted, thereby reducing the yield.

In the process of the invention, PEG having a molecular weight of 200-20,000, preferably 2,000-12,000, especially 3,000-6,000, is used, but PEG 3000 is preferred. The PEG concentration in the farthest precipitation step is 2-6% by weight, preferably about 20%. 4% by weight and in the second step 6-20% by weight, preferably approx. 12% by weight. The pH during the first precipitation step may be 6.0 - 8.5, preferably 6.2 - 6.6, especially approx.

6.4, and in the second step 5.0 - 8.5, preferably 6.0 - 6.6, in particular approx. 6.3.

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ίο

The temperature in the farthest precipitation step may be 6 -22 ° C, preferably 18 - 22 ° C. In the second precipitation step, the temperature is also preferably 18 to 22 ° C (room temperature).

In the extraction of the present Factor VIII concentrate, cryoprecipitate from human blood plasma or other Factor II-containing blood fractions can be used, as well as plasma fractions from animal species, for example pigs.

The process according to the invention will be described in more detail below with the aid of some examples which illustrate the preparation of the concentrate. For preparation, the MF solution may be subjected to heat treatment for 10 hours at 6 ° C in the presence of a suitable stabilizer such as a mixture of glycine and sucrose to obtain hepatitis protection and / or lyophilized. .

EXAMPLE 1

Cryoprecipitate from 600 ml of human blood plasma containing 20,240 units Factor VIII: C is replated in 28 ml of citrate / glucose buffer and freed from prothrombin complex by adsorption with ft ftll ^ O ^. Then 4 wt% PEG 3000 is added. The pH is adjusted to 6.4 with 0.5 M HCl and the mixture is incubated for 30 minutes at room temperature. Precipitated protein 25 is removed by centrifugation and lysine HCl is added to a cancer entry of 0.55 mol / l. An additional 8% by weight of PEG 3000 is added and the pH is adjusted to 6.3 with 0.1 M NaOH. After incubation for 45 minutes at room temperature, the precipitate is isolated by centrifugation and resuspended in citrate / glucose-NaCl, pH 7.8. Thus, the recharged precipitate containing 112 units of factor VIII: C and 9 mg of total protein has a specific activity of 12 units of factor VIII: C (A H F). mg of protein, and the yield of Factor

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11 from plasma is 20%. No solubility determination was made, but it is assumed that the solubility is at least 200 units Factor VIII: C / ml, cf. Examples 5 and 6.

5 These results are listed in Table 5 below, together with results from comparison trials without the addition of light. The table shows that the process according to the invention can achieve a significant increase in specific activity and a reduction of the content of immunoglobulin 10 G in relation to PEG / PEG precipitates without the presence of salts.

EXAMPLE 2 30 ml of cryo-reading obtained analogously to Example 1 and adsorbed with M 2 Q 4, 4% by weight of PEG 3000 is added.

15 µM is allowed to 6.4 with 0.5 M HCl, and the mixture is incubated 30 imimutes at room temperature. Precipitated protein is removed by centrifugation and "saline" is added. Then an additional 8% by weight of PEG 3000 is added and the pH is adjusted to 6.3- with 0.1 M NaOH. After incubation for 45 minutes at room temperature, the precipitate is isolated by centrifugation and redissolved in citrate / glucose-NaCl, pH 7.8.

Table 6 shows the results obtained for various salts. No solubility determinations were made, but it is assumed that the solubility is at least 200 units Factor VIII: C / ml, cf. Examples 5 and 6.

DK 157170 B

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DK 157170 B

EXAMPLE 3

Cryoprecipitate from 2.5 L plasma is redissolved in 100 ml citrate / glucose buffer and freed from prothrombin complex by adsorption with Al 2 O 2. 4% by weight of PEG 5,000 is added. The pH is adjusted to 6.4 with 0.5M HCl and the mixture is incubated 30 mlnuts at room temperature. Precipitated precipitate is removed by centrifugation and arginine HCl is added to a concentration of 0.40 mol / l. An additional 8 weight S PEG 3000 is added and the pH is adjusted to 6.3 with 0.1M 10 NaOH. After incubation 45 minutes at room temperature, the precipitate is isolated by. centrifugation and redissolve in 20 ml of citrate / sucrose / NaCl, pH 7.8. The concentrate contains 360 units of Factor VIII: C and 52 mg of protein (specific activity 7 units / mg). No determination of the solubility of the concentrate was made, but it is assumed to be at least 200 units / ml, cf. Examples 5 and 6.

