U.S. patent application number 12/339597 was filed with the patent office on 2009-07-16 for substantially animal protein-free recombinant furin and methods for producing same.
This patent application is currently assigned to BAXTER INTERNATIONAL INC.. Invention is credited to Roland Geyer, Leopold Grillberger, Meinhard Hasslacher, Artur Mitterer, BARBARA PLAIMAUER, Manfred Reiter, Simone Von Fircks.
Application Number | 20090181423 12/339597 |
Document ID | / |
Family ID | 40524590 |
Filed Date | 2009-07-16 |
United States Patent
Application |
20090181423 |
Kind Code |
A1 |
PLAIMAUER; BARBARA ; et
al. |
July 16, 2009 |
SUBSTANTIALLY ANIMAL PROTEIN-FREE RECOMBINANT FURIN AND METHODS FOR
PRODUCING SAME
Abstract
The present invention relates to recombinant furin (rFurin) and
methods for producing rFurin. More specifically, the invention
relates to substantially animal protein-free rFurin and methods for
producing substantially animal protein-free rFurin.
Inventors: |
PLAIMAUER; BARBARA; (Vienna,
AT) ; Von Fircks; Simone; (Vienna, AT) ;
Grillberger; Leopold; (Vienna, AT) ; Hasslacher;
Meinhard; (Vienna, AT) ; Geyer; Roland;
(Vienna, AT) ; Mitterer; Artur; (Orth/Donau,
AT) ; Reiter; Manfred; (Vienna, AT) |
Correspondence
Address: |
BAXTER HEALTHCARE CORPORATION
ONE BAXTER PARKWAY, DF2-2E
DEERFIELD
IL
60015
US
|
Assignee: |
BAXTER INTERNATIONAL INC.
DEERFIELD
IL
BAXTER HEALTHCARE SA
WALLISELLEN
|
Family ID: |
40524590 |
Appl. No.: |
12/339597 |
Filed: |
December 19, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61018152 |
Dec 31, 2007 |
|
|
|
Current U.S.
Class: |
435/68.1 ;
435/219 |
Current CPC
Class: |
A61P 7/02 20180101; A61P
7/04 20180101; A61P 43/00 20180101; C12N 9/6454 20130101 |
Class at
Publication: |
435/68.1 ;
435/219 |
International
Class: |
C12P 21/06 20060101
C12P021/06; C12N 9/50 20060101 C12N009/50 |
Claims
1. A composition comprising substantially animal protein-free
recombinant furin.
2. The composition of claim 1 comprising recombinant furin at an
activity of at least about 10000 U furin/mL and CHO host cell
protein at a concentration less than about 11 .mu.g protein/mL.
3. The composition of claim 1 comprising recombinant furin at an
activity of at least about 10000 U furin/mL and CHO host cell
protein at a concentration less than about 1.0 ng protein/U furin
activity.
4. The composition of claim 1 comprising recombinant furin at an
activity of at least about 10000 U furin/mL and CHO host cell DNA
at a concentration less than about 14 ng DNA/mL.
5. The composition of claim 1 comprising recombinant furin at an
activity of at least about 10000 U furin/mL and CHO host cell DNA
at a concentration less than about 0.5 pg DNA/U furin activity.
6. The composition of claim 1 comprising recombinant furin at an
activity of at least about 100 U/.mu.g and CHO host cell protein at
a concentration less than about 11 .mu.g protein/mL.
7. The composition of claim 1 comprising recombinant furin at an
activity of at least about 100 U/.mu.g and CHO host cell protein at
a concentration less than about 1.0 ng protein/U furin
activity.
8. The composition of claim 1 comprising recombinant furin at an
activity of at least about 100 U/.mu.g and CHO host cell DNA at a
concentration less than about 14 ng DNA/mL.
9. The composition of claim 1 comprising recombinant furin at an
activity of at least about 100 U/.mu.g and CHO host cell DNA at a
concentration less than about 0.5 pg DNA/U furin activity.
10. A method of making substantially animal protein-free
recombinant furin comprising the step of growing a host cell
transformed or transfected with a polynucleotide encoding furin in
serum-free medium under conditions that permit secretion of the
furin into the medium.
11. The method of claim 10 comprising the step of adapting the host
cell to grow in medium with increasingly lower concentrations of
serum until all serum is removed from the medium.
12. The method of claim 10 comprising transferring the host cell
from growth in medium comprising serum to growth in serum-free
medium.
13. The method of claim 11 or 12 wherein the host cell is a CHO
cell.
14. A method of using the composition of claim 1 comprising the
step of contacting a pro-protein with the composition under
conditions to cleave a pro-peptide from the pro-protein to form a
mature protein.
15. The method of claim 14 wherein the mature protein is von
Willebrand Factor.
16. The method of claim 14 wherein the mature protein is Factor
VIII.
Description
[0001] This application claims priority of U.S. Provisional
Application No. 61/018,152, filed Dec. 31, 2007, which is
incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The present invention generally relates to recombinant furin
(rFurin) and methods for producing rFurin. More specifically, the
invention relates to substantially animal protein-free rFurin and
methods for producing substantially animal protein-free rFurin.
BACKGROUND OF THE INVENTION
[0003] Active or mature proteins are usually present in very low
amounts in living organisms. Therefore, their pro-proteins or
pro-enzymes are preferably activated in vitro by contacting them
with activation enzymes (e.g. proteases). Pro-proteins (or protein
precursors) are inactive proteins that become active by one or more
posttranslational modifications and, in particular, by the cleavage
of a pro-peptide from the pro-protein. Examples of pro-proteins
include, for example, pro-insulin, prothrombin, pro-von Willebrand
Factor (pro-VWF), and the like.
[0004] Von Willbrand factor (VWF) is a blood glycoprotein involved
in coagulation. VWF is deficient or defective in von Willebrand
disease and is involved in a large number of other diseases,
including thrombotic thrombocytopenic purpura, Heyde's syndrome,
and possibly hemolytic-uremic syndrome. VWF is a glycoprotein
circulating in plasma as a series of multimers ranging in size from
about 500 to 20,000 kD. Multimeric forms of VWF are composed of 250
kD polypeptide subunits linked together by disulfide bonds. VWF
mediates the initial platelet adhesion to the sub-endothelium of
the damaged vessel wall, and it is believed that only the larger
multimers of VWF exhibit hemostatic activity. VWF multimers having
large molecular masses are stored in the Weibel-Pallade bodies of
endothelial cells and are liberated upon stimulation. Liberated VWF
is then further processed by plasma proteases to result in low
molecular weight forms of VWF.
[0005] In humans the removal of the pro-peptide is almost complete,
whereas, in mammalian cell lines with a high level of recombinant
VWF expression, this process is not very efficient. Therefore, cell
culture supernatants from such recombinant cell lines usually
comprise a mixture of mature VWF and VWF precursors, like pro-VWF.
In order to obtain mature VWF, it is therefore necessary to convert
the VWF precursors, in particular pro-VWF, into mature VWF. This
process is usually achieved by cleaving the pro-peptide with a
protease.
[0006] Current conventional methods produce mature VWF by either
incubating its pro-form with proteases in a liquid phase, whereby
the maturation itself (i.e., the cleavage of the pro-peptide from
the pro-protein) occurs in an unbound state in free solution, or as
described, for example, in WO 00/49047, by immobilizing the
protease on a solid carrier, which is contacted and incubated with
a preparation comprising pro-VWF (see e.g. WO 00/49047). However,
these methods comprise various disadvantages over the methods
according to the present invention.
[0007] Industrially, VWF and, in particular, recombinant VWF (rVWF)
is synthesized and expressed together with recombinant Factor VIII
(rFVIII) in a genetically engineered Chinese Hamster ovary (CHO)
cell line. The function of the co-expressed rVWF is to stabilize
rFVIII in the cell culture process. rVWF is synthesized in the cell
in the pro-form, containing a large pro-peptide attached to the
N-terminus. Upon maturation in the endoplasmic reticulum and Golgi
apparatus, the pro-peptide is cleaved by the action of the cellular
protease furin and the mature protein is secreted as a homopolymer
of identical subunits, consisting of dimers of the expressed
protein. However, the maturation is typically incomplete, leading
to a product comprising a mixture of pro-VWF and mature VWF.
[0008] Previous publications have shown that pro-VWF can be
converted to mature VWF by in vitro treatment with furin or
furin-like proteases (Schlokat et al., Biotechnol. Appl. Biochem.
24: 257-267, 1996; Preininger et al., Cytotechnology 30: 1-15,
1999; and EP 0775750A). In particular, EP 0775750A suggests the
co-expression of furin and VWF recombinantly so that the maturation
of VWF may occur in situ.
[0009] Recombinant furin (rFurin) transforms pro-rVWF
(pro-recombinant von Willebrand factor) to rVWF by cleaving the
Arg741-Ser742 peptide bond. This maturation step is part of a rVWF
production process for the treatment of von Willebrand Disease Type
B and part of the manufacturing process for recombinant Factor
VIII-half life (rFVIII-HL). Furin belongs to the family of the
pro-protein convertases and is dependent on calcium (Ca.sup.2+).
Furin specifically cleaves the C-terminal peptide bond of arginine
within a specific sequence, containing arginine at positions -1 and
-4. This sequence can be found in numerous human proteins, showing
that furin plays a major role in the maturation of a number of
human pro-proteins.
[0010] The production of activated proteins is of high clinical and
diagnostic importance. For example, active or mature proteins, like
mature VWF, may be used to control blood coagulation. The present
invention provides improved recombinant furin (rFurin) which is
substantially animal protein-free rFurin for the subsequent
production of activated proteins. More specifically, the present
invention provides substantially animal protein-free rFurin for
transforming pro-VWF into mature VWF.
SUMMARY OF THE INVENTION
[0011] The present invention provides recombinant furin (rFurin),
which is substantially animal protein-free recombinant furin
(rFurin), and methods for producing same. Such rFurin is
substantially free of other proteins which may normally be
associated with the production of rFurin, such as serum proteins
and host cell proteins. This rFurin allows for the subsequent
production of mature proteins with high specific activity and high
purity without side effects associated with protein contaminant in
the rFurin preparation. More specifically, this rFurin allows for
the production of mature VWF with high specific activity and high
purity. Accordingly, the invention provides methods for selection
and adaptation of recombinant host cells to chemically-defined
medium, expression of rFurin which is secreted into cell culture
supernatant, and purification of rFurin after cell removal.
[0012] The substantially animal protein-free rFurin of the
invention includes preparations or compositions of rFurin
comprising host cell protein in a concentration which ranges
between about 0.1 to 0.6 ng protein or less/Unit furin activity or
between about 2 and 11 .mu.g protein or less/mL and essentially
lacking contaminating proteins from serum in the culture medium. In
one aspect, the substantially animal protein-free rFurin
encompasses preparations of rFurin comprising contaminating host
cell DNA in a concentration between about 0 to 0.4 pg DNA or
less/Unit furin activity or between about 0 and 24 ng DNA or
less/mL and essentially lacking contaminating proteins from serum
in the culture medium.
[0013] The invention includes compositions comprising substantially
animal protein free recombinant furin at an activity of at least
10000 U furin/mL and host cell protein at a concentration less than
about 11 .mu.g protein/mL. Such compositions may also comprise host
cell protein at a concentration less than about 1.0 ng protein/U
furin activity. In one aspect, the host cell protein is from a CHO
cell.
[0014] In another aspect, the invention includes compositions
comprising substantially animal protein free recombinant furin at
an activity of at least 10000 U furin/mL and host cell DNA at a
concentration less than about 14 ng DNA/mL. In various aspects,
such compositions also comprise host cell DNA at a concentration
less than about 0.5 pg DNA/U furin activity. In one aspect, the
host cell DNA is from a CHO cell.
[0015] The invention also includes compositions comprising
substantially animal protein free recombinant furin at a specific
furin activity of at least about 100 U/.mu.g and host cell protein
at a concentration less than about 11 .mu.g protein/mL. Such
compositions may also comprise host cell protein at a concentration
less than about 1.0 ng protein/U furin activity. In one aspect, the
host cell protein is from a CHO cell.
[0016] The invention further includes compositions comprising
substantially animal protein free recombinant furin at a specific
furin activity of at least about 100 U/.mu.g and host cell DNA at a
concentration less than about 14 ng DNA/mL. Such compositions may
also comprise host cell DNA at a concentration less than about 0.5
pg DNA/U furin activity. In one aspect, the host cell DNA is from a
CHO cell.
