Note: Descriptions are shown in the official language in which they were submitted.
CA 02293401 1999-12-10
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FILE, Pttd-tl~ TH iR
T~-~wTRAN~L~ATI~~~
A Method for Depleting Viral and Molecular
Pathogens in a Biological Material
The invention relates to a method for depleting
viral and molecular pathogens in a biological material
containing one or several biological substances to be
recovered.
The production of therapeutic proteins and
preparations, in particular of immunoglobulin, by
extraction from human or animal tissues or liquids,
such as blood or plasma, as well as from continuously
growing transformed mammalian cells frequently carries
the risk of a potential contamination by pathogens,
such as viruses, virus-like particles or prions.
Therefore, measures must be taken to prevent any
pathogens possibly present from being transmitted to
human beings.
Human blood or plasma, respectively, may e.g.
contain viruses causing diseases such as AIDS,
hepatitis B or other hepatitis diseases. With plasma
proteins derived from plasma pools the risk of
transmitting infectious agents, such as viruses, is
very low because of the selection of blood or plasma
donations and the production method. Suitable measures,
such as excluding high-risk blood donors from donating
blood as well as analyzing blood or plasma donations
which make it possible to identify infectious donations
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and exclude them from further distribution, do allow an
elimination of most of the infectious donations, yet in
most instances not all can be found. Existing assaying
systems for detecting infectious viruses in biological
materials cannot always completely eliminate concerns
regarding a potential transmission of pathogens, since
on account of the broad spectrum of infecious pathogens
existing it is impossible to assay the starting
material for all viruses or molecular pathogens that
may be present in a sample. Moreover, most of the tests
do not identify the virus itself, but rather identify
antibodies developed against that virus so that during
the so-called "diagnostic window" a detection of a
contamination is not possible. For some groups of
viruses, moreover, a reliable or sufficiently sensitive
detection method does not exist. Although newly
developed assaying methods, in particular nucleic acid
amplification methods, such as, e.g., PCR, are highly
sensitive and specific, they can only be applied for
pathogens whose nucleic acid sequence is known. In
those cases in which the human pathogens are known yet
a sensitive method of detecting them does not exist,
there remains the doubt that a negative result is
obtained merely on account of a too low virus content
which is below the sensitivity limit of the assaying
system.
Therefore, specific removal and/or inactivation
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methods for depleting viruses have been developed for
the production of pharmaceutical and therapeutic
products so that infectious particules are no longer to
be expected in the final product.
Various inactivation methods are based on a
physical-chemical treatment by means of heat and/or
chemicals. The methods particularly used are the
thermal treatment, pasteurizing, treatment of the
protein solution with ~i-propiolactone and UV light,
treatment with a combination of a solvent and a
detergent (so-called S/D method) or exposing the
protein solution to light after the addition of a
photodynamic substance. With these methods, a virus
inactivation of up to 106 log steps has been reached.
The efficiency of the inactivation method may, however,
vary depending on the type of virus present. Although
S/D-treated blood products are considered safe in terms
of a transmission of HCV, HBV or HIV, non-enveloped
viruses, such as HAV or parvovirus, are not inactivated
by these methods (Prowse C., Vox Sang. 67 (1994), 191-
196) .
With biological products, heat treatment methods
preferably are carried out either in solution (EP-
0 124 506), in the dry state (EP-0 212 040 or
WO 82/03871) or in the moistened state (EP-0 324 729).
This may often result in losses because of the
thermolability of many biological substances.
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The type of the inactivation method used may also
have an influence on the products, and a stabilisation
to minimize the loss of protein thus frequently is
required. Moreover, some inactivation methods must be
followed by purification steps so as to remove
chemicals added.
Methods for virus depletion particularly include
chromatographic methods, filtration of protein
solutions via a membrane filter, or adsorption of
viruses on a solid phase and subsequent removal of the
solid phase, as described in EP-0 679 405. However, it
has been found that although the treatment with a solid
phase, such as, e.g., with Aerosil~, does allow for a
removal of HIV up to 4 log steps from an
immunoglobulin-containing solution, the loss of IgG may
be up to 42% (Gao et al., Vox Sang. 64 (1993), 204-
209). With such high losses, such a method appears
rather unsuitable for application on a large technical
scale.
