CA2124690C

CA2124690C - Composition comprising coagulation factor viii formulation, process for its preparation and use of a surfactant as stabilizer

Info

Publication number: CA2124690C
Application number: CA002124690A
Authority: CA
Inventors: Thomas Osterberg; Angelica Fatouros
Original assignee: Genetics Institute LLC
Current assignee: Genetics Institute LLC
Priority date: 1992-10-02
Filing date: 1993-10-01
Publication date: 2007-09-11
Anticipated expiration: 2013-10-01
Also published as: HU211670A9; NO942033L; EP1016673B1; ATE198277T1; JPH07501560A; EP0627924B1; SK65994A3; US5733873A; DE69329795D1; ES2154650T3; FI111443B; DE69329795T2; EP0627924A1; EP1016673A1; AU5288393A; MX9306133A; EP1652534A2; CZ290342B6; DK0627924T3; NZ256921A

Abstract

The present invention relates to novel composition comprising coagulation factor VIII and a non-ionic surfactant such as block copolymers, e.g. polyoxamers or polyoxyethylene (20) sorbitan fatty acid esters e.g. polysorbate 20 or polysorbate 80 as stabilizer. The composition can also comprise sodium chloride, calcium chloride, L-histidine and/or sugars or sugar alcohols.
The invention also relates to the use of a non-ionic surfactant as stabilizer for a composition comprising coagulation factor VIII.

Description

COMPOSITION COMPRISING COAGULATTON FACTOR iTIII
FORMULATION, PROCESS FOR TI5 PREPARATION AND USE OF A
SURFACTANT AS STABILIZER.

The present invention relates to a novel formulation comprising coagulation factor VIII and a non-ionic surfactant such as block co-polymers, e.g.
polyoxamers or polyoxyethylene (20) sorbitan fatty acid esters e.g.
polysorbate 20 or polysorbate 80. The composition can also comprise sodium cliloride, calcium chloride, L-histidine and/or sugars and/or sugar alcohols.
Haemophilia is an inherited disease which has been known for centuries but it is only within the last three decades that it has been possible to differentiate between the various forms; haemophilia A, haemophilia B and haemophilia C. Haemophilia A is the most frequent form. Tt affects only males with an incidence of one or two individuals per 10 000 live-born males. The disease is caused by strongly decreased level or absence of biologicalty active ooagulation factor VM (antihaemophilic factor) which is a protein normally present in plasma. The clinical manifestation of haemophilia A is a strong bleeding tendency and bef re treatment with factor VIII concentrates was introduced, the mean age of those patients was less than 20 years. Concentrates of factor VM obtained from plasma have been available for about three decades. This has improved the situation for treatment of haenlophilia patients considerably and given them possibility to live a normal life.

Therapeutic factor VII][ concentrates have until now been prepared by fractionation of plasma. However, there are now methods available for production of factor VIII in cell culture using recombinant DNA techniques as reported in e.g. J Gitschier et al. Nature 312,330-37,1984 and EP 160 457.
Factor VIII concentrates derived from human plasma contain several fragmented fully active factor VIII forms (Andersson et al, Proc. Natl. Acad.
5ci. USA, Vol 83,2979-83, May 1986). The smallest active form has a . = _ . .,. . _ . . .. , . . , .. . .. . . .,.

212469~ =

molecular mass of 170 kDa and consists of two chains of 90 kDa and 80 kDa held together by a metal ion bridge. Reference is here made to EP 197 901.
Kabi Pharmacia has developed a recombinant factor VIII product which corresponds to the 170 kDa plasma factor VIII form in therapeutic factor VIII
concentrates. The truncated recombinant factor VIII molecule is termed r-VIII SQ and is produced by Chinese Hamster Ovary (CHO) cells in a cell culture process in serum free medium at finite passage.

The specific activity of r-VIII SQ could be more than 12 000 IU/mg protein and preferably more than 14 000 IU/ mg. Activity of about 15 000 ItJ/mg has been measured. About 10 000 IU VIII.C per mg protein has earlier been known for our r-VIII SQ.

Recombinant factor VIII SQ is indicated for treatment of classical haemophilia. The dosage is similar to the dosage of the plasma factor VIII
concentrates. Due to the high concentration now obtainable only small voluines are needed for injection.

