U.S. patent application number 16/853444 was filed with the patent office on 2020-08-06 for virus filtration of liquid factor vii compositions.
The applicant listed for this patent is Novo Nordisk Healthcare AG. Invention is credited to Jesper Christensen, Erik Halkjaer, Thomas Budde Hansen, Nina Johansen, Turid Preuss, Lene Vaedele Madsen Tomoda.
Application Number | 20200248150 16/853444 |
Document ID | / |
Family ID | 1000004766122 |
Filed Date | 2020-08-06 |
United States Patent
Application |
20200248150 |
Kind Code |
A1 |
Christensen; Jesper ; et
al. |
August 6, 2020 |
Virus filtration of liquid factor VII compositions
Abstract
The present invention relates to a novel method for improving
the viral safety of liquid Factor VII compositions, in particular
those comprising active Factor VII polypeptides (a Factor VIIa
polypeptide).
Inventors: |
Christensen; Jesper;
(Gentofte, DK) ; Halkjaer; Erik; (Copenhagen N,
DK) ; Preuss; Turid; (Smoerum, DK) ; Hansen;
Thomas Budde; (Copenhagen N, DK) ; Tomoda; Lene
Vaedele Madsen; (Lyngby, DK) ; Johansen; Nina;
(Roedovre, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novo Nordisk Healthcare AG |
Zurich |
|
CH |
|
|
Family ID: |
1000004766122 |
Appl. No.: |
16/853444 |
Filed: |
April 20, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16145565 |
Sep 28, 2018 |
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16853444 |
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15286068 |
Oct 5, 2016 |
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16145565 |
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14753551 |
Jun 29, 2015 |
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15286068 |
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13349980 |
Jan 13, 2012 |
9102762 |
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14753551 |
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12173475 |
Jul 15, 2008 |
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13349980 |
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11439828 |
May 23, 2006 |
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12173475 |
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PCT/EP04/53206 |
Dec 1, 2004 |
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11439828 |
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60528763 |
Dec 11, 2003 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Y 304/21021 20130101;
C12N 9/6437 20130101; C12N 2740/13063 20130101; C12N 7/00 20130101;
C07K 14/745 20130101 |
International
Class: |
C12N 7/00 20060101
C12N007/00; C07K 14/745 20060101 C07K014/745; C12N 9/64 20060101
C12N009/64 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 1, 2003 |
DK |
PA 2003 01775 |
Claims
1. A method for removing viruses from a liquid composition of
recombinant Factor VII comprising one or more Factor VII
polypeptides having a concentration in the range of 0.01 to 5
mg/mL, the method comprising subjecting the liquid composition to
nanofiltration using a nanofilter having a pore size of 80 nm or
less, wherein 50-100% of the Factor VII polypeptides in the
composition subjected to the nanofilter are in an activated form
(FVIIa) prior to nanofiltration.
2. The method according to claim 1, wherein the liquid composition
has a pH of in the range of 7.0-9.5.
3. The method according to claim 2, wherein the pH is in the range
of 8.3-8.7.
4. The method according to claim 1, wherein the pore size of the
nanofilter is 50 nm or less.
5. The method according to claim 1, wherein the membrane of the
nanofilter is manufactured from one or more materials selected from
the group consisting of: cuprammonium regenerated cellulose,
hydrophilic polyvinylidene fluoride (PVDF), composite PVDF, surface
modified PVDF, and polyether sulfone.
6. The method according to claim 1, wherein the pore size of the
nanofilter is in the range of 10-30 nm.
7. The method according to claim 1, wherein the liquid Factor VII
composition is obtained from a cell culture supernatant.
8. The method according to claim 1, wherein the liquid composition
is serum-free.
9. The method according to claim 1, wherein the Factor VII
polypeptide(s) is/are produced by cell culture in the presence of
bovine or foetal calf serum.
10. The method according to claim 1, wherein the Factor VII
polypeptide(s) is/are produced by cell culture in Chinese Hamster
Ovary (CHO) cells in a medium free from any components of animal
origin.
11. The method according to claim 1, wherein the concentration of
the Factor VII polypeptide(s) in the liquid composition is in the
range of 0.05-2.0 mg/mL.
12. A method for removing viruses from a liquid composition of
recombinant Factor VII comprising one or more Factor VII
polypeptides having a concentration in the range of 0.01 to 5
mg/mL, wherein 50-100% of the Factor VII polypeptides in the
composition are in an activated form (FVIIa) prior to
nanofiltration, the method comprising the steps of (i) combining
the composition with a detergent, and (ii) subjecting the solution
to nanofiltration using a nanofilter having a pore size of 80 nm or
less.
13. The method of claim 1, wherein 70-100% of the Factor VII
polypeptides in the composition subjected to the nanofilter are the
activated form of Factor VII.
14. The method of claim 1, wherein 80-100% of the Factor VII
polypeptides in the composition subjected to the nanofilter are the
activated form of Factor VII.
15. A method for removing viruses from a liquid composition of
recombinant Factor VII comprising one or more Factor VII
polypeptides having a concentration in the range of 0.01 to 5
mg/mL, the method comprising subjecting the liquid composition to
nanofiltration using a nanofilter having a pore size of 50 nm or
less, wherein 50-100% of the Factor VII polypeptides in the
composition subjected to the nanofilter are in an activated form
(FVIIa) prior to nanofiltration.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of pending U.S.
application Ser. No. 16/145,565, filed Sep. 28, 2018, which is a
continuation of U.S. patent application Ser. No. 15/286,068, filed
Oct. 5, 2016 (abandoned), which is a continuation of U.S. patent
application Ser. No. 14/753,551, filed Jun. 29, 2015 (abandoned),
which is a continuation of U.S. patent application Ser. No.
13/349,980, filed Jan. 13, 2012 (U.S. Pat. No. 9,102,762), which is
a continuation of U.S. patent application Ser. No. 12/173,475,
filed Jul. 15, 2008 (abandoned), which is a continuation of U.S.
patent application Ser. No. 11/439,828, filed May 23, 2006
(abandoned), which is a continuation of International Patent
Application No. PCT/EP04/53206, filed Dec. 1, 2004, which claims
priority from Danish Patent Application No. PA 2003 01775, filed
Dec. 1, 2003; and to U.S. Patent Application No. 60/528,763, filed
Dec. 11, 2003; the contents of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a novel method for
improving the viral safety of liquid Factor VII compositions, in
particular those comprising active Factor VII polypeptides (a
Factor VIIa polypeptide).
BACKGROUND OF THE INVENTION
[0003] A variety of Factors involved in the blood clotting process
have been identified, including Factor VII (FVII), a plasma
glycoprotein. Haemostasis is initiated by the formation of a
complex between Tissue Factor (TF) being exposed to the circulating
blood following an injury to the vessel wall, and Factor VIIa which
is present in the circulation in an amount corresponding to about
1% of the total Factor VII protein mass. Factor VII exists in
plasma mainly as a single-chain zymogen which is cleaved by FXa
into its two-chain, activated form, Factor VIIa. Recombinant
activated Factor VIIa (rFVIIa) has been developed as a
pro-haemostatic agent. The administration of rFVIIa offers a rapid
and highly effective pro-haemostatic response in haemophilic
subjects with bleedings, who cannot be treated with other
coagulation Factor products due to antibody formation. Also
bleeding in subjects with Factor VII deficiency or subjects having
a normal coagulation system but experiencing excessive bleeding can
be treated successfully with Factor VIIa.
[0004] The purification and handling of Factor VII must be careful,
due the possibility for degradation of the molecule. Factor VII and
Factor VIIa, being large molecules (approx. molecular weight 50
kD), are susceptible to mechanical degradation by shear forces
during purification and filtration. Further, Factor VIIa is an
active proteolytic enzyme that degrades other proteins including
Factor VIIa. Degradation of Factor VIIa mainly involves cleavage in
the heavy chain of Factor VIIa, particularly at amino acids no. 290
and 315 in the molecule. Finally, methionine residues in Factor VII
and Factor VIIa may be oxidized.
[0005] An object of the present invention is to provide a method
for the removal or inactivation of viruses from liquid Factor VII
compositions by which method the integrity of the Factor VII
constituents is substantially preserved.
[0006] WO 96/00237 discloses a method of virus-filtration of a
solution that contains a macromolecular, e.g. a protein such as the
plasma protein Factor IX.
