Note: Descriptions are shown in the official language in which they were submitted.
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A Process for the Preparation of a Virus Inactivated Fraction
Containing Factor VIII by means of Chromatographic Methods
This inventions is related to a process for the
preparation of a virus inactivated fraction containing factor
VIII by means of chromatographic methods.
Factor VIII is a vital material that plays an important
role in blood clotting. Blood clotting disorders can thus be
treated by administration of factor VIII. Therefore, a large
need exists for administrable factor VIII preparations.
Numerous attempts have been made to isolate factor VIII in a
highly enriched form from natural sources. Thus,
chromatographic methods for the purification of factor VIII
from cryoprecipitates are already known. The latter is a
fraction which is available by treatment of plasma in the
cold. EP 367 840 B1 pertains to a chromatographic method for
the isolation of factor VIII from blood plasma without a
preliminary cryoprecipitation. The fraction containing factor
VIII is separated by chromatographic separation on a
hydrophilic chromatographic material modified by ion-
exchanging groups . EP 0 238 701 pertains to a method for the
preparation of an ultra-pure, infectious antihemophilic
factor, where the pretreated fractions are a cryoprecipitate
liberated from fibrinogen, globulin, albumins, and other
interfering ingredients by means of ethanol precipitation.
EP-A-0 343 275 describes chromatographic separation with ion
exchangers after virus inactivation of the cryoprecipitate
fraction. EP 0 173 242 A describes a method for obtaining
factor VIII preparations by chromatography on anion-
exchanging materials which are solely based on carbohydrates,
the carbohydrate matrix being modified by DEAF groups. In
particular, DEAF sepharose and DEAF cellulose are described as
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being useful. In GB-A-1,178,958, a purification of factor VIII
using ECTEOLA cellulose columns is described. The modified
cellulose contains basic substituents introduced by reaction
of epichlorohydrin and triethanolamine. The above mentioned
prior art makes use either of chromatographic separation in
the form of batch processes or of column chromatography.
Although fairly good results are obtained with these methods,
it still remains desirable for economic as well as ethical
reasons to increase the yield of biologically valuable factor
VIII.
Hence, the technical problem underlying this invention
consists in providing a method which, proceeding from the
prior art, allows for an improved preparation of factor VIII
with respect to yield and biological activity.
Surprisingly, the problem has been solved by a process
proceeding from a cryoprecipitate after dissolution thereof or
blood plasma, optionally followed by an aluminum hydroxide
treatment, and employing a separation step using membrane
chromatography on membranes or compact disks of the porous
hydrophilic polymers poly(glycidyl methacrylates) or
hydrophilized polystyrene.
The process of the invention can be performed with
commercial cryoprecipitate or blood plasma. Preferably, the
thawed cryoprecipitate is treated with aluminium hydroxide for
further pre-purification of the sample in order to
preconcentrate factor VIII.
Preferably, a virus inactivation is carried out prior to
the actual chromatographic purification on materials arranged
within or on membranes. This virus inactivation is performed
according to the method described in EP 131 740 A1 by
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treatment with biocompatible organic solvents (detergents),
Triton~ X-100/TNBP, preferably Tween~/TNBP (tri-n-butyl
phosphate). Good results will also be obtained with sodium
cholate/TNPB. Preferably, detergents are employed in amounts
of up to 15o by weight. Wherein a virus inactivation is
performed by treatment of said fraction with a di- or trialkyl
phosphate and a non-ionic surfactant.
The chromatographic separation step for the purification
of factor VIII in the sample may be carried out either on
base materials modified by ion-exchanging groups,
particularly anion exchangers, or on materials modified by
immunoaffinity ligands. It is critical that the mentioned
materials are arranged in membranes. Particularly useful are
membranes as well as compact disks made of porous
poly(glycidyl methacrylates) and/or hydrophilized
polystyrene.
A membrane suitable for separation consists of compact
disks made of polymer carriers. The base materials of the
membranes or disks are provided with corresponding anion-
exchanging groups or immunoaffinity ligands. Particularly
anion-exchanging groups such as quaternary amines or
diethylaminoethyl groups are considered as ion-exchanging
groups. Suitable ca n on exchangers are in principle weakly
and strongly acidic ration exchangers, such as materials
which are modified by sulfonic acid or phosphoric acid
groups.
The ion-exchanging groups can be bound to the base
material fibers with or without a so-called spacer.
Materials provided with spacers are also referred to as
tentacle materials. In DE 42 04 694 corresponding spacers
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and ligands are mentioned. For example, a glucosamine
residue can serve as a spacer as well. Anion-exchanging
groups such as DEAF or quaternary amines may also be bound
at the membranes made of porous poly(glycidyl methacrylate)
or the other materials mentioned. The binding of the anion-
exchanging groups takes place either directly to the
material forming the membrane or else through a spacer as
well, e.g. a glucosamine residue.
In another embodiment of the process of the invention
affinity membrane chromatography with immobilized substances
having a high affinity for factor VIII is used.