The structure and biochemistry of recombinant factor VITY-products in general have been described by Kaufman Tibtech, Vol 9,1991 and Hematology, 63, 155-65, 1991. The structure and biochemistry of r-VIII SQ
have been described in WO 91/09122. The stability of proteins is generally a problem in pharmaceutical industry.

It has often been' solved by drying of the protein in different drying processes, such as freeze drying. The protein has thereafter been distributed and stored in dried form.
The solution before drying or freeze-drying, the dried material and the reconstituted product should all be stable, so that not too much activity is lost during the drying process, the storage or during handling.

Factor VIII which has been fractionated from plasma is normally sold as lyophilized powder which should be reconstituted with water.

A formulation with. a low amount of protein will generaliy loose activity during purification, sterile manufacturing, in the package and during the administration. This problem is usually solved by the addition of human albumin which reduces the activity loss of the active protein considerably.
Human albumin functions as a general stabilizer during purification, sterile manufacturing and freeze-drying (see review by Wang et al., J. of Parenteral Sci. and Tech. Vo142, Number 2S, supplement. 1988). Human albumin is S also a good cake-former in a formulation for freeze-drying. The use of albumin for stabilization of factor VIII is known and is currently used in all highly purified factor VM products on the market.
However, it is not desirable to add human albumin to a therapeutic protein manufactured by recombinant DNA technology. In addition, the use of human albumin as a formulation excipient often limits the use of many of the most powerful and sensitive analytical methods for protein characterization.

There is a need for albumin free formulations containing factor VIII and especially recombinant factor VIII which are stable during drying or freeze-drying, in solution and as a solution after reconstitution.

Several solutions have been proposed for the stabilization of different probeins:

EP 35 204 (Cutter) discloses a method for imparting thermal stability to a protein composition in the presence of a polyol.

EP 381345 (Corint) discloses an aqeous liquid of a peptide, desmopressin, in the presence of carboxymethylcellulose.

In WO 89/09614 (Genentech), a stabilized formulation of human growth hormone comprising glycine, inannitol and a buffer is disdosed and in a preferred embodiunent a non-ionic surfactant such as polysorbate 80 is added. The non-ionic surfactant is added for reduced aggregation and denaturation. The formulation has an increased stability in a lyophilized formulation and upon reconstitution.

EP 268 110 (Cetus) discloses a solution comprising a particular protein, interleulcin-2, which is dissolved in an inert carrier medium comprising a non-ionic polymeric detergent as a solubilizer/stabilizer. The preferred 4~.. a.~ PCI'/SE93/00793 detergents are octylphenoxy polyethoxy ethanol compounds, polyethylene glycol monostearate compounds and polyethylene sorbitan fatty acid esters.
US 4 783 441 (Hoechst)disdoses an aqueous solution comprising a protein, such as insulin and a surface active substance.

US 4 165 370 (Coval) discloses a gamma globulin solution and a process for the preparation thereof.lhe solutions contains polyethylene glycol (PEG). A
non-ionic surfactant can be added to the solution.
In EP 77 870 (Green Cross) the addition of amino acids, monosaccarides, oligosaccarides or sugar alcohols or hydrocarbon carboxylic acid to improve stability of a solution containing factor VIII is disclosed and the addition of sugar alcohol or disaccarides to an aqueous solution of factor VIII for increasing stability during heat treatment has been disclosed in EP 117 064 (Green Cross).

WO 91/10439 (Octopharma) clauns stable injectable solution of factof VIII or factor IX which comprises a disaccaride, preferably saccarose and one or more amino acids.

EP 315 968 and EP 314 095 (Rorer) claim stable formulations of factor VIII
with different ionic strength.

Proteins are different with regard to physico-chemical properties. When preparing a pharmaceutical preparation which should be physico-chemical acceptable, and stable for a long time, consideration can not only be taken to the physiological properties of the protein but also other aspects must be considered such as the industrial manufacture, easy handling for the patient and safety for the patient. The results of these aspects are not predictable when testing different formulations and there often is a unique solution for each protein.