[0007] WO 98/37086 discloses removal of viruses from plasma-derived
protein solutions by nanofiltration using a membrane having an
average pore size of 15 nm.
[0008] Tomokiyo et al., Vox Sanguinis, 2003, 84, 54-64, disclose
the large-scale production of human plasma-derived activated Factor
VII concentrate. The method of production involves the step of
virus-filtration of a solution comprising inactive Factor VII.
BRIEF DESCRIPTION OF THE INVENTION
[0009] In a broad aspect, the preset invention relates to methods
for the removal and/or inactivation of viruses from Factor VII
composition. The term "virus" as used herein means any
ultramicroscopic infectious agent that replicates itself only
within cells of living hosts, or noninfectious particles derived
thereof. In one embodiment the virus is infectious. In one
embodiment the virus is a non-infectious virus particle.
[0010] A first aspect of the present invention relates to a method
for removing viruses from a liquid Factor VII composition, said
method comprising subjecting said solution to nanofiltration using
a nanofilter having a pore size of at the most 80 nm.
[0011] A second aspect of the present invention relates to a method
for removing viruses from a liquid Factor VII composition, said
composition comprising one or more Factor VII polypeptides, at
least 5% of said one or more Factor VII polypeptides being in the
activated form, said method comprising subjecting said solution to
nanofiltration using a nanofilter having a pore size of at the most
80 nm.
[0012] A third aspect of the invention relates to a method for
removing viruses from a liquid Factor VII composition, said
composition comprising one or more Factor VII polypeptides, said
liquid composition being substantially serum-free, said method
comprising subjecting said solution to nanofiltration using a
nanofilter having a pore size of at the most 80 nm.
[0013] A further aspect of the invention relates to a method for
removing viruses from a liquid Factor VII composition, said
composition comprising one or more Factor VII polypeptides, said
method comprising subjecting said solution to nanofiltration using
a nanofilter having a pore size of at the most 80 nm, said
nanofilter having a membrane manufactured from one or more
materials selected from cuprammonium regenerated cellulose,
hydrophilic polyvinylidene fluoride (PVDF), composite PVDF, surface
modified PVDF, and polyether sulfone.
[0014] A further aspect of the invention relates to a method for
inactivating viruses in a liquid Factor VII composition, said
composition comprising one or more Factor VII polypeptides, the
method comprising the step of combining said composition with a
detergent.
[0015] A further aspect of the invention relates to a method for
high-level elimination of the presence of active viruses in a
liquid Factor VII composition, the method comprising the steps of
(i) inactivating viruses, and (ii) removing viruses.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic illustration of a system suitable for
methods of the invention. The system includes a pressure tank (1)
with a supply of compressed air, a pre-filter (2) for removing
particles that would otherwise clog the virus filter, a pressure
gauge (P), a virus filter (3), and a pool tank (4).
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention provides methods for removing or
inactivating viruses, including non-enveloped viruses, from a
liquid Factor VII composition which typically comprises a
significant ratio of activated and thereby proteolytically active
Factor VII polypeptides. The method includes the step of subjecting
the liquid Factor VII composition to nanofiltration using a
nanofilter having a pore size of at the most 80 nm.
[0018] The method is particularly useful for the removal of
enveloped viruses as well as non-enveloped viruses such as Murine
Leukemia virus (enveloped) which may be removed by filters with a
pore size around 50 nm, and Porcine Parvovirus (non-enveloped)
which may be removed by filters with a pore size around 20 nm.
[0019] The liquid Factor VII compositions, e.g. those comprising a
significant ratio of activated Factor VII polypeptides, can in
principle be prepared from the dry Factor VII constituents, but are
more typically obtained from large-scale production processes, e.g.
processes involving recombinant techniques. In such processes a
cell culture supernatant is typically harvested and subsequently
subjected to one or more processing steps to obtain the desired
protein, including, without limitation, centrifugation or
filtration to remove cells that were not immobilized in the
carriers; affinity chromatography, hydrophobic interaction
chromatography; ion-exchange chromatography; size exclusion
chromatography; electrophoretic procedures (e.g., preparative
isoelectric focusing (IEF), differential solubility (e.g., ammonium
sulfate precipitation), or extraction and the like. See, generally,
Scopes, Protein Purification, Springer-Verlag, New York, 1982; and
Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH
Publishers, New York, 1989. Purification of Factor VII polypeptides
may also involve, e.g., affinity chromatography on an anti-Factor
VII antibody column (see, e.g., Wakabayashi et al., J. Biol. Chem.
261:11097, 1986; and Thim et al., Biochem. 27:7785, 1988) and
activation by proteolytic cleavage, using Factor XIIa or other
proteases having trypsin-like specificity, such as, e.g., Factor
IXa, kallikrein, Factor Xa, and thrombin. See, e.g., Osterud et
al., Biochem. 11:2853 (1972); Thomas, U.S. Pat. No. 4,456,591; and
Hedner et al., J. Clin. Invest. 71:1836 (1983). Alternatively, a
Factor VII polypeptide may be activated by passing it through an
ion-exchange chromatography column, such as Mono Q.RTM. (Pharmacia)
or the like.
[0020] The methods of the present invention are particularly useful
for large-scale production processes. By the term "large-scale" is
typically meant methods wherein the volume of the liquid Factor VII
polypeptide compositions is at least 100 L, such as at least 500 L,
e.g. at least 1000 L, or at least 5000 L. This is not to be
limiting in any way, as the present invention will also work for
liquid Factor VII polypeptide compositions of less than 100 L.
[0021] It has now been realized that nanofiltration may be applied
even after the Factor VII polypeptide bulk has been partially or
fully activated.
[0022] Thus, the methods of the invention are applicable as one of
the steps of the overall purification process for the Factor VII
polypeptide, typically one of the final steps of the purification
process.
[0023] More specifically, a typical purification process starting
from harvested material from the a fermentation broth (or from
human (or mammalian) plasma) can be outlined as follows:
TABLE-US-00001 Possible stages Purification step for virus
filtration Harvest .dwnarw. 1 Capture .dwnarw. 2 Intermediate
purification .dwnarw. 3 Polishing .dwnarw. 4 Drug substance
[0024] The content of Factor VII polypeptide in the activated form
is initially (i.e. from the harvest step) typically around 2%, and
increases in the course of the purification process to 90% or more
before the polypeptide is obtained as a drug substance.
[0025] The liquid Factor VII composition subjected to
nanofiltration comprises one or more Factor VII polypeptides in a
suitable solvent. The solvent is typically water or an aqueous
mixture/solution, such as pure water, an aqueous buffer, a
water/ethanol mixture, a water/DMSO mixture, or an aqueous salt
solution, e.g. saline, a urea solution or guanidine solution. A
suitable aqueous liquid may also comprise a detergent
(surfactant).
[0026] In interesting embodiments, the liquid Factor VII
composition is obtained, or originates, from a cell culture
supernatant, e.g. a cell culture supernatant obtained as disclosed
in WO 02/29084. In one embodiment, the liquid Factor VII
composition is serum-free, i.e. free from animal-derived
components. Thus, the cell cultures may be cultivated in a medium
lacking animal derived components.
[0027] An attractive variant hereof is the one where the Factor VII
polypeptide(s) is/are produced by cell culture in CHO cells, e.g.
in CHO cells in a medium free from any components of animal origin,
or a medium lacking animal-derived components and lacking proteins
("protein-free").
[0028] The medium for CHO cells may be any commercially available
protein-free CHO medium lacking animal-derived components or an
in-house produced medium for CHO cells.
[0029] In some embodiments, the cells used in practicing the
present invention are adapted to suspension growth in medium
lacking animal-derived components, such as, e.g., medium lacking
serum. Such adaptation procedures are described, e.g., in
Scharfenberg, et al., Animal Cell Technology Developments towards
the 21St Century, E. C. Beuvery et al. (Eds.), Kluwer Academic
Publishers, pp. 619-623, 1995 (BHK and CHO cells); Cruz,
Biotechnol. Tech. 11:117-120, 1997 (insect cells); Keen,
Cytotechnol. 17:203-211, 1995 (myeloma cells); Berg et al.,
Biotechniques 14:972-978, 1993 (human kidney 293 cells). In a
particularly embodiment, the host cells are BHK 21 or CHO cells
that have been engineered to express human Factor VII or a Factor
VII polypeptide and that have been adapted to grow in the absence
of serum or animal-derived components.