The substances having affinity for factor VIII are
immobilized at the carrier by means of chemically reactive
groups. Preferably, the reactive group will attack at the
end of a spacer rather than directly at the carrier
material. Immobilization of the substances for factor VIII
takes place through binding at reactive groups such as
tosyl, tresyl, hydrazid and others. Corresponding procedures
are known from T. M. Phillips "Affinity chromatography" in
"Chromatography" (E. Heftmann, ed.), 5th ed. Elsevier,
Amsterdam 1992.
In another preferred embodiment materials are employed
for the separation of factor VIII that can ensure a
hydrophobic interaction. Hydrophobic materials which are
employed are acyclic and/or cyclic alkyl chains, for
instance C1 to C18 alkyl chains, and aromatic substances.
Suitable materials providing hydrophobic interaction
preferably include those having graded hydrophobicity.
Hydrophobicity can be graded by introduction of polar groups,
such as protic-polar or aprotic-polar groups, for example
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hydroxy, amino, cyano groups. Preferably, it is adapted
corresponding to the respective separation conditions.
Virus inactivation may also take place by heat
treatment. Preferably, the eluated sample containing factor
VIII is subjected to a pasteurization step following a first
membrane chromatographic step. A corresponding procedure is
proposed in DE-A-4 318 435. Therein, fraction enriched with
factor VIII are contacted with di- or trialkyl phosphates and
optionally wetting agents in the presence of stabilizing
agents and simultaneously or subsequently treated for a
period of 5 hours to 30 hours at elevated temperatures
ranging from 55°C to 67°C. It may be of advantage to combine
the two methods of virus inactivation, the treatment with
detergents and with heat.
In order to remove the chemicals employed in the
pasteurization step, a second membrane chromatography may
follow. Preferably, the separation of the added stabilizing
agents takes place by means of a membrane modified by DEAF or
quaternary ammonium compounds that are arranged on the
surface of the chromatographic carrier material through a
spacer. In addition, it is possible to arrange the
corresponding ligands on the surface of the carrier material
without a spacer. The stabilizing agents are not retarded by
this anion exchange material under the conditions chosen,
whereas factor VIII is adsorbed on the chromatographic
material.
Thereafter, factor VIII is eluated with an aqueous
solvent system exhibiting gradually increasing salt
concentrations.
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The fraction containing factor VIII thus obtained is
concentrated, filled and optionally lyophilized by
conventional methods. Preferably, factor VIII is applied from
a solution with low ionic strength in the first membrane
chromatographic separation step.
Preferably, the aqueous system has an ionic strength
corresponding to a 0 to 150 mM sodium chloride solution. At
such ionic strengths, factor VIII is still adsorbed on the
chromatographic material, whereas more weakly binding
impurities can be washed out with aqueous systems of the
same ionic strength.
In another embodiment of the process according to the
invention, purification of the adsorbed material may be
performed with an aqueous system having an ionic strength
corresponding to a 200 to 400 mM sodium chloride solution.
Desorption of factor VIII and elution of this fraction then
are carried out with an aqueous system having an ionic
strength corresponding to a 500 to 1500 mm sodium chloride
solution, while the pH value is maintained within a range of
4 to 9. If cation-exchange chromatography is performed, it
preferably takes place at a pH value < 6, whereas anion-
exchange chromatography is rather carried out at higher pH
values of above 6.
If purification of factor VIII is performed by
immunoaffinity membrane chromatography, then, unlike with
the above mentioned method using anion-exchange materials,
elution is carried out with chaotropic reagents or highly
concentrated salt solutions. Preferably, elution takes place
with concentrations of chaotropic reagents or salts which
are sufficient to disrupt the binding between the substance
having high affinity to factor VIII and factor VIII itself.
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The concentrations of the mentioned materials in the
respective elution systems depends on the strength of the
affinity between factor VIII and the corresponding binding
component. As a result, elution can take place with aqueous
solutions with lower denaturating potency. Preferably,
aqueous solutions having concentrations of from 1 to 6 M of
urea, especially from 2 to 4 M of urea, or correspondingly
highly concentrated salt solutions are employed for elution
of factor VIII from the immunoaffinity membrane.
In hydrophobic interaction chromatography, the sample is
applied in an aqueous solution with very high ionic
strength, such as, for example, highly concentrated ammonium
sulfate (up to a concentration of 4 M) or sodium chloride
(up to a concentration of up to 5 M). In particular, elution
is carried out gradually or continuously with salt solutions
of lower ionic strengths. An aqueous solution with organic
solvents, particularly a diluted alcoholic solution, may
also be used as solutions with lower ionic strengths for the
elution of the samples in hydrophobic interaction membrane
chromatography.
The procedure according to the invention surprisingly
ensures a quick and uncomplicated purification of factor
VIII which is obtained at the same time in high purity and
high yields. In addition, the specific activity of the
factor VIII obtained in this way is quite high which may be
accounted for by the low denaturation of the active factor
in the process according to the invention.