[0017] The invention also includes methods of making a composition
comprising substantially animal protein-free recombinant furin
described herein. Such methods comprise the step of adapting the
host cells to growth in medium with increasingly lower
concentrations of serum until all serum is removed from the medium.
In another aspect, the methods comprise the step of transferring
the host cell from growth in medium comprising serum to growth in
serum-free medium. In an exemplary aspect, the host cell is a CHO
cell.
[0018] The invention includes methods of using a composition
comprising substantially animal protein-free recombinant furin
described herein. Such uses comprise the step of contacting a
pro-protein with the composition under conditions to cleave a
pro-peptide from the pro-protein to form a mature protein. The
rFurin can be used in the formation of any mature protein from a
pro-protein that is cleaved by furin. In one aspect, the mature
protein is von Willebrand Factor. In another aspect, the mature
protein is Factor VIII. In addition, the invention contemplates
that the rFurin of the invention is useful for both in vitro and in
vivo processing of any pro-protein that it cleaves.
BRIEF DESCRIPTION OF THE DRAWING
[0019] A further illustration of the invention is given with
reference to the accompanying drawings, which are set out below in
FIGS. 1-18.
[0020] FIG. 1 depicts the expressed active rFurin protease
construct in one embodiment of the invention. The rFurin construct
is truncated at the C-terminal end at AA 577 to remove the Cys-rich
transmembrane and cytosol domains.
[0021] FIG. 2 depicts a pedigree of the generation of the
CHO/rFurin clone #488-3.
[0022] FIG. 3 shows a pedigree of the generation of the CHO/rFurin
clone #289-20.
[0023] FIG. 4 sets out a comparison of the graphical distribution
of the rFurin producers in the cell populations of PMCB#01 and
PMCB#04. 80.74% of the cells in PMCB#04 express rFurin; 74.06% of
the cells in PMCB#01 express rFurin.
[0024] FIG. 5 shows a "Doehlert Matrix" where five temperatures
were combined with three pH values, resulting in seven combinations
of temperature and pH.
[0025] FIG. 6 shows a surface plot analysis of the data in
reference to the volumetric productivity. The coordinates of the
data in FIG. 6 are marked as points. The surface shows the assumed
correlation of the single data.
[0026] FIG. 7 shows a contour plot which illustrates the influence
of temperature and pH on the volumetric productivity. The dots
indicate the conditions (pH/temp.) which had been tested
experimentally.
[0027] FIG. 8 shows a surface plot which is a three-dimensional
illustration demonstrating the strong influence of the temperature
and the weak influence of the pH on the volumetric
productivity.
[0028] FIG. 9 shows a surface plot which illustrates the modeled
correlation three-dimensionally; it demonstrates the quadratic
relationship and shows clearly a maximum for the growth rate at
36.5.degree. C.
[0029] FIG. 10 sets out an analysis of the data in reference to
specific productivity. There is a similar correlation of the
specific productivity with temperature and pH as seen for the
volumetric productivity.
[0030] FIG. 11 shows that by decreasing the temperature from
37.degree. C. to 35.1.degree. C., the volumetric productivity could
be increased from approx. 200 kU/L/d to 540 kU/L/d.
[0031] FIG. 12 shows SDS-page and silver-stain for rFurin. The band
patterns of the Capto-MMC eluates of campaign ORFU06002 and
ORFU07002 correlate to a high degree; all samples show a prominent
Furin band at approx. 60 kDa. A trend to slightly lower molecular
weight of the Furin bands is visible in samples of campaign
ORFU06002 from batches MMC01 to MMC08 (FIG. 12, lanes 1-8).
[0032] FIG. 13 shows the Western blot analysis of samples using a
monoclonal anti-Furin antibody.
[0033] FIG. 14 shows the specific band patterns for rFurin from
isoelectric focusing (IEF) and subsequent Western blotting of
rFurin samples of campaign ORFU06002.
[0034] FIG. 15 shows the specific band patterns for rFurin from
isoelectric focusing (IEF) and subsequent Western blotting of
rFurin samples of campaign ORFU07002.
[0035] FIG. 16 shows Western blot results for rFurin from
isoelectric focusing (IEF) and subsequent Western blotting of
rFurin samples of campaign ORFU07002.
[0036] FIG. 17 shows Furin Reverse Phase HPLC for samples from
campaign ORFU06002 (Capto-MMC eluates). Samples were tested with C4
RP-HPLC in order to establish a fingerprint pattern for rFurin.
[0037] FIG. 18 shows Furin Reverse Phase HPLC for samples from
campaign ORFU07002 (Capto-MMC eluates). Samples were tested with C4
RP-HPLC in order to establish a fingerprint pattern for rFurin.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention relates to the development and
production of a recombinant host cell line that is capable of
growing in serum-free medium and secreting active recombinant furin
(rFurin) into the cell culture supernatant. The host cell line
selected for transfection of a plasmid encoding recombinant furin
is in one aspect the same as used for expression of recombinant
Factor VIII and recombinant VWF. The resulting rFurin is then
purified so that is substantially free of animal protein.
[0039] Furin, also known as PACE, PACE4, PC1/PC3, PC2, PC4 and
PC5/PC6, belongs to the group of the subtilisin-like serine
proteases, which play an important role in the cleavage of
proproteins, especially in the secretory synthesis (Van de Ven et
al., Crit. Rev. Oncogen. 4:115-136, 1993). Pro-proteins are
post-translationally, intracellularily processed to their mature
form by the endogenous protease in the Golgi apparatus. The
protease cleavage site comprises a recognition sequence which is
characterized by the amino acid sequence Arg-X-Lys/Arg-Arg. The
protease furin cleaves proproteins specifically after this
consensus sequence (Hosaka et al., J. Biol. Chem. 266:12127-12130,
1991).
[0040] The DNA and amino acid sequence of human and murine furin,
as well as further proteins with subtilisin-like protease function
have been identified (Roebroek et al., Mol. Biol. Rep. 11: 117-125,
1986; Roebroek et al., EMBO J. 5:2197-2202, 1986; Barr et al., DNA
Cell Biol. 10:319-328, 1991; Van den Ouweland et al., Nucleic Acids
Res. 17:7101-7102, 1989; Van den Ouweland et al., Nucleic Acids
Res. 18:664, 1990; Smeekens et al., 1990, J. Biol. Chem.
265:2997-3000; Smeekens et al., Proc. Natl. Acad. Sci. USA. 88;
340-344, 1991; Kiefer et al., DNA Cell Biol. 10: 757, 1991;
Nakayama et al., J. Biol. Chem. 267:5897-5900, 1992; and Hatsuzawa
et al., J. Biol. Chem. 265: 22075-22078, 1990). The human furin
gene encodes a protein consisting of 794 amino acids, certain
functions being allocatable to individual characteristic regions: a
catalytic center, a middle domain, a cystine-rich region, a
transmembrane domain, and a cytoplasmatic domain (Van de Ven et
al., Crit. Rev. Oncogen. 4:115-136, 1993). In one aspect, the human
furin polypeptide is set out in GenBank Accession No: EAX02111
(National Center for Biotechnology Information, U.S. National
Library of Medicine, Bethesda, Md.). However, the worker of
ordinary skill in the art will appreciate that any protein having
furin biological activity, i.e., the ability to cleave pro-proteins
(e.g., pro-VWF to produce mature VWF) can be produced by the
methods described herein.
[0041] Intact furin is incorporated into the membrane system of the
Golgi apparatus where it is functionally active (Bresnahan et al.,
J. Cell Biol. 111:2851-2859, 1990). A truncated form of the
over-expressed native furin of 75-80 kD could be detected in cell
supernatant as secreted protein (Wise et al., Proc. Natl. Acad.
Sci. USA 87: 9378-9382, 1990). This naturally secreted furin is
known as "shed furin" (Vidricaire et al., Biochem. Biophys. Res.
Comm. 195:1011-1018, 1993) and is cleaved N-terminally of the
transmembrane portion (Vey et al., J. Cell Biol. 127:1829-1842,
1994).
[0042] Furin truncated by genetic engineering, in which the
encoding part of the transmembrane and cytoplasmatic domains has
been deleted, can also be expressed and secreted correspondingly.
Such N-terminal deletions have been described for amino acids
714-794 (Leduc et al., J. Biol. Chem. 267:14304-14308, 1992, Molloy
et al., J. Biol. Chem. 267:16396-16402, 1992); for amino acids
716-794 ("Sol-PACE") (Wasley et al., J. Biol. Chem. 268:8458-8465,
1993; and Rehemtulla et al., Blood 79:2349-2355, 1992); and for
amino acids 705-794 (Hatsuzawa et al., J. Biol. Chem.
267:16094-16099, 1992). Furin mutants additionally comprising a
deletion of the cystine-rich region have also been described
(Hatsuzawa et al., J. Biochem. 101:296-301, 1992; Creemers et al.,
J. Biol. Chem. 268:21826-21834, 1993).
[0043] The endoproteolytic activity of furin and its selectivity
for basic amino acids was first determined in experiments with
pro-von Willebrand factor (pro-vWF). Pro-vWF consists of a
propolypeptide with 741 amino acids and mature von Willebrand
factor (vWF) with 2050 amino acids (Verweij et al., EMBO J.
5:1839-1847, 1986). The liberation of mature vWF from pro-vWF
results from a proteolytic cleavage after Arg763. Transfection of
pro-vWF cDNA in eukaryotic expression vectors results in the
production of equimolar amounts of the 360 kD pro-vWF and of the
260 kD mature vWF in the cell culture supernatant. vWF is probably
processed into its mature form in transfected cells, by
endogenously occurring furin (Wise et al., Proc. Natl. Acad. Sci.
USA 87:9378-9382, 1990, Van de Ven et al., Mol. Biol. Rep.
14:265-275, 1990).
[0044] Among additional pro-proteins which are cleaved by furin or
by subtilisin-like enzymes, respectively, are a series of hormones
and growth factors (e.g., proactivin A, hepatocyte-growth factor),
plasma proteins (albumin, factor VII, factor IX, factor X),
receptors (insulin pro-receptor), viral proteins (e.g. HIV-1 gp160,
influenza virus haemagglutinin) as well as bacterial proteins
(diphteria toxin, anthrax toxin) (Decroly et al., J. Biol. Chem.
269:12240-12247, 1994; Stieneke-Grober et al., EMBO J.
11:2407-2414, 1992; Barr, Cell 66:1-3, 1991, Wasley et al., J.
Biol. Chem. 268:8458-8465, 1993; Klimpel et al., Proc. Natl. Acad.
Sci. USA 89:10277-10281, 1992; Tsuneoka et al., J. Biol. Chem.
268:26461-26465, 1993; Bresnahan et al., J. Cell. Biol.
111:2851-2859, 1990; Hosaka et al., J. Biol. Chem. 266:12127-12130,
1991; and Vey et al., J. Cell. Biol. 127:1829-1842, 1994). The
rFurin of the present invention is contemplated for use in cleaving
these pro-proteins as well.
[0045] By co-expression of the nucleic acid sequences encoding
intact furin and a pro-protein in eukaryotic cell cultures, an
increased processing of the pro-proteins has been achieved in vivo.
This has been demonstrated, e.g., for pro-factor IX (Wasley et al.,
J. Biol. Chem. 268:8458-8465, 1993) and for pro-vWF (WO 91/06314;
Van de Ven et al., Mol. Bio. Rep. 14:265-275, 1990; and Rehemtulla
et al., Blood 79:2349-2355, 1992). The present invention
contemplates that the rFurin of the invention is useful for both in
vitro and in vivo processing of any pro-protein that it
cleaves.
[0046] Beside the co-expression of intact furin with pro-proteins,
truncated furin has been expressed together with pro-proteins.
Furin deletion mutants have been demonstrated as enzymatically
active when co-expressed in vivo and as secreted; the enzymatic
activity of such deletion mutants could be detected inter alia in
the processing of pro-factor IX (Wasley et al., J. Biol. Chem.
268:8458-8465, 1993) and pro-vWF (Rehemtulla et al., Blood 79:
2349-2355, 1992). Co-expression experiments with furin deletion
mutants have shown that the transmembrane and the cytoplasmatic
parts of the protein are not essential to the catalytic function
(Rehemtulla et al., Proc. Natl. Acad. Sci. USA 89: 8235-8239,
1992).