A widely used chromatographic method for isolating
biological substances is anion exchange chromatography.
The possibility of depleting viruses by this separation
method has also been described in the literature. For
instance, virus depletion at an anion exchange
chromatography for purifying vWF under conditions under
which vWF, yet not the virus, binds to the anion
exchanger has been examined (Burnouf-Radosevich, Vox
CA 02293401 1999-12-10
San. 62 (1992), 1-11). By thoroughly washing the column
prior to elution, depending on the respective virus, it
was possible to recover vWF with a virus depletion of
from 1.5 to 5 powers of ten.
Zolton et al. (Vox Sang. 49 (1985), 381-389)
examined the virus depletion rate in case of anion
exchange chromatography for purifying gamma-globulins
under conditions under which gamma-globulins do not
bind to an anion exchanger. In these methods, DEAE
Sepharose was used as anion exchanger at a pH of 7.5.
The infectivity of a starting solution to which
hepatitis B virus had been admixed could be eliminated
by means of this anion exchange chromatography. By this
experiment, a depletion of the hepatitis B virus by the
factor 3000 was effected. However, nothing could be
said about the depletion rate of other viruses at pH
7.5. What was interesting is that at a pH of below 7.2,
viruses appeared in the effluent of the anion
exchanger, so that this method generally has been
considered not to be applicable in the neutral or
weakly acidic pH range.
EP 506 651 describes a multi-step method of
recovering a preparation containing IgA, IgG and
Transferrin, a reduction of the virus titer having been
obtained in each individual method step. During the
extraction and precipitation step with 12% ethanol, a
virus reduction by a factor of 105 could be attained.
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During the adsorption step, the proteins were bound to
an anion exchanger, washed, and eluted again. With this
step, virus reduction was at a factor of 103.
Burnouf (Dev. Biol. Stand. 81 (1993), 199-209)
reported that during a purification of factor VIII,
parainfluenza virus and HIV-1 could be depleted by 4
and 3 powers of ten, respectively, by means of an anion
exchange step. When purifying vWF via anion exchange
chromatography, a depletion rate of PRV (porcine
pseudorabies virus) of 5 log steps has been reported.
Mitra et al. (Curr. Stud. Hematol. Blood Transfus.
56 (1989), 34-43) show that when purifying IgG from
plasma according to the plasma fractionating scheme of
Cohn-Oncley by a sequence of precipitating steps in the
presence of certain concentrations of ethanol and
defined pH values in the cold (-5°C), a virus depletion
of >5 and >8 log steps, respectively, of murine C-virus
and HIV, respectively, could be obtained. In this paper
it is also reported that a 25s ethanol solution at a
physiological pH could be highly virucidal. According
to Mitra et al., however, a combination of the ethanol
treatment with an ion exchange chromatography is
neither shown nor suggested.
Hamman et al. (Vox Sang. 67 (1994), 72-77) show
that in the course of producing a factor VIII
concentrate, a depletion of the virus activity or virus
concentration, respectively, of merely 1 to 2 log steps
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can be attained by means of an ion exchange
chromatography.
Therefore, there exists a demand for an industrial-
ly applicable method for a guaranteed separation of
viruses from protein solutions so as to reduce the
risks of infectivity for patients who are treated with
pharmaceutical or therapeutical preparations of animal
or human origin or prepared from cell cultures by a
genetical engineering method.
Likewise, there is a demand for virus-safe,
pathogen-free biological products in which it is
already ensured by their preparation method that no
pathogen is transmitted and that the method is gentle
enough so as to leave the biological activity of the
products largely unaffected.
Thus, it is an object of the present invention to
provide an improved method of depleting viral and
molecular pathogens in a biological material.
