FI100107B - A method for preparing a combination of coagulation enhancing factor VIII fragments - Google Patents

Description

100107

A method for preparing a combination of coagulation enhancing factor VIII fragments

The present invention relates to a process for the preparation of a coagulation-enhancing combination of a factor VIII N-terminal fragment having a molecular weight of 92 to 210 kD and a factor VIII C-terminal fragment having a molecular weight of Θ0 to 70 kD.

Factor VIII is a protein that occurs naturally in the blood.

It participates as a cofactor in the conversion of FX to activated FX (FXa). The presence of FVIII increases the amount of FXa generation by about 100-fold (Dieijen et al., J. Biol. Chem. 156, p. 3433, 1981). FVIII deficiency (hemophilia A) manifests as severe bleeding.

The role of FVIII in the coagulation cascade is shown in the following diagram:

Cofactors: FX

FVIII 2 +

Ca FlXa

Phospholipids / F ui FXa

Thrombin or FXa can activate FVIII and thrombin, FXa or protein can inactivate it.

Hemophilia A patients can be treated with FVII I either prophylactically or acutely in cases of bleeding.

FVII I can be recovered from human plasma, which contains approximately 1 ppm of protein FVIII. This method can produce only limited amounts of FVIII and is thus only desired for the biosynthetic production of 2,100,107 FVIII in cell culture. Three groups of researchers have been successful here (Wood et al., Nature 312, p. 330, 1980, Toole et al., Nature 312, p. 342, 1984 and Truet et al., DNA 4, p. 333, 1985).

2 mg of foreign protein / ml can be produced in cell culture. This would correspond to 20,000 units / ml FVIII. This level goes far beyond what has been described in the literature about FVIII.

One reason is that FVIII is a very large protein with a molecular weight of about 330 kD (Vehar et al., Nature 312, p.

337, 1984).

FVIII purified from blood plasma or cell culture comprises a fragment termed a factor VIII light chain or FVIII-LC having a molecular weight of about 80 kD and a fragment termed FVIII heavy chain or FVII I-HC having a molecular weight of 92 kD. -210 kD. Fragments are prepared from a 330 kD protein by proteolytic cleavage such that the 80 kD FVIII-LC is the C-terminal fragment, while the FVIII-HC is the N-terminal fragment, the size of which depends on the degree of cleavage.

Thus, it is known that the coagulation-enhancing FVIII contains an 80 kD fragment and a fragment with a molecular weight of 92 kD or more (Fulcher and Zimmerman, Symposium on FVIII,

Scripps Clinic, 1982).

It would be advantageous to prepare FVIII from smaller fragments if these fragments could be combined in vitro to enhance FVIII. These fragments could be more easily produced biosynthetically in large quantities because of their smaller size relative to intact FVIII. In addition, the preparation of FVIII fragments would be advantageous in view of subsequent purification from cell culture, since the formation of the combination alters the yield and molecular weight of the product. Since it is possible to purify the fragments as well as the combination, a purer end product can be obtained.

It 3 100107

Experiments have shown that a simple combination of fragments does not produce coagulation enhancing products. Nor does the literature describe methods or conditions that would allow FVII I fragments to be converted to coagulation-enhancing combinations.

The present invention is based on the finding that a coagulation enhancing combination is produced by reacting a non-coagulating FVI II heavy chain (FVIII-HC, N-terminal fragment) with a non-coagulating FVII I light chain (FVIII-LC, C-terminal fragment). in the presence of a complexing agent. This is completely surprising since the literature describes the purification of FVIII-LC and FVIII-HC, cf. DK Patent Application 5387/85, without describing any attempt to create a combination, despite the fact that creating an effect by combining would be of great theoretical and practical importance.

Burke et al (Abstract 14, p. 111, Research in Clinic and Laboratory 16, 1986) were able to prepare coagulation-efficient FVIII in vivo in cells transfected with DNA for both FVIII-LC and FVIII-HC. However, it was not possible to obtain clotting activity by mixing culture supernatants containing FVIII-LC and FVIII-HC, respectively, although different conditions were experimented with.

FVIII-LC can be purified from human plasma and has no coagulation activity (see WO 86/02838). FVII I-HC fragments can also be purified from blood plasma (Truett et al., DNA 4, p. 333, 1985). These fragments also have no coagulation activity.

