U.S. patent application number 08/598264 was filed with the patent office on 2002-06-06 for removal of viruses from protein solutions by ultrafiltration.
Invention is credited to BERNHARDT, DIETER, GRONER, ALBRECHT, NOWAK, THOMAS.
Application Number | 20020068368 08/598264 |
Document ID | / |
Family ID | 7753509 |
Filed Date | 2002-06-06 |
United States Patent
Application |
20020068368 |
Kind Code |
A1 |
BERNHARDT, DIETER ; et
al. |
June 6, 2002 |
REMOVAL OF VIRUSES FROM PROTEIN SOLUTIONS BY ULTRAFILTRATION
Abstract
The invention relates to the removal of viruses from aqueous
solutions, as a rule protein solutions, by ultrafiltration. This
entails the viruses to be removed being increased in size by
incubation with a high molecular weight receptor binding thereto,
preferably a specific antibody, so that, on the one hand, the
separation effect is improved and, on the other hand, a larger pore
diameter which can now be chosen for the filters used also makes it
possible for smaller viruses to be separated from larger protein
molecules present in protein solutions, and, where appropriate, the
filtration rate is increased.
Inventors: |
BERNHARDT, DIETER; (COLBE,
DE) ; GRONER, ALBRECHT; (SEEHEIM, DE) ; NOWAK,
THOMAS; (STAUFENBERG-MAI, DE) |
Correspondence
Address: |
FINNEGAN HENDERSON FARABOW
GARRETT & DUNNER
FRANKLIN SQUARE BLDG
1300 I ST NW SUITE 700
WASHINGTON
DC
200053315
|
Family ID: |
7753509 |
Appl. No.: |
08/598264 |
Filed: |
February 7, 1996 |
Current U.S.
Class: |
436/536 |
Current CPC
Class: |
C07K 1/34 20130101 |
Class at
Publication: |
436/536 |
International
Class: |
G01N 033/536 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 9, 1995 |
DE |
P 195 04 211.5 |
Claims
1. A method for removing viruses from protein solutions, which
comprises the viruses to be removed being increased in size by
binding to ligands and being held back by filtration.
2. The method as claimed in claim 1, wherein the ligands are
antibodies or parts thereof which still bind to viruses, which ay
be modified chemically or by genetic engineering.
3. The method as claimed in claim 1, wherein the ligands are
monoclonal antibodies or parts thereof which still bind to
viruses.
4. The method as claimed in claim 1, wherein the ligands or parts
thereof which still bind to viruses are modified chemically or by
genetic engineering.
5. The method as claimed claim 1, wherein the ligands, antibodies,
monoclonal antibodies or parts thereof binding viruses are added
before the filtration.
6. The method as claimed in claim 1, wherein an incubation of step
takes place before the filtration to permit virus-ligand
binding.
7. The method as claimed in claim 1 wherein hollow fiber filters or
membrane filters of defined pore size are used.
8. The method as claimed in claim 1, wherein the filtration is
carried out via the tangential flow method.
9. A method for removing unwanted substances from liquid substance
mixtures, which comprises the substances to be removed being
increased in size by binding to ligands and being held back by
filtration.
Description
[0001] The invention relates to the removal of viruses aqueous
solutions, as a rule protein solutions, by ultrafiltration. This
entails the viruses to be removed being increased in size by
incubation with high molecular weight ligands binding thereto,
preferably specific antibodies, so that, on the one hand, the
separation effect is improved and, on the other hand, a larger pore
diameter which can now be chosen for the filters used also makes it
possible for smaller viruses to be separated from larger protein
molecules present in protein solutions, and, where appropriate, the
filtration rate is increased.
[0002] Proteins purified and concentrated from human plasma are
used for therapy and prophylaxis of human diseases. These products
are prepared from plasma pools consisting of about 10,000
individual donations. Since some of these donations may potentially
be contaminated with human pathogenic viruses such as HIV-1/2,
hepatitis B virus, hepatitis C virus and other viruses, there is
the possibility of infection being caused by administration of the
plasma proteins. In order to minimize this contamination hazard,
donations are obtained only from healthy donors who are
additionally tested for infection markers (antibodies against HIV 1
and HIV 2, UBsAg, antibodies against HCV and elevated liver
function test results (ALT)); positive donations are rejected and
not used for obtaining plasma proteins. The purification and
concentration steps used in the industrial preparation of plasma
proteins and, in particular, steps specifically introduced into the
production to eliminate and/or inactivate viruses lead to plasma
proteins with a very high safety standard.
[0003] In order to increase the safety of plasma proteins even
further, there have been investigations of the use of filtration
methods, for example dead-end and tangential flow filtration, in
order to eliminate any viruses present in the protein solution.