-According to the invention, this object is achieved
by a method of the initially described kind which is
characterized in that the biological material is
admixed with an organic solvent, the biological
material with the organic solvent admixed thereto is
contacted with an ion exchanger, wherein the pathogens
are adsorbed on the ion exchanger material, and at
least one of the biological substances to be recovered
does not interact or interacts only slightly with the
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ion exchanger material, and the ion exchanger with the
pathogens adsorbed thereon is separated from the
biological material, a virus-depleted preparation of
the biological substance being recovered.
Surprisingly it has been found that in the presence
of the organic solvent, the reduction factor of the ion
exchange step was substantially increased as compared
to that described in the prior art. Thus, Hamman et al.
(supra) found a reduction factor of merely 1 log step
in the ion exchange step.
Therefore, it could not be expected that in a
single-step method, i.e., an adsorption method, a high
reduction factor could be expected without further
elution. Likewise, it has been surprising that the
presence of the solvent substantially changes the
adsorption specificity of the ion exchanger, whereby
pathogens are bound, while biological substances, in
particular proteins, substantially remain unbound.
Thus, with the method of the invention it has been
possible e.g. for HAV to attain a reduction factor of
>5.95 log steps, which corresponds to a complete
depletion, while in contrast thereto Hamman et al.
attained a reduction factor of merely 1 log step for
HAV by means of an ion exchange step.
It has been known that a depletion treatment with
12% ethanol can assist in the virus reduction, yet it
has been highly surprising that an organic solvent is
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also suitable to be applied simultaneously with an ion
exchange treatment for virus depletion.
Within the scope of the present invention it has
been shown that, particularly with the anion exchange
chromatography or the adsorption on an anion exchanger,
respectively, particular depletion rates can be
achieved, preferably in combination with materials
based on carbohydrates or synthetic polymers, such as,
e.g. DEAE-Sephacel~, DEAE-Sephadex~, DEAE-Sepharose
CL6B~, DEAE-Sepharose Fast Flow~,~ QAE-Sephadex~, Q-
Sepharose Fast Flow~, Q-Sepharose High Performance, Q-
Sepharose Big Beads° (all from Pharmacia);
DEAE-Tris-Acryl~, DEAE-Spherodex~, Q-Hyper-D~ (all from
Sepracor);
Macroprep DEAE~, Macroprep Q° (all from BioRad);
DEAE-Toyopearl~, QAE-Toyopearl~, Toyopearl Super-Q~
(all from Tosohaas);
Protein PAK DEAE° (Waters);
Fractogel EMD-TMAE~, Fratogel EMD-DEAE~, Fractogel EMD-
DMAE°, Licrospher 1000 TMAE~, Licrospher 1000 DEAE~ and
Licrospher 4000 DMAE~ (all from MERCK) .
Particularly preferred (primarily when processing
immunoglobulin) is an anion exchanger of the DEAF type,
in particular of the DEAE-Sephadex type.
Subsequently, the pathogens adsorbed on the ion
exchanger are removed by separating the ion
exchanger/pathogen complex from the biological
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material. In this manner, the adsorbate is immediately
separated from the biological material in a simple
manner and without treatment with an elution buffer.
Preferably, the complex is separated by passing the
biological material through a permeable filter, in
particular a deep bed filter. Separation of the complex
may also be effected by sedimentation, in particular
centrifugation. Preferably, separation of the ion
exchanger is also effected in the presence of the
solvent.
Within the scope of this invention, by biological
substances, substances of biological origin are meant,
which are recovered e.g. from body fluids, such as
blood or plasma, or may be isolated from the culture
supernatants of recombinant cells, wherein these
biological substances to be recovered within the scope
of the present invention or whose virus contamination
is to be depleted, respectively, may particularly be
used therapeutically, prophylactically or
diagnostically or as pharmaceutical preparations,
respectively. These biological substances may, e.g., be
proteins/peptides, carbohydrates or lipids, in
particular biologically active substances, such as
immunoglobulins or blood factors. Yet also other
classes of substances which may be formed by
prokaryotic or eukaryotic cells are to be summarized as
"biological substances".