According to the invention, one or more divalent metal ions are used as complexing agents. Examples of suitable substances of this type 2-2 + 2+ are Mn, Ca and Co. Other suitable complexing agents include FlXa and FX and substances that react with R-SH and / or R-S-S-R compounds. If desired, mixtures of these substances can be used.

The FVIII-LC or FVII I light chain is a fragment derived from the C-terminal domain of full-length FVIII. The molecular weight of the fragment is typically about 80 kD, but may be 70 kD or less. A fragment with a molecular weight of Θ0 kD has immunological reactivity to FVIII-LC antigen in the described assay and has no reactivity to FVIII-HC antigen.

The FVIII-HC or FVIII light chain is a fragment derived from the N-terminal domain of full-length FVIII. The molecular weight of the fragment is typically 92 kD, but may be less or up to 210 kD. Plasma-purified FVIII-HC consists of a mixture with a molecular weight of 92-210 kD. A fragment having a molecular weight greater than or equal to 92 kD has immunological reactivity to the FVII I-HC antigen in the described assay, but has no reactivity to the FVI I I-LC antigen in the assay.

Coagulation-enhancing FVIII is a protein capable of shortening the clotting time of hemophilia A plasma in a clotting assay. In addition, the coagulation-enhancing FVIII is capable of increasing FXa formation in the Coatest assay (compare below) and thus altering the chromogenic substrate. Coagulation activity is expressed as FVIII: C.

The prothrombin combination consists of coagulation factors containing ΪΓ -carboxy-glutamic acid, i. FII, FVII, FIX, protein, or activated forms of these coagulation factors (Davie et al., Advances in Enzymology 48, p. 277, 1979).

Coatest Assay for FVIII In this assay, FVIII: C was measured in a system consisting of 2+ FIXa, FX and Ca, and phospholipid (PL) (Rosen et al. 100 1007 ai, Thromb Haemostas 54, p. 81Θ, 1985). FXa is formed in an amount that depends on the amount of FVIII: C. The assay was performed as described below: 1. 50 ul of sample was mixed with 50 ul of activating reagent (mixture of FIXa / FX and PL). Incubation time 10 min, 22 C.

2. 25 ul of 25 mM CaCl 2 was added and the mixture was incubated for 20 min at 22 ° C.

3. 50 of chromogenic substrate (S2222) for FX was added.

4. After 15 min of incubation, citric acid was added and the Eg of the sample was read.

It is not possible to monitor the enzymatic activation of FVIII in the Coatest thesis because FVII I is fully activated in the assay by incubation with FIXa / FX, PL and Ca. Immunological quantification of FVIII-LC (FVII I-LC antigen <Ag) was measured in a specific immunoassay (Nordfang et al., Thromb Haemostas 53, p. 346, 1985). Human inhibitor antibody was plated on microplates, a sample was added, and bound FVIII-LC was detected with a peroxidase-labeled fragment of human inhibitor IgG F (ab '> 2). Normal human plasma was used as a standard.

FVII I-HC antigen (Ag) was measured by a specific inhibition test. The canine inhibitor antibody was applied to microplates with loose wells. Sample and I-labeled FVIII-HC were added. The amount of FVIII-HC in the sample determines the amount of bound I-FVIII-HC. The standard is FVIII concentrate (FVIII Nordisk), which is determined to contain 1 FVIII-HC unit per FVI I I: C unit.

6 100107

The amount of FVIII-LC and FVIII-HC defined by the immune test is expressed in relative terms. This means that the ratio between unit FVIII: C, unit FVIII-LCAg, unit FVIII-HCAg for different FVII I types is not 1: 1: 1. However, it is assumed that the units of the different assays are comparable, but there may be differences between the FVII I: C unit, the FVII I-LCAg unit, and the FVII I-HCAg unit for the FVII I sample, where all protein is coagulation enhancer in the Coatest.

Determination of molecular weight

Molecular weight is determined by modified SDS-PAGE (CLaemm-li, Nature 227, p. 680, 1970).

Preparation of FVIII A sample of FVIII for control tests was prepared from FVIII concentrate (see WO 84/03628) by affinity chromatography on goat anti-von Willebrandt Factor Sepharose (Truett et al., DNA 4, p. 333, 1985).

