WO1989009784A1

WO1989009784A1 - Production of heat-stable factor viii concentrate

Info

Publication number: WO1989009784A1
Application number: PCT/AU1989/000154
Authority: WO
Inventors: Adrian Malcolm Oates
Original assignee: Commonwealth Serum Laboratories Commission
Priority date: 1988-04-08
Filing date: 1989-04-07
Publication date: 1989-10-19
Also published as: AU3258389A

Abstract

A method for preparation of a heat-stable Factor VIII concentrate is characterised by subjecting a controlled pore glass (CPG) eluate to a diafiltration step.

Description

PRODUCTION OF HEAT-STABLE FACTOR VIII CONCENTRATE

This invention relates to the production of heat-stable Factor VIII concentrate, and in particular it relates to the production of a Factor VIII concentrate which is able to withstand "superheating", such as heating up to and including 80°C for 72 hours.

The production of both "intermediate-purity" and "high-purity" Factor VIII concentrates for clinical use in haemophilia is a well known and described by Newman, J. et.al., British Journal of Haemotoloαv, 21, 1-20 (1971); Smith, J.K. and Bidwell, E., Clinics in Haemotoloσy. £, 183-206 (1979); Margolis, J. and Rhoades, P., Vox Sang. 36. 369-374 (1979). Essentially the technique has been based on the separation of a cryoprecipitate containing Factor VIII from other plasma proteins by freezing and subsequent controlled thawing of plasma. Purification of the resultant cryoprecipitate to remove contaminants has included adsorption with silica-based chromatographic media or aluminium hydroxide. Margolis and Rhoades (supra) proposed the preparation of high purity Factor VIII from crude cryoprecipitate by controlled pore glass (CPG) chromatography. Factor VIII activitys was found to be associated with a high molecular weight protein recovered in the void volume of a column of CPG with contaminating proteins, especially fibrinogen, being adsorbed onto the large internal silica surface.

In a practical application of this CPG chromatographic technique, a crude concentrate of Factor VIII, i.e. absorbed washed cryoprecipitate as described by Margolis and Rhoades (supra), is treated with controlled pore glass (CPG) of 500A nominal pore size, equilibrated with Tris -NaCl - citrate amino acid buffer. At 120-200 mesh size the internal surface area of the CPG granules is many hundreds of times larger than the external surface. Furthermore, being composed of almost pure Si0₂, it acts as a powerful adsorbent of plasma proteins, especially of fibrinogen. The internal surface is not accessible to the large molecules carrying Factor VIII activity which can be recovered in the void volume. CPG can thus be used in a combination of absorption and permeation chromatography. It is also chemically inert, reusable and easy to clean and sterilize. Adsorbed protein is normally removed with warm 1-2% Sodium Dodecyl Sulphate (SDS) and at intervals the CPG can be fired at

700°C to remove all traces of organic matter. The system is, therefore, ideally suited for large-scale production of Factor VIII. The degree of purification depends on the relative amounts of CPG and crude starting material. For example, a product containing 1.0 units Factor VIII per mg protein can be obtained by passing 500ml of crude concentrate (approximately 15,000 Factor VIII units) through 2,000ml of CPG. An alternative, more rapid procedure is to stir approximately 1 litre of crude concentrate (400-500 donor units = 25,000-35,000 units of Factor VIII) with 2 litres of equilibrated CPG, pour the slurry into a 10 x 50cm column and collect the effluent. The Factor VIII remaining in the void volume is then recovered by passing 1-1.2 litres of buffer through the column, after which the column is washed with SDS and water. The resulting 2.0-2.4 1 of concentrate is then frozen for final processing. Normally two such harvests are thawed, acidified with HC1 to pH 6.7 and centrifuged at 8°C to remove insoluble precipitate. The supernatant is then passed through an 0.22μ membrane filter and dispensed in 20-35 mis aliquots for freeze-drying.

