DK173863B1 - Immunosorbent - Google Patents

Description

DK 173863 B1

The present invention relates to an immunosorbent suitable for removing FVIII inhibitor antibodies in blood.

Hemophilia A is a congenital disease caused by a lack of coagulation-5 factor VIII: C. This factor is found in blood plasma and can be purified

partly from blood. Preparations containing this factor (AHF) may be administered to hemophilia A patients so that the patients' blood is able to coagulate. Preparation of this type of preparation is e.g. described in U.S. Patent No. 3,652,530 and Application No. WO 84/03628. In these preparations Factor VIII: C

the protein typically 0.1% of the total amount of protein. Higher purity factor VIII: C can be obtained by affinity chromatography (Zimmerman et al., U.S. Patent No. 4,361,509, Fass et al. Blood 59, 394, 1982).

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The factor VIII: C protein is not yet fully characterized, but part of the structure is known. (L. W. Hoyer, Blood 58, 1, 1981; M.

Weinstein et al., Proc. Natl. Acad. Sci., USA 78, 5137, 1981; G. Kuo et al., Thronib. Haemostas. 50, 262, 1983). The molecular weight is approx. 300 20 kD.

It is known that 10-20% of hemophilia A patients not only lack Factor VIII: C but also form antibodies to Factor VIII: C. This type of patient is called inhibitor patients, and the antibodies in these 25 patients are called inhibitor antibodies because they inhibit the coagulation activity of Factor VIII: C (H.R. Roberts & R.

Cromartie, Progress in Clinical and Biological Research 150, 1, 1984). The presence of these antibodies renders the administration of the AHF preparation ineffective, as Factor VIII: C is neutralized and AHF administration induces elevated antibody levels.

Inhibitor antibodies can be used as reagents in immunoassays to measure Factor VIII: C antigen (VIIIsCAg) (B. Dinesen, C. Feddersen, Thromb. Res. 31, 707, 1983, O. Nordfang et al., Thromb. Haemostas.

50, 111, 1983). A VIII: CAg unit is defined as the content of 1 ml of normal human plasma.

DK 173863 B1 2

Inhibitor patients have so far sought treatment: a) For non-specific treatment with activated prothrombin complex preparations (FEIBA®, Autoplex®). These preparations contain an unknown component (perhaps Factor Vila, see U. Hedner and W. Kisiel, J. Clin. Invest. TI, 1837, 1983), which can cause plasma to coagulate despite high levels of inhibitor. However, activated prothrombin complex preparations cannot help all inhibitor patients and patients are not cured of their inhibitor. In addition, there is a high risk of blood clots when using this type of preparation.

b) It has been shown that inhibitor formation can be "exhausted" by administration of very large doses of AHF (100-200 units / kg daily).

This makes it possible to induce immune tolerance to Factor VIII

15 (H. H. Brackman & J. Gormsen, Lancet, pp. 933, 1977). After this type of treatment, the patient can be treated with AHF preparations like other bleeding patients. However, high dose AHF treatment is also nonspecific, as very large doses of extraneous protein are administered and patients cannot utilize the coagulation activity of AHF 20 preparations during treatment.

At the beginning of the treatment phase, the inhibitor level rises, after which it is reduced to 0. Over time, bleeding can not be prevented with AHF during treatment, but bleeding can be partially stopped with the above-mentioned FEIBA® (factor eight inhibitor bypass activity) 25 preparations.

This treatment is very expensive (typically 600,000 US $ per patient, see S. Stenbjerg et al., Thromb. Res. 34, 533, 1984) and is therefore low in prevalence.

The invention is based on the surprising finding that a component of plasma fractions having Factor VIII: CAg reactivity and little or no Factor VIII: C coagulation activity is reactive with 7 of 7 antibodies from inhibitor patients.

In an embodiment of the invention, the immunosorbent is characterized in that the protein with FVIII: CAg activity is 80/77 kD part of FVIII: C.

5 By Kuo et al. it appears that the 80/77 kD fragment exhibits VIII: CAg activity against a particular inhibitor antibody (zHI) and no coagulation activity. From there, however, one could not expect the fragment, as demonstrated below, to be able to block the inhibitory activity of 7 of 7 inhibitor antibodies.