Preparation of FVIII-LC (Sample A) FVIII-LC can be purified from blood plasma by various methods (as described e.g. in WO 86/02838). High concentration FVIII-LC isolated by affinity chromatography with Nordiocton monoclonal 47 IgG prepared as described by Nordfang et al., Thrombosis and Haemostasis, Vol. 54, pp. 586-590, 1985, Nordiocto diluted in 200 ml of buffer A (0.02 M imidazole, 0.15 M NaCl, 10 mM EDTA, pH 7.4) 110 units / ml VI I-LCAg concentration, incubated overnight with 7 ml of 47 IgG Sepharose (adjusted with 9 mg IgG / ml). The incubation mixture was poured onto the column and the eluate was collected. The gel was washed with 40 ml of buffer A and 40 ml of buffer A with a total amount of 0.65 M NaCl. FVIII-LC was eluted with 40 ml of 20 mM imidazole / 0.65 M NaCl / 10 mM EDTA / 50% ethylene glycol / pH 7.4.

The 4 ml peak fraction was dialyzed against 50 mM imidazole / 0.15 m NaCl / 10% glycerol / 0.02% NaN3 / pH 7.4. The concentration of FVII I components in the dialyzed samples is shown in Table 1.

7 100107 Preparation of FVIII-HC (lane B) FVIII-HC is prepared from sample FVII I by affinity chromatography on monoclonal 56 IgG Sepharose (prepared as described by Nordfang et al., Thrombosis and Haemostasis, Vol. 54, p. 586- 590, 1985. 56 IgG Sepharose binds to the FVIII-LC / FVII I-HC combination via FVIII-LC 25 ml of FVIII sample containing 405 FVII I-HCAg units / ml was incubated overnight. 5 ml of 56 IgG Sepharose (adjusted with 4 mg of 56 IgG / ml) The incubation mixture was poured onto a column and the eluate was collected and the gel was washed with 5 ml of buffer B (20 mM imidazole / 0.15 M NaCl / 10% glycerol). / 0.1 M lysine / pH 7.4) containing 0.35 M CaCl 2 The gel was then washed with 15 ml of buffer B with a total NaCl content of 0.65 M, followed by washing with 5 ml of buffer B. with 10 mM EDTA and 0.02%

NaNg (EDTA buffer). The gel was dried and incubated for 1 hour at room temperature with EDTA buffer. After incubation, FVIII-HC was eluted with 5 ml of EDTA buffer. The 2 ml peak fraction was dialyzed against 50 mM imidazole / 0.15 M NaCl / 10% glycerol / 0.02% NaNg / pH 7.4. The concentration of FVII I components in the dialyzed sample is shown in Table 1.

Table 1 FVII I fragments in dialyzed FVIII-LC sample and FVIII-HC sample FV111: C FVIII-LCAg FVIII-HCAg units / ml units / ml units / ml FVIII-LC (sample A) <0.01770 1 .8 FVIII-HC (sample B> <0.01 0.1 2000 Samples A and B were analyzed by SDS-PAGE, see the attached figure where lane 1 corresponds to sample A (FVIII-LC) 4 FVIII-LCAg units, θ 100107 lane 2 contains molecular weight markers, and lane 3 corresponds to sample B (FVIII-HC) Θ FVII I-HCAg

The method according to the invention is illustrated below by means of some exemplary embodiments.

Example 1 Samples A and B were each diluted 10-fold in buffer C (50 mM imidazole, 0.15 M NaCl, 0.1% BSA, pH 7.4).

20 μl of A (diluted 1:10) was mixed with 20 μl of B 1:10, 3 μl of 0.15 M MnCl and 40 μl of buffer C. After 4 hours of incubation at 22 ° C: The incubation mixture contained 1200 m units of FVIII: C / ml as measured by Coatest.

The following experiments were performed for comparison purposes

Koe A

The experiment was performed as described in the example, except that 3 buffer C was added instead of 3 0.15 M MnCl 2. After 4 hours of incubation at 22 ° C, this incubation mixture contained less than 5 m units of VIII: C / ml as measured by Coatest.

Koe B

Sample A was diluted 40-fold in buffer C. Θ0 ml of A (diluted 1:40) was mixed with 3 μl of 0.15 M MnCl 2. After 48 hours of incubation, the incubation mixture contained less than 5 units of VIII: C / ml. Similarly, the incubation mixture with Sample B contained less than 5 m units of VIII: C / ml.

Koe C

80 μl of FVII I sample diluted 1000-fold in buffer C was mixed with 3 μl of 0.15 M MnCl 2. After 48 hours of incubation, 179 m units of VIII: C / ml were measured.

