FR2650393A1 - Method for the purification of factor VIII (F VIII) by ion exchange chromatography - Google Patents

Abstract

The invention makes it possible to obtain a factor VIII concentrate having a specific activity and a high yield, which concentrate is free of foreign protein of non-human origin. Factor VIII obtained by any process is deposited on a column containing an anion exchange gel preequilibrated with a first buffer. After having charged the factor VIII onto the column, the gel is washed with a second buffer until an optical density less than 0.1 is obtained. The purified factor VIII is then released from the gel with a 3rd buffer. After chromatography, the specific activity is 30/1600 IU/mg.

Description

 Human factor VIII concentrate, obtained from human plasma or by genetic engineering, is used to supplement its deficiency in hemophiliac A.

 Current techniques allow the purification of F VIII with specific activities (international units of F VIII per gram of protein) ranging from 1 to 3000 and very variable yields (80-350 international units / liter of plasma) according to the method used . The higher the purification factor, the higher the yield in units of F VIII, per liter of plasma drops. At the present time, only one technique, based on immunopurification, makes it possible to obtain antihaemophilic concentrate A with a very high purification factor. The disadvantage of this process is the release of murine monoclonal antibodies into the final product, which in some cases would result in anaphylactic shock.

The object of the invention is therefore to obtain a factor concentrate
VIII having a high specific activity and yield, concentrated free of foreign proteins and of non-human origin.

1. RAW MATERIAL
. - F VIII extract prepared from cryoprecipitate after absorption of aluminum overhydroxide (Al (OH) 3)
- F VIII extract prepared from cryoprecipitate after absorption on aluminum hydroxide (Al (OH) 3) and precipitation of fibrinogen at pH 6.5 and 10 C.

 F VIII extract prepared from cryoprecipitate after precipitation with 2 M glycine and 1.5 M sodium chloride (NaCl)

 - factor VIII obtained by any other process.

 In all four cases, F VIII is in buffer 1 and can undergo a viral inactivation treatment by the detergent solvent method, before chromatography.

2. CHROMOTOGRAPHY
F VIII is deposited on a column containing a gel, anionic exchanger, type Q Sepharose Fast Flow pre-equilibrated with buffer 1.

 The contact time F III, gel is 100 minutes - or - 20.

After loading antihemophilic F III onto the column, the gel is washed with buffer 2 until the optical density is less than 0.1. F. VIII, freed from impurities, is then unhooked from the gel by the buffer 3. The washing rates had elution are greater, 2 to 4 times the absorption rate.

<tb> Cryo <SEP> extract <SEP> in <SEP> Cryo <SEP> extract <SEP> in <SEP> Cryo <SEP> extract <SEP> in
<tb><SEP> water <SEP> ppi <SEP> (41 / Kg) <SEP> the <SEP> buffer <SEP> 1 <SEP> (41, / Kg)
<tb> water <SEP> ppi <SEP> (41 / kg) <SEP> I <SEP> I
<tb><SEP> I <SEP>
<tb> Absorption <SEP> Al (OH) 3 <SEP> Precipitation <SEP> Absorption
<tb><SEP> to <SEP> to <SEP><SEP> glycine <SEP> 2M <SEP> on <SEP> Al (OH) 3
<tb><SEP> v
<tb> Precipitation <SEP> Precipitation
<tb> to <SEP> pH <SEP> 6, <SEP> and <SEP> to <SEP> 10 <SEP> C <SEP> to <SEP> NaCl <SEP>1.5'A
<tb><SEP> F <SEP> F <SEP> VIII <SEP> in <SEP> Buffer <SEP> 1
<tb><SEP> Inactivation <SEP> viral
<tb><SEP> Chronotograpliie
<tb><SEP> Chromotography <SEP> on <SEP> Q <SEP> Sepharose <SEP> Fast <SEP> Flow
<tb><SEP> Pre-Balanced <SEP> in <SEP> Buffer <SEP> 1
<tb><SEP> Wash <SEP> of <SEP> freeze <SEP> with <SEP> buffer <SEP> 2 <SEP> (about <SEP> S <SEP> vol. <SEP> of <SEP> freeze)
<tb><SEP> Elution <SEP> with <SEP> tarpon <SEP> r <SEP> (approximately <SEP> 1.5 <SEP> vol. <SEP> of <SEP> gel)
<tb><SEP> t
<tb><SEP> Ultrafiltration
<tb><SEP> Concentration
<Tb>