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The recovery of virus-depleted preparations of the
biological substances from the supernatant of the
adsorbate or from the filtrate, respectively, commonly
is effected by further processing or purification of
the biological substances or fractions of the
biological material, respectively, precipitations,
chromatographic purification methods, a filtration,
diafiltration, formulation i.a. being effected.
Within the scope of the present invention, the
biological material may be a plasma fraction, an
immunoglobulin-containing plasma fraction, preferably
CORN fraction II+III, a plasma protein-containing
fraction comprising blood factors, such as, e.g.,
factor II, factor VII, factor VIII, factor IX, factor
X, factor XI, protein C, protein S, vWF, a concentrate
comprising one of the said blood factors, a supernatant
of a hybridoma cell line, a cell culture supernatant of
transformed or infected mammalian cells or an extract
of an animal or human tissue. The parameters for
carrying out the method will each be adapted to the
type and nature of the biological material used and to
the contaminating pathogens possibly present therein.
The optimal parameters, such as pH, temperature, period
of incubation for carrying out the method, type of
organic solvent used in the method according to the
invention, in dependence on the type of pathogen, the
specificity of the ion exchanger, and the nature of the
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biological material used (purity of solution, protein
concentration in the solution) may be found by the
skilled artisan on the basis of his general knowledge.
The method according to the invention is
particularly suitable to deplete molecular and/or viral
pathogens in a biological material, viral pathogens
both of the group of lipid-enveloped and of the group
of non-lipid-enveloped viruses being effectively
removed. Among them are in particular viruses such as
HAV, HBV, HCV, HGV, HEV, HDV, HIV, CMV or parvovirus.
Within the scope of the present invention, by
organic solvents in particular such solvents are to be
understood which do not trigger any substantial
denaturing processes when being admixed with the
biological materials under the conditions chosen. Among
them are particularly solvents, such as methanol,
ethanol, or other biologically compatible alcohols.
The optimal concentration of the respective solvent
- just like slight deviations in the optimal chromatog-
raphic conditions, which may possibly be caused by the
presence of the solvent - will be easy to be determined
for any skilled artisan by simple experiments. In
general, the solvent will be employed at a concentra-
tion of from 5 to 20 % by volume, preferably at a
concentration of from 10 to 15 o by volume, in
particular at a concentration of approximately 12 to 14
o by volume.
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Preferably, a mono- or polyvalent alcohol is
employed as the organic solvent, ethanol being
particularly preferred. According to the invention,
particularly preferred ethanol concentrations are
between 12 and 14 o by volume, in particular between 13
and 14 % by volume.
Preferably, the ion exchange treatment is carried
out at low temperatures, temperatures of below 10°C or
below 5°C being preferred. It has been shown that the
anion exchange treatments are particuarly suitable at a
temperature of between 0 and -10°C for the method
according to the invention.
It has also been shown that the method according to
the invention, in contrast to prior art reports, in
particular in contrast to the results of Zolton et al.
(1985), can also be successfully employed at pH values
of below 7.5 or 7.2, respectively, i.e. in the neutral
and acidic range, respectively.
A preferred method variant of the method according
to the invention thus consists in that the treatment of
the biological material with the ion exchanger, in
particular with the anion exchanger, is carried out at
a pH of from 5.6 to 7.2, preferably between 6.0 and
6.4, in particular at pH 6.2.
Treatment of the aqueous solution of the biological
material with the anion exchanger preferably is carried
out for a period of time of from 1 minute to 20 hours,
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in particular for 4 to 8 hours, wherein incubation may
either be effected in the batch method or in a
continuous flow system.
With the method according to the invention, a
biological material may be obtained which is safely
free from molecular and/or viral pathogens, wherein the
pathogens are subtantially completely removed. Thus,
depending on the added ethanol concentration, a
substantially complete virus removal has been found. By
the single-step method according.'to the ivnention, a
pathogen reduction factor of preferably >5.5 log steps,
particularly preferred >7.0 log steps, is attained.