[0030] In an alternative embodiment, the Factor VII polypeptide(s)
is/are produced by cell culture in the presence of bovine or fetal
calf serum.
[0031] According to one aspect of the invention, a feature is that
a significant ratio, i.e. at least 5%,such as at least 7%, e.g. at
least 10%, of the one or more Factor VII polypeptides are in the
activated form (i.e. the bioactive, cleaved form of a Factor VII
polypeptide (i.e. a Factor VIIa polypeptide)). In further
embodiments, the Factor VIIa polypeptide represents 5-70%, such as
7-40%, e.g. 10-30%, of the mass of the one or more Factor VII
polypeptides. In other embodiments, the Factor VIIa polypeptide
represents 50-100%, such as 70-100%, e.g. 80-100%, of the mass of
the one or more Factor VII polypeptides. In still other
embodiments, the Factor VIIa polypeptide represents 20-80%, such as
30-70%, e.g. 30-60%, of the mass of the one or more Factor VII
polypeptides.
[0032] In most embodiments, the solution comprises a Factor VII
polypeptide in inactivated form as well as a bioactive Factor VIIa
polypeptide, i.e. the Factor VIIa polypeptide represents less than
100% of the mass of the one or more Factor VII polypeptides. In the
most typical embodiment, the composition comprises a(n) (activated)
Factor VIIa polypeptide that corresponds to an (inactive) Factor
VII polypeptide, i.e. the Factor VIIa polypeptide is the Factor VII
polypeptide in the activated form. In other embodiment, the Factor
VIIa polypeptide is somewhat different from the activated form of
the inactivated Factor VII polypeptide. It should of course be
understood that the composition in particular embodiments may
comprise more than one Factor VII polypeptide and more than one
Factor VIIa polypeptide.
[0033] The term "one or more Factor VII polypeptides" encompasses
wild-type Factor VII (i.e. a polypeptide having the amino acid
sequence disclosed in U.S. Pat. No. 4,784,950), as well as variants
of Factor VII exhibiting substantially the same or improved
biological activity relative to wild-type Factor VII. The term
"Factor VII" is intended to encompass Factor VII polypeptides in
their uncleaved (zymogen) form, as well as those that have been
proteolytically processed to yield their respective bioactive
forms, which may be designated Factor VIIa. Typically, Factor VII
is cleaved between residues 152 and 153 to yield Factor VIIa. The
term "Factor VIIa" specifically means an activated (i.e. bioactive,
cleaved) Factor VII polypeptide. Thus, "Factor VIIa" is a subgroup
relative to "Factor VII". The term "inactive Factor VII"
specifically means Factor VII not being Factor VIIa.
[0034] The term "Factor VII polypeptide" also encompasses
polypeptides, including variants, in which the Factor VIIa
biological activity has been substantially modified or somewhat
reduced relative to the activity of wild-type Factor VIIa, as well
as Factor VII derivatives and Factor VII conjugates. These
polypeptides include, without limitation, Factor VII or Factor VIIa
into which specific amino acid sequence alterations have been
introduced that modify or disrupt the bioactivity of the
polypeptide. The term "Factor VII derivative" as used herein, is
intended to designate wild-type Factor VII, variants of Factor VII
exhibiting substantially the same or improved biological activity
relative to wild-type Factor VII and Factor VII-related
polypeptides, in which one or more of the amino acids of the parent
peptide have been chemically and/or enzymatically modified, e.g. by
alkylation, glycosylation, PEGylation, acylation, ester formation
or amide formation or the like. This includes but is not limited to
PEGylated human Factor VIIa, cysteine-PEGylated human Factor VIIa
and variants thereof. Non-limiting examples of Factor VII
derivatives includes GlycoPegylated FVII derivatives as disclosed
in WO 03/31464 and US Patent applications US 20040043446, US
20040063911, US 20040142856, US 20040137557, and US 20040132640
(Neose Technologies, Inc.); FVII conjugates as disclosed in WO
01/04287, US patent application 20030165996, WO 01/58935, WO
03/93465 (Maxygen ApS) and WO 02/02764, US patent application
20030211094 (University of Minnesota).
[0035] The term "PEGylated human Factor VIIa" means human Factor
VIIa, having a PEG molecule conjugated to a human Factor VIIa
polypeptide. It is to be understood, that the PEG molecule may be
attached to any part of the Factor VIIa polypeptide including any
amino acid residue or carbohydrate moiety of the Factor VIIa
polypeptide. The term "cysteine-PEGylated human Factor VIIa" means
Factor VIIa having a PEG molecule conjugated to a sulfhydryl group
of a cysteine introduced in human Factor VIIa.
[0036] The biological activity of Factor VIIa in blood clotting
derives from its ability to (i) bind to Tissue Factor (TF) and (ii)
catalyze the proteolytic cleavage of Factor IX or Factor X to
produce activated Factor IX or X (Factor IXa or Xa,
respectively).
[0037] For the purposes of the invention, biological activity of
Factor VII polypeptides ("Factor VII biological activity") may be
quantified by measuring the ability of a preparation to promote
blood clotting using Factor VII-deficient plasma and
thromboplastin, as described, e.g., in U.S. Pat. No. 5,997,864 or
WO 92/15686. In this assay, biological activity is expressed as the
reduction in clotting time relative to a control sample and is
converted to "Factor VII units" by comparison with a pooled human
serum standard containing 1 unit/mL Factor VII activity.
Alternatively, Factor VIIa biological activity may be quantified by
(i) measuring the ability of Factor VIIa (or the Factor VII
polypeptide) to produce activated Factor X (Factor Xa) in a system
comprising TF embedded in a lipid membrane and Factor X. (Persson
et al., J. Biol. Chem. 272:19919-19924, 1997); (ii) measuring
Factor X hydrolysis in an aqueous system ("In Vitro Proteolysis
Assay", see below); (iii) measuring the physical binding of Factor
VIIa (or the Factor VII polypeptide) to TF using an instrument
based on surface plasmon resonance (Persson, FEBS Letts.
413:359-363, 1997); (iv) measuring hydrolysis of a synthetic
substrate by Factor VIIa (or a Factor VII polypeptide) ("In Vitro
Hydrolysis Assay", see below); or (v) measuring generation of
thrombin in a TF-independent in vitro system.
[0038] Factor VII variants having substantially the same or
improved biological activity relative to wild-type Factor VIIa
encompass those that exhibit at least about 25%, such as at least
about 50%, such as at least about 75%, such as at least about 90%
of the specific activity of Factor VIIa that has been produced in
the same cell type, when tested in one or more of a clotting assay,
proteolysis assay, or TF binding assay as described above. In one
embodiment the biological activity is more than 80% of the
biological activity of recombinant wild type human Factor VIIa. In
another embodiment the biological activity is more than 90% of the
biological activity of recombinant wild type human Factor VIIa. In
a further embodiment the biological activity is more than 100% of
the biological activity of recombinant wild type human Factor VIIa.
In a further embodiment the biological activity is more than 120%
of the biological activity of recombinant wild type human Factor
VIIa. In a further embodiment the biological activity is more than
200% of the biological activity of recombinant wild type human
Factor VIIa. In a further embodiment the biological activity is
more than 400% of the biological activity of recombinant wild type
human Factor VIIa.
[0039] Factor VII variants having substantially reduced biological
activity relative to wild-type Factor VIIa are those that exhibit
less than about 25%, such as less than about 10%, such as less than
about 5%, such as less than about 1% of the specific activity of
wild-type Factor VIIa that has been produced in the same cell type
when tested in one or more of a clotting assay, proteolysis assay,
or TF binding assay as described above. Factor VII variants having
a substantially modified biological activity relative to wild-type
Factor VII include, without limitation, Factor VII variants that
exhibit TF-independent Factor X proteolytic activity and those that
bind TF but do not cleave Factor X.
[0040] Variants of Factor VII, whether exhibiting substantially the
same or better bioactivity than wild-type Factor VII, or,
alternatively, exhibiting substantially modified or reduced
bioactivity relative to wild-type Factor VII, include, without
limitation, polypeptides having an amino acid sequence that differs
from the sequence of wild-type Factor VII by insertion, deletion,
or substitution of one or more amino acids.