100107 g of 80 wood FVI I I samples diluted 1000-fold in buffer C were mixed with 3 buffer C. After 48 hours of incubation, 140 m units of FVIII: C / ml were measured.

Example 2 Samples A and B were each diluted 10-fold in buffer C. 20 μl of A was mixed with 20 μl of B (diluted 1/10), 3 μl of 2.2 M CaCl 2 and 40 μl of buffer C. After 12 days of incubation at 22 ° C, the incubation mixture contained 1300 m units of VIII: C / ml.

The following experiment was performed on a control

The assay was repeated as described above, except that 3 μl of buffer C 3 μl instead of 2.2 M CaCl 2 was added. After 12 days of incubation at 22 ° C, the incubation mixture contained less than 5 m units of VIII: C / ml.

Example 3 Samples A and B were each diluted 100-fold in buffer C. 100 μl of A and 100 μl of B were mixed, 50 μl of the mixture was tested in Coateet as described above. 1.5 m units of the mixture were measured. 50 of the mixture was further tested in modified Coateet by pre-incubation with FIXa / FX for 1 hour before the addition of PL. This increased the Coatest active ratio to 3.0 m / ml.

The following experiment was performed on a control FVIII sample diluted 30,000-fold in buffer C.

A 50 ul diluted sample was tested in Coateet as described above. 3.4 m units / ml was measured.

A 50 μl diluted FVI11 sample was further tested with a modified Coatest by 1 hour preincubation with FIXa / FX before the addition of PL. For the diluted FVII I sample, 3.8 m units / ml was measured.

10 100107

Example 4

When the experiment was performed as described in Example 1, 1000 m units of VIII: C / ml were measured after 24 hours of incubation. When 40 μl of FIXa / FX 40 μl of buffer C was added instead of 1600 m units / ml after 24 hours of incubation.

The following experiment was performed on a control

The experiment was performed as described in Example 1, the first comparative experiment. After 24 hours of incubation, less than 5 m units / ml of the incubation mixture was measured.

The second experiment was performed as follows: 40 μl of FVII I sample diluted 500-fold in buffer C was mixed with 40 μl of FIXa / FX and 3 μl of 0.15 M Mn. After 24 hours of incubation, 120 m units / ml. When 40 μl of FIXa / FX was replaced with 40 μl of buffer C, 170 μl / ml was measured. In addition, when 3 μl of 2 + '0.15 M Mn 3 μl of buffer C was replaced, 140 m units were measured.

Example 5 Samples A and B were each diluted 20-fold in buffer C. 20 μl of A 1/20 were mixed with 20 μl of B 1/20 and 40 μl of FIXa / FX and 3 μl of 0.15 M CaCl 2. After 24 hours of incubation at 22 ° C, the incubation mixture contained 199 mU FVIII: C / ml. If FIXa / FX was replaced with 40 μl of buffer C, then 43 mU FVIII: C / ml was measured after 4 hours of incubation.

Example 6 FVIII-LC and FVII I-HC samples containing 800 units of FVIII-LC: Ag / ml and 850 units of FVI11-HC: Ag / ml, respectively.

each was diluted 3-fold. 20 ul FVIII-LC

2 + 1/3 was mixed with 20 FVIII-HC 1/3 and 10 Me. In the mixture-2 + '2+ 2+' 2+ sa A Me was 25 m Mn. In mixture B Me was 250 mM Ca and in 2 + 2+ 2 + mixture C Me was 25 mM Mn and 250 mM Ca. After 24 hours of incubation, mixture A contained 10.3 units of FVIII: C / ml, mixture B contained 4.0 units and mixture C contained 12.9 units of 11,100,107 FVIII: C / ml. After 144 hours of incubation, mixtures A, B and C contained 6.6 units, 6.5 units and 11.9 units of FVIII: C / ml, respectively.

Example 7 COS cells were transfected with plasmid PSVF6-80 expressing the Θ0 kD chain, cf. DK Patent Application 0428/87. To the culture-derived supernatant containing 870 units of FVII I-LC: Ag / ml, plasma-purified FVIII-2 + HC was added to a final concentration of 20 FVII I-HC: Ag units / ml and Mn to a final concentration of 5 mM. After incubation for 22 hours at 22 ° C, the mixture contained 137 m units of FVIII: C / ml. When FVIII-HC and Mn were added to a final concentration of FVII I-LC: Ag / ml of 1000 m units of plasma-purified FVIII-LC, respectively, the incubation mixture contained 33 m units of FVIII: C / ml after 24 hours of incubation. . When M-2 + M alone and not FVIII-HC was added to the culture supernatant, the mixture contained less than 2.5 m units of FVIII: C / ml after 24 hours of incubation.