[0047] WO 91/06314 discloses the recombinant expression of furin in
prokaryotic and eukaryotic cells, the preparation of furin fusion
proteins, deletion mutants and fragments, the purification of
recombinantly prepared furin, and the potential use of purified
furin for the processing of proproteins in vitro in general. WO
92/09698 describes the expression of PACE (furin), the
co-expression with inactive precursors of proteins, such as, e.g.,
pro-vWF, as well as the preparation of fusion proteins.
Stieneke-Grober et al. (EMBO J. 11:2407-2414, 1992) describe the in
vitro cleavage of influenza virus HA protein by means of purified
furin. Decroly et al. (J. Biol. Chem. 269:12240-12247, 1994)
describe the in vitro cleavage of HIV gp160 by means of furin.
[0048] In experiments with C-terminally shortened furin, the
cleavage of pro-albumin and complement Pro-C3 (Oda et al., Biochem.
Biophys. Res. Commun. 189:1353-1361, 1992), anthrax toxin (Klimpel
et al., Proc. Natl. Acad. Sci. USA 89:10277-10281, 1992),
diphtheria toxin (Tsuneoka et al., J. Biol. Chem. 268: 26461-26465,
1993) and pro-factor IX (Wasley et al., J. Biol. Chem.
268:8458-8468, 1993, Bristol et al., Biochemistry 33:14136-14143,
1994) has been carried out successfully in vitro.
[0049] The rFurin of the present invention, therefore, is
contemplated for use in the in vivo and in vitro processing of
pro-proteins as described above. In one aspect, the rFurin of the
invention is especially useful in the in vitro processing of
pro-VWF and pro-factor IX. However, its use is not to be construed
as limited to the processing of said proteins. In a further aspect
the rFurin of the invention is particularly useful in the in vitro
processing of recombinant pro-proteins.
[0050] A further aspect of the present invention is the
co-culturing of cells which express pro-vWF and rFurin. Thus,
pro-vWF in the cell culture supernantant is cleaved in vitro into
its active form by rFurin which is also present in the cell culture
supernatant. Processed vWF is subsequently isolated from the
culture and purified, as discussed in U.S. Pat. No. 6,210,929,
incorporated herein by reference. For co-culturing, all the common
expression systems can be used, and various systems for expressing
pro-vWF and rFurin may be combined with each other. In one aspect,
an expression system is used in which both pro-vWF and rFurin are
expressed in host cells of the same origin.
[0051] The term "host cell" is used to refer to a cell which has
been transformed, or is capable of being transformed with a nucleic
acid sequence and then of expressing a selected gene of interest.
The term includes the progeny of the parent cell, whether or not
the progeny is identical in morphology or in genetic make-up to the
original parent, so long as the selected gene is present.
[0052] The invention includes any host cells or hosts known in the
art for recombinant protein production. Therefore, the cells in the
present invention can be derived from any source. In one aspect,
the invention includes eukaryotic and prokaryotic host cells. In
another aspect, the invention includes plant cells, animal cells,
fish cells, amphibian cells, avian cells, insect cells, and yeast
cells. In one aspect, exemplary yeast cells include Pichia, e.g. P.
pastoris, and Saccharomyces e.g. S. cerevisiae, as well as
Schizosaccharomyces pombe, Kluyveromyces, K. zactis, K. fragilis,
K. bulgaricus, K. wickeramii, K. waltii, K. drosophilarum, K.
thernotolerans, and K. marxianus; K. yarrowia; Trichoderma reesia,
Neurospora crassa, Schwanniomyces, Schwanniomyces occidentalis,
Neurospora, Penicillium, Totypocladium, Aspergillus, A. nidulans,
A. niger, Hansenula, Candida, Kloeckera, Torulopsis, and
Rhodotorula. Exemplary insect cells include Autographa californica
and Spodoptera frugiperda, and Drosophila.
[0053] In a further aspect, the host cells are mammalian cells,
including primary epithelial cells (e.g., keratinocytes, cervical
epithelial cells, bronchial epithelial cells, tracheal epithelial
cells, kidney epithelial cells and retinal epithelial cells) and
established cell lines and their strains (e.g., 293 embryonic
kidney cells, BHK cells, HeLa cervical epithelial cells and PER-C6
retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK
cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa
229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS180 cells,
LS174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24
cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-1 cells,
LLC-MK.sub.2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1
cells, Y-1 cells, LLC-PK.sub.1 cells, PK(15) cells, GH.sub.1 cells,
GH.sub.3 cells, L2 cells, LLC-RC 256 cells, MH.sub.1C.sub.1 cells,
XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives
thereof), fibroblast cells from any tissue or organ (including but
not limited to heart, liver, kidney, colon, intestines, esophagus,
stomach, neural tissue (brain, spinal cord), lung, vascular tissue
(artery, vein, capillary), lymphoid tissue (lymph gland, adenoid,
tonsil, bone marrow, and blood), spleen, and fibroblast and
fibroblast-like cell lines (e.g., Chinese hamster ovary (CHO)
cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells,
citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510
cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells,
Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299
cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells,
MiCl.sub.1 cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7
cells, Vero cells, DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells,
SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells,
NOR-10 cells, C.sub.3H/IOTI/2 cells, HSDM.sub.1C.sub.3 cells,
KLN205 cells, McCoy cells, Mouse L cells, Strain 2071 (Mouse L)
cells, L-M strain (Mouse L) cells, L-MTK (Mouse L) cells, NCTC
clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indian
muntjac cells, SIRC cells, C.sub.II cells, and Jensen cells, or
derivatives thereof).
[0054] Exemplary mammalian cells include varieties of CHO, BHK,
HEK-293, NS0, YB2/3, SP2/0, and human cells such as PER-C6 or
HT1080, as well as VERO, HeLa, COS, MDCK, NIH3T3, Jurkat, Saos,
PC-12, HCT 116, L929, Ltk-, W138, CV1, TM4, W138, Hep G2, MMT, a
leukemic cell, an embryonic stem cell or a fertilized egg cell. In
one aspect of the invention, an exemplary host cell is a CHO cell.
In a further aspect of the invention, the medium is used to culture
CHO cells in suspension.
[0055] Host cells can be engineered to express a protein in a
variety of ways known in the art, including but not limited to
insertion of exogenous nucleic acid encoding the desired protein,
optionally as part of an expression vector, insertion of an
exogenous expression control sequence such that it causes increased
expression of the host cell's endogenous gene encoding the desired
protein, or activation of the host cell's endogenous expression
control sequence(s) to increase expression of endogenous gene
encoding the desired protein.
[0056] Cultures of host cells can be prepared according to any
methods known in the art, and methods of growing such host cells
and recovering recombinant protein produced by the cells, whether
from the cells or culture medium, are known in the art. Such
culturing methods may involve addition of chemical inducers of
protein production to the culture medium. Exemplary host cells and
procedures are described below.
[0057] A nucleic acid encoding a furin polypeptide is inserted into
an appropriate expression vector using standard molecular biology
techniques. In one aspect, the nucleic acid encodes the human furin
polypeptide as set out in GenBank Accession No: EAX02111 (National
Center for Biotechnology Information, U.S. National Library of
Medicine, Bethesda, Md.), however the worker of ordinary skill in
the art will appreciate that any protein having furin biological
activity, i.e., the ability to cleave pro-VWF to produce mature
VWF, can be produced by the methods described herein. In a further
aspect, a C-terminally truncated, fully secreted rFurin was
designed by deleting nucleotides encoding amino acids 578 to 794
comprising the cystine-rich, the transmembrane, and the cytoplasmic
domains. In an even further aspect, a tail of amino acids may be
added to aid in purification processes. In yet another aspect, a
tail of 10 histidine residues was added after amino acid 577, with
or without interjacent four glycine residues serving as a flexible
linker.
[0058] Expression vectors optionally may include a promoter, one or
more enhancer sequences, an origin of replication, a
transcriptional termination sequence, a complete intron sequence
containing a donor and acceptor splice site, a sequence encoding a
leader or signal sequence for polypeptide secretion, a ribosome
binding site, a polyadenylation sequence, a polylinker region for
inserting the nucleic acid encoding the polypeptide to be
expressed, and/or a selectable marker element. Each of these
sequences is discussed below.
[0059] Optionally, the vector may contain a "tag"-encoding
sequence, i.e., an oligonucleotide sequence located at the 5' or 3'
end of the furin polypeptide coding sequence; the oligonucleotide
molecule encodes polyHis (such as hexaHis), or another "tag" such
as FLAG, HA (hemaglutinin influenza virus) or myc for which
commercially available antibodies exist. This tag is typically
fused to the polypeptide upon expression of the polypeptide, and
can serve as a means for detection or affinity purification of the
furin polypeptide from the host cell.
[0060] Suitable vectors include, but are not limited to, cosmids,
plasmids, or modified viruses, but it will be appreciated that the
vector system must be compatible with the selected host cell. In
one aspect, the vector is a plasmid. In a further aspect, the
plasmid is pUC-based cloning vector. Other vectors that can be used
in the invention include expression vectors, replication vectors,
probe generation vectors, sequencing vectors, and retroviral
vectors. Vectors contemplated by the invention include, but are not
limited to, microorganisms such as bacteria transformed with
recombinant bacteriophage, plasmid, phagemid, or cosmid DNA
expression vectors; yeast transformed with yeast expression
vectors; insect cell systems infected with viral expression vectors
(e.g., baculovirus); plant cell systems transfected with virus
expression vectors (e.g., Cauliflower Mosaic Virus, CaMV; Tobacco
Mosaic Virus, TMV) or transformed with bacterial expression vectors
(e.g., Ti or pBR322 plasmid); or even animal cell systems.
[0061] Mammalian expression vectors typically comprise an origin of
replication, a suitable promoter, and also any necessary ribosome
binding sites, polyadenylation site, splice donor and acceptor
sites, transcriptional termination sequences, and 5' flanking
nontranscribed sequences. DNA sequences derived from the SV40 viral
genome, for example, the SV40 origin, early promoter, enhancer,
splice, and polyadenylation sites may be used to provide the
required expression control elements. Exemplary eukaryotic vectors
include pcDNA3, pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene)
pSVK3, pBPV, pMSG, pSVL, and pVITRO3.
[0062] Nucleic acid can be transferred into host cells by any means
known in the art, e.g. through liposome-mediated transfer,
receptor-mediated transfer (ligand-DNA complex), electroporation,
microinjection of DNA, cell fusion, DEAE-dextran, calcium chloride,
calcium phosphate precipitation, microparticle bombardment,
infection with viral vectors, lipofection, transfection, or
homologous recombination.
[0063] The term "transformed" or "transfected" as used herein
refers to a host cell modified to contain an exogenous
polynucleotide, which can be integrated into the chromosome of the
host cell or maintained as an episomal element. It is contemplated
that in certain aspects of the methods provided, the host cell is
transfected in a "transfection step." The method may comprise
multiple transfection steps. In addition, other methods known in
the art for introducing exogenous polynucleotides into a host cell,
including for example, electroporation and cell fusion which are
not technically "transformation" are within the definition of the
term "transformation" for purposes of this description.
[0064] The invention also provides methods for culturing, i.e.
growing, host cells under conditions that result in rFurin protein
expression. Such methods include the step of recovering the rFurin
produced by the host cells from the culture medium. In an exemplary
aspect, the host cells are grown in a chemically defined,
serum-free medium. Because serum is a biochemically undefined
material, contains many components which have not been fully
identified, differs from lot to lot, and is frequently contaminated
with microorganisms, such as viruses and mycoplasma, the presence
of serum in the recombinant production of rFurin is undesirable.
Furthermore, the presence of animal proteins in serum in the
culture media can require lengthy purification procedures.
[0065] The invention therefore provides a biochemically defined
culture medium, essentially free from animal protein, for culturing
cells recombinantly transfected with a human furin gene. The
components of the medium are mostly inorganic, synthetic or
recombinant and as such are not obtained directly from any animal
source.
[0066] The cell culture medium of the present invention may
comprise one or more replacement compounds and can comprise one or
more replacement compounds which can be metal binding compounds
and/or can comprise one or more complexes comprising one or more
replacement compounds. In some embodiments, the medium can comprise
one or more complexes, said complex comprising one or more
transition elements or salts or ions thereof complexed one or more
replacement compounds which can be metal-binding compounds. In some
embodiments, the medium is capable of supporting the culture of
cells in vitro and permits transfection of cells cultured
therein.