In plasma circulating factor VM is stabilized by association with its carrier protein, the von Willebrand factor (vWF). In plasma and also in conventional intermediate purity factor VIII concentrates the ratio vWF to factor VIII is at least 50:1 on a weight basis. In very high purity factor VIII

WO 94/07510 2124,620 PCT/SE93/00793 concentrates, with a specific activity of more than 2 000 IU per mg protein, the ratio vWF to factor VM is about 1:1 (w/w) and essentially alI factor VIII
is bound to vWF. Despite this stabilization further protection by the addition of albumin is required in order to achieve an acceptable stability during 5 lyophilization and storage.

All super pure preparations on the market are stabilized with albumin (human serum albumin).
There is a now a demand for injectable factor VIII without albumin and containing a nlinimum of additives.

We have now developed a new formulation which solves the above mentioned problems for factor VIII.

To our great surprise we have found that factor VIII, which is a very sensitive protein, can be stabflized without albumin, when a non-ionic surfactant is added.

Thus the present invention relates to a composition cnrnprising a coagulation factor VIII and a non ionic surfactant as stabilizer. Our factor VM is highly purified, i.e. has a specific activityof more than 5000 IU/mg protein, and the composition is stabilized without the addition of albumin, When factor VM is recombinant it can be either in its full-length form or as a deletion derivative such as SQ derivative.
The amount of faztor VI1I is from 10 to 100 000 ItJ/ml, preferably 50 to 10 0001U/mI.
The non-ionic surfactant is preferably chosen from block co-polymers such as a poloxamer or polyoxyethylene (20) fatty acid ester, such as polysorbate 20 or polysorbate 80. Tween 80 has been used as polysorbate 80.
The non-ionic surfactant should be present in an amount above the critical micelle concentration (CMC). See Wan and Lee, Journal of Pharm Sci, 63, 136,1974.
The polyoxyethylene (20) fatty acid ester is thus prefe~,:;Cbly in an amount of at least 0.01 mg/ml. The amount could e.g. be between 0.02 and 1 mg/ml.
The composition can also comprise sodium or potassium chloride, preferably in an amount of more than 0.1 M.

._.___..__- _....._._...._...._.:.:e.........-:,...-.zz..r..aars:'.:.rJt.
...... ibT0. ]a ....zrnl ....5.._' ..T..-.~.._.i ....e':.i',f'~f~vt..'~c~ t .::' 1 . ~..e.
a..,.i:;..= :1.-:'ti . aYl~.~'s f vnfr iaZ~r ._ . .. ... .. . .. .. ... .. .. .... ....... :_F''.,. ...

WO 94/07510 PC'T/SE93/00793 The composition comprises preferably a calcium salt such as calcium chloride or calcium gluconate preferably in an amount of more than 0.5 mM
and an amino acid such as L-histidine in an amount of more than 1 mM.
The amount could e.g. be chosen between 0.05 and 500 mM.
Mono-or disaccarides such as sucrose or sugar alcohols could be added e.g.
in an amount of 1 to 300 mg/ml.

The composition comprises preferably L-histidine and sucrose. The ratio sodium chloride to L-histidine in the composition is preferably more than 1:1.

The composition could comprise i) 10-100 000 IU/ml of recombinant factor VIII
ii) at least 0.01 mg/ml. of a polyoxyethylene (20) fatty acid ester iii) sodium chloride, preferably in an amount of more than 0.1 M.
iv) calcium salt such as calcium chloride or calciunn gluconate preferably in an amount of more than 0.5 mM.
v) an amino acid such as L-histidine in an amount of more than 1 mM.' To this composition could mono-or disaccarides or sugar alcohols, preferably sucrose be added.
The composition could be in a dried form, preferably"lyophilized or in aqeous solution before or after drying. The dried product is reconstituted with sterile water for injection or a buffer solution.
The claimed composition can also be a stable aqeous solution ready for use.
The invention also relates ot compositions in which the specific activity of r-Vffi SQ is more than 12 000 ILJ / mg protein, preferably more than 14 000 IL7 / mg.