EXAMPLE 4

Cryoprecipitate from 2.5 L of plasma is recharged in 100 ml of 20 citrate / glucose buffer jpg liberated for prothrombin complex by adsorption with A ^ O ^. 4 weight of PEG 3000 is added. The pH is adjusted to 6.4 with 0.5M HCl and the mixture is incubated for 30 minutes at room temperature. Precipitated protein is removed by centrifugation and lysine-HCl is added to a concentration of 0.55 mol / l. An additional 8% by weight of PEG 3000 is added and the pH is adjusted to 6.3 with 0.1M NaOH. After incubation 45 min. at room temperature, the precipitate is isolated by centrifugation and redissolved in 5 ml of citrate / sucrose / NaCl, pH 7.8. The concentrate contains 30 460 units. Factor VIII: C and 30 mg of protein (specific activity 15 units / mg). No solubility of the concentrate was determined, but it is assumed to be at least 200 units / ml, cf. Examples 5 and 6.

DK 157170B

EXAMPLE 5

Cryoprecipitate from 80 kg of plasma is redissolved in 2500 ml of citrate buffer and freed from prothrombin complex by adsorption with A ^ O ^ · 4% by weight of PEG 3000 is added.

The pH is adjusted to 6.4 with 0.5 M HCl and the mixture incubates for 30 minutes at room temperature. Precipitated protein is removed by centrifugation and lysine HCl is added to a concentration of 0.55 mol / l. An additional 8% by weight of PEG 3000 is added and the pH is adjusted to 6.3 with 0.1 M 10 NaOH. After incubation for 45 min. at room temperature, the precipitate is isolated by centrifugation and redissolved in 60 ml of citrate / sucrose / NaCl. The solution is sterile filtered and dispensed into 15 vials. After freezing and freeze drying, each vial contains 1000 units.

15 AHF, with a specific activity of 37 units / mg protein.

As they are a total of 15,000 units of AHF extracted from 60 ml of solution, it can be stated that the solubility is at least 250 units / ml.

EXAMPLE 6 Cryoprecipitate from 13 L of plasma is redissolved in 525 ml of citrate / glucose buffer and freed from prothrombin complex by adsorption with A ^ O ^. 4% by weight of PEG 3000 is added. The pH is adjusted to 6.4 with 0.5M HCl and the mixture is incubated for 30 minutes at room temperature. Precipitated protein 25 is removed by centrifugation and lysine HCl is added to a concentration of 0.55 mol / ml. first An additional 8% by weight of PEG 3000 is added and the pH is adjusted to 6.3 with 0.1 M NaOH. After 45 min of incubation at room temperature, the precipitate is isolated by centrifugation and redissolved in 2.1 ml of 30 citrate / sucrose / NaCl, pH 7.8. The 3.1 ml concentrate contains 2180 units. Thus, FVIIIîC and 81 mg of protein solubility are 703 units / ml and the specific

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Activity 27 units / mg.

EXAMPLE 7 In this example, it is examined whether the salted-in effect of glycine could be enhanced by the addition of NaCl, which raises the ionic strength, and whether NaCl, a known salting agent, has sufficient salting effect alone during a PEG precipitation to to a concentrate according to the invention.

To further vary the process conditions, it was chosen to effect the precipitation at low temperature (6-9 ° C).

50 g of cryoprecipitate is redissolved in 200 ml of dest. t ^ O at room temperature. 12.3 ml of 1.3% A₂O ^ and 7.9 g of PEG 3000 (3%) are added. The pH is adjusted to 6.7 with the IM HAc agitated to 9 ° C. After cooling down, precipitate 15 is removed by centrifugation for 15 minutes at 9 ° C. An additional 8¾ PEG, 2M glycine (13%) and 14% NaCl are added, after which the pH is adjusted to 5.7 deg and the mixture is cooled down to 6 ° C. The burad drop is isolated by centrifugation for 15 min at 6 ° C and redissolved in 50 ml (1/5 cryo vol.) Of 5 mM citrate, 0.13 20 M NaCl, 0.1 M glycine pH 7.3.

FV111 and the protein content of the redissolved concentrate were low. Therefore, with varying NaCl and glycine concentrations, it was investigated whether the salting expected was due to glycine. As can be seen from DK 157170 B 'below; 16 Table, FVIII precipitates only slightly without NaCl. However, NaCl addition alone does not cause its own protein precipitation (such as PEG addition). On the contrary, NaCl appears to be saline-like. At the low temperature, glycine helps to precipitate FVIII, but the high specific activity cannot be achieved without NaCl addition.