[0067] According to one aspect of the invention, a transition
element is preferably selected from the group consisting of
scandium, titanium, vanadium, chromium, manganese, iron, cobalt,
nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum,
technetium, rubidium, rhodium, palladium, silver, cadmium,
lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium,
platinum, gold, mercury, and actinium, or salts or ions thereof,
and is preferably an iron salt. Suitable iron salts include, but
are not limited to, FeCl.sub.3, Fe(NO.sub.3).sub.3 or FeSO.sub.4 or
other compounds that contain Fe.sup.+++ or Fe.sup.++ ions.
[0068] Metal binding compounds in the medium include any
macromolecules which can interact with or bind with transition
elements and facilitate their uptake by cells. Such
interaction/binding can be covalent or non-covalent in nature. The
metal-binding compound used in this aspect of the invention is
preferably selected from the group consisting of a polyol, a
hydroxypyridine derivative,
1,3,5-N,N',N''-tris(2,3-dihydroxybenzoyl)amino-methylbenzene,
ethylenediamine-N,N'-tetramethylenephosphonic acid, trisuccin, an
acidic saccharide (e.g., ferrous gluconate), a glycosaminoglycan,
diethylenetriaminepentaacetic acid, nicotinic acid-N-oxide,
2-hydroxy-nicotinic acid, mono-, bis-, or tris-substituted
2,2'-bipyridine, a hydroxamate derivative (e.g. acetohydroxamic
acid), an amino acid derivative, deferoxamine, ferrioxamine, iron
basic porphine and derivatives thereof, DOTA-lysine, a texaphyrin,
a sapphyrin, a polyaminocarboxylic acid, an
alpha.-hydroxycarboxylic acid, a polyethylenecarbamate, ethyl
maltol, 3-hydroxy-2-pyridine, and IRC011. In one aspect, the
metal-binding compound is a polyol such as sorbitol or dextran, and
particularly sorbitol. In a related aspect, the metal-binding
compound is a hydroxypyridine derivative, such as
2-hydroxypyridine-N-oxide, 3-hydroxy-4-pyrone,
3-hydroxypypyrid-2-one, 3-hydroxypyrid-2-one, 3-hydroxypyrid-4-one,
1-hydroxypyrid-2-one, 1,2-dimethyl-3-hydroxypyrid-4-one,
1-methyl-3-hydroxypyrid-2-one, 3-hydroxy-2(1H)-pyridinone, ethyl
maltol or pyridoxal isonicotinyl hydrazone. The metal binding
compounds of the present invention can also bind divalent cations
such as Ca.sup.++ and Mg.sup.++.
[0069] The culture medium of the present invention may comprise one
or more ingredients selected from the group consisting of adenine,
ethanolamine, D-glucose, heparin, a buffering agent,
hydrocortisone, insulin, linoleic acid, lipoic acid, phenol red,
phosphoethanolamine, putrescine, sodium pyruvate,
tri-iodothyronine, thymidine, L-alanine, L-arginine, L-asparagine,
L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine,
L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,
L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,
L-tyrosine, L-valine, N-acetyl-cysteine, biotin, choline chloride,
D-Ca.sup.++-pantothenate, folic acid, i-inositol, niacinamide,
pyridoxine, riboflavin, thiamine, vitamin B.sub.12, Pluronic F68,
recombinant insulin, a calcium salt, CuSO.sub.4, FeSO.sub.4,
FeCl.sub.3, Fe(NO.sub.3).sub.3, KCl, a magnesium salt, a manganese
salt, sodium acetate, NaCl, NaHCO.sub.3, Na.sub.2HPO.sub.4,
Na.sub.2SO.sub.4, a selenium salt, a silicon salt, a molybdenum
salt, a vanadium salt, a nickel salt, a tin salt, ZnCl.sub.2,
ZnSO.sub.4 or other zinc salts, wherein each ingredient is added in
an amount which supports in vitro cell culture.
[0070] In another aspect, the culture medium of the invention may
optionally further comprise one or more supplements selected from
the group consisting of one or more cytokines, soy peptone, one or
more yeast peptides and one or more plant peptides (most preferably
one or more of rice, aloe vera, soy, maize, wheat, pea, squash,
spinach, carrot, potato, sweet potato, tapioca, avocado, barley,
coconut and/or green bean, and/or one or more other plants), e.g.,
see international application no. PCT/US97/18255, published as WO
98/15614.
[0071] The culture medium of the present invention may also
optionally include one or more buffering agents to maintain an
optimal pH. Suitable buffering agents include, but are not limited
to, N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid]
(HEPES), MOPS, MES, phosphate, bicarbonate and other buffering
agents suitable for use in cell culture applications. A suitable
buffering agent is one that provides buffering capacity without
substantial cytotoxicity to the cells cultured. The selection of
suitable buffering agents is within the ambit of ordinary skill in
the art of cell culture.
[0072] The above-described media components when admixed together
in solution form a complete culture medium of the present
invention. A complete medium is suitable for use in the culture of
a variety of mammalian cells, as described in more detail herein.
Based on the information obtained herein, and knowledge possessed
by those of ordinary skill in the art, one of ordinary skill in the
art can obtain operative media formulations without undue
experimentation.
[0073] Initially and prior to adaptation for growth in a chemically
defined serum-free medium, host cells may be grown in standard
media well known to one of ordinary skill in the art. The media
will usually contain all nutrients necessary for the growth and
survival of the cells. Suitable media for culturing eukaryotic
cells are, Roswell Park Memorial Institute (RPMI) medium 1640 (RPMI
1640), Minimal Essential Medium (MEM), and/or Dulbecco's Modified
Eagle Medium (DMEM), DMEM/F12, and ExCell 325 medium, all of which
may be supplemented with serum and/or growth factors as indicated
by the particular cell line being cultured. Notably, however, the
invention provides that the serum in the media is then removed from
the culture to obtain host cells that can grow in serum-free
medium. Thus, the invention provides optimal media for culturing
host cells under serum-free conditions for maximal production of
rFurin. In a further aspect, the cells are grown in serum-free
medium in suspension culture. Recipes for the various media of the
invention are provided in the Examples herein.
[0074] In one aspect, an antibiotic or other compound useful for
selective growth of transformed cells is added as a supplement to
the media. The compound to be used will be dictated by the
selectable marker element present on the plasmid with which the
host cell was transformed. Selectable markers that confer
resistance to particular drugs that are ordinarily toxic to an
animal cell can be used in the methods and compositions of the
invention. For example, where the selectable marker element is
kanamycin resistance, the compound added to the culture medium will
be kanamycin. Other compounds for selective growth include
ampicillin, tetracycline, geneticin, neomycin, zeomycin (zeo);
puromycin (PAC); Blasticidin S (BlaS), and GPT. Additional
selectable markers are known in the art and useful in the
compositions and methods of the invention.
[0075] Metabolic enzymes that confer cell survival or induce cell
death under prescribed conditions can also be used in the methods
and compositions of the inventions. Examples include, but are not
limited to: dihydrofolate reductase (DHFR); herpes simplex virus
thymidine kinase (TK), hypoxanthine-guanine
phosphoribosyltransferase (HGPRT), and adenine
phosphoribosyltransferase (APRT), which are genes which can be
employed in cells lacking TK, HGPRT or APRT, respectively. The
worker of ordinary skill in the art will appreciate, however, that
a rFurin product of the invention will be essentially free of these
added proteins.
[0076] The medium can be used to culture any host cells or hosts
known in the art for recombinant protein production. In one aspect
of the invention, an exemplary host cell is a CHO cell. In a
further aspect of the invention, the medium is used to culture CHO
cells in suspension.
[0077] When the recombinant protein of interest is secreted into
the medium by the host cells, the medium can be harvested
periodically, so that the same host cells can be used through
several harvest cycles. Culture medium may be added in a batch
process, e.g. where culture medium is added once to the cells in a
single batch, or in a fed batch process in which small batches of
culture medium are periodically added. Medium can be harvested at
the end of culture or several times during culture. Continuously
perfused production processes are also known in the art, and
involve continuous feeding of fresh medium into the culture, while
the same volume is continuously withdrawn from the reactor.
Perfused cultures generally achieve higher cell densities than
batch cultures and can be maintained for weeks or months with
repeated harvests. Thus, chemostat cultures and batch reefed
cultures are both suitable for the manufacturing of rFurin, as are
other culture methods known in the art.
[0078] A variety of culture systems are known in the art, including
T-flasks, spinner and shaker flasks, roller bottles and
stirred-tank bioreactors. Roller bottle cultivation is generally
carried out by seeding cells into roller bottles that are partially
filled (e.g., to 10-30% of capacity) with medium and slowly
rotated, allowing cells to attach to the sides of the bottles and
grow to confluency. The cell medium is harvested by decanting the
supernatant, which is replaced with fresh medium.
Anchorage-dependent cells can also be cultivated on microcarrier,
e.g. polymeric spheres, that are maintained in suspension in
stirred-tank bioreactors. Alternatively, cells can be grown in
single-cell suspension.
[0079] The amount of rFurin produced by a host cell can be
evaluated using standard methods known in the art. Such methods
include, without limitation, Western blot analysis,
SDS-polyacrylamide gel electrophoresis, non-denaturing gel
electrophoresis, High Performance Liquid Chromatography (HPLC)
separation, immunoprecipitation, ELISA, and/or activity assays such
as DNA binding gel shift assays. The invention also contemplates
that specific productivity (expressed as amount of
protein/cell/day) of rFurin can be evaluated using standard methods
as known in the art and as described herein.
[0080] "Substantially animal protein-free rFurin" is defined as
encompassing preparations of rFurin comprising host cell protein in
a concentration which ranges from between about 0.1 to 0.6 ng
protein or less/Unit furin activity or between about 2 and 11 .mu.g
protein or less/mL and essentially lacking contaminating proteins
from serum in the culture medium. In one aspect, the substantially
animal protein-free rFurin encompasses preparations of rFurin
comprising host cell DNA in a concentration which ranges from
between about 0 to 0.4 pg DNA or less/Unit furin activity or
between about 0 and 24 ng DNA or less/mL and essentially lacking
contaminating proteins from serum in the culture medium. In one
aspect, host cells expressing rFurin are grown in a
chemically-defined, serum-free medium. Alternatively, the cells may
be grown in medium with serum and purified according to methods
provided herein.
[0081] Host cells expressing rFurin are cultured in suspension in a
medium free of animal (including human) derived substances under
chemostat conditions. The cells are removed by filtration and the
rFurin containing cell culture supernatant is concentrated by
ultrafiltration and purified by ion exchange chromatography to
result in a solution of rFurin with an activity of at least about
1000 Units/ml, of at least about 2000 Units/ml, of at least about
3000 Units/ml, of at least about 4000 Units/ml, of at least about
5000 Units/ml, of at least about 6000 Units/ml, of at least about
7000 Units/ml, of at least about 8000 Units/ml, of at least about
9000 Units/ml, of at least about 10000 Units/ml, of at least about
15000 Units/ml, of at least about 20000 Units/ml, of at least about
25000 Units/ml, of at least about 30000 Units/ml, of at least about
35000 Units/ml, of at least about 40000 Units/ml, of at least about
45000 Units/ml, of at least about 50000 Units/ml, of at least about
55000 Units/ml, of at least about 60000 Units/ml, of at least about
65000 Units/ml, of at least about 70000 Units/ml, of at least about
75000 Units/ml, of at least about 80000 Units/ml, of at least about
85000 Units/ml, of at least about 90000 Units/ml, of at least about
95000 Units/ml, of at least about 100000 Units/ml, of at least
about 120000 Units/ml, of at least about 140000 Units/ml, of at
least about 160000 Units/ml, of at least about 180000 Units/ml, of
at least about 200000 Units/ml, and of at least about 500000
Units/ml, and up to more than 500000 U/ml.