The daimed composition can be prepared by mixing factor VIII with a non-ionic surfactant in an aqeous solution, -preferably together with an amino acid such as Uhistidine, sodiuni salt, sucrose and a calcium salt or by eluating factor VIII from the last purification step with a buffer containing a non-ionic surfactant in an aqeous solution, preferably together with an amino acid such as L-histidine, sodium salt, sucrose and a calcium salt.

The invention also relates to the use of a non ionic surfactant preferably chosen from block co-polymers, preferably a poloxamer or polyoxyethylene (20) fatty acid ester, preferably polysorbate 20 or polysorbate 80, as stabilizer for a composition comprising coagulation factor VIIC.

An amino acid is used to buffer the system and it protects also the protein in the amorphous phase. A suitable buffer could be L-histidine, lysine and/or arginine. L-Histidine has primarily been chosen because of the good buffer capacity of L-histidine around pH 7.

Sucrose or sugar alcohol can also be added for the protection of the protein., Calcium (or divalent metal ions), here added as calcium chloride (CaCl2) but other salts such as calcium gluconate, calcium glubionate or calcium gluceptate can also be used, is necessary for the maintenance of the association of factor VIII heavy and light chain.

The data presented in the examples indicate that r-VIII SQ is stable for at least 12 months when stored at 5f3 C.

The following examples iliustrate the invention and show stability data for different formulations, all falling under the patent protection, a protection which is not limited to these examples.
The following figures are iilustrating the invention:

Figure 1 HPLC gelfiltration, Example 10A, stored 5 months at 250C.
Figure 2 HPLC gelfiltration, Example I Oti, stored 5 months at 300C.
SUBSTITUTE Sh1EET

EXPERIMENTAL
Material and methods The production of recombinant factor VM SQ (r-VIII SQ) was essentially performed as described in patent WU..91 /09122, example 1-3. A DHFR
defident CHO celline (DG44N.Y.) was electroporated with an expression vector containing the r-VIII SQ gene and an expression vector containing the dihydrofolate-reductase gene. Following selection on selective media :10 surviving colonies were amplified through growth in stepwise increasing amounts of rriethotrexate. Supernatant from the resulting colonies were individually scTeened for V.III:C activity. A production done was chosen and this was subsequently adapted to serum firee suspension growth in a defined medium and finally a large scale fermentation process was developed.
Supernantant is collected after certain time periods and further purified as described below.

The clarified conditioned medium was pH adjusted and applied to a S-Sepharose FF column. After washing, factor VM was eluated with a salt buffer containing 5 mM CaCI2,.

Immunoadsorption was carried out on an immunoaffinity resin where the ligand was a monoc]onal antibody (8A4) directed towards the heavy chain of Factor VIII. Before loading to the column the S-eluate was treated with 0,3 % TNBP and I% Octoxynol9..
The column was equilibrated, washed and factor VIII was eluated with a buffer containing 0,05 M CaC12 and 50 % ethylene glycol.

The mAb-eluate was loaded on a Q-Sepharose FF column., equilibrated with the elut3on buffer in the immunoaffinity step. After washing, factor VM was eluated with 0,05 M L-histidine, 4 mM CaClz, 0,6 M NaCl, pH 6,8.

The Q-eluate was applied to a gel filtration column (Superdex 200 p.g.).
Equilibration and elution was carried out with a formulation containing ,35 sodium chloride, L-hist-idine, calcium chloride and polysorbate 80.
*Trade-mark The protein peak was collected and the solution was formulated before freeze drying.

The VIII:C activity and the concentration of the inactive components were adjusted by diluting with an appropi-iate buffer. The solution was then sterile filtered (0,22 m), dispensed ansi freeze-dried. Samples from each composition were frozen and stored at - 70 oC. These samples were thawed and used as references during the assay of VIII:C.

The coagulant activity VIII:C was assessed by a chromogenic substrate assay (Coatest*Factor VIII, Chromogenix AB, Molndal, Sweden). Activated factor X
(Xa) is generated via the intrinsic pathway where factor VIII:C acts as cofactor. Factor Xa is then determined by the use of a synthetic chromogenic substrate, S-2222 in the presence of a thrombin inhibitor 1-2581 to prevent hydrolysis of the substrate by thrombin. The reaction is stopped with acid, and the VIII:C, which is proportional to the release of pNA (para-nitToaniline), is determined photometrically at 450 nm against a reagent blank. The unit of factor VIII:C is expressed in international units (IU) as defined by the current Internatzonal Concentrate Standard (IS) established by :20 WHO.