[0041] Non-limiting examples of Factor VII variants having
substantially the same biological activity as wild-type Factor VII
include S52A-FVIIa, S60A-FVIIa (Lino et al., Arch. Biochem.
Biophys. 352: 182-192, 1998); Factor VIIa variants exhibiting
increased proteolytic stability as disclosed in U.S. Pat. No.
5,580,560; Factor VIIa that has been proteolytically cleaved
between residues 290 and 291 or between residues 315 and 316
(Mollerup et al., Biotechnol. Bioeng. 48:501-505, 1995); oxidized
forms of Factor VIIa (Kornfelt et al., Arch. Biochem. Biophys.
363:43-54, 1999); Factor VII variants as disclosed in
PCT/DK02/00189; and Factor VII variants exhibiting increased
proteolytic stability as disclosed in WO 02/38162 (Scripps Research
Institute); Factor VII variants having a modified Gla-domain and
exhibiting an enhanced membrane binding as disclosed in WO
99/20767, US patents U.S. Pat. Nos. 6,017,882 and 6,747,003, US
patent application 20030100506 (University of Minnesota) and WO
00/66753, US patent applications US 20010018414, US 2004220106, and
US 200131005, U.S. Pat. Nos. 6,762,286 and 6,693,075 (University of
Minnesota); and Factor VII variants as disclosed in WO 01/58935,
U.S. Pat. No. 6,806,063, US patent application 20030096338 (Maxygen
ApS), WO 03/93465 (Maxygen ApS) and WO 04/029091 (Maxygen ApS).
[0042] Non-limiting examples of Factor VII variants having
increased biological activity compared to wild-type Factor VIIa
include Factor VII variants as disclosed in WO 01/83725, WO
02/22776, WO 02/077218, WO 03/27147, WO 03/37932; WO 02/38162
(Scripps Research Institute); and Factor VIIa variants with
enhanced activity as disclosed in JP 2001061479
(Chemo-Sero-Therapeutic Res Inst.).
[0043] Non-limiting examples of Factor VII variants having
substantially reduced or modified biological activity relative to
wild-type Factor VII include R152E-FVIIa (Wildgoose et al., Biochem
29:3413-3420, 1990), S344A-FVIIa (Kazama et al., J. Biol. Chem.
270:66-72, 1995), FFR-FVIIa (Hoist et al., Eur. J. Vasc. Endovasc.
Surg. 15:515-520, 1998), and Factor VIIa lacking the Gla domain,
(Nicolaisen et al., FEBS Letts. 317:245-249, 1993).
[0044] Explicit examples of Factor VII polypeptides include,
without limitation, wild-type Factor VII, L305V-FVII,
L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII,
V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII,
V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII,
V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII,
K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII,
V158D/M298K-FVII, and S336G-FVII, L305V/K337A-FVII,
L305V/V158D-FVII, L305V/E296V-FVII, L305V/M298Q-FVII,
L305V/V158T-FVII, L305V/K337A/V158T-FVII, L305V/K337A/M298Q-FVII,
L305V/K337A/E296V-FVII, L305V/K337A/V158D-FVII,
L305V/V158D/M298Q-FVII, L305V/V158D/E296V-FVII,
L305V/V158T/M298Q-FVII, L305V/V158T/E296V-FVII,
L305V/E296V/M298Q-FVII, L305V/V158D/E296V/M298Q-FVII,
L305V/V158T/E296V/M298Q-FVII, L305V/V158T/K337A/M298Q-FVII,
L305V/V158T/E296V/K337A-FVII, L305V/V158D/K337A/M298Q-FVII,
L305V/V158D/E296V/K337A-FVII, L305V/V158D/E296V/M298Q/K337A-FVII,
L305V/V158T/E296V/M298Q/K337A-FVII, S314E/K316H-FVII,
S314E/K316Q-FVII, S314E/L305V-FVII, S314E/K337A-FVII,
S314E/V158D-FVII, S314E/E296V-FVII, S314E/M298Q-FVII,
S314E/V158T-FVII, K316H/L305V-FVII, K316H/K337A-FVII,
K316H/V158D-FVII, K316H/E296V-FVII, K316H/M298Q-FVII,
K316H/V158T-FVII, K316Q/L305V-FVII, K316Q/K337A-FVII,
K316Q/V158D-FVII, K316Q/E296V-FVII, K316Q/M298Q-FVII,
K316Q/V158T-FVII, S314E/L305V/K337A-FVII, S314E/L305V/V158D-FVII,
S314E/L305V/E296V-FVII, S314E/L305V/M298Q-FVII,
S314E/L305V/V158T-FVII, S314E/L305V/K337A/V158T-FVII,
S314E/L305V/K337A/M298Q-FVII, S314E/L305V/K337A/E296V-FVII,
S314E/L305V/K337A/V158D-FVII, S314E/L305V/V158D/M298Q-FVII,
S314E/L305V/V158D/E296V-FVII, S314E/L305V/V158T/M298Q-FVII,
S314E/L305V/V158T/E296V-FVII, S314E/L305V/E296V/M298Q-FVII,
S314E/L305V/V158D/E296V/M298Q-FVII,
S314E/L305V/V158T/E296V/M298Q-FVII,
S314E/L305V/V158T/K337A/M298Q-FVII,
S314E/L305V/V158T/E296V/K337A-FVII,
S314E/L305V/V158D/K337A/M298Q-FVII,
S314E/L305V/V158D/E296V/K337A-FVII,
S314E/L305V/V158D/E296V/M298Q/K337A-FVII,
S314E/L305V/V158T/E296V/M298Q/K337A-FVII, K316H/L305V/K337A-FVII,
K316H/L305V/V158D-FVII, K316H/L305V/E296V-FVII,
K316H/L305V/M298Q-FVII, K316H/L305V/V158T-FVII,
K316H/L305V/K337A/V158T-FVII, K316H/L305V/K337A/M298Q-FVII,
K316H/L305V/K337A/E296V-FVII, K316H/L305V/K337A/V158D-FVII,
K316H/L305V/V158D/M298Q-FVII, K316H/L305V/V158D/E296V-FVII,
K316H/L305V/V158T/M298Q-FVII, K316H/L305V/V158T/E296V-FVII,
K316H/L305V/E296V/M298Q-FVII, K316H/L305V/V158D/E296V/M298Q-FVII,
K316H/L305V/V158T/E296V/M298Q-FVII,
K316H/L305V/V158T/K337A/M298Q-FVII,
K316H/L305V/V158T/E296V/K337A-FVII,
K316H/L305V/V158D/K337A/M298Q-FVII, K316H/L305V/V158D/E296V/K337A
-FVII, K316H/L305V/V158D/E296V/M298Q/K337A-FVII,
K316H/L305V/V158T/E296V/M298Q/K337A-FVII, K316Q/L305V/K337A-FVII,
K316Q/L305V/V158D-FVII, K316Q/L305V/E296V-FVII,
K316Q/L305V/M298Q-FVII, K316Q/L305V/V158T-FVII,
K316Q/L305V/K337A/V158T-FVII, K316Q/L305V/K337A/M298Q-FVII,
K316Q/L305V/K337A/E296V-FVII, K316Q/L305V/K337A/V158D-FVII,
K316Q/L305V/V158D/M298Q-FVII, K316Q/L305V/V158D/E296V-FVII,
K316Q/L305V/V1581/M298Q-FVII, K316Q/L305V/V158T/E296V-FVII,
K316Q/L305V/E296V/M298Q-FVII, K316Q/L305V/V158D/E296V/M298Q-FVII,
K316Q/L305V/V158T/E296V/M298Q-FVII, K316Q/L305V/V158T/K337A/ M
298Q-FVII, K316Q/L305V/V158T/E296V/K337A-FVII,
K316Q/L305V/V158D/K337A/M298Q-FVII, K316Q/L305V/V158D/E296V/K337A
-FVII, K316Q/L305V/V158D/E296V/M298Q/K337A-FVII,
K316Q/L305V/V158T/E296V/M298Q/K337A-FVII, F374Y/K337A-FVII,
F374Y/V158D-FVII, F374Y/E296V-FVII, F374Y/M298Q-FVII,
F374Y/V158T-FVII, F374Y/S314E-FVII, F374Y/L305V-FVII,
F374Y/L305V/K337A-FVII, F374Y/L305V/V158D-FVII,
F374Y/L305V/E296V-FVII, F374Y/L305V/M298Q-FVII,