[0082] In another aspect the purified solution of recombinant furin
of the invention has a specific activity of at least about 10
U/.mu.g protein, at least about 20 U/.mu.g protein, at least about
30 U/.mu.g protein, at least about 40 U/.mu.g protein, at least
about 50 U/.mu.g protein, at least about 60 U/.mu.g protein, at
least about 70 U/.mu.g protein, at least about 80 U/.mu.g protein,
at least about 90 U/.mu.g protein, at least about 100 U/.mu.g
protein, at least about 120 U/.mu.g protein, at least about 140
U/.mu.g protein, at least about 160 U/.mu.g protein, at least about
180 U/.mu.g protein, at least about 200 U/.mu.g protein, at least
about 250 U/.mu.g protein, at least about 300 U/.mu.g protein, at
least about 350 U/.mu.g protein, at least about 400 U/.mu.g
protein, at least about 450 U/.mu.g protein, at least about 500
U/.mu.g protein, at least about 550 U/.mu.g protein, at least about
600 U/.mu.g protein, at least about 650 U/.mu.g protein, at least
about 700 U/.mu.g protein, at least about 750 U/.mu.g protein, at
least about 800 U/.mu.g protein, at least about 850 U/.mu.g
protein, at least about 900 U/.mu.g protein, at least about 950
U/.mu.g protein, and at least about 1000 U/.mu.g protein.
[0083] In another embodiment, the purified solution of rFurin in
the invention contains host cell protein at a concentration of less
than about 20.0 .mu.g/ml, less than about 19.0 .mu.g/ml, less than
about 18.0 .mu.g/ml, less than about 17.0 .mu.g/ml, less than about
16.0 .mu.g/ml, less than about 15.0 .mu.g/ml, less than about 14.0
.mu.g/ml, less than about 13.0 .mu.g/ml, less than about 12.0
.mu.g/ml, less than about 11.0 .mu.g/ml, less than about 10.5
.mu.g/ml, less than about 10.0 .mu.g/ml, less than about 9.5
.mu.g/ml, less than about 9.0 .mu.g/ml, less than about 8.5
.mu.g/ml, less than about 8.0 .mu.g/ml, less than about 7.5
.mu.g/ml, less than about 7.0 .mu.g/ml, less than about 6.5
.mu.g/ml, less than about 6.0 .mu.g/ml, less than about 5.5
.mu.g/ml, less than about 5.0 .mu.g/ml, less than about 4.5
.mu.g/ml, less than about 4.0 .mu.g/ml, less than about 4.0
.mu.g/ml, less than about 3.5 .mu.g/ml, less than about 3.0
.mu.g/ml, less than about 2.5 .mu.g/ml, less than about 2.0
.mu.g/ml, less than about 1.5 .mu.g/ml, less than about 1.0
.mu.g/ml, less than about 0.5 .mu.g/ml, less than about 0.4
.mu.g/ml, less than about 0.3 .mu.g/ml, less than about 0.2
.mu.g/ml, less than about 0.1 .mu.g/ml, and about 0 .mu.g/ml.
[0084] In another aspect, the purified solution of rFurin in the
invention contains host cell protein at a concentration of less
than about 1.0 ng protein/U rFurin, less than about 0.95 ng
protein/U rFurin, less than about 0.90 ng protein/U rFurin, less
than 0.85 ng protein/U rFurin, less than about 0.80 ng protein/U
rFurin, less than about 0.75 ng protein/U rFurin, less than about
0.70 ng protein/U rFurin, less than about 0.65 ng protein/U rFurin,
less than about 0.60 ng protein/U rFurin, less than about 0.55 ng
protein/U rFurin, less than about 0.50 ng protein/U rFurin, less
than about 0.45 ng protein/U rFurin, less than 0.40 ng protein/U
rFurin, less than about 0.35 ng protein/U rFurin, less than about
0.30 ng protein/U rFurin, less than about 0.25 ng protein/U rFurin,
less than about 0.20 ng protein/U rFurin, less than about 0.15 ng
protein/U rFurin, less than about 0.10 ng protein/U rFurin, less
than about 0.05 ng protein/U rFurin, less than about 0.04 ng
protein/U rFurin, less than about 0.03 ng protein/U rFurin, less
than about 0.02 ng protein/U rFurin, less than about 0.01 ng
protein/U rFurin, and about 0 ng protein/U rFurin.
[0085] The examples herein below demonstrate the invention using
CHO host cells to produce rFurin, however, the worker of ordinary
skill will realize that any host cell type can be similarly adapted
for producing the rFurin of the invention. CHO cells have been
widely used in the production of recombinant proteins, and
engineered CHO cells (those in which a CHO cell line is transfected
with a product gene and a selectable marker gene) are routinely
grown in culture medium containing serum. However, the use of serum
poses a number of problems. Serum is an expensive commodity, which
is not readily available in amounts required for commercial
production. Serum is also a biochemically undefined material and
contains many components which have not been fully identified nor
their actions determined. Thus serum will differ from batch to
batch, possibly requiring testing to determine levels of the
various components and their effect on the cells.
[0086] In addition, serum is frequently contaminated with
microorganisms such as viruses and mycoplasma many of which may be
harmless, but still represent an additional unknown factor.
Furthermore, the presence of animal proteins in culture media can
require lengthy purification procedures. In particular, the
presence of bovine antibodies in bovine serum albumin (BSA) makes
purification of the desired antibodies expressed by the recombinant
CHO cell line extremely difficult. Removal of bovine antibody from
medium prior to use is possible, but this removal and the
additional product testing required after removal adds greatly to
the cost of production of the product. Consequently, there are
benefits in using a culture medium devoid of animal components
which will support cellular growth, especially of CHO cells. While
CHO cells do not readily grow in serum-free conditions, the present
invention provides rFurin grown in CHO cells under serum-free
conditions.
[0087] Engineered CHO cells are also difficult to grow in
suspension. It is highly desirable to achieve growth in suspension
when using the cells to express a product like rFurin. For the
production of such a biological protein on a commercial scale, it
is desirable to be able to support growth in fermenters of a
considerable size. A suitable medium is also required to support
the cells so that they may grow in large production conditions.
Such suitable media are set out in the Examples herein. The worker
of ordinary skill in the art will appreciate that any methods of
culturing cells in the art can be used in culturing the host cells
comprising rFurin as set out in the invention. Non-limiting
examples of culture methods are provided in the Examples
herein.
[0088] The invention also provides purification methods that are
carried out after cells are grown in serum-free medium to remove
CHO cell proteins from rFurin. The worker of ordinary skill in the
art will appreciate that any methods of protein purification known
in the art can be used in the purification of rFurin from the
culture medium. Non-limiting examples of purification methods are
provided in the Examples herein. Accordingly, rFurin which is
essentially substantially free from all animal source protein can
be produced. The substantially animal protein-free rFurin is
optionally stored frozen until use.
EXAMPLES
[0089] Additional aspects and details of the invention will be
apparent from the following examples, which are intended to be
illustrative rather than limiting. Example 1 describes the
construction of a rFurin expression plasmid and host cell
transfection; Example 2 describes the processes of adapting the
rFurin-expressing CHO cell clones to growth in serum-free
conditions; Example 3 describes a process of optimization for
manufacturing rFurin in animal-protein free medium; Example 4
describes the purification of rFurin; Example 5 sets out the
downstream processing (concentration and purification) and analysis
of the large scale production of rFurin; and Example 6 demonstrates
the safety, sterility, and stability testing that is performed to
determine and maintain the quality of the host cell bank.
Example 1
Construction of a Recombinant Furin Expression Plasmid and Host
Cell Transfection
[0090] A detailed description of the furin progenitor plasmids used
to construct a rFurin expression plasmid designated #556 is set out
in Table 1. Expressed under control of a constitutive
cytomegalovirus (CMV) promoter, the mature rFurin contains the
catalytic domain, the P domain, and a small portion of the
cystine-rich domain whereas regions located C-terminal to amino
acid 577 are removed leading to a fully secreted active
protease.
[0091] A description of the construction of the DHFR-vector used as
the selection plasmid is depicted in Table 2. For the development
of stably expressing CHO/rFurin cell clones designated # 488-3 and
# 289-20, CHO cells lacking a functional endogenous DHFR gene were
co-transfected with plasmids # 556 and # 73 employing calcium
phosphate co-precipitation. Clones secreting high levels of rFurin
were selected in several rounds of subcloning and amplification
using the DHFR/MTX selection system.
TABLE-US-00001 TABLE 1 Description of furin plasmid generation
Plasmid Description Comments # 177 Original plasmid obtained from
Wim Carried out in the University of J. M. van de Ven (University
of Leuven, Belgium. Leuven, Belgium). A pUC18-based plasmid
containing the 4.0 kbp EcoRI fragment of the human furin cDNA
(2.385 kbp) with additional sequences of the 5'- and
3'-untranslated regions (UTR). # 180 Human full-length furin
expression The 2.8 kbp SmaI/AvrII vector. fragment of plasmid #
177, comprising the complete furin cDNA (2.385 kbp) and in addition
~50 bp of the 5'-UTR and ~400 bp of the 3'-UTR, was cloned into the
expression vector # 55. # 55 Eukaryotic expression plasmid from The
.beta.-galactosidase NotI- Clontech (Palo Alto, CA, USA) which
cassette was removed and a has been modified to contain a multiple
cloning site was multiple cloning site instead of the .beta.-
inserted instead having galactosidase cDNA. The plasmid amongst
other restriction sites provides a human cytomegalovirus also the
unique sites for SmaI immediate early (CMV IE) gene and AvrII.
promoter and enhancer, the RNA splicing signals from the SV40
genome consisting of the late viral protein gene 16s/19s splice
donor and acceptor sequences, and the SV40 polyadenylation signal.
The original vector pCMV.beta. is a pUC19 derivative containing the
E. coli ~3.4 kbp .beta.- galactosidase cDNA inserted into the NotI
site. # 229 Human furin lacking the C-terminal Between the SauI and
AvrII sequences from position 578 to 794 sites, an appropriate
spanning the cysteine-rich, the trans- reannealed membrane and the
cytoplasmic oligodesoxynucleotide-linker regions. After glycine
577, 4 additional was inserted coding for 4 glycines as `spacer`
and 10 histidine glycines, 10 histidines followed residues were
introduced. by a stop codon. # 378 Derivative of plasmid # 229. The
12 bps coding for 4 Truncated furin after glycine 577 glycines
located on a SauI/ containing 10 histidines but without 4 Hind III
fragment were removed glycines. by PCR. The modified fragment was
then religated into the plasmid-backbone. # 556 Derivative of
plasmid # 378. Deletion of 30 bps coding for Truncated furin after
glycine 577 the 10 histidine residues by which is devoid of any
additional PCR. The modified SauI/Hind heterologous sequences. III
fragment was relegated into the plasmid-backbone.
TABLE-US-00002 TABLE 2 Description of DHFR plasmid generation
Plasmid Description Comments # 29 The original plasmid named
Constructed outside Baxter. pAdD26SV(A)-3 was obtained from H. J.
Hauser (GBF, Braunschweig, Germany). This plasmid contains the full
length murine dihydrofolate reductase (DHFR) cDNA behind an
adenovirus major late promoter. # 73 Murine DHFR-cDNA under control
of the The PstI fragment comprising SV40 early promoter. the DHFR
cDNA and the SV40 polyadenylation signal of plasmid # 29 was cloned
into the PstI site of plasmid # 53. Due to the cloning strategy,
plasmid # 73 contains two polyadenylation signals. # 53 Eukaryotic
expression vector from The .beta.-galactosidase NotI- Clontech
(Palo Alto, CA, USA) which has cassette was removed and a been
modified to contain a multiple multiple cloning site was inserted
cloning site instead of the .beta.- instead having amongst other
galactosidase cDNA. The plasmid restriction sites also a unique
provides a simian virus 40 (SV40) early site for PstI. gene
promoter and enhancer, the RNA splicing signals from the SV40
genome consisting of the late viral protein gene 16s/19s splice
donor and acceptor sequences, and the SV40 polyadenylation signal.
The original vector pSV40.beta. is a pUC19 derivative containing
the E. coli ~3.4 kbp .beta.- galactosidase cDNA inserted into the
NotI site.
[0092] Transfected CHO cells were grown in DHFR-medium, composed of
DMEM NUT MIX F12 (1:1) without hypoxanthine, thymidine and glycine
supplemented with Hepes, L-glutamine, Penicillin-Streptomycin, and
with 5% or 10% dialyzed, gamma-irradiated FBS (=5% DHFR, 10% DHFR).
Dialyzed and gamma-irradiated FBS was purchased from Life
Technologies with full documentation of certificates of analysis,
origin and irradiation. The preparation of gamma-Trypsin solution
(1 mg/ml) was performed at Baxter in Orth, Austria, by the
department of Media Preparation/PCC.