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The 80/77 kD fragment with VIII: CAg reactivity from plasma fractions can be recovered in several ways, for example by affinity chromatography, hydrophobic interaction chromatography or cation exchange. As starting material, any VIII: CAg-containing fraction can in principle be used, but a cryosupematant or a plasma fraction ion obtained by precipitating redissolved cryoprecipitate by 2-6% by weight, preferably approx. 4% by weight PEG. The latter is particularly convenient because this precipitate is not normally used during the further plasma fractionation.

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The invention is not limited to the use of the fragments 80/77 kD and 70/67 kD of Factor VIII: C. In Figs. Ib below shows that even smaller fragments of Factor VIII: C are reactive against inhibitor antibodies. Likewise, it can be envisaged that other inhibitor antibodies may have reactivity against other parts of the Factor VIII: C molecule. For example. Factor VIII: C isolated from blood plasma contains a fragment with a molecular weight of 92 kD (Zimmerman et al., U.S. Patent No. 4,361,509).

To the extent that such fragments are capable of blocking inhibitor antibodies, as shown in Table 5, they will be encompassed by the invention.

The invention is also not limited to the use of plasma isolated Factor VIII: C fragments. From the DNA sequence of Factor 35 VIII: C it will be possible to prepare sub-fragments of the FVIII: C gene. These sub-fragments may be inserted into suitable vectors (eg plasmids or viruses). These vectors can be inserted into suitable host cells (eg E. coli, yeast, CHO, COS or other mammalian cells) so that the cells are capable of producing subfragments of Factor VIII: C. It can be predicted that such fragments are capable of blocking inhibitor antibodies as shown in Table 5, without containing significant amounts of VIII: C coagulation activity, since they have the same molecular structure as the corresponding peptides from blood plasma. They can therefore be used for the preparation of the immunosorbent according to the invention.

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Reactivity or antibodies

Several authors (HP Muller et al., Blood 5J3, 1000, 1981; B. Sola et al., Proc. Natl. Acad. Sci., USA 79, 1983, 1982) have described the preparation of monoclonal antibodies to Factor VIII: C. These antibodies can be obtained after immunization of mice with Factor VIII, by fusing spleen cells with tumor cells, as described by K6hler & Milstein (Nature 256, 495, 1975). Using this technique, monoclonal antibodies to Factor VIII: C, termed 42 IgG, 47 IgG and 56 IgG, respectively, have been prepared.

FIG. 1a shows a solid phase of immunoisolation with monoclonal antibodies. Plastic beads were coated with antibody to mouse immunoglobulin / monoclonal antibody. After washing, the beads 25 were incubated with 125 I-labeled Factor VIII: C. Factor VIII: C prepared as described by E. Tuddenham et al-, J. Lab. Clin. With. 93, 40, 1979. After washing, the beads were extracted with SDS sample buffer and the eluates were run on SDS gel. The figure shows the autoradiogram of SDS gel: Track 1: Tracer. (125 I-labeled Factor VIII: C); Lane 2: normal IgG mouse; Lane 3: 42 IgG; Lane 4:47 IgG; Lane 5:56 IgG.

It can be seen from FIG. let these antibodies bind the 80/77 kD portion 35 of Factor VIII: C (lanes 3, 4, 5). This is the same moiety that binds to a human inhibitor antibody, zHI.

___________ ηηΛ i ni i r- \ T ^ DK 173863 B1 6

FIG. Ib shows a solid phase immunoisolation performed as an experiment in Figs. la against zHI bound to sphere, and against a degraded VIII: C sample. It can be seen from FIG. Ib that a degraded sample can produce 5 peptides other than the 80/77 kD doublet with VIII: CAg activity against zHI (70/67 kD and other degradation products).

FIG. Figure 2 shows that 7 of 7 inhibitor antibodies are capable of blocking the binding of zHI in VIII: CAg inhibition assay. Since the zHI 10 antibody binds the 80/77 kD doublet, the other inhibitor antibodies must also have reactivity against this fragment of Factor VIII: C. The assay was performed as follows: Wells in microtiter plates were coated with zHI immunoglobulin. After washing, plasma containing Factor VIII: C was added, and after another wash, inhibitor immunoglobulin was added along with peroxidase-labeled zHI antibody. 100% binding of the peroxidase-labeled zHI is achieved by adding buffer instead of inhibitor immunoglobulin.

Affinity chromatography of VIII: CAg 20

Upon absorption of AHF against monoclonal 47 IgG coupled to Sepharose, it has been found that only VIII: CAg and not coagulation active Factor VIII: C (Table 1) are adsorbed. This is surprising, "as the antigen is present on both Factor VIII: C and VIII: CAg.