Example 8 25 trees of FVIII-LC were mixed in buffer C with 25 μl of FVIII-HC, 7 μl of MnCl 2 and 10 μl of oxidant reducing agent to obtain the final concentrations of the individual components shown in Table 2. FVIII: C was measured 5 hours after incubation at 20 ° C.

12 100107

Table 2 Recombination of FVIII-LC and FVIII-HC in the presence of an oxidizing agent.

FVI I I-LC: Ag FVIII-HC: Ag mM oxidant pel-% FVIII: C ratios / ml units / ml MnCl2 in the tea FVIII-LC:

To Ag after 5 hours 11 11 5 none 4.9 11 11 5 15 / μM DTT 16.8 11 11 5 150 7uM ME 18.8 11 11 5 30 / μM Cys 17.4 11 112 5 15 / μM Cys 68.8 11 11 0 none <0.5

At the given concentrations of DDT (dithiothreitol), ME (mercaptoethanol) and Cys (cysteine), there is an equilibrium between the oxidizing and reducing forms of the aqueous buffer.

Example 9 FVIII-HC was prepared as described above and thus modified by mixing 50 μm of mercaptoethanol into EDTA-e create intestinal buffer and dialysis buffer. After dilution, recombination with FVI I I-HC samples was allowed to incubate in buffer C at room temperature with the addition of mercaptoethanol to 35 μM, MnCl 2 to 5 mM, and FVIII-LC 22 FVIII-LC:

Up to Ag unit / ml.

13 100107

Table 3 shows recombination with two types of FVIII samples.

Table 3 FVIII-HC FVIII-HC: Ag units / ml% FVIII: C in a recombination mixture to FVI I I-HC: Ag after 20 hours Sample 1, prepared as described above 2.59 31 Sample 2, prepared as described above 3.31 27 Sample 3, prepared with ME as described in this example 3.00 89

Example 10

The incubation mixture of the following composition was prepared in buffer C from individual FVII I fragments: 60 units / ml FVIII-LC, 60 units / ml FVIII-HC, 50 mM CaCl 2, 2 units / ml FX.

After incubation for 20 hours at room temperature, 6.9 FVII I: C units / ml were measured. From the corresponding incubation mixture without FX, 0.59 FVII I: C units / ml was measured after 20 hours of incubation at 20 ° C.

Claims (11)

100107 A process for the preparation of a coagulation enhancing complex between an N-terminal fragment of factor VIII having a molecular weight of 92 to 210 kD and a C-terminal fragment of factor VIII having a molecular weight of 80 to 70 kD, wherein the individual naturally occurring amino acids in one or both fragments may, if desired. be omitted or replaced by other amino acids, characterized in that the two fragments are reacted with each other in the presence of a coagulant. The method of claim 1, wherein the N-terminal fragment is a heavy chain having a molecular weight of about 92 kD and the C-terminal fragment is a light chain having a molecular weight of about 80 kD. A method according to claim 1, characterized in that one or more cys amino acids in at least one of the fragments are replaced by serine. Process according to Claim 3, characterized in that the fragment having a molecular weight of 92 to 210 kD or the fragment having a molecular weight of 80 to 70 kD, or both, have been treated with an agent which shows reactivity with the group -SH and / or - For SS-containing compounds. Method according to Claim 4, characterized in that one or both of the fragments is produced biosynthetically. Process according to any one of Claims 1 to 5, characterized in that the complexing agent comprises one or more divalent metal ions. Process according to Claim 6, characterized in that the complexing agent is Mn 2+, Ca 2+ or Co 2+. Il 100107 Process according to any one of Claims 1 to 5, characterized in that the coagulant promoter is a mixture of a divalent metal ion and a component of the prothrombin complex, preferably FX. Process according to any one of Claims 1 to 5, characterized in that the complexing agent is a mixture consisting of a divalent metal ion and one or more components FIX, FIXa, FX or FXa. Process according to any one of Claims 1 to 5, characterized in that the complexing agent is a substance which shows reactivity towards compounds containing the group -SH and / or -S-S-. Process according to any one of Claims 1 to 5, characterized in that the complexing agent is a mixture of a divalent metal ion and a substance which shows reactivity towards compounds containing the group -SH and / or -S-S-.