F374Y/L305V/V158T-FVII, F374Y/L305V/S314E-FVII,
F374Y/K337A/S314E-FVII, F374Y/K337A/V158T-FVII,
F374Y/K337A/M298Q-FVII, F374Y/K337A/E296V-FVII,
F374Y/K337A/V158D-FVII, F374Y/V158D/S314E-FVII,
F374Y/V158D/M298Q-FVII, F374Y/V158D/E296V-FVII,
F374Y/V158T/S314E-FVII, F374Y/V158T/M298Q-FVII,
F374Y/V158T/E296V-FVII, F374Y/E296V/S314E-FVII,
F374Y/S314E/M298Q-FVII, F374Y/E296V/M298Q-FVII,
F374Y/L305V/K337A/V158D-FVII, F374Y/L305V/K337A/E296V-FVII,
F374Y/L305V/K337A/M298Q-FVII, F374Y/L305V/K337A/V158T-FVII,
F374Y/L305V/K337A/S314E-FVII, F374Y/L305V/V158D/E296V-FVII,
F374Y/L305V/V158D/M298Q-FVII, F374Y/L305V/V158D/S314E-FVII,
F374Y/L305V/E296V/M298Q-FVII, F374Y/L305V/E296V/V158T-FVII,
F374Y/L305V/E296V/S314E-FVII, F374Y/L305V/M298Q/V158T-FVII,
F374Y/L305V/M298Q/S314E-FVII, F374Y/L305V/V158T/S314E-FVII,
F374Y/K337A/S314E/V158T-FVII, F374Y/K337A/S314E/M298Q-FVII,
F374Y/K337A/S314E/E296V-FVII, F374Y/K337A/S314E/V158D-FVII,
F374Y/K337A/V158T/M298Q-FVII, F374Y/K337A/V158T/E296V-FVII,
F374Y/K337A/M298Q/E296V-FVII, F374Y/K337A/M298Q/V158D-FVII,
F374Y/K337A/E296V/V158D-FVII, F374Y/V158D/S314E/M298Q-FVII,
F374Y/V158D/S314E/E296V-FVII, F374Y/V158D/M298Q/E296V-FVII,
F374Y/V158T/S314E/E296V-FVII, F374Y/V158T/S314E/M298Q-FVII,
F374Y/V158T/M298Q/E296V-FVII, F374Y/E296V/S314E/M298Q-FVII,
F374Y/L305V/M298Q/K337A/S314E-FVII,
F374Y/L305V/E296V/K337A/S314E-FVII,
F374Y/E296V/M298Q/K337A/S314E-FVII, F374Y/L305V/E296V/M298Q/K337A
-FVII, F374Y/L305V/E296V/M298Q/S314E-FVII,
F374Y/V158D/E296V/M298Q/K337A-FVII,
F374Y/V158D/E296V/M298Q/S314E-FVII,
F374Y/L305V/V158D/K337A/S314E-FVII,
F374Y/V158D/M298Q/K337A/S314E-FVII,
F374Y/V158D/E296V/K337A/S314E-FVII,
F374Y/L305V/V158D/E296V/M298Q-FVII,
F374Y/L305V/V158D/M298Q/K337A-FVII,
F374Y/L305V/V158D/E296V/K337A-FVII,
F374Y/L305V/V158D/M298Q/S314E-FVII,
F374Y/L305V/V158D/E296V/S314E-FVII,
F374Y/V158T/E296V/M298Q/K337A-FVII,
F374Y/V158T/E296V/M298Q/S314E-FVII,
F374Y/L305V/V158T/K337A/S314E-FVII,
F374Y/V158T/M298Q/K337A/S314E-FVII,
F374Y/V158T/E296V/K337A/S314E-FVII,
F374Y/L305V/V158T/E296V/M298Q-FVII,
F374Y/L305V/V158T/M298Q/K337A-FVII,
F374Y/L305V/V158T/E296V/K337A-FVII,
F374Y/L305V/V158T/M298Q/S314E-FVII,
F374Y/L305V/V158T/E296V/S314E-FVII,
F374Y/E296V/M298Q/K337A/V158T/S314E-FVII,
F374Y/V158D/E296V/M298Q/K337A/S314E-FVII,
F374Y/L305V/V158D/E296V/M298Q/S314E-FVII,
F374Y/L305V/E296V/M298Q/V158T/S314E-FVII,
F374Y/L305V/E296V/M298Q/K337A/V158T-FVII,
F374Y/L305V/E296V/K337A/V158T/S314E-FVII,
F374Y/L305V/M298Q/K337A/V158T/S314E-FVII,
F374Y/L305V/V158D/E296V/M298Q/K337A-FVII,
F374Y/L305V/V158D/E296V/K337A/S314E-FVII,
F374Y/L305V/V158D/M298Q/K337A/S314E-FVII,
F374Y/L305V/E296V/M298Q/K337A/V158T/S314E-FVII,
F374Y/L305V/V158D/E296V/M298Q/K337A/S314E-FVII, S52A-Factor VII,
S60A-Factor VII; R152E-Factor VII, S344A-Factor VII, Factor VIIa
lacking the Gla domain; and P11Q/K33E-FVII, T106N-FVII,
K143N/N145T-FVII, V253N-FVII, R290N/A292T-FVII, G291N-FVII,
R315N/V317T-FVII, K143N/N145T/R315N/V317T-FVII; and Factor VII
having substitutions, additions or deletions in the amino acid
sequence from 233Thr to 240Asn, Factor VII having substitutions,
additions or deletions in the amino acid sequence from 304Arg to
329Cys.
[0045] In some embodiments, the Factor VIIa polypeptide is human
Factor VIIa (hFVIIa), such as recombinantly made human Factor VIIa
(rhFVIIa). In other embodiments, the one or more Factor VII
polypeptides comprise a Factor VII sequence variant. In some
embodiments, the one or more Factor VII polypeptides have a
glycosylation different from wild-type Factor VII.
Nanofiltration
[0046] The liquid Factor VII composition is subjected to
nanofiltration using a nanofilter having a pore size of at the most
80 nm. The pore size of the nanofilter is more particularly at the
most 50 nm, e.g. at the most 30 nm, such as in the range of 10-30
nm.
[0047] The term "pore size" typically means the size of the
smallest viruses that are withheld by the filter.
[0048] Examples of suitable commercially available nanofilters are
Asahi Planove 15 N, Asahi Planove 20 N, Asahi Planova 35 N, and
Asahi Planova 75 N, all from Asahi Chemical, Tokyo, Japan;
Millipore NFR, Millipore NFP, Millipore Viresolve 70, and Millipore
Viresolve 180, all from Millipore; and Pall DV20, Pall DV 50, Pall
Omega VR 100 K; and Bemberg Microporous Membrane-15 nm
(BMM-15).
[0049] The nanofilter membrane may, e.g., be manufactured from one
or more materials selected from cuprammonium regenerated cellulose,
hydrophilic polyvinylidene fluoride (PVDF), composite PVDF, surface
modified PVDF, polyether sulfone and similar materials. In one
embodiment, the material is selected from polyvinylidene
fluoride-based materials and polyether sulfone-based materials.
[0050] The nanofiltration may be conducted by in the tangential
filtration mode or in the dead-end filtration mode as will be
understood by the skilled artisan. In one embodiment, the
nanofiltration is conducted in the dead-end filtration mode.
[0051] The pH value of the liquid Factor VII composition upon
nanofiltration is not considered particularly critical. Thus, the
pH value is normally given by in view of the conditions applied in
the process steps immediately preceding the nanofiltration step. In
some embodiments, the pH value is adjusted so that the liquid
composition has a pH of in the range of 5.5-10, such as in the
range of 7.0-9.5, e.g. in the range of 7.6-9.4, such as in the
range of 7.7-9.3, e.g. in the range of 8.0-9.0 or in the range of
8.3-8.7. In one embodiment the pH is in the range of 5-7. In one
embodiment the pH is higher than 9.5, such as in the range of
9.5-10.
[0052] Furthermore, the concentration of the Factor VII polypeptide
in the liquid composition is typically also given by the preceding
process steps, but will normally lie in the range of 0.01-5 mg/mL,
such as in the range of 0.05-2.0 mg/mL.