However, the antigen appears more available on the coagulation-inactive VIII: CAg than on Factor VIII: C.

TABLE 1

Specific VIII: CAg adsorption to 47 IgG coupled to Sepharose AHF unbound EG / NaCl eluate

Immunosorbent VIII: C VIIIiCAq VIII: C VIII; CAq_VIH; CVIII; CAq 47 IgG / Cl 2B 88 270 90 90 0.32 138 35 Control Cl 2B 88 270 77 248 0.14 0.09 DK 173863 B1 7

Table 2 shows that a variety of salts can be used with ethylene glycol (EG) to elute FVIII: CAg from immunosorbent with monoclonal antibody. MgCl2, which is sparingly soluble in EG, can be used for elution without the presence of EG.

TABLE 2

Effect of various salts on ethylene glycol elution

from 56 IgG

10 ------------------------------------------------- ----

Sample VIII: CAg enh.

AHF (Nordiocto® 210

Flow 140 15 eluate, 50% EG / saturated NaCl 22, 50% EG / saturated KCl 19, 50% EG / 2 M Kl 3.6, 50% EG / 2 M CaCl2 2.2, 2M MgCl2 22 20 -, 50 % EG / saturated NaAc 24

With 56 IgG coupled to sepharose, it is possible to purify VIII: CAg from cryosupernatant. Cryosupernatant contains 0.4 VIII: CAg 25 units / ml, and less than 1 ppm of the protein is VIII: CAg. Table 3 shows that in one step an 8,000-fold purification can be obtained so that a preparation with a specific VIII: CAg activity of 61 units can be prepared. VIII: CAg / mg protein. This is a significantly higher specific activity than that found in existing AHF-30 preparations.

In the manufacture of high purity (HP) preparations from less purified AHF, e.g. a 4% PEG precipitate (US 3,652,330, WO 84/03628). This precipitate contains VIII: CAg, and as shown in Table 3, it is possible to use this precipitate as starting material for VIII: CAg purification. With just 0.25 ml of immunosorbent, it is possible to purify 920 VIII: CAg units from 140 ml of redissolved 4% PEG precipitate. The obtained preparation has a specific VIII: CAg activity of 6,900 units. VIII: CAg / mg protein.

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With human inhibitor IgG coupled to Sepharose Cl 2B, it is also possible to purify VIII: CAg from redissolved 4% PEG precipitate (Table 3). However, the yield of VIII: CAg is less than with the use of monoclonal antibody, as 50% ethylene glycol with 2.5M NaCl is not able to quantitatively elute the bound VIII: CAg. The experiments are described in more detail in Examples 2-4 below.

TABLE 3

Purification of VIII: CAg from various starting materials by 56 IgG and human inhibitor IgG

Fraction VIII: CAg protein spec.act.

_enh. _mg, Ε28ο_enh./mg (Ex. 2) 20 Kryosup, 500 ml 200 30,000 0.007

Cryosup flow 500 ml 100 30,000 0.003 EG / NaCl eluate from 5.6 IgG, 2 ml_39_0,64_61 (Ex. 3) 25 4% PEG bdf., 140 ml 1500 5,200 0.29 4% PEG bdf. flow-through, 140 ml 340 5,200 0.07 EG / NaCl eluate from 56 IgG, 1 ml_920_0,131 6900_ 30 (Ex. 4) 4% PEG bdf., 110 ml 1000 3,400 0.29 4% PEG bdf. flow-through, 110 ml 340 3,400 0.10 EG / NaCl eluate from human 35 inhibitor, 1 ml_ 68_0.082 830_ 9 DK 173863 B1

Factor VIII: CAg can be prepared by adding a Factor VIII: CAg-containing solution, e.g. plasma fraction, is treated with an immunosorbent comprising antibodies specific for Factor VIII: CAg, bound to solid particles, whereupon the bound VIII: CAg is desorbed by elution with a buffer and worked up into a preparation.

As immunosorbent, human inhibitor antibodies or monoclonal antibodies can be used, preferably 56 IgG or 47 IgG.

Hydrophobic interaction chromatography of VIII: CAg and cation exchange VIII: CAg can be purified from VIII: CAg-containing solutions, e.g. plasma fractions without the use of affinity chromatography with 15 antibodies. Thus, 77/80 kD Factor VIIIzCAg has been found to be extremely basic and hydrophobic. These properties can be used for the extraction of Factor VIII: CAg from plasma fractions by hydrophobic interaction chromatography and cation exchange, cf. also Example 5 below.