Filter units for eliminating viruses are produced by various
companies (DiLeo, A. J. et al. Biologicals 21, 275-286 (1993);
DiLeo A. J. et al. Biologicals 21, 287-296 (1993); Burnout, T. et
al., Vox Sang. 67, 132-138 (1994)). Thus, for example, Asahi
Chemical Industry Co. Ltd., Tokyo, Japan, produces filter units
stating a defined (average) pore size, while, for example,
Millipore Corp., Bedford, Mass., USA, produces filter units stating
a nominal molecular weight cut off
[0004] It has emerged from our investigations that viruses are held
back at different rates by filters with different pore sizes,
depending on the diameter of said viruses (HIV: 80-100 nm: HCV:
40-60 nm; HBV: 40-45 nm; picornaviruses: 24-30 nm; parvoviruses:
18-25 am): (I) filters with an average pore diameter of 75 nm
essentially retain HIV, while the other specified viruses are found
in the filtrate; (II) filters with an average pore diameter of 35
nm retain HIV completely and HCV and HBV to a large extent, while,
for example, picornaviruses and parvoviruses are found in the
filtrate; (III), filters with an average pore diameter of 15 nm
retain HIV, HCV and HBV and, to a large extent, for example
picornaviruses and parvoviruses. Filtration through a filter with
an average pore diameter of 15 nm leads to a general increase in
the virus safety of plasma proteins. However, since most plasma
proteins have such a high molecular weight that they cannot be
filtered through a 15 nm filter, i.e. are likewise held back, only
filters with an average pore diameter of 35 nm (or a nominal
molecular weight cut off of 70,000, D to 100,000 D) are suitable
for filtering most plasma proteins, but these do not remove to an
adequate extent at least picornaviruses (such as, for example,
hepatitis A virus) and parvoviruses (such as the human pathogenic
parvovirus B 19) from plasma proteins.
[0005] The object therefore was to achieve an adequate, i.e.
complete, retention even of small viruses by filtration, and to
make filtration methods also applicable to those proteins which
resemble simply in terms of their size a small virus. In addition,
it was intended to increase the filtration rate as far as
possible.
[0006] The object is achieved by the present invention un that the
viruses to be removed are increased in size by binding to high
molecular weight ligands, preferably specific antibodies,
particularly preferably monoclonal antibodies, in principle of all
subclasses, but preferably subclass IgG or IgM or parts thereof
still capable of binding, which are, where appropriate, modified or
enlarged by genetic engineering, to such an extent that they can be
held back by filtration. The increase in size can also be achieved
by aggregate formation. It is in fact possible with this method to
separate relatively large proteins such as factor VIII or von
Willebrand factor from such viruses of increased size by
filtration, it now being possible to choose the pore size such that
the proteins pass through and the viruses of increased size are
held back. It is moreover possible by choice of a larger pore
width, which is now possible, to increase the filtration rate. In
the most general form, the present invention makes it possible to
increase the size of any constituents of an aqueous solution by
binding to high molecular weight ligands to such an extent that
separation is then possible from the now smaller constituents in a
filtration step.
[0007] Further examples of ligands which can be used for the
purpose of the present invention for specific viruses are listed
below:
1 Antibodies, possibly modified HIV CD4 receptor HIV Sialic acid
(.apprxeq. derivatives, for Influenzaviruses example
sialooligosaccharides) Heparan sulfate HSV C3d complement
receptor/complement EBV receptor 2 (CR2) Acetylcholine receptor
Rabiesviruses ICAM-1 (intracellular adhesion Rhinoviruses
molecule-1) Gangliosides Paramyxoviruses IgA receptor HBV Epidermal
Growth Factor Receptor Vaccinia Beta adrenergic receptor Reovirus
Serotype 3 Immunoglobulins superfamily protein Poliovirus H-2
antigens Semliki Forest Virus
[0008] Human pathogenic viruses such as, for example, HBV, HCV,
HIV, picornaviruses and parvoviruses may, despite selection of
donors, be present in a plasma pool. These viruses bind to
antibodies present in the protein solution, in particular either to
antibodies present in the plasma pool during incubation of the
low-cryo plasma or of the resuspended cryoprecipitate at 2.degree.
C. to 37.degree. C. for 15 minutes to 36 hours, preferably at
2.degree. C. to 8.degree. C. for a period of 2 to 36 hours, in
particular 4 to 18 hours, or at 10.degree. C. to 25.degree. C. for
a period of 15 minutes to 18 hours, preferably 30 minutes to 6
hours, or to antibodies specifically added shortly before the
filtration to the plasma protein solution to be filtered, which
antibodies can be used in native form or a form modified chemically
or by genetic engineering (for example low-cryo plasma,
immunoglobulin fractions, purified immunoglobulins of human or
animal origin, monoclonal antibodies) during incubation of the
protein solution at 2.degree. C. to 37.degree. C. for 15 minutes to
36 hours, preferably at 2.degree. C. to 8.degree. C. for a period
of 2 to 36 hours, in particular 4 to 18 hours, or at 10.degree. C.
to 30.degree. C. for a period of 15 minutes to 8 hours, preferably
30 minutes to 4 hours. The virus-antibody complexes formed in this
way can be removed from the plasma protein solution by filtration,
for example dead-end filtration or, preferably, tangential flow
filtration.