As noted by Smith and Bidwell (supra), concentrates containing Factor VIII prepared by these prior methods have only been satisfactorily sterilised by filtration through membranes which can remove all bacteria and their spores. However, viral contaminants are not removed by this procedure. Thus, the need to manufacture Factor VIII concentrates for intravenous administration to people with haemophilia A, capable of being heated to high temperatures to inactivate contaminating blood-borne viruses, is of paramount importance. In view of this, investigations have been carried out to determine the ability of Factor VIII concentrates to withstand superheating, that is heating at up to and including 80°C for 72 hours. As a result of these investigations, it has been found that superheating known Factor VIII concentrates, including those purified by CPG chromatography, results in gross insolubility and complete loss of Factor VIII activity.

According to the present invention, there is provided a method of preparing a heat stable Factor Vlll concentrate, which is characterised by subjecting a controlled pore glass (CPG) eluate to a diafiltration step. This invention also extends to a heat-stable Factor VIII concentrate prepared by this method. Preferably the CPG eluate is prepared by the known steps of cryoprecipitation followed by CPG chromatography as described above. Preferably also, the diafiltration step is performed on a hollow membrane filter with a 100,000 molecular weight exclusion limit, such as an Amicon Hollow Fibre Diafiltration Unit (Model No. HIP 100-20), using an equilibration buffer containing sucrose, for example a Tris-NaCl-citrate buffer containing 1.5% sucrose. Alternatively, the buffer exchange can be performed on a gel-filtration media such as Sephadex G25, followed by concentration on a Hollow-Fibre Diafiltration Unit.

It has been found that by performing a simple diafiltration step on the controlled pore glass eluate against a Tris buffer, pH6.9 containing NaCl, Tris-sodium citrate, calcium chloride and sucrose as a stabilising agent, a material that is incompatible with superheating is removed. Solubilisation of the starting cryoprecipitate in the above buffer instead of amino acid/sodium citrate buffer and proceeding through the CPG chromatography process still results in gross insolubility and a >80% loss of Factor VIII activity. It thus appears that diafiltration removes the substance/s causing this instability. Preliminary data on pilot scale indicates Factor VIII yields >150 U/kg plasma after superheating. Solubility is in <4 minutes and the product appears slightly turbid with a few fibres and floccular particles.

The process of the present invention therefore enables the production of a superheat-stable Factor Vlll concentrate from controlled pore glass eluate, a product which may serve to lower the incidence of infection with Hepatitis nonAnonB and other blood-borne viruses in patients. Further details of the process of this invention will be apparent from the following Examples which describe the presently preferred method of preparing a heat-stable Factor VIII concentrate.

EXAMPLE 1

Part A: Diafiltration of controlled pore glass eluate,

Starting material:

Controlled pore glass eluate in citrate/saline/amino acid buffer. Diafiltration Unit:

Amicon Hollow Fibre Diafiltration Unit. Model No.

HIP 100-20. Product No. 1831. Equilibration buffer: lOmM Tris, pH 6.9 lO M Tri-sodium citrate lOOmM sodium chloride

1.5% sucrose

1.2mM calcium chloride Procedure:

1. Diafilter the CPG eluate on a hollow-fibre diafiltration unit against 5 sample volumes of the equilibration buffer, i.e. until 5 sample volumes have passed as permeate.