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Hydrophobic chromatography can be performed at pH 6 - 9.5, but the binding is strongest at high pH, e.g. pH 8.5. A hydrophobic gel such as phenyl-Sepharose (Pharmacia) is used. The binding to the phenyl-Sepharose can take place without the addition of salt, but is significantly increased by the addition of NaCl. 0.3M NaCl addition to the cryosupernatant is appropriate as it will not interfere with the subsequent recovery of other plasma proteins such as albumin and immunoglobulin G. Using 4% PEG precipitate from redissolved cryoprecipitate, higher NaCl concentrations can be used. The bound Factor VIII: CAg is eluted with a buffer, e.g. under the conditions set forth in Example 5 below.

Cation exchange can be carried out at a pH below 8.0, giving the best bond. A suitable value is pH 5.5, as the pH of the cryosupernatant can be lowered thereto without damaging VIII: CAg.

DK 173863 B1 10

A strong cation exchanger can be used, such as "Whatman®SE53". The bound Factor VIII: CAg is eluted with a buffer, e.g. under the conditions set forth in Example 5 below.

With both of the above methods, a very selective purification of Factor VIIl: CAg is obtained, and a very pure eluate. A particularly convenient method for further concentration of VIII: CAg activity is to combine a hydrophobic chromatography with a cation exchange, as described above and illustrated in Examples 5 and 10 6 below. In this case, a weaker cation exchanger, such as "CM fast flow sepharose", is preferred.

As can be seen from Example 5 and Table 4, these two purification methods utilize different properties of VIII: CAg and give a combined 1100-fold purification from the cryosupernatant. These purification methods can be used, as affinity chromatography, on all VIII: CAg-containing solutions, e.g. plasma fractions, cf. Table 4 showing both purification of cryosupernatant and the precipitate from 4% PEG precipitation of redissolved cryoprecipitate.

TABLE 4

Purification of VIII: CAg by hydrophobic interaction chromatography and cation exchange Fraction VIII: CAg protein spec.act.

_enh._mg, E2Bq enh. / mg (Example 5)

Kryosup. 1100 160,000 0.007 30 Flow from Phenyl Sepharose 390 160,000 0.002

Eluate from phenyl-Sepharose 440 2,100 0.21

Flow from SE 53 50 2,000 0.04 35 Eluate from SE 53_300_37,5_8,0 11 DK 173863 B1 4% PEG bdf. 798 3,000 0.26

Flow from Phenyl-Sepharose 36 2,400 0.01

Eluate from 5 phenyl-Sepharose 560 83 6.7

Passage from SE 53 64 83 0.77

Eluate from SE 53_380_1.83 210

Effect of VIII: CAg 10

In vitro coagulation inhibition experiments suggest that the purified VIII: CAg will also have an in vivo effect. In coagulation inhibition assay, the coagulation inhibitory effect of inhibitor IgG on normal plasma is measured. The dilution which gives 50% inhibition is indicated in Bethesda units (C.K. Kasper et al. Thromb.

Diath. Haemorr. 3_4, 869, 1975). Table 5 shows a comparative experiment in which normal plasma was incubated with approx. 3 Bethesda units inhibitor IgG from 6 different inhibitor patients as well as as a control of the previously described zHI IgG. The experiments were performed with and without the presence of a preparation containing 100 VIII: CAg unit / ml. The table shows that the purified VIII: CAg has a pronounced inhibitor-suppressing effect on all the antibodies compared to the experiments without VIII: CAg. Thus, the Bethesda titer of all antibodies is reduced by more than 25%. This shows that the inhibitory effect of all the 7 antibodies tested is due to reactivity against the 80/77 kD part of Factor VIII: C.

Table 5 Attenuation of coagulation inhibition by 80/77-VIII: Cag 30 ----------------------------------- ------------------------

Inhibitor IgG% VIII: C after% VIII: C after incubation incubation without VIII; Cag_med VIII: Cag HZ (zHI) 0 82 35 AJ 6 89 KB 17 61 DK 173863 B1 12 E O 71 THL 10 95 KH 5 80 BE 6 76 5 Normal IgG_100_102_

The above 7 antibodies were deposited on 30.10.1984 at the Statens Seruminstitut, Copenhagen under no. 50-KR-306.

Use of VIII: CAg as immunosorbent

It is possible to remove Factor IX inhibitor antibodies from the blood of a Factor IX inhibitor patient by passing the blood to a column to which Factor IX is linked, C. Freiburghaus, Thromb.