The optimal adsorption conditions during the ion
exchange chromatography may easily be optimized by any
skilled artisan by referring to the teaching of the
present invention, depending on the biological
substance to be recovered and on the virus to be
adsorbed, respectively, and depending on the respective
organic solvent used.
The present method is also excellently suited for a
combination with further pathogen-inactivating or
pathogen-depleting steps, such as thermal and/or
detergent treatment, radiation treatment or filtration,
nanofiltration according to Austrian application A
780/96 being particularly preferably combinable with
the method according to the invention.
The present method is particularly suited for
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producing immunoglobulin preparations, yet it may also
be specifically employed for the recovery of
pharmaceutical preparations, blood factors, such as
factor II, factor IIa, factor VII, factor IX, factor X,
protein S, protein C or vWF.
The present invention will be explained in more
detail by way of the following examples, without,
however, being restricted thereto.
E x a m p 1 a s .
E x a m p 1 a 1 . Virus depletion in IgG-
containing solutions (at
present considered by Applicant to be the best mode
of carrying out the invention)
An immunoglobulin-containing COHN-fraction II+III
was adjusted with ethanol to a final concentration of
10% to 14°s, each, and the solution was brought to a
temperature of -3°C to -1°C. Subsequently, the solution
was admixed with the test viruses stated in Table 1.
Per g of protein, 0.5 g of DEAF-Sephadex A-50 were
admixed, the pH was adjusted at 6.2 ~ 0.1, and
incubation was effected by keeping constant the
temperature and the pH for 6 hours under stirring.
Subsequently, the exchanger suspension was removed by
deep bed filtration, and the virus titer in the
filtrate was determined. The virus depletion rate,
expressed as the decrease of the infectious virus
particles in powers of ten, is summarized in Table 1
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and was determined by means of virus titration or PCR.
As is apparent from this table, this method - depending
on the test virus used - leads to a depletion by 2 to
more than 6 powers of ten. The yield of IgG in the
supernatant was >70%. In control experiments in which
the incubation of the test virus was carried out
analogously without the addition of the anion
exchanger, no virucidal effect of the ethanol could be
found under these conditions.
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T a b 1 a 1
IgG i.v.
OAl
Method
DEAE-Se
hadex
and
Filtration
Ethanol 10% 11% 12% 13%
14%
Yield 100% 100% 100% 100%
97%
IRF to
PRV n.d. n.d. 2.95 >5.44 >5.40
MVM 2.76 3.73 >6 76 n.d. >6.27
TBE n.d. n.d. 5.00 5.96 n.d.
BVDV n.d. n.d. 1.80 2.61 >4.35
HCV n.d. >3.94 >5.32 n.d. n.d.
HAV n.d. 3.91 >5.95 n.d. n.d.
HGV n.d. n.d. 4.03 n.d. n.d.
HIV n.d. n.d. >4.30 n.d. n.d.
ERV n.d. n.d. >5.10 n.d. n.d.
PRV - Pseudorabies virus
MVM - Minute Virus of Mice/parvovirus
TBE - Tick-borne encephalitis virus
BVDV - Bovine diarrhea virus
HCV - Hepatitis C virus
HAV - Hepatitis A virus
HGV - Hepatitis G virus
HIV-1 = Human Immunodeficiency Virus-1
ERV - Equine rhinopneumonitis virus
n.d. - not determined
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E x a m p 1 a 2 . Depletion of parvovirus from
IgG-containing solutions
An immunoglobulin-containing COHN fraction III
having an ethanol content of 12% was admixed in an
analogous manner, as described in Example 1, with
parvovirus B19. The total load in the B19-admixed
starting material was 1011.3 DNA copies/ml. The DEAE-
Sephadex adsorption step with subsequent filtration was
carried out as described in Example 1. DNA copies/ml
were determined in the filtrate by means of PCR, as
described in Austrian application A 780/96. No
parvovirus-specific DNA could be detected in the
filtrate. Taking into consideration the detection
limits, a virus reduction factor of >7.4 log steps was
achieved with the method according to the invention.
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