The recovery of VIII:C is calculated as the percentage of VIII:C in the reconstituted solution divided by the VIII:C in the frozen and thawed solution for freeze-drying writh appropriate adjustment for dilutions.
:25 Soluble aggregates were determined by gel filtration. A prepacked Superdez 200 HR 10/30 column (Pharmacia) was used with a fluoroscence detector (exitation wavelerlgth 280 nin), emission wavelength 340 nm). The reconstituted preparation were analysed. Evaluation of results from 30 gelfiltration was done by visual exam-ination of the chromatograms, or by integration of the peak areas if aggregates were found.

Recovery over freeze drying is expressed in % yield of frozen reference.
*Trade-mark . . .

10 PCr/SE93/00793 Example 1. Comparison between albumin and non-ionic surfactant.
Recombinant factor VIII was prepared according to the method described under Experimental.

Two ml of the solution was lyophilized and thereafter reconstituted in an amount of 5 ml of sterile water for injections.

The compositions were the following :
10 lA IB 1C 1D
IrHistidine, mM 50 50 50 50 Sodium chloride, M 0,6 0,6 0,6 0,6 Calcium chloride, mM 4 4 4 4 Polysorbate 80, % - - 0.02 -PEG 9:000, % 0.1 0.1 - -Albumin, k - 1 - 1 VII'i:C charged ICT/ml 250 250 250 250 Recovery, IU/ml after reconstit. 83 197 232 222 This example shows that there was no difference in the"recovery of factor Vffi:C when the non ionic surfactant or albumin was used.

Example 2, Comparison between different strengths of non ionic surfactant Recombinant factor VID[ was prepared according to the method described under Experimental.

Two ml of the solution was lyophilized and thereafter reconstituted in an amount of 2 ml of sterile water for injections.

The compositions were the following :

L-fiistidine / L-Glutamate equiunolar amount, mg /snl 10 10 10 Sodium chloride, % 2 2 2 Caldum chloride, mg/ml 0.1 0.1 0.1 Polysorbate 80, % - 0,001 0,01 VIILC charged IU/ml 300 300 300 Reoovery, IU/n-d after reannstit.
Initial 69 133 228 35 h* 43 140 222 7h* 49 133 204 * stored as reconstituted solution at ambient temperature It is here dearly shown the surprisingly good stabilizang effect on factor VIIi when a non ionic surfactant is used.

:Y . =.:....=,Yl1J'. .-,:.:.. .._. . ... ... . .,. . . .. .. -Example 3, Variation of non-ionic surfactant concentration.
Recombinant factor VIII was prepared according to the method described under Experimental.
Two ml of the solution was lyophilized and thereafter reconstituted in an amount of 5 inl of sterile water for injections.

L-Histidine, mM 50 50 50 50 50 Sodium chloride, M 0.34 0.34 0.34 0.34 0.34 Calcium chloride, mM 4 4 4 4 4 Polysorbate 80, % 0.01 0.02 0.03 0.04 0.05 Reaoveiy, after reconstit., % 91 90 93 99 100 Results from this example indicate that the recovery of factor VIiI (VIII:C) was very high after reconstitution and good for all concentrations of polysorbate 80 used.

Example 4. Variation of sodium chloride concentration Recombinant factor VIII was prepared according to the method described under Experimental.
Two ml of the solution was lyophilized, stored at different temperatures for up to 6 months (mon) and thereafter reconstituted in an amount of 5 ml of sterile water for injections.

L-Histidine.mM 50 50 Sodium chloride, M 0.3 0.6 Calcium cliloride, mM 4 4 PEG-4000 % 0.1 0.1 (Polyethylene glycol) Polysorbate 80, % 0.025 0.025 Recovery, %, initial 85 86 stored at 80C
3 mon 88 87 4 mon 87 83 6 mon 87 83 stored at 250C, 1 mon 92 93 3 mon 87 79 4 mon 84 81 6 mon 85 85 -stored at 370C 1 mon 88 90 3 mon 80 80 4 xnon 80 77 6 mon 81 80 stored at 500C 1 mon 84 89 3 mon 77 77 4 mon 73 70 0,3 or 0,6 M sodium chloxide showed very good stability. Both formulations were stable for 6 months at 37 C.