[0093] A pedigree of the generation of the CHO/rFurin clone # 488-3
is depicted in FIG. 2. Clone CHO/rFurin # 488-3 was obtained from
initial clones which underwent two rounds of subcloning in 10% DHFR
selection medium before entering amplification in selection medium
supplemented with 100 nM MTX in which one round of subcloning in
medium containing unchanged MTX concentration was performed. Clone
# 448-3 was expanded for freezing. The CHO/rFurin clone # 289-20
was likewise prepared and expanded. However, clone #289-20 is a
successor clone derived from clone #488-3. A pedigree of the
generation of the CHO/rFurin clone # 289-20 is depicted in FIG.
3.
[0094] Furin activity was measured in the conditioned medium of
clones, which were cultured for 24 hrs in serum-free DHFR medium.
Cell clones which showed high furin activity (U/10.sup.6 cells per
24 hrs) were selected. Selected high producer clones were expanded
for the preparation of freeze stock ampules, and used for splitting
for the next cloning round. Isolation and identification of high
producer cell clones was performed. Cell densities were analyzed
using the Casy cell-counter. Furin expression levels of up to
200-300 U/10.sup.6 cells per 24 hrs were achieved for clone #
488-3. Furin expression levels of up to 400 U/10.sup.6 cells per 24
hrs were achieved for clone # 289-20.
Example 2
Adapting the Recombinant Furin Expressing Cell Clones to Growth in
Serum-Free Conditions
[0095] The strategy for cell line adaptation and selection is to
adapt the cell line to a serum- and protein-free cell line in
either gradually in a step-wise dilution or abruptly. The purpose
of this study was to find a CHO cell population growing under
serum-free conditions, which was stably producing rFurin. The CHO
cell clone #488-3 was used as starting material. The rFurin
expressing cell clone CHO #488-3 was changed over to serum-free
conditions in three parallel conducted adaptations as set out in
detail below.
[0096] The serum depletion process started in spinner flasks with
use of microcarriers to find a means to hold back cells in the
phase of adaptation, since in that phase cells usually show slow
growth. By using this method, it was possible to avoided, during
subsequent media changes, diluting the cells to such concentrations
where growth could be inhibited.
[0097] Three variants of an in-house developed medium, BAP, BAS,
and BCS, (as shown in Table 3) were used during the course of this
study.
TABLE-US-00003 TABLE 3 In-house media formulations of BAP, BAS and
BCS JDE Concentration Components Item No. [g/kg] BAP BAS BCS
DMEM/F12 0200437 11.745 X X X L-Glutamine 0200444 0.600 X X .sup.
X.sup.1 Phenol red 0200425 0.008 X X X sodium salt Putrescine
0200233 0.0036 -- -- X dihydrochloride Iron (II) sulfate 0200231
0.0006 -- -- X heptahydrate Ethanolamine 0200426 0.00153 X X X
Synperonic F68 0200172 0.250 X X .sup. X.sup.2 Soy peptone 0200171
2.50 X -- -- Sodium bicarbonate 0301012 2.00 X X X WFI F124 ad 1 kg
X X X (water for injection) .sup.1Concentration of L-Glutamine in
BCS: 0.900 g/kg .sup.2Concentration of Synperonic F68 in BCS: 1.00
g/kg
[0098] Depending on the purpose of the respective experiment these
media variants were provided with different supplements, as listed
in Table 4.
TABLE-US-00004 TABLE 4 Media and their supplements Put.sup.1
Glut.sup.2 Synp.sup.3 Fe.sup.4 Zn.sup.5 (mg/l) (mg/l) (mg/l) (mg/l)
(mg/l) ExCell 325PF CHO -- 600 -- -- 1.0 or 5.0 BAS 3.6 300 750 0.6
5.0 BCS -- -- -- -- 1.0 or 5.0 .sup.1Putrescine dihydrochloride
.sup.2L-Glutamine .sup.3Synperonic F68 .sup.4Iron(II) sulfate
heptahydrate .sup.5Zinc(II) sulfate heptahydrate
[0099] The following tables give an overview of media and reagents,
which were used in the course of this study. Table 5 summarizes
media and reagents which were used for the establishment of the
pre-master cell bank clones PMCB#01 and the PMCB#04.
TABLE-US-00005 TABLE 5 Media and Reagents Used for the
Establishment of PMCB#01 and PMCB#04 Description Lot Number MEDIA
10% DHFR medium #061005/09 BAP + 5% FBS M/MAB-05/009, M/MAB-05/013
BAS + Put + Glut + Synp + Fe + 5% FBS M/MAB-06/019.sup.4 BAS + Put
+ Glut + Synp + Fe M/MAB-06/012.sup.1, M/MAB-06/017, M/MAB-06/031,
M/MAB-06/036, M/MAB-06/044, M/MAB-06/048.sup.4, M/MAB-06/058.sup.4,
M/MAB-06/061.sup.4 BAS + Put + Glut + Synp + Fe + Zn M/MAB-06/063,
M/MAB-06/069, M/MAB-06/072, M/MAB-06/074.sup.4, M/MAB-06/076.sup.4,
M/MAB-06/079.sup.4 BCS + Zn M/CLD-06/001 REAGENTS Gamma-Trypsin
Solution 1 mg/ml GT_04002_1 N1 Buffer pH 7.3 N1_05001_2
Dimethylsulfoxide 219102 (Baxter Mat.No.: 33000000001/JDE 0200407)
Cytopore 2 Carrier Cyt2_24_06_001.sup.1 Na.sub.2HCO.sub.3-Solution
R/CBL/05/005.sup.1 .sup.1only applied to PMCB#01 .sup.4only applied
to PMCB#04
[0100] Table 6 summarizes media and reagents which were used in the
course of sub-cloning and establishing of the corresponding
evaluation cell banks (ECBs) of the sub-clones #488-3/CJ06-19/5F10
(5F10) and #488-3/CJ06-19/1E8 (1E8).
TABLE-US-00006 TABLE 6 Lot numbers of media and reagents used for
the establishment of subclones 5F10 and 1E8 Description Lot Number
MEDIA 10% DHFR Anzuchtmedium #061005/09 BAP + 5% FBS M/MAB-05/009,
M/MAB-05/013 ExCell + 5% FBS + Glut M/MAB-05/017 ExCell + Glut
M/MAB-05/016, M/MAB-05/022, M/MAB-06/025, M/MAB-06/035,
M/MAB-06/054, M/MAB-06/057, M/MAB-06/060 ExCell + Glut + Zn
M/MAB-06/067, M/MAB-06/071, M/MAB-06/104, M/MAB-06/142 ExCell +
Glut/BAS + Put + Glut + Synp + Fe (1:1) M/MAB-06/062 ExCell +
Glut/BAS + Put + Glut + Synp + Fe + Zn (1:1) M/MAB-06/070 ExCell +
Glut/BCS (1:1) M/MAB-06/077 ExCell + BCS + Glut + Zn M/MAB-06/090,
M/MAB-06/103, M/MAB-06/106 BCS + Zn M/MAB-06/105, M/MAB-06/110,
M/MAB-06/116, M/MAB-06/143, M/MAB-06/129, M/MAB-06/149 REAGENTS
Gamma-Trypsin Solution 1 mg/ml GT_04002_1 Trypsin-Inhibitor
Solution 1 mg/ml TI_04001_1 N1 Buffer pH 7.3 N1_05001_2
Dimethylsulfoxid 219102 (Baxter Mat.No.: 33000000001/JDE 0200407)
Na.sub.2HCO.sub.3-Solution R/CBL/05005
[0101] The lot numbers of all supplements, which were added to the
media, are referenced in the appropriate manufacturing protocol of
the corresponding medium. Other media additives are listed in Table
7.
TABLE-US-00007 TABLE 7 Other Media Additives Description
Manufacturer Catalog No. Lot No. Provided by Aqua Bidest Fresenius
B230673 SBV 093 Manufacturer Aqua Bidest Fresenius B230673 TDV 252
Manufacturer Aqua Bidest Fresenius B230673 TKV 261 Manufacturer
Aqua Bidest Fresenius B230673 UCV 282 Manufacturer Cytopore 2
Carrier Pharmacia Biotech 17-1271-03 249933 Pilot Plant II DMEM NUT
MIX F12.sup.2 Gibco 041-90163M 3097428 Recombinant Cell Lines
ExCell 325PF CHO JRH 14340 4N0597 Manufacturer ExCell 325PF CHO JRH
14340 5L0191 Manufacturer ExCell 325PF CHO SAFC Biosciences 14340C
5M0775 Manufacturer FBS.sup.1,2 Gibco 10603-017 3092829A
Recombinant Cell Lines FBS.sup.1 JRH 12303 1A0348 Manufacturer
Glutamin.sup.2 Gibco 25030-024 3096452 Recombinant Cell Lines
Glutamine Sigma G-5763 117 H 00655 Manufacturer Hepes-Puffer.sup.2
Gibco 15630-056 3094125 Recombinant Cell Lines Paraformaldehyde
Sigma P-6148 043 K 0653 Manufacturer Paraformaldehyde Sigma P-6148
045 K 0703 Manufacturer Paraformaldehyde Sigma P-6148 118 H 0987
Manufacturer Penic./Streptomycin.sup.2 Gibco 15140-122 1276184
Recombinant Cell Lines Putrescine Sigma P5780 22 K 2615
Manufacturer Synperonic F68 Serva 35724 01137 Manufacturer
.sup.1animal derived materials: for certificates refer to appendix
.sup.2ingredients of "10% DHFR medium", only used in the beginning
of experiment SF05-80
[0102] Adaptation to serum-free conditions was performed in
T-flasks or in spinner flasks in conjunction with cell retention
(centrifugation and the like) by weaning off the cells from fetal
bovine serum (FBS).
[0103] Suspension cultures in T-flasks were incubated at
36.+-.2.degree. C. and 7.5.+-.1.0% CO.sub.2. The culture in spinner
flasks was performed in Techne and Bellco spinners without carriers
operating at 36.+-.2.degree. C. with 80 rpm and 130 rpm,
respectively.
[0104] The subcloning of the CHO/rFurin cell clone #488-3,
subsequent to its adaptation to serum-free medium conditions,
resulted in a CHO cell clone, growing in suspension in serum-free
in-house medium, stably producing rFurin in a large amount. The
procedure was based on the limited dilution method. Briefly, the
cell suspension was diluted so that 100 .mu.l of the suspension
contains a cell. The wells of a 96-well plate were filled with 100
.mu.l of this suspension. In theory, each well contains one cell
for clonal development. As these single cells start to grow, clones
develop. Thus, every newly generated cell can be traced back to the
first original cell in the well. The clones were expanded in
24-well plates, then in T25 flasks, then in T75 flasks, and then in
T175 flasks.
[0105] During culture, in process controls (IPC) were performed to
monitor growth conditions and to measure rFurin expression. Cell
densities during culture were measured using the Casy instrument.
The Nucleo counter instrument was applied for the detection of cell
nuclei after CTX extraction. Determination of cell density and
viability after thawing was performed with trypan blue exclusion
method using a hemacytometer. Cell density and viability was also
analyzed by using an automated trypan blue exclusion method
performed by the Cedex instrument.
[0106] The supernatants of the cell cultures were used to determine
the amount and activity of expressed rFurin. Fluorescence activated
cell sorting (FACS) analysis was used to see the ratio of producer
to non-producer cells in a given cell population. Morphology and
growth behavior of cells were determined by optical control.
Additionally, the supernatant of the cell cultures was also
examined to monitor medium conditions, such as the determination of
the pH value and the residual concentration of glucose, glutamine,
lactate, and ammonium. These analyses were performed by means of a
NOVA instrument.
[0107] In the course of this study, two pre-master cell banks
(PMCBs) PMCB#01 and PMCB#04 were produced and two cell clone lines
were established as set out below. Evaluation cell banks (ECB) (see
Table 15) were also generated.
Preparation and QC Testing of PMCB#01
[0108] One vial of the CHO/rFurin #488-3 (ECB#01) was thawed in BAS
medium containing additives (Put, Glut, Synp and Fe) and 5% FBS.
The cells were cultured cells for five days in a T175. The cells
were then adapted to serum-free conditions as set out below.
[0109] Cells were prepared in 150 ml growth medium (BAS with Put,
Glut, Synp and Fe) containing 5% FBS, 0.2 g/l. Cytopore 2 carriers
were used for the adaptation to serum-free conditions. Having
inoculated the 5-day old cell suspension resulting in a starting
cell density of about 2.0.times.10.sup.5 cells/ml, the cells
attached to the porous carriers within the first hours. Thus, the
cells were kept in the spinner flask, while the growth medium was
exchanged to reduce the serum concentration in two steps, from 5%
to 3.8% to 0% on day 05. During the following culture period the
cells got adapted to the serum-free medium conditions. The cells
detached from the surface of the carriers and continued their
growth in suspension. The cell density and the viability of the
suspension cells increased continuously. Every two to three days
the suspension cells were split in a ratio of 1:2.