2. Prefilter the diafiltered material through a 3, 0.45, 0.22μ filter sandwich.

3. Sterile filter the diafiltered material through a sterile 3, 0.45, 0.22μ filter sandwich.

4. Dispense as 35ml fills into 65ml bottles. Part B: Freezing/Freeze drying of the product.

1. Load product directly onto the freeze drying shelf preferably at 20°C but within the range of 15-25°C.

2. Close the door of the freeze dryer.

3. Lower the freeze dryer shelf temperature to -5°C±1 over 60 minutes preferably at a rate of 0.42°C per minute.

4. Hold the freeze dryer shelf temperature at -5°C±1 for at least 120 minutes.

5. Lower the freeze dryer shelf temperature to -50°C over 90 minutes at a rate of 0.5°C per minute.

6. Hold the freeze dryer shelf temperature at -50°C for 1 to 12 hours.

7. Apply vacuum. When the chamber pressure reaches 0.1 Torr continue as below.

8. Increase the freeze dryer shelf temperature to -22°C over 4 hours at a rate of 7°C per hour.

9. Increase the freeze dryer shelf temperature to 20°C over 4 hours at a rate of 1.11°C per hour.

10. Hold the freeze dryer shelf temperature at 20°C for 2.5 hours.

11. Increase the freeze dryer shelf temperature to 30°C over 30 minutes at a rate of 0.33°C per minute. 12. Hold the freeze dryer shelf temperature at 30°C for 18 hours.

13. Increase the freeze dryer shelf temperature to 48°C over 60 minutes at a rate of 0.3°C per minute.

14. Hold the freeze dryer shelf temperature at 48°C for 2 hours.

15. Stopper the product under vacuum.

16. Heat the product at up to and including 80°C for 72 hours.

EXAMPLE 2

Part A: Gel Filtration of Controlled Pore Glass Eluate.

Starting Material:

Controlled Pore Glass Eluate in Citrate/Saline/Amino Acid buffer.

Gel Filtration Media: Sephadex G25 (fine) Column Dimensions:

Bed Height - 20 cm

Bed Diameter - 5 cm Linear Flow Rate - 20 mls/cm²/hr Equilibration Buffer:

10 mM Tris, pH 6.9 10 mM Tri-sodium Citrate 100 mM Sodium Chloride ι.5% Sucrose

1.2 mM Calcium Chloride Procedure :

1. Pass the Controlled Pore Glass Eluate through the gel-filtration column at the defined flow rate. The sample volume should lie within 0.15-0.25 of the gel bed volume.

2. Monitor elution of the protein peak by UV absorbance and conductivity.

3. Start collecting the protein peak as soon as the UV absorbance begins to rise. Finish collecting the protein peak when twice the sample volume has been collected or the conductivity meter begins to deviate from baseline.

4. Concentrate the buffer exchanged protein peak to the desired potency on a Hollow-fibre Diafiltration unit as used in Example 1.

5. Continue with finishing of the product as per Example 1, Step 2.

Part B is performed as per Example 1.

Claims

CLAIMS :

1. A method for preparation of a heat-stable Factor VIII concentrate, which is characterised by subjecting a controlled pore glass (CPG) eluate to a diafiltration step.

2. A method according to claim 1, wherein said CPG eluate is prepared by cryoprecipitation of plasma to separate a cryoprecipitate containing Factor VIII from other plasma proteins, followed by controlled pore glass chromatography of the cryoprecipitate.

3. A method according to claim 1 or claim 2, wherein said diafiltration step is performed on a hollow membrane filter with a 100,000 molecular weight exclusion limit against an equilibration buffer containing sucrose.

4. A method according to claim 1 or claim 2, wherein said CPE eluate is subjected to buffer exchange using an equilibration buffer containing sucrose, followed by concentration on a hollow membrane filter with a 100,000 molecular weight exclusion limit.

5. A method according to claim 3 or claim 4, wherein said equilibration buffer is a Tris-NaCl-citrate buffer containing 1.5% sucrose.

6. A method according to any of claims 1 to 5, wherein said concentrate is sterilized by filtration subsequent to said diafilitration step.

7. A method according to claim 6, wherein said concentrate is freeze-dried subsequent to said sterilization step.

8. A method according to claim 7, wherein said freeze-dried concentrate is heated at up to and including 80°C for 72 hours to inactivate blood-borne viruses.

9. A heat-stable Factor VIII concentrate prepared by the method of any of claims 1 to 9.

10. A heat-stable Factor VIII concentrate which is able to withstand heating at up to and including 80°C for 72 hours.