Example S. Variation of L-Histidine concentration Recombinant factor VIII was prepared according to the method described under Experimental.
2,2 ml of the solution was lyophilized, stored at different temperatures for up to 3 months (mon) and thereafter reconstituted in an amount of 5 ml of sterile water for injections.

L-Histidine, mM 46 59 Sodium chloride, M 0.31 031 Calcium chloride, mM 3,7 3,7 PEG-4000 % 0.091 0.091 (Polyethylene glycol) Polysorbate 80, % 0.364 0.364 Recovery, %
stored at 80C, Initial 78 84 3 mon 70 76 stored at 250C, 1 mon 3 mon 69 74 stored at. 370C 1 mon 76 85 3 mon 61 48 stored at 500C 1 mon 60 73 3 mon 44 48 This example shows that these different amounts of L-histidine does not effect the stability.

...... ..._:.,... ..1.'......., h ir.,. .
...,. . . .. . .., ........ . . : i .i.;....., ........_.::A.:. -... . . ..:;=.,.'. .h.4..:'l. ....,....:=. ,.
,:e......=.5. ...,...... ..... J.'... . i',4x=S" '......, V..~~,~x <..u~i. _ ._l.v4= . . .i . . .

Example 6 Recombinant factor VM was prepared according to the method described under Experimental.

L-Histidine, mM 65 65 Sodium chloride. M 0.3 0.3 Caldum c.hloride, mM 4 4 10 PEG4000 % 0 0.1 Tween 80, % 0.025 0.025 These solutions were freezed/thawed 1, 5 and 10 times and the recovery was the following:
IU/ml IU/ml cold 298 291 1 freezing 293 293 These studies sho*ed that VIII:C was stable after repeated freeze-thawing and that PEG-4000, which is thought to act as cryoprotectant, is not necessary in this formulation.

ExaMle 7. Variation of pH
Recombinant factor VIII was prepared according to the method described under Experimental.
2,2 ml of the solution was lyophilized and thereafter reconstituted in an amount of 5 ml of sterile water for injections.

L-Histidine, mM 65 65 65 65 Sodium chloride, M 0,3 0,3 0,3 0,3 Calcium chloride, mM 4 4 4 4 Polysorbate 80, % 0.025 0.025 0.025 0.025 pH 6.0 6.5 7.0 7.5 Recovery, Initial 74 70 78 79 3 hours* 73 80 78 77 *stored as reconstituted solution at ambient temperature This example shows that a pH is of no significant importance between 6.0 and 7.5 approx.

Examvle 8 Addition of sucrose Recombinant factor VIII was prepared according to the method described under Experimental.
2,2 inl of the solution was lyophilized and thereafter reconstituted in an amount of 5 ml of sterile water for injections.

L-Histidine, mM 58 20.5 Sodium chloride. M 0.3 0.3 Calcium chloride, mM 3,7 3,7 Sucrose, mM 0 13.3 Polysorbate 80, % 0.025 0.025 Sucrose was added to the solution B after the final purification step before lyophilization.

WO 94/07510 2124690 PC.'I'/SE93/00793 The recovery after freeze-drying was 76 % for A and 87 % for B. The same activity was found 4 hours after reconstitution stored at room temperature.
This study indicated that the addition of sucrose is favourable for the recovery of VIII:G over freeze-drying.

Examnle 9. Variation of calcium salt Recombinant factor Vffi was prepared according to the method described under Experimental.

Two ml of the solution was lyophilized and thereafter reconstituted in an amount of 5 ml of sterile water for injections.

L-Histidine, muM 23 23 23 23 .Sodium chloride, M 0,34 0,34 0.34 0,34 Caldum chloride, mM 4 4 0.15 0.15 Polysorbate, % 0.025 0.025 0.025 0.025 Sucrose, mM - 10 - 10 Caldumgluconate, mM 0 0 6 6 Recovery,%, Initial 63 74 74 78 4 hours* 60 73 73 77 *stored as reconstituted solution at ambient temperature _ This example shows that CaC12 can be substituted by Calcium gluconate.