[0053] The nanofiltration process may be conducted using a
filtration system as illustrated in FIG. 1.
[0054] The process may be conducted as in the following
illustrative example: The pressure tank (1) is filled with water
for injection (WFI), and the pressure in the tank is raised to 3.5
bars before the virus filter (3), and the filter is flushed for 10
minutes. The pressure is reduced to 2 bars and the virus filter (3)
is flushed for another 10 minutes. The pressure tank (1) is emptied
from WFI and the process is optionally repeated with a buffer
before the liquid Factor VII composition is filled into the
pressure tank (1). The pressure is raised to 2 bars and is kept
substantially constant during the filtration. The virus filter (3)
may subsequently be tested for integrity by standard
procedures.
[0055] The filtrate is collected in a pool tank and can further be
processed in order to obtain a pharmaceutical composition
comprising a Factor VIIa polypeptide as a drug substance.
[0056] This being said, it is typically advantageous to apply a
pre-filtration step before the nanofiltration step in order to
remove larger particles, aggregates, etc. that would otherwise
cause the nanofilter to become clogged. Such a pre-filter typically
has a pore size of at in the range of 0.05-0.5 pm. In one
embodiment the pre-filter is Millipore NFR filter.
[0057] Alternatively to using air pressure, a liquid pump placed
after the pressure tank may provide the necessary pressure for the
filtration.
[0058] If the nanofiltered liquid Factor VII composition comprises
inactive Factor VII polypeptides, the composition may subsequently
be subjected to an activation step, e.g. as described in Bjorn. S.
& Thim, L. Res. Disclosures (1986) 269, 564-565, Pedersen, A.
H. & al., Biochemistry (1989), 28, 9331-9336, and Tomokiyo, K.
& al., Vox Sang. 84, 54-64 (2003).
[0059] Further processing of the composition and final formulation
as a pharmaceutical product may be conducted as disclosed in
Jurlander, B. & al., Seminars in Thrombosis and Hemostasis 27,
4, 373-383 (2001).
Nanofiltration of Serum-Free Liquid Factor VII Polypeptide
Compositions
[0060] One separate aspect of the invention, which may include some
or all of the above characteristics, relates to a method for
removing viruses from a liquid Factor VII composition, said
composition comprising one or more Factor VII polypeptides, said
liquid composition being substantially serum-free, said method
comprising subjecting said solution to nanofiltration using a
nanofilter having a pore size of at the most 80 nm.
[0061] An attractive variant hereof is the one where the Factor VII
polypeptide(s) is/are produced by cell culture in CHO cells, e.g.
in CHO cells in a medium free from any components of animal
origin.
[0062] This aspect of the invention is not particularly limited to
liquid Factor VII compositions in which a certain proportion of the
Factor VII polypeptide(s) is/are in the activated form. However,
the conditions mentioned above for the first aspect of the
invention also applies for this, the second aspect of the
invention, mutatis mutandis.
Nanofiltration of Liquid Factor VII Polypeptide Compositions via
Particular Filters
[0063] Another separate aspect of the invention, which may include
some or all of the above characteristics, relates to a method for
removing viruses from a liquid Factor VII composition, said
composition comprising one or more Factor VII polypeptides, said
method comprising subjecting said solution to nanofiltration using
a nanofilter having a pore size of at the most 80 nm, said
nanofilter having a membrane manufactured from one or more
materials selected from cuprammonium regenerated cellulose,
hydrophilic polyvinylidene fluoride (PVDF), composite PVDF, surface
modified PVDF, and polyether sulfone.
[0064] In one embodiment, the material is selected from
polyvinylidene fluoride-based materials and polyether sulfone-based
materials.
[0065] This aspect of the invention is not particularly limited to
liquid Factor VII compositions in which a certain proportion of the
Factor VII polypeptide(s) is/are in the activated form. However,
the conditions mentioned above for the first aspect of the
invention also applies for this, the third aspect of the invention,
mutatis mutandis.
Virus Inactivation by Addition a Detergent
[0066] In another aspect, the present invention also relates to a
method for inactivating viruses in a liquid Factor VII composition,
said composition comprising one or more Factor VII polypeptides,
the method comprising the step of combining said composition with a
detergent.
[0067] In some embodiments, the detergent is selected from
non-ionic detergents such as those selected from octylphenoxy
polyethoxyethanol, polysorbates, poloxamers, polyoxyethylene alkyl
ethers, polyethylene/polypropylene block co-polymers,
polyethyleneglycol (PEG), polyoxyethylene stearates, and
polyoxyethylene castor oils. Illustrative examples hereof are
non-ionic detergents are Triton X-100, Tween.RTM., polysorbate 20,
polysorbate 60, polysorbate 80, Brij-35 (polyoxyethylene dodecyl
ether), poloxamer 188, poloxamer 407, PEG8000, Pluronic.RTM.
polyols, polyoxy-23-lauryl ether, Myrj 49, and Cremophor A.
[0068] A particularly useful detergent is a octylphenoxy
polyethoxyethanol of the formula
p-((CH.sub.3).sub.3CH.sub.2C(CH.sub.2).sub.2)--C.sub.6H.sub.4--O--(CH.sub-
.2CH.sub.2O).sub.n--H wherein n is in the range of 5-15, in
particular one where n is 9-10, such the detergent Triton
X-100.
[0069] In one embodiment, the detergent is combined with the liquid
Factor VII composition to obtain a concentration of the detergent
in the composition of in the range of 0.01-0.5% by weight, such as
in the range of 0.05-0.4% by weight, such as in the range of
0.05-0.3% by weight, such as in the range of 0.05-0.2% by weight,
such as in the range of 0.05-0.1% by weight.
[0070] In a further embodiment, the detergent is combined with the
composition at a temperature of in the range of 2-12.degree. C.,
such as in the range of 2-9.degree. C.
[0071] For most purposes, it is found undesirable to include a
trialkylphosphate detergent, thus, the detergent may be
substantially free of trialkylphosphate solvents such as
tri(n-butyl) phosphate.
[0072] In one particular embodiment, the method comprises the steps
of combining the Factor VII polypeptide composition with Triton
X-100 to a concentration of 0.05-0.2% by weight at a temperature in
the range of 2-9.degree. C., with the proviso that detergent is
substantially free of trialkylphosphate solvents such as
tri(n-butyl)phosphate.
[0073] This aspect of the invention is not particularly limited to
liquid Factor VII compositions in which a certain proportion of the
Factor VII polypeptide(s) is/are in the activated form. However,
the conditions mentioned above for the first aspect of the
invention also applies for this, the fourth aspect of the
invention, mutatis mutandis.
Combination of Virus Inactivation Steps
[0074] In a still further aspect, the present invention relates to
a method for high-level elimination of the presence of active
viruses in a liquid Factor VII composition, the method comprising
the steps of (i) inactivating viruses by the method defined under
"Virus inactivation by addition a detergent", and (ii) removing
viruses by the any of the methods defined herein under
"Nanofiltration", in any order.
[0075] In one embodiment, the step of inactivating viruses precedes
the step of removing viruses.
[0076] Even though the individual steps are believed to be
sufficient for the purpose of eliminating the presence of active
viruses, the two methods can be considered as at least partially
"orthogonal" in the sense that certain viruses may be more
difficult to eliminate by one of the methods, whereas the same of
the certain viruses can more easily be eliminated by the other
method, and vice versa. Thus, combination of the two methods will
provide an even higher level of safety for the patient for which
the Factor VII polypeptide is intended, and not the least for the
medical doctor prescribing the Factor VII polypeptide medicament,
and for the regulatory authorities approving the medicament. Thus,
the combination of the two methods may have a high commercial
value.
[0077] As described above the present invention relates to the
removal or inactivation of virus particles. The reduction of the
amount of virus particles at a particular process step is usually
described in log-units (log 10 logarithm, or log.sub.10) , wherein
the reduction factor is calculated as the amount of virus particles
after the step relative to the amount of virus particles before the
process step.
[0078] E.g. if 10.sup.6 virus particles are found before a step and
10.sup.2 are found after the step, the reduction is 10.sup.4, or 4
log.sub.10.
[0079] The total reduction of virus particles from the complete
process is described in the same way and may be calculated by
addition of the virus clearance from each step in the process, the
word "clearance" meaning both removal of virus and inactivation of
virus
[0080] For a specific virus clearance step to be effective, it is
preferred to have a virus reduction of at least 4 log.sub.10.