15 Haemostas. 50, 208, 1983.

In contrast, it is not possible to specifically remove Factor VIII inhibitor antibodies in a similar manner. There are several reasons for this: 20 l) The molecular weight of Factor VIII in AHF preparations is usually 1000-20,000 kD, as the preparation also contains Factor VIII: RAg. Therefore, Factor VIII binds very poorly to gels.

2) By coupling of Factor VIII complex, Vlll: Cag can be coupled via VIII: RAg or other parts of Factor VIII. Thus, VIII: CAg will be easily washed off the coupled gel during use.

These conditions will not apply to VIII: CAg purified as described here.

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Therefore, VIII: CAg bound to a suitable solid matrix, such as a sepharose gel, will be well suited as an immunosorbent, e.g. in extracorporeal specific adsorption therapy of Factor VIII inhibitor patients.

The preparation of the immunosorbent according to the invention is illustrated in the following examples, from which some of the results are already given in the preceding tables.

Example 1 5 5 mg 47 IgG are coupled to 5 ml Sepharose 4B activated with 0.5 g CNBr.

After blocking with 1 M glycine, pH 8.5 and washing with elution buffer cycle, the gel is incubated overnight with 100 ml of AHF (Nordocto®), which contains 400 VIIIrCAg units / ml. The gel is washed on 10 column with 20 ml of buffer A (20 mM imidazole, 10 mM CaCl 2, 0.15 M NaCl, 0.02% NaN 3, pH = 7.35) and 100 ml of buffer A with 0.5 M NaCl. The gel is eluted with buffer A with 0.5 M NaCl in 50% EG. 6 ml eluate contains 7200 units. VIII: CAg. FIG. 3 shows SDS-PAGE of elution fractions.

Judging by blackening intensities, more than 25% of the protein is 15 in the eluate of 80/77 kD protein.

Example 2 0.5 mg of 56 IgG is coupled to 0.5 ml of Sepharose 28 / Cl. After blocking and eluting with the elution buffer cycle, the gel is incubated overnight with 500 ml of cryosupernatant (plasma after cryoprecipitation) containing 200 VIII: CAg units (Spec, act .: 0.007 VIII: CAg units / mg).

The immunosorbent is isolated from the cryosupernatant by passing the incubation mixture through a column prepared from a 25 ml disposable syringe. The passage contains 100 VIII: CAg enh. After washing with 2 ml of buffer B (50 mM imidazole, 0.15 M NaCl, 0.02% NaN3, pH = 7.35) and 100 ml of buffer B with 2.5 M NaCl, elute with 2.5 ml of buffer B with 2.5 M NaCl in 50% EG. The eluate contains 39 VIII: CAg enh. (Spec, act. 61 units / mg) and 0.5 units. VIII: C coagulation activity, cf. also 30 Table 3 above.

Example 3

Dissolved cryoprecipitate is absorbed with Al 2 O 3 and precipitated with 4% PEG, 35 as described in Patent Application No. WO 84/03628. The precipitate is redissolved by stirring for 45 minutes. with 1/4 cryovolume buffer B with DK 173863 B1 14 0.5 M NaCl and 10 mM EDTA. Turbidities are removed by centrifugation and the redissolved precipitate with 1,500 VIII: CAg units. (Spec, act .: 0.29 units / mg) is inked overnight with 0.25 ml "Sepharose 4B" gel coupled with 5 mg 56 IgG / ml. The gel is collected as described in Example 2 and the pass contains 340 VIII: CA The gel is washed with 3 ml of buffer B and 2 ml of buffer B with 2.5 M NaCl. Elute with 1.1 ml of buffer B with 2.5 M NaCl in 50% ethylene glycol. The eluate contains 920 VIII: CAg units, and the specific activity is 6900 units / mg, cf. also Table 3 above.

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Example 4

A 4% PEG precipitate from AHF process containing 1000 VIll: CAg enh. redissolved in buffer B with 0.5 M NaCl and 10 mM EDTA. After reconstitution, incubate overnight with 0.25 ml of Sepharose Cl 2B gel coupled with 10 mg of human inhibitor IgG / ml. The gel was collected as described in Example 2 and the pass contained 340 VIII: CA

The gel is washed and eluted as described in Example 3. The eluate contains 68 VIII: CAg units and the specific activity is 830 units / mg, cf. also Table 3 above.