Effect of glycine and varying NaCl concentration% + 13% glycine-glycine

NaCl__ mg * »FVIII: C Spec. act. mg *% FVIII: C spec.

protein yield unit / mg protein yield unit / mg 0 5.86 5 0.22 6.50 14.4 0.58 5 3.96 26 1.74 10 2.26 58 6.73 3.89 15.9 1 , 07 12 2.18 41 4.90 14 0.88 17 5.02 2.82 5.2 0.49

Kryopræc. 100 0.58 38 PEG /

Al 3 O supernatant 73 0.77 * determined as E ^ qq EXAMPLE 8 (Determination of factor VIII: RP content)

It is apparent from Zimmermann, U.S. Patent No. 4,361,509, 10 that in order to achieve the separation of the two components 1 Factor VIII complex must be resorted to specific purification methods, such as immunosorption or ion exchange chromatography.

In the foregoing Examples, where starting material 15 is used (uploaded) cryoprecipitate thus containing 17

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—R * i

In the Factor VIII complex, such purifications are not carried out at any time, and it is therefore evident to the average skilled person that the Factor VIII concentrates obtained in the Examples still contain both coagulation active Factor VIII: C as the von Willebrand Factor Factor VIII: RP.

However, no measurements of Factor VIΓΙ: RP content were made and it is therefore not possible to include numerical values for this in the existing examples.

However, Factor VIII: C and Factor 10 VIIIrRP have been carried out on a number of AHF preparations according to the invention, which are marketed under the designation "NORDIOCTif" and prepared analogously to Example 5, ie in unit doses of 1000 units Factor VIII: C ( + 20?).

The following results were obtained:

Preparation FVIII: RP, Enh / Dose FVIIIîC, Enh / Dose 44102 727 847 44203 936 1028 44301 82'5 1161 44302 942 1109 44401 566 909 44402 808 1034 15 FVIII-RP is determined by rocket immune electrophoresis with A antibody D By default, a pool of normal plasma (<bl Enh FVIII-RP / ml) is used.

FVIIIîC is determined by one-step coagulation assay. By default, a pool of normal plasma (y in Enh 20 FVIIIîC / ml) is used.

Claims (9)

1. Concentrate of the anti-haemophilic factor VIII (AHF) substantially free of denatured AHF and others in the plasma proteins present in the concentrate, but not of factor VIII: RP, know the e-5 by showing a solubility in an aqueous injection medium of 200 - 500 units of AHF / ml and a specific activity of 3.85 - 50 units of AHF / mg of protein derived from the starting material of the concentrate and that it can be prepared by purifying a solution 10 of a cryoprecipitate from blood plasma or a second factor VII-containing blood fraction, for removing prothrombin and subsequent fractionation with polyethylene glycol, PEG, comprising a step of precipitating the majority of fibrinogen by 2-6% by weight of PEG 200-20,000 and a subsequent step, at which the supernatant from the aforementioned PEG precipitate is precipitated by 6-20 wt.% PEG 20-20,000 in the presence of a salting agent selected from amino acids, preferably basic, carbohydrates and sugar alcohol. is where the separated precipitate with 20 concentrate content of AHF is extracted. A process for preparing an AHF concentrate according to claim 1 or 2 wherein a solution of a cryoprecipitate from blood plasma or other factor VIII-containing blood fraction is purified to remove prothrombin 25 and then fractionated with polyethylene glycol, PEG, comprising a step of precipitation. of the majority of fibrinogen by 2-6 wt% PEG 200-20,000 and a subsequent step of precipitating AHF, which is suitable in that the supernatant from the aforementioned 30 PEG precipitate is precipitated by 6-20 wt% PEG 200-20,000 in the presence of a salting agent selected from amino acids, preferably basic, carbohydrates and sugar alcohols, upon which the precipitated precipitate with concentrated AHF content is recovered. DK 157170B Process according to claim 3, characterized in that lysine-HCl or arginine-HCl is used as a salting agent. A process according to claim 3, characterized in that a carbohydrate, preferably a mono- or disaccharide, is used as a salting agent. Process according to claim 5, characterized in that sucrose is used as a salting agent. 6. A process according to claims 3-6, characterized in that, in the first step, the PEG is added to a concentration of approx. 4 weight of the solution. Method according to claims 3-7, characterized in that in the second precipitation step PEG corresponding to a total concentration of approx. 12 weight-? Ô. Process according to claim 4, characterized in that lysine-HCl or arginine-HCl is added in an amount corresponding to a lysine or arginine concentration of 0,1 - 0,9, preferably 0,3 - 0. , 8 mol / l. A process according to claims 3-9, characterized in that the pH during the first precipitation step is 6.0 - 8.5, preferably 6.2 - 6.6, especially approx. 6.4, and during the second precipitation step are 5.0 - 8.5, preferably 6.0 - 6.6, in particular approx. 6.3.