[0081] In one embodiment of the present invention, a filtration
step reduces the amount of virus particles with at least about 4
log.sub.10. In one embodiment of the present invention, a
filtration step reduces the amount of virus particles with at least
about 5 log.sub.10.
[0082] In one embodiment of the present invention, a step of
combining said FVII composition with a detergent inactivates at
least about 4 log.sub.10 of virus. In one embodiment of the present
invention, a step of combining said FVII composition with a
detergent inactivates at least about 5 log.sub.10 of virus.
[0083] The determination of the amount of virus particles is known
to the person skilled in the art and may be measured in standard
TCID.sub.50 assays (Tissue culture infectious dose 50% endpoint per
mL), plaque assays or PCR assays. TCID.sub.50 and plaque assays may
be used to measure the concentration of infectious particles,
whereas the PCR assays may be used to measure both infectious and
non-infectious inactivated virus particles
Embodiments of the Present Invention:
[0084] 1. A method for removing viruses from a liquid Factor VII
composition, said composition comprising one or more Factor VII
polypeptides, at least 5% of said one or more Factor VII
polypeptides being in the activated form, said method comprising
subjecting said solution to nanofiltration using a nanofilter
having a pore size of at the most 80 nm. [0085] 2. The method
according to embodiment 1, wherein as at least 7%, e.g. at least
10%, of the one or more Factor VII polypeptides are in the
activated form. [0086] 3. The method according to embodiment 1,
wherein the activated form of the Factor VII polypeptide represents
5-70%, such as 7-40%, e.g. 10-30%, of the mass of the one or more
Factor VII polypeptides. [0087] 4. The method according to
embodiment 1, wherein the activated form of the Factor VII
polypeptide represents 50-100%, such as 70-100%, e.g. 80-100%, of
the mass of the one or more Factor VII polypeptides. [0088] 5. The
method according to embodiment 1, wherein the activated form of the
Factor VII polypeptide represents 20-80%, such as 30-70%, e.g.
30-60%, of the mass of the one or more Factor VII polypeptides.
[0089] 6. The method according to any of the preceding embodiments,
wherein the liquid composition has a pH of in the range of 7.0-9.5,
e.g. in the range of 7.6-9.4, such as in the range of 7.7-9.3, e.g.
in the range of 8.0-9.0 or in the range of 8.3-8.7. [0090] 7. The
method according to any of the preceding embodiments, wherein the
concentration of the Factor VII polypeptide(s) in the liquid
composition is in the range of 0.01-5 mg/mL, such as in the range
of 0.05-2.0 mg/mL. [0091] 8. The method according to any of the
preceding embodiments, wherein the pore size of the nanofilter is
at the most 50 nm, e.g. at the most 30 nm, such as in the range of
10-30 nm. [0092] 9. The method according to any of the preceding
embodiments, wherein the membrane of the nanofilter is manufactured
from one or more materials selected from cuprammonium regenerated
cellulose, hydrophilic polyvinylidene fluoride (PVDF), composite
PVDF, surface modified PVDF, and polyether sulfone. [0093] 10. The
method according to any of the preceding embodiments, wherein the
liquid Factor VII composition is obtained, or originates, from a
cell culture supernatant. [0094] 11. The method according to any of
the preceding embodiments, wherein the liquid composition is
substantially serum-free. [0095] 12. The method according to any of
the embodiments 1-10, wherein the Factor VII polypeptide(s) is/are
produced by cell culture in the presence of bovine or foetal calf
serum. [0096] 13. The method according to any of the preceding
embodiments, wherein the Factor VII polypeptide(s) is/are produced
by cell culture in CHO cells. [0097] 14. The method according to
embodiment 13, wherein the Factor VII polypeptide(s) is/are
produced by cell culture in CHO cells, in a medium free from any
components of animal origin. [0098] 15. A method for removing
viruses from a liquid Factor VII composition, said composition
comprising one or more Factor VII polypeptides, said liquid
composition being substantially serum-free, said method comprising
subjecting said solution to nanofiltration using a nanofilter
having a pore size of at the most 80 nm. [0099] 16. The method
according to embodiment 15, wherein the liquid Factor VII
composition is obtained, or originates, from a cell culture
supernatant. [0100] 17. The method according to any of the
embodiments 15-16, wherein the Factor VII polypeptide(s) is/are
produced by cell culture in CHO cells. [0101] 18. The method
according to embodiment 17, wherein the Factor VII polypeptide(s)
is/are produced by cell culture in CHO cells, in a medium free from
any components of animal origin. [0102] 19. The method according to
any of the embodiment 15-18, wherein at least 5% of said one or
more Factor VII polypeptides are in the activated form. [0103] 20.
The method according to any of the embodiments 15-19, wherein the
liquid composition has a pH of in the range of 7.0-9.5, e.g. in the
range of 7.6-9.4, such as in the range of 7.7-9.3, e.g. in the
range of 8.0-9.0 or in the range of 8.3-8.7. [0104] 21. The method
according to any of the embodiments 15-20, wherein the
concentration of the Factor VII polypeptide(s) in the liquid
composition is in the range of 0.01-5 mg/mL, such as in the range
of 0.05-2.0 mg/mL. [0105] 22. The method according to any of the
embodiments 15-21, wherein the pore size of the nanofilter is at
the most 50 nm, e.g. at the most 30 nm, such as in the range of
10-30 nm. [0106] 23. The method according to any of the embodiments
15-22, wherein the membrane of the nanofilter is manufactured from
one or more materials selected from cuprammonium regenerated
cellulose, hydrophilic polyvinylidene fluoride (PVDF), composite
PVDF, surface modified PVDF, and polyether sulfone. [0107] 24. A
method for removing viruses from a liquid Factor VII composition,
said composition comprising one or more Factor VII polypeptides,
said method comprising subjecting said solution to nanofiltration
using a nanofilter having a pore size of at the most 80 nm, said
nanofilter having a membrane manufactured from one or more
materials selected from cuprammonium regenerated cellulose,
hydrophilic polyvinylidene fluoride (PVDF), composite PVDF, surface
modified PVDF, and polyether sulfone. [0108] 25. The method
according to embodiment 24, wherein the material is selected from
polyvinylidene fluoride-based materials and polyether sulfone-based
materials. [0109] 26. A method according to any of the embodiments
24-25, wherein at least 5% of the one or more Factor VII
polypeptides is/are in the activated form. [0110] 27. The method
according to any of the embodiments 24-26, wherein the liquid
composition has a pH of in the range of 7.0-9.5, e.g. in the range
of 7.6-9.4, such as in the range of 7.7-9.3, e.g. in the range of
8.0-9.0 or in the range of 8.3-8.7. [0111] 28. The method according
to any of the embodiments 24-27, wherein the concentration of the
Factor VII polypeptide(s) in the liquid composition is in the range
of 0.01-5 mg/mL, such as in the range of 0.05-2.0 mg/mL. [0112] 29.
The method according to any of the embodiments 24-28, wherein the
pore size of the nanofilter is at the most 50 nm, e.g. at the most
30 nm, such as in the range of 10-30 nm. [0113] 30. The method
according to any of the embodiments 24-29, wherein the liquid
Factor VII composition is obtained, or originates, from a cell
culture supernatant. [0114] 31. The method according to any of the
embodiments 24-30, wherein the liquid composition is substantially
serum-free. [0115] 32. The method according to any of the
embodiments 24-31, wherein the Factor VII polypeptide(s) is/are
produced by cell culture in the presence of bovine or foetal calf
serum. [0116] 33. The method according to any of the embodiments
24-32, wherein the Factor VII polypeptide(s) is/are produced by
cell culture in CHO cells. [0117] 34. The method according to
embodiment 33, wherein the Factor VII polypeptide(s) is/are
produced by cell culture in CHO cells, in a medium free from any
components of animal origin. [0118] 35. A method for inactivating
viruses in a liquid Factor VII composition, said composition
comprising one or more Factor VII polypeptides, the method
comprising the step of combining said composition with a detergent.
[0119] 36. The method according to embodiment 35, wherein the
detergent is a octylphenoxy polyethoxyethanol of the formula
p-((CH.sub.3).sub.3CH.sub.2C(CH.sub.2).sub.2)--C.sub.6H.sub.4--O--(CH.sub-
.2CH.sub.2O).sub.n--H wherein n is in the range of 5-15. [0120] 37.