[0110] An evaluation cell bank (ECB) was then prepared. On culture
day 28, cells having a viability of greater than 60% were
transferred to a new experiment. The culture was grown in 200-300
ml BAS medium (with Put, Glut, Synp and Fe) in a Bellco spinner
without carriers. According to the determined CASY cell density,
the suspension culture was split every two to three days to a
starting cell density of about 2.0.times.10.sup.5 cells/ml. After
16 days, when the cell culture reached a viability of greater than
80%, the evaluation cell bank (ECB) consisting of six vials of
cells was produced.
[0111] One vial of the ECB was thawed in a new experiment. The
cells were grown for four days in a T175 in BAS medium with Put,
Glut, Synp, Fe and Zn. Cells were then transferred to a Bellco
spinner containing up to 600 ml BAS medium with Put, Glut, Synp, Fe
and Zn. Again, every two to three days the suspension culture was
split to a starting cell density of about 2.0.times.10.sup.5
cells/ml. On culture day 13, 143 ml of the cell suspension were
removed for the preparation of the PMCB#01, consisting of 20 vials.
The cells were expanded and quality control tests were performed on
PMCB#01.
Preparation and QC Testing of PMCB#04
[0112] One vial of the CHO/rFurin #488-3 (ECB#01) was thawed in BAS
medium containing containing Put, Glut, Synp, Fe and 5% FBS. Half
of the six day old culture was transferred to a new experiment for
adaptation to serum-free conditions.
[0113] Here, the adaptation to serum-free conditions was not
performed step by step, but rather was carried out abruptly. A
Bellco spinner flask was prepared with BAS medium (with addition of
Put, Glut, Synp and Fe) containing no FBS and no carriers. The
cells were inoculated with a starting cell density of about
2.5.times.10.sup.5 cells/ml. Due to the sudden serum-free
conditions, the doubling time of the cells decreased to a rather
low level. To avoid diluting the cells to such a concentration
where growth could be possibly inhibited, the medium was changed by
spinning down the cell suspension. The cell pellet was resuspended
in fresh growth medium. Culture splits were performed when the cell
density was greater than 4.0.times.10.sup.5 cells/ml. After having
reached a minimum viability of about 50% at culture day 15, the
cells started to recover and, from culture day 32 and on, their
viability increased to between 85-90%. On culture day 61, the
adapted cells were frozen as an ECB consisting of 15 vials.
[0114] One vial of the ECB was thawed in a new experiment in
serum-free BAS medium comprising Put, Glut, Synp and Fe. After the
culture was cultured in T175 flasks for seven days, it was
transferred to a Bellco Spinner, where the cells grew for two
further days. Then, the addition of Zn was tested. About 100 ml of
the centrifuged cell suspension were resuspended in serum-free BAS
medium containing Put, Glut, Synp, Fe and Zn. The suspension was
cultured in a Bellco Spinner. According to the determined CASY cell
density, every two to three days the suspension culture was split
to a starting cell density of about 2.0.times.10.sup.5
cells/ml.
[0115] For inoculum preparation and to be able to produce a large
amount of a homogenous cell suspension for the generation of the
PMCB#04, the cell culture was scaled up to 1000 ml in a Bellco
Spinner. On culture day 02, 465 ml of the cell suspension were used
to produce the PMCB#04, consisting of 20 ampules. The cells were
expanded and quality control tests were performed on PMCB#04.
[0116] FIG. 4 sets out a comparison of the graphical distribution
of the rFurin producers in the cell populations of PMCB#01 and
PMCB#04. 80.74% of the cells in PMCB#04 express rFurin. 74.06% of
the cells in PMCB#01 express rFurin.
[0117] CHO/rFurin #488-3 subclones CJ06-19/5F10 and CJ06-19/1E8
were then generated. An ampule of the CHO/rFurin clone #488-3 was
thawed and the culture was passaged in a T175 flask. On culture day
17, three different media were tested, BAP medium (developed by
Baxter), CD-CHO (provided by Gibco) and ExCell 325 PF CHO (provided
by JRH), each of them contained 5% FBS. After four days of growth
in T175 flasks, cells grown in ExCell medium were adapted to
serum-free conditions.
[0118] The cells were weaned off from serum in small steps. The
whole procedure ranged over three experiments. First, the
anchorage-dependent cells were initially cultured in T175 flasks in
ExCell 325 PF CHO containing 5% FBS. The serum was then slowly
reduced to 0.5% on culture day 13. During the next 13 culture days
two splits were performed, wherein the serum concentration was
further reduced to 0.25% and the viability decreased to lesser than
70%. The cells lost their anchorage-dependent behavior, showed more
and more spherical shape, and started to grow in suspension.
[0119] The last step of serum reduction occurred in a Techne
Spinner in ExCell 325 PF CHO medium containing 0.25% FBS and no
carriers. After a culture period of 23 days, the serum
concentration reached 0%. Cells were split and cultured to keep all
relevant parameters in their given range. The cell density moved
between 0.15 to 0.9.times.10.sup.6 cells/ml. The viability dropped
to less than 40% during the first week, but then returned to values
of greater than 90%. On culture day 42, the adapted cells were
frozen in the ECB/CJ06-20 consisting of 20 vials.
[0120] The cells were then subcloned. A cell suspension was diluted
with preconditioned ExCell 325 PF CHO in such a way, that 100 .mu.l
of the suspension theoretically contained 0.5-1.0 cells. In the
subcloning experiment, five 96-well plates were filled with 100
.mu.l of this cell suspension per well. The day after the seeding
of the cells, the wells were searched for single cells under the
microscope. Wells containing one cell were marked and observed
further. Addition and exchange of preconditioned ExCell 325 PF CHO
were performed when necessary. When the cell died or in the absence
of cell division during the next two weeks, the relevant well was
excluded from the experiment.
[0121] Two single cells showed growth. The evolved clones, having
reached an appropriate size, were transferred into a well of a
24-well plate. Here, the exchange of preconditioned ExCell 325 PF
CHO was also performed according to the growth and the requirements
of the culture. The subclones CJ06-19/5F10 and CJ06-19/1E8 were
transferred into a T25 flask on culture day 07 and day 10,
respectively.
[0122] The ECBs were prepared in ExCell medium. These ECBs present
the source material for further investigations concerning the two
subclones, such as a re-adaptation from expensively purchased media
to a more economical formulation, self-developed by Baxter. ECB of
sub clone CJ06-19/5F10, CJ06-42, was expanded in ExCell 325 PF CHO
medium from a T25, to a T75, to a T175, and then into a Bellco
Spinner. The ECB/CJ06-63 consisting of 10 vials were frozen from a
culture out of the T175 on day 21. ECB of subclone CJ06-19/1E8,
CJ06-43, was also expanded in ExCell 325 PF CHO medium. The culture
was expanded from a T25, to a T75, to a T175, and then into a
Bellco Spinner. The ECB/CJ06-64 consisting of 10 vials were frozen
from a culture out of the T175 on day 18.
[0123] The ECBs (Subclones 5F10 and 1E8) were adapted to BCS medium
as set out below. The subclone CJ06-19/5F10 of ECB clone CJ06-19
was thawed in experiment CJ06-66, and then by adding BCS medium to
ExCell medium in an increasing volume, the cells were weaned from
ExCell medium and acquired the ability to grow in BCS medium. The
subclone CJ06-19/1E8 of ECB clone CJ06-19 was simultaneously
adapted to BCS medium in a similar manner.
[0124] Table 8 shows all serum-free cell banks which were prepared
in the course of this study.
TABLE-US-00008 TABLE 8 Summary of serum-free cell banks of rFurin
expressing CHO cell clone #488-3 Cell density/ Thawing Test No.:
Date Name Amp Amp. Medium contr. PMCB#01 DE06-02 10.03.06
ECB/DE06-02 1.0 .times. 10.sup.7 6 BAS + Put + Glut + Synp + Fe ok
CJ06-73 12.05.06 PMCB#01 1.5 .times. 10.sup.7 20 BAS + Put + Glut +
Synp + Fe + Zn ok PMCB#04 CJ06-51 11.04.06 ECB/CJ06-51 1.2 .times.
10.sup.7 15 BAS + Put + Glut + Synp + Fe ok SK06-65 12.05.06
PMCB#04 1.5 .times. 10.sup.7 20 BAS + Put + Glut + Synp + Fe + Zn
ok Sub Clones CJ06-20 27.02.06 ECB/CJ06-20 1.1 .times. 10.sup.7 20
ExCell + Glut n.d. CJ06-63 14.04.06 Klon: CJ06-19/5F10 1.0 .times.
10.sup.7 10 ExCell + Glut ok ECB/CJ06-63 SK06-58 10.05.06 Klon:
CJ06-19/5F10 1.0 .times. 10.sup.7 5 ExCell + Glut/ ok ECB/SK06-58
BAS + Put + Glut + Synp + Fe(1:1) SK06-83 19.06.06 ECB/SK06-83 1.5
.times. 10.sup.7 15 ExCell + Glut/ ok BAS + Put + Glut + Synp + Fe
+ Zn (1:1) SK06-151 03.11.06 Klon: CJ06-19/5F10 1.0 .times.
10.sup.7 5 BCS + Zn n.d. ECB/SK06-151 CJ06-64 14.04.06 Klon:
CJ06-19/1E8 1.0 .times. 10.sup.7 10 ExCell + Glut ok ECB/CJ06-64
SK06-59 10.05.06 Klon: CJ06-19/1E8 1.0 .times. 10.sup.7 5 ExCell +
Glut/ ok ECB/SK06-59 BAS + Put + Glut + Synp + Fe (1:1) SK06-84
19.06.06 ECB/SK06-84 1.5 .times. 10.sup.7 15 ExCell + Glut/BCS
(1:1) ok SK06-152 03.11.06 Klon: CJ06-19/1E8 1.0 .times. 10.sup.7 7
BCS + Zn n.d. ECB/SK06-152
[0125] A comparison of PMCB#01 and PMCB#04 and subclones 5F10 and
1E8 is set out in Table 9.
TABLE-US-00009 TABLE 9 Comparison of PMCB#01, PMCB#04 and Subclones
5F10 and 1E8 Average values of Cell Evaluated Cell density x
Viability Furin act. FACS .mu. g q.sub.p Q.sub.p population
Experiments period [.times.10.sup.6 cells/ml] [%] [U/ml] [%]
(d.sup.-1] [h] [U/(10.sup.6 * d)] [U/(ml * d)] PMCB#01 CJ06-69
29.04.-17.05.06 1.16 84.4 274.87 71.30 0.494 35.94 123.0 77.1
PMCB#04 SK06-49, 26.04.-17.05.06 1.10 94.4 287.03 72.02 0.614 28.52
207.7 96.8 SK06-63 Clone CJ06-66, 21.04.-17.05.06 0.59 94.3 105.57
83.30 0.523 37.43 135.0 40.8 5F10 SK06-52 Clone 1E8 CJ06-67,
21.04.-17.05.06 0.59 94.8 95.55 88.34 0.512 39.01 114.4 35.7
SK06-53
[0126] The cell populations cultured in the experiments CJ06-69 as
well as SK06-49 and SK06-63 represent the precursor cultures of the
cell lines for the PMCBs. These cells were grown in BAS medium
(with the addition of Put, Glut, Synp, Fe and Zn), and were frozen
as PMCB#01 and PMCB#04, respectively.
[0127] The subclones 5F10 and 1E8 were grown in the commercially
available medium ExCell 325 PF CHO provided by JRH. Because the
ability of growing in medium based on the BAS formulation was
preferred, PMCB#01 and PMCB#04 were chosen for further production
of rFurin.
[0128] As presented in Table 10, the cells of PMCB#04 showed better
growth behavior in comparison to PMCB#01. Viabilities and growth
rates were greater in PMCB#04, and generation doubling times were
lower. The cell-specific as well as the volumetric production ratio
of rFurin was also greater throughout the evaluated period of time.
In a further experiment, the data concerning the viabilities was
confirmed. One ampule of each of the PMCBs was thawed into a T175
flask containing BCS medium with Zn as additive. As shown in Table
10 the viability of PMCB#04 was again greater than that of
PMCB#01.