Example 5 (see Table 4) 2.6 L of cryosupernatant containing 1100 VIII: CA add 5 mM 25 ethylene glycol bis- (beta-aminoethyl ether) N, N'-tetraacetic acid (EGTA) / 0.3 M NaCl and adjust the pH to 8.5. 180 ml of Phenyl-Sepharose (Pharmacia) is added and incubated for 1 hour. The gel is collected on a column and washed with 400 ml of 5 mM imidazole / 0.45 M NaCl / pH 7.4 and eluted with 250 ml of 50% ethylene glycol / 5 mM imidazole / pH 7.4. The eluate 30 contains 440 units VIII: CAg with a specific activity of 0.21 units / mg protein.

The pH is adjusted to 5.5 and 25 ml of Whatman® SE53 cation exchangers are added. After 30 minutes, collect the ion exchanger on a column and wash with 50 ml of 50 mM phosphate / 5 mM EGTA / pH 7.4. VIII: CAg is eluted with 35 ml of 1 M NaCl / 50 mM phosphate / 5 mM EGTA / pH 7.4. The eluate contains 300 VIII.-CAg units. with a specific activity of 8.0 units / mg protein 5 Example 6 500 g 4% PEG precipitate from AHF process according to application WO 84/03628 (from 300 l plasma) redissolved in 3.7 l 50 mM phosphate / 0.75 M NaCl / 5 mM EDTA / pH 8.5. The pH is adjusted to 8.5 with 0.5 M NaOH and the solution 10 after filtration through filter paper contains 17,500 VIII: CA and 190,000 mg of protein. The redissolved precipitate is passed through a column of 250 ml of phenyl-Sepharose gel at a flow rate of 3.7 l / h.

The phenyl-Sepharose is washed with 1.3 l of 25 mM phosphate / 5% ethylene glycol / pH 7.4 at a flow of 3.7 l / h. 13,500 VIII: CAg enh.

15 with 2,000 mg protein (Spec. Act. 6.8 units / mg prot.) Is eluted in 1.3 l 25 mM phosphate / 65% ethylene glycol / pH 7.4. The eluate is added to NaCl to a concentration of 50 mM and the pH is adjusted to 7.0. The eluate is passed through a cation exchange column with 6.25 ml of "CM-fast flow Sepharose" (Pharmacia) at a flow rate of 500 ml / hour. The column is washed with 60 ml of 10 mM phosphate / 50 mM NaCl / pH 7.3 at a flow of 500 ml / hour.

10,800 VIII: CAg enh. with 4.9 mg protein (specific activity 2,200 units / mg) eluting in 18 ml 5 mM phosphate / 0.5 M NaCl / 7 1/2% sucrose, pH 7.3 (600 VIII: CAg units / ml) .

The eluate is mixed with human albumin to a concentration of 0.5%, sterile filtered and dispensed into 3 bottles each containing 6 ml.

After freeze-drying, the preparation is heat-treated at 68 ° C for 72 hours.

The contents of each bottle are dissolved in 18 mg of sterile water and contain 190 VIII: CAg / ml.

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Example 7 VIII: CAg is prepared as described in Example 6, except using 50 mM NaHCO 3 0.5 M NaCl, pH 7.3 to elute the 35 CM ion exchanger. The pH of the eluate is adjusted to 8.5, and 4800 VIII: CAg DK 173863 B1 16 units. in 3 ml of eluate is coupled to 1 ml of CNBr-activated "Sepharoae 4B". The coupled gel is blocked with 1 M glycine pH 8.5.

200 μΐ VIII: CAg-Seph. 4B gel was incubated for 2 hours at 37 ° C with 6.4 ml of plasma from a Factor VIII inhibitor patient. For the incubation, the plasma contained inhibitor antibodies in an amount of 22 BU / ml.

After the incubation, the amount of inhibitor was reduced to 1.8 BU / ml. After reactivation with 3M NH 4 SCN, the 200 μΐ VIII: CAg-Seph. 4B gel again incubated for 2 hours at 37 ° C with 6.4 ml of plasma from the same 10 Factor VIII inhibitor patient. During this incubation, the inhibitor titer was reduced to 3.5 BU / ml.

Claims (2)

An immunosorbent for removing FVIII inhibitor antibodies, characterized in that it contains a protein having FVIII: CAg activity. Immunosorbent according to claim 1, characterized in that the protein with FVIII: CAg activity is the 80/77 kD part of FVIII: C. 10