WO 94/07510 PC1'/SE93/00793 Example 10 Recombinant factor Vffi was prepared according to the method described under Experimental.
2,2 ml of the solution was lyophilized and thereafter reconstituted in an amount of 5 ml of sterile water for injections. VIII:C per vial in reconstituted preparation was about 1000 IU.

l0A lOB
L-Histidine, mM 14.7 58 Sodium chloride. M 0.31 0.31 Calcium chloride, mM 3.7 3.7 Sucrose, mM 19.9 -Polysorbate 80, % 0.025 0.025 Recovery, IU/nml after reconstitution Initial 213 198 4h,25oC 213 198 24,25 oC 201 182 Recovery, %
Initial 92 91 5 months, 250C 88 -5 months, 300C 76 85 12 months, 70C 89 97 The recovery was good when part of the L-histidine was substituted by sucrose.

These formulations were studied by gelfiltration after 5 months'storage at 250C and 300C, respectively and the results are shown in figures 1 and 2. .
The only peaks to be seen is the peak at 42, indicating factor Vffi:C and the peak at 70 which is histidine. Aggregates is to be found earlier than 40. From figure 1 it can be seen that no detectable amount of aggregates was found after 5 months at 250C for 10A. Figure 2 shows a small amount of aggregates which is less than 2 % after 5 months at 300C for 10B.

SUBSTITUTE SHEET

Example 11 Recombinant factor V1II was prepared according to the method described under Experimental.
2,2 Ynl of the solution was lyophilized and thereafter reconstituted in an amount of 5 ml of sterile water for injections. VIII:C per vial in reconstituted preparation was about 500 IU.

L-Histidine, mM 14.7 58 Sodium chloride, M 031 0.31 Calcium chloride, mM 3.7 3.7 Sucrose, mM 19.9 -Polysorbate 80, % 0.025 0.025 Recovery, IU/ml after reconstitution Initial 98 105 4h,250C 96 103 24, 25oC 93 101 Recovery, %
Initial 91 93 stored at 250C, 5 mon 89 87 25 stored at 300C, 5 mon 76 79 stored at 70C 12 mon 88 89 Both formulations showed good stability.
These formulations were studied by gelfiltration and the results were similar as shown in Figures 1 and 2.
No aggregation was formed when the formulations had been stored for 5 months at 250C and 300C, respectively.

WO 94/07510 PC.'I'/SE93/00793 Example 12 Recombinant factor VIII was prepared according to the method described under Experimental.

5 2 ml of the solution was lyophilized, stored at different temperatures for up to 3 months (mon) and thereafter reconstituted in an amount of 4 ml of sterile water for injections. VIII:C per vial in reconstituted preparation was about 500 ILT.

Mannitol, mg/ml 20 20 L-Histidine, mg/ml 2,67 2.67 Sodium chloride, mg/ml 18 18 Caldum chloride, mM 3,7 3,7 Polysorbate 80, mg/ml 0.23 0.23 Reeovery, , %
initial 91 93 stored at. 70C 5 mon 90 85 An acceptable stability was achieved after five months at7oC.

Claims

CLAIMS:

1. A composition comprising:

recombinant coagulation factor VIII with a specific activity of more than 5,000 IU/mg protein, and at least 0.01 mg/ml of a non-ionic surfactant as a stabilizer of the recombinant coagulation factor VIII.

2. The composition according to claim 1, in which factor VIII is stable without the addition of albumin.

3. The composition according to claim 1 or 2, in which factor VIII is full-length or a deletion derivative of recombinant factor VIII with retained coagulant activity.

4. The composition according to claim 3, in which factor VIII is contained in a concentration of 10 to 100,000 IU/ml.

5. The composition according to any one of claims 1-4, in which the non-ionic surfactant is present in a concentration greater than a critical micelle concentration.

6. The composition according to any one of claims 1-5, in which the non-ionic surfactant is chosen from block co-polymers.