The method according to embodiment 36, wherein the detergent is one
where n is 9-10, such as Triton X-100. [0121] 38. The method
according to embodiment 35, wherein the detergent is selected from
the list consisting of Tween.RTM., polysorbate 20, polysorbate 60,
and polysorbate 80. [0122] 39. The method according to any of the
embodiment 35-38, wherein the detergent is combined with the liquid
Factor VII composition to obtain a concentration of the detergent
in the composition of in the range of 0.01-0.3% by weight, such as
in the range of 0.05-0.2% by weight. [0123] 40. The method
according to any of the embodiments 35-39, wherein the detergent is
combined with the composition at a temperature of in the range of
2-12.degree. C., such as in the range of 2-9.degree. C. [0124] 41.
The method according to any of the embodiments 35-40, wherein the
detergent is substantially free of trialkylphosphate solvents such
as tri(n-butyl)phosphate. [0125] 42. A method for high-level
elimination of the presence of active viruses in a liquid Factor
VII composition, the method comprising the steps of (i)
inactivating viruses by the method defined in any of the
embodiments 35-41, and (ii) removing viruses by the any of the
methods defined in any of the embodiments 1-35, in any order.
[0126] 43. The method according to embodiment 42, wherein the step
of inactivating viruses precedes the step of removing viruses.
EXAMPLES
Example 1
Serum-free Production of Factor VII
[0127] The following experiment was performed to produce Factor VII
in large pilot-scale culture.
[0128] A CHO K1 cell line transformed with a Factor VII-encoding
plasmid was adapted to growth in suspension culture in a medium
free of animal derived components. A bank of the adapted cells was
frozen. Cells from the bank were propagated in spinner bottles in
suspension culture in medium free of animal derived components. As
the cell number increased, the volume was gradually increased by
addition of new medium. When the volume had reached 4 L, and the
cell number had reached .apprxeq.0.8 * 10.sup.6/ml, the contents of
the spinner bottles were transferred into a 50 L stirred tank
reactor (seed reactor). As the cell number increased in the 50 L
reactor, the volume was gradually increased by addition of new
medium. When the volume had reached 50 L, and the cell number had
reached 1.times.10.sup.6/ml, the contents of the 50 L reactor were
transferred into a 500 L stirred tank reactor (production reactor).
The 500 L reactor contained macroporous Cytopore 1 carriers
(Amersham Biosciences) within which the cells became immobilized
within 24 hours after inoculation. The volume in the 500 L reactor
was gradually increased by addition of new medium as the cell
number increased. When the volume had reached 450 L, and the cell
number had reached 2.times.10.sup.6/ml, the production phase was
initiated and a medium change was performed every 24 hours:
Agitation was stopped to allow for sedimentation of the
cell-containing carriers, and 80% of the culture supernatant was
then harvested and replaced with new medium. The harvested culture
supernatant was filtered to remove non-trapped cells and cell
debris and was then transferred for further processing. The 50 L as
well as the 500 L bioreactor was instrumented for control of
temperature, dissolved oxygen (sparging of oxygen through
microsparger), agitation rate, headspace aeration rate and pH
(downwards control by addition of CO.sub.2 gas to headspace).
Furthermore, the 500 L bioreactor was instrumented for control of
dissolved CO.sub.2. Online CO.sub.2 measurement was performed by
means of an YSI 8500 CO.sub.2-instrument. The level of CO.sub.2 was
controlled by sparging of atmospheric air into the liquid through a
tube according to the CO.sub.2 signal. The sparging rate was set to
0 L/min per L of culture liquid when the CO.sub.2 concentration was
at or below the set-point, and to 0.01-0.05 L/min per L of culture
liquid when the CO.sub.2 concentration was above the set-point. The
set-point for dissolved CO.sub.2 was 160 mmHg. As mentioned, no
base was added to the bioreactor to control pH upwards. During the
production phase the cell density reached 1-2.times.10.sup.7
cells/ml, and the Factor VII concentration in the daily harvest
10-20 mg/L. The pCO.sub.2 was maintained within the range of
150-170 mmHg. The pH was kept above 6.70, even though no base was
added.
Example 2
Filtration of Eluate from the Capture Step
[0129] Protein Solution to be Filtered : 25 L of FVII solution from
the Capture Step, with the following Characteristics [0130]
Concentration of FVII/FVIIa : 630 mg/L [0131] 1,7% of oxidized
forms of FVII [0132] Degree of activation (i.e. percentage of
FVIIa) : not analysed [0133] Degradation : <2,2% The Filtration
was Conducted Essentially as Described herein with Reference to
FIG. 1: [0134] Filter : Millipore NFR, 0,08 m2 [0135] Pressure : 2
bar
Properties of the Filtrate:
[0135] [0136] Concentration of FVII/FVIIa : 610 mg/L, i.e. : yield
of FVII : 96,8% [0137] 1,5% of oxidized forms of FVII [0138] Degree
of activation (i.e. percentage of FVIIa) : not analysed. [0139]
Degradation : <2,2%
Example 3
Filtration of Eluate from the Capture Step
[0140] Protein Solution to be Filtered :185 ml of FVII Solution
from the Capture Step, with the following Characteristics [0141]
Concentration of FVII/FVIIa : 82 mg/L [0142] 3,4% of oxidized forms
of FVII [0143] Degree of activation (i.e. percentage of FVIIa) :
19%. [0144] Degradation : <3% The Filtration was Conducted
Essentially as Described herein with Reference to FIG. 1: [0145]
Filter : Pall DV50, 0,0017 m2 [0146] Pressure : 2 bar
Properties of the Filtrate:
[0146] [0147] Concentration of FVII/FVIIa : 77,1 mg/L, i.e. : yield
of FVII : 94% [0148] 4,1% of oxidized forms of FVII [0149] Degree
of activation (i.e. percentage of FVIIa) : 20%. [0150] Degradation
: <3%
Example 4
Filtration of Eluate from the Capture Step
[0151] Protein Solution to be Filtered : 108 ml Step 1 Eluate with
the following Characteristics : [0152] Concentration of FVII/FVIIa
: 320 mg/L [0153] 3,7% of oxidized forms of FVII [0154] Degree of
activation (i.e. percentage of FVIIa) 3,3% [0155] Degradation :
<0,5% The filtration was Conducted Essentially as Described
herein with Reference to FIG. 1: [0156] Filter : Asahi Planova 20
N, 0,001 m2 [0157] Pressure : 0,8 bar
Properties of the Filtrate:
[0157] [0158] Concentration of FVII/FVIIa : 310 mg/L, i.e. : yield
of FVII : 100% [0159] 3,7% of oxidized forms of FVII [0160] Degree
of activation (i.e. percentage of FVIIa) : not analysed, [0161]
Degradation : <0,5%
Example 5
Filtration of FVII Bulk Drug Substance
[0162] Protein Solution to be Filtered : 98 ml of FVIIa Bulk
Substance, with the following Characteristics [0163] Concentration
of FVII/FVIIa : 1460 mg/L [0164] 2,1% of oxidized forms of FVII
[0165] Degree of activation (i.e. percentage of FVIIa) : >90%.
[0166] Degradation : 11,9% The filtration was Conducted Essentially
as Described herein with Reference to FIG. 1: [0167] Filter :
Millipore NFP, 0,0017 m2 [0168] Pressure : 2 bar
Properties of the Filtrate:
[0168] [0169] Concentration of FVII/FVIIa : 1320 mg/L, i.e. : yield
of FVII : 90,4% [0170] 2,3% of oxidized forms of FVII [0171] Degree
of activation (i.e. percentage of FVIIa) : not analysed, as the
degree of activation in the solution to be filtered is 98% [0172]
Degradation : 12,3%
Example 6
Virus Removal
[0172] [0173] 50 ml of a Factor VII polypeptide solution (see
Example 1) from the capture step comprising a Murine Leukemia
Virus, titer YY plaque-forming units (pfu). The Filtration is
Conducted Essentially as Described herein with Reference to FIG. 1:
[0174] Filter: Millipore NFR, yy cm2 [0175] Pressure: YY bar [0176]
Virus titer in the filtrate: xx pfu [0177] Calculated clearance
factor: xx.
* * * * *