* Cryo = cryoprecipitate
** Water ppi = water for injections. 3. BUFFER
Tapon i = Trisodium citrate 0.02M + or - 0.005 pH 7.0 + or - 0.4 Glycine 0.02M + or - 0.01
NaCl 0.96M + or - 0.01
CaCl2 0.0025M + or - 0.0005 Tapon 2 = Tris 0.02M e or - 0.005 pH 7.1 + or - 0.2 Glycine 0.02M - or - 0.01
NaCl 0.06M + or - 0.01
CaCl2 0.05M + or - 0.002 Tapon 3 = Tris 0.02M + or - 0.005 pH 7.1 + or - 0.2 Glycine 0.02M + or - 0.01
NaCl 0.06M + or - 0.01
CaCl2 0.02M + or - 0.05 4.FLOOR FLOW AND DEBITS

<tb> F <SEP> VII <SEP> cryo <SEP> F <SEP> VII <SEP> after <SEP> F <SEP> VII <SEP> cryo <SEP>
<tb> and <SEP> precipitation <SEP> precipitation
<tb> to <SEP> pH <SEP> 6.5 <SEQ> glycine, <SEP> NaCl
<tb> Volume <SEP> of
<tb> F <SEP> VIII
<tb> 4 <SEP> - <SEP> 6 <SEP> 8 <SEP> - <SEP> 12 <SEP> 1.5 <SEP> - <SEP> 2
<tb> Volume <SEP> of <SEP> gel
<Tb>
Contact time of F VIII with gel = 100 minutes + or - 20.

Washing and elution rates = 2 - 4 X absorption rate. 5. RESULTS OF PURIFICATION
Specific activity avantchro, matography =
of the order of 1 IU / mg of protein
Specific activity after chromatography =
30 - 1600 tMg of protein,
according to the quality of the raw material.

 The protein assay is performed according to the Bradford method.

6. INDUSTRIAL REALIZATION
Gel: Q Sepharose Fast Flow (201), Pharmacia.

Column: KS37QA 37x1j with inputs and outputs either in 1/2 "or 1/4",
Pharmacia.

Pump: Serra R411, 4.20 mA with 1/2 "inlet and 1/2" or 1/4 "outlet,
Pharmacia.

Factor VIII deposited: 40 liters in buffer 1 pH 7.0 + or - 0.4 with a protein concentration up to 20 g -1. Flow rate of F VIII: 27 1, / h.

(A total contact time of F VIII on the gel, 90 minutes is essential).

Washing of F VIII on the gel: with buffer 2 and a flow rate of 50 l / h.

Elution of F VIII: with buffer 3 and a flow rate of 50 h.

 After each use the gel is washed with 2M NaCl, 0.2M NaOH, and distilled water, then stored either in buffer 1 (for short storage) or in buffer 1 at 20 r ethanol for long storage times.

 The invention makes it possible to obtain, on an industrial scale, an antihemophilic concentrate A (factor VIII) indicated for the treatment of all haemorrhages of hemophilia A in hemophilia A without or with an inhibitor F VIII, and this with a specific activity that can be very high and a yield of the order of 200 - 30Q units per liter of plasma.

Claims (1)

The invention makes it possible to obtain a concentrate of factor VIII having a high specific activity and a high yield, concentrated free of foreign proteins and of non-human origin. The method uses the following raw materials - F VIII extract prepared from cryoprecipitate after absorption on aluminum hydroxide (A1 (OH) 3). - F VIII extract prepared from cryoprecipitate after absorption on aluminum hydroxide (Al (OH) 3) and precipitation of fibrinogen at pH 6.5 and 100 C. - extract of F VIII prepared from cryoprecipitate after precipitation with 2 M glycine and 1.5 M sodium chloride (NaCl) - factor VIII obtained by any other method. The F VIII is deposited on a column containing a gel, anionic exchanger, type Q Sepharose Fast Flow pre-equilibrated with buffer 1. After loading antihemophilic F VIII on the column, the gel is washed with buffer 2 and this up to the optical density is less than 0.1. F VIII, free of impurities; is then unhooked from the gel by the buffer 3. The washing and elution rates are higher, 2 to 4 times the absorption rate. Result of the purification - specific activity before chromotography = of the order of 1 IU / mg of protein. - specific activity after chromotography =  30 - 1600 IU / mg of protein,  according to the quality of the raw material.  The protein assay is performed according to the Bradford method.