7. The composition according to any one of claims 1-6, which further comprises sodium or potassium chloride in an amount of more than 0.1 M.

8. The composition according to any one of claims 1-7, which further comprises a calcium salt in an amount of more than 0.5 mM.

9. The composition according to claim 8, wherein the calcium salt is calcium chloride or calcium gluconate.

10. The composition according to any one of claims 1-9, which further comprises an amino acid in an amount of more than 1 mM.

11. The composition according to claim 10, wherein the amino acid is L-histidine.

12. The composition according to any one of claims 1-9, which further comprises mono- or disaccarides.

13. The composition according to any one of claims 1-8, which further comprises L-histidine and sucrose.

14. The composition according to any one of claims 1-6, which further comprises sodium chloride and L-histidine at a ratio of more than 1:1.

15. The composition according to any one of claims 1-3, comprising:

i) 10-100,000 IU/ml of recombinant factor VIII, ii) at least 0.01 mg/ml of a polyoxyethylene (20) fatty acid ester, iii) sodium chloride in an amount of more than 0.1 M, iv) a calcium salt in an amount of more than 0.5 mM, and v) an amino acid in an amount of more than 1 mM.

16. The composition according to claim 15, wherein the calcium salt is calcium chloride or calcium gluconate; and the amino acid is L-histidine.

17. A composition obtained by drying the composition according to any one of claims 1-15, which is dried.

18. The composition according to claim 17, which is lyophilized.

19. The composition according to any one of claims 1-16, which is in a stable aqueous solution ready for use.

20. The composition according to claim 3, in which the deletion derivative factor VIII is deletion derivative factor VIII SQ with a specific activity of more than 12,000 IU/mg protein.

21. A process for the preparation of the composition according to claim 1, which comprises:

mixing factor VIII with a non-ionic surfactant in an aqueous solution, together with an amino acid, a sodium salt, sucrose and a calcium salt.

22. A process for the preparation of the composition according to claim 1, which comprises:

eluating factor VIII from a last purification step with a buffer containing a non-ionic surfactant in an aqueous solution, together with an amino acid, a sodium salt, sucrose and a calcium salt.

23. Use of a non-ionic surfactant chosen from block co-polymers, poloxamer, polyoxyethylene (20) fatty acid ester, polysorbate 20 or polysorbate 80, as stabilizer for a composition comprising coagulation factor VIII with a specific activity of more than 5,000 IU/mg protein.

24. The composition according to claim 6, in which the block co-polymers are a poloxamer or polyoxyethylene (20) fatty acid ester.

25. The composition according to claim 6, in which the block co-polymers are polysorbate 20 or polysorbate 80.

26. The composition according to claim 12, in which the saccaride is sucrose or a sugar alcohol.

27. A pharmaceutical composition for treating haemophilia, that is free from human serum albumin and is:
(a) an aqueous solution containing:

(i) 10-100,000 IU/ml of recombinant coagulation factor VIII having a specific activity of more than 5,000 IU/mg protein, and (ii) a non-ionic surfactant in an amount above a critical micelle concentration (CMC) thereof and within the range of from 0.01 to 1 mg/ml, where the non-ionic surfactant stabilizes the recombinant coagulation factor VIII, or (b) a dry product that is obtained by drying the aqueous solution (a) or forms the aqueous solution (a) when reconstituted by adding sterile water or a buffer solution.

28. The composition according to claim 27, which further contains sodium chloride in an amount of more than 0.1 M.

29. The composition according to claim 27 or 28, which further contains a calcium salt selected from the group consisting of calcium chloride, calcium gluconate, calcium glubionate and calcium gluceptate, for maintaining an association of factor VIII heavy and light chains.

30. The composition according to any one of claims 27-29, which further comprises an amino acid selected from the group consisting of L-histidine, lysine and arginine to buffer the composition.

31. The composition according to any one of claims 27-30, wherein the recombinant coagulation factor VIII
is a truncated recombinant coagulation factor VIII having a molecular mass of 170 kDa and consisting of two chains of 90 kDa and 80 kDa held together by a metal bridge.

32. The composition according to any one of claims 27-31, wherein the non-ionic surfactant is polysorbate 20 or polysorbate 80.