EXAMPLE 1
[0009] Bovine parvovirus (BPV; ATCC VR-767), as model virus for the
human pathogenic parvovirus B 19, was grown in diploid fetal bovine
lung cells in EME medium containing 5% FCS and then separated from
cells and cell debris by low-speed centrifugation (2000 g, 15
minutes, 4.degree. C.); the virus-containing supernatant was
divided into aliquots and stored at -70.degree. C. until
investigated. Porcine parvovirus (PPV; ATCC VR-742) was
investigated for comparison; PPV was grown and isolated like BPV
but in a permanent porcine kidney cell line (IB-RS-2 D10; ATCC CRL
1835).
[0010] The following test mixtures were mixed, incubated at
20.degree. C. for 1 hour and then filtered through BMM process
filter PLANOVA.TM. 35 (from Asahi Chemical Industry Co. Ltd.,
Tokyo, Japan) in accordance with the instructions of the
manufacturing company. The infectiosity titer (CCID.sub.50: cell
culture infectious dose 50%) was determined in the starting
material and in the filtrate after filtration.
2 Test A Test B Test C Test D Test E Protein Albumin Albumin
Albumin Albumin Albumin solution (5%) (5%) (5%) (5%) (5%) (260 ml)
Virus BPV BPV BPV PPV PPV material (30 ml) Antibody- PBS Human
Human Human Human con- (no anti- serum serum serum serum taining
bodies) (B19- (B19- (B19- (B19- solution positive/ negative/
positive/ negative/ (30 ml) ELISA) ELISA) ELISA) ELISA) Infection
10.sup.5.2 10.sup.4.2 10.sup.5.1 10.sup.6.4 10.sup.6.2 titer before
filtra- tion (CCID.sub.50) Infection 10.sup.4.9 <10.sup.0.5
10.sup.4.3 10.sup.6.0 10.sup.5.9 titer after filtra- tion
(CCID.sub.50)
[0011] Since BPV cross-reacts serologically with B19 (Bernhardt, D.
et al., Tierrztl. Umschau 49, 481-483 (1994)), antibodies against
B19 from human plasma also bind to BPV but not to PPV. The
antigen-antibody complexes produced during the incubation are so
large that, in contrast to uncomplexed antigen, they cannot be
filtered.
EXAMPLE 2
[0012] A licensed poliovirus vaccine for oral immunization
(Oral-Virelon.RTM.; live attenuated vaccine) was suspended in a
protein solution comprising 10% fetal calf serum in DMEM; purified
immunoglobulin (Beriglobin.RTM.) was added to part of this virus
suspension, and the same volume of PBS was added to the other part.
After incubation at 15.degree. C. for 2 hours, the samples were
filtered (Sartocon.RTM.-Micro, 100,000 D nominal molecular weight
cut off) Since the immunoglobulins neutralize polioviruses, so that
an infectiosity assay in this part of the test provides no
information about a reduction in concentration by the
filtration,virus in samples of the retentates and of the filtrates
was, after a pH shift (pH 4, 10 minutes; then centrifugation
through a sucrose cushion of 25% (w/w) sucrose at 20,000 g, 45
minutes, 4.degree. C. and resuspension of the pellet in PBS pH
7.2), detected in a dot-blot on nitrocellulose. The resuspended
samples were initially diluted 1:2 in tris/glycine buffer pH 8.3
and then further diluted in a 1:3 dilution series; 100 .mu.l
portions of each dilution were applied to a nitrocellulose filter
(pore size 0.4 .mu.m), the membrane was blocked with skimmed milk
powder (3%), incubated with antiserum against polioviruses (from
rabbit) at 37.degree. C. for 1 hour and then incubated further with
POD-labeled anti-rabbit antibodies. The bound antibodies were
visualized with 4-chloro-1-naphthol/H.sub.2O.sub.2 (dot-blot
procedure as described by Cardosa, M J & Tio, P. H., Bull. WHO,
69, 741-745, 1991).
3 Test A Test B Protein solution 10% of FCS in 10% of FCS in (260
ml) DMEM DMEM Virus material Poliovirus Poliovirus (30 ml)
Antibody-contain- purified immu- PBS ing solution (30 noglobulins
ml) (Beriglobin) Dot blot titer 1024 1024 before filtration Dot
blot titer <2 256 after filtration
[0013] The poliovirus-antibody complexes cannot, in contrast to
uncomplexed antigens, be filtered; removal from the protein
solution can therefore be achieved by adding immunoglobulins.
* * * * *