TABLE-US-00010 TABLE 10 Thawing experiments of PMCB#01 and PMCB#04
cell density Cell [.times.10.sup.7 cells/ viability population
experiment vial] [%] PMCB#01 CJ06-77 [75] 1.05 68.7 PMCB#04 SK06-67
[76] 0.99 77.4
[0129] Thus, PMCB#04 was chosen as the source material for the
future production of a rFurin Master Cell Bank (MCB) and all
further working cell banks (WCBs). The PMCB#04 consisting of 20
vials was established in compliance with the current Good Tissue
Practice regulations. Quality Control (QC) testing was performed in
accordance to requests of the ICH-Guideline Q5D. PMCB#04 was chosen
for producing rFurin in manufacturing processes.
[0130] The growth medium is free of human or animal derived
substances and is self-developed. Having the cells removed by
filtration, the rFurin containing supernatant is concentrated by
ultrafiltration. After purification by exchange chromatography, the
activity of the rFurin solution is aimed to be at least 200 Units
rFurin/ml.
[0131] Testing for mycoplasma, viruses, extraneous agents,
sterility, and expression efficiency was carried out. The criteria
for selection are high Furin activity (e.g., Furin protein, ELISA)
and homogeneity of the cell population in immunoflourescence
(FACS), performed on different subclones in comparison to initial
clones #488-3 and #289-20, respectively. In addition, the impurity
profiles have to be compared qualitatively (e.g. by UV-peak
patterns after RP HPLC or by SDS-PAGE/Coomassie techniques).
Example 3
Optimization for Manufacturing Recombinant Furin in Animal
Protein-Free Medium
[0132] This example describes the development and optimization
process for the culture of the rFurin expressing CHO clone #488-3.
Specific medium optimization with regard to amino acids, glucose,
and NaHCO3 concentration was carried out, which resulted in
increased cell growth rates and higher productivities of the
fermentation process. Optimization for inline controlled process
parameters was carried out with the optimized medium formulation
for pO2 (10%, 20% and 50%), and a factorial experiment was carried
out to determine optimum pH (range 7.1-7.3) and temperature (range
35.1.degree. C.-37.9.degree. C.), which resulted in a significant
yield improvement for CHO clone #488-3 when fermentation was
carried out at lower temperatures between 35.degree.-36.degree.
C.
[0133] As possible production modes, chemostat cultures and batch
reefed cultures were compared, indicating that both process types
are suitable for the manufacturing of rFurin, and give comparable
yields with identical parameter settings. Specific experiments were
carried out to investigate the influence of agitation types and
rates in different bioreactor setups. It was shown that the
specific growth rates and expression rates, cell densities and
therefore volumetric productivities are strongly influenced by
variations in the bioreactor setup, and that under conditions of
increased agitation rates the yields could be significantly
increased. Altogether, the influence of the main parameters of the
rFurin upstream process are well characterized with regard to their
effect, and a high yielding process could be transferred to the
pilot plant for preclinical and clinical manufacturing. The details
are set out below.
[0134] A subclone # 488-03 was developed in-house which was adapted
to a serum- and insulin-free medium. The following experiments were
carried out in a FBS-free and insulin-free medium (BACD-medium) as
the basic medium formulation (see Table 11).
TABLE-US-00011 TABLE 11 Components of the basic medium
(BACD-medium) Concentration Component [g/kg] DMEM/F12 (1:1) 11.76
L-Glutamine 0.6 Ethanolamine 0.00153 Synperonic 0.25
Putrescine.cndot.2HCl 0.0036 FeSO4.cndot.7H2O 0.0006
CuSO4.cndot.5H2O 0.00000125 NaHCO3 2.0
[0135] In order to improve cell growth and to provide optimal
conditions for cell propagation, an amino acid analysis of the
supernatant of a chemostat culture was performed (see Table 12). As
a result, three essential amino acids were added to the medium,
namely methionine (10 mg/L), leucine (40 mg/L) and phenylalanine
(10 mg/L). Fermentation was performed in a 1.5 L bioreactor at
37.degree. C., pH 7.15 and a pO.sub.2 of 20%.
TABLE-US-00012 TABLE 12 Amino acid analysis by HPLC Amino acids
FUR_06/10_M04_K06 FUR_06/10_M04_K07 BACD_24_06_014_026 relative
peak area relative peak area Peak area [%] [%] [%] Aspartic acid
100.00 24.04 9.88 Glutamic acid 100.00 57.78 24.89 Serine 100.00
14.37 15.35 Asparagine 100.00 15.03 19.17 H.sub.2O Glycine 100.00
103.55 90.58 Glutamine 100.00 19.26 23.14 Histidine 100.00 45.59
45.32 Threonine 100.00 63.09 61.39 Arginine 100.00 72.22 74.25
Alanine 100.00 1513.36 1518.46 Proline 100.00 58.54 55.36 Tyrosine
100.00 46.37 47.63 Cystine 100.00 53.43 56.39 Valine 100.00 42.75
42.11 Methionine 100.00 13.23 11.51 Isoleucine 100.00 48.39 48.79
Leucine 100.00 23.58 20.63 Lysine HCl 100.00 41.85 40.65
Phenylalanine 100.00 34.99 33.00 Tryptophan %: 100 69.1 68.8
BACD_24_06_014_026 . . . basic medium (s. materials and methods)
FUR_06/10_M04_K06 . . . Fermentation lot, 6.sup.th day of culture
FUR_06/10_M04_K07 . . . Fermentation lot, 7.sup.th day of culture
The peak area of the respective amino acid in the HPLC-diagram of
the basic medium was set to 100%. By comparison to the peak area
obtained on day 6 and 7 of the chemostat culture the decrease of
the respective amino acid was determined.
[0136] Due to low glutamine concentrations in the supernatant of
the culture, glutamine was added to the medium (300 mg/L) to give a
final concentration of glutamine at 900 mg/L. After the addition of
glutamine to the medium the growth rate increased from 0.55
d.sup.-1 to 0.67 d.sup.-1 (see Tables 13 and 14). By the addition
of these three amino acids mentioned above (namely methionine (10
mg/L), leucine (40 mg/L) and phenylalanine (10 mg/L)) to the
medium, the growth rate of the cells could be increased again (0.69
d.sup.-1) (see Table 15). The volumetric productivity went up to
approximately 267 kU/L/d (=+13%) and the specific productivity
showed an increase of 16%. The supplementation of the medium with
glutamine, methionine, leucine and phenylalanine showed a positive
effect on cell growth, and volumetric and specific activity, and
was therefore retained for further medium preparation.
TABLE-US-00013 TABLE 13 Fermentation data of the chemostat culture
FUR_06/10-M04 from day 6 to day 9 of culture Experiment glc gln pH
D CC .mu. Furin P qP FUR_06/10-M04 [g/L] [g/L] NOVA [1/d] [1E6/mL]
[1/d] [IU/mL] [kIU/L/d] [IU/C/d] * 10.sup.6 Day 6-9 1.58 0.10 7.16
0.494 2.35 0.548 306.55 151 64
TABLE-US-00014 TABLE 14 Fermentation data of the chemostat culture
FUR_06/10-M04 after the supplementation of the medium with of 300
mg/L glutamine on day 12 Experiment FUR_06/10- glc gln pH D CC .mu.
Furin P qP M04 [g/L] [g/L] NOVA [1/d] [1E6/mL] [1/d] [IU/mL]
[kIU/L/d] [IU/C/d] * 10.sup.6 Day 15-21 1.47 0.35 7.16 0.668 2.27
0.673 352.68 236 104
TABLE-US-00015 TABLE 15 Fermentation data of the chemostat culture
FUR_06/10-M04 after the addition of methionine (10 mg/L), leucine
(40 mg/L) and phenylalanine (10 mg/L) on day 22 to the medium
Experiment glc gln pH D CC .mu. Furin P qP FUR_06/10-M04 [g/L]
[g/L] NOVA [1/d] [1E6/mL] [1/d] [IU/mL] [kIU/L/d] [IU/C/d] *
10.sup.6 Day 26-29 1.37 0.38 7.13 0.713 2.20 0.695 373.80 267
121
[0137] To check whether the supplemented amounts of the amino acids
in the medium were sufficient, another amino acid analysis of a
chemostat culture (10 L) was performed. The culture had been
supplied with 300 mg/L glutamine and the respective amounts of the
three amino acids. The samples were drawn on day 7 and 15 of
culture, and the culture conditions were similar to those mentioned
above. The results (see Table 16 where only the data for
methionine, leucine and phenylalanine are shown) confirmed that
sufficient amounts of the supplemented amino acids were present in
the fermentation broth.
TABLE-US-00016 TABLE 16 Relative amount of methionine, leucine and
phenylalanine in a chemostat culture (FUR 06/17_F04) supplemented
with glutamine (300 mg/L), methionine (10 mg/L), leucine (40 mg/L)
and phenylalanine (10 mg/L) Amino acid Peak area [%] relative peak
area [%] relative peak area [%] BACD-24-06-030-057 FUR
06/17_F04-K07 FUR 06/17_F04-K15 Methionine 100.0 47.5 61.7 Leucine
100.0 50.3 65.4 Phenylalanine 100.0 50.1 65.2
[0138] Reduction of the NaHCO.sub.3 concentration and increase of
the glucose concentration. The influence of high dissolved CO.sub.2
concentrations in the cell culture on growth and productivity was
investigated. Due to large scale production in a bioreactor, a
greater CO.sub.2 concentration in the cell culture can be expected
than in 2.5-32 L bioreactors. Therefore, two fermentation runs were
carried out in parallel, one run with a CO.sub.2 concentration of
approximately 7.5% and the other one with a CO.sub.2 concentration
of approximately 12%. The CO.sub.2 concentration was adjusted by
varying the CO.sub.2 fraction in the head space flow. The CO.sub.2
concentration in the cell culture was measured by analyzing drawn
samples with the NOVA instrument. The fermentation was carried out
at 37.degree. C., at a pH of 7.15 and with a pO.sub.2 of 20%.
[0139] A CO.sub.2-concentration of 11-12% had a negative influence
on cell growth and productivity. At this CO.sub.2-concentration, a
growth rate of 0.29 d.sup.-1 was reached over an interval of 12
days at a cell count of 1.1.times.10.sup.6 cells/mL and a dilution
rate of 0.30 d.sup.-1. In the fermentation run with approx. 7.5%
CO.sub.2, a growth rate of 0.52 d.sup.-1 was reached at a cell
count of 1.49.times.10.sup.6 cells/mL and a dilution rate of 0.53
d.sup.-1 in the same interval. At high CO.sub.2 concentrations, the
viability was reduced to 86.1%, compared to 95.9% at 7.5% CO.sub.2.
Additionally, the volumetric productivity was reduced to
approximately 36% and the specific productivity to 50%. Due to the
high CO.sub.2 concentration, the specific glucose uptake rate was
decreased as well (-39%). The negative influence of an increased
CO.sub.2 concentration (11-12%) on cell growth and productivity was
quite obvious and, therefore, it is optimal to carry out the
fermentation at 7.5% CO.sub.2.
[0140] As set out above, experiments showed that when the
concentration of CO.sub.2 was increased to 12% in the 1,000 L
bioreactor, a strong decrease in the performance of the CHO-Furin
clone 488-3 can be expected. Therefore, it was decided to reduce
the NaHCO.sub.3 concentration in the medium from 2 g/L to 1.5 g/L.
A lesser amount of NaHCO.sub.3 in the medium also decreased the
buffer capacity of the medium and, therefore, two fermentation runs
(10 L) were compared, one with 3.15 g/L glucose and 2 g/L
NaHCO.sub.3 in the medium (FUR.sub.--06/24_F01) and another one
with 4.65 g/L glucose and 1.5 g/L NaHCO.sub.3
(FUR.sub.--06/26_F04).
[0141] To simulate the conditions of a large scale bioreactor
(1,000 L), the concentration of dissolved CO.sub.2 during
fermentation was adjusted to 7-8% in the fermenter with 2 g/L
NaHCO.sub.3 and to 6-7% in the fermenter with 1.5 g/L NaHCO.sub.3
by constant CO.sub.2 gassing in the head space. The growth rates of
both cultures were similar (0.58 and 0 56 d.sup.-1) and the
cultures showed comparable volumetric productivities and
viabilities. However, the specific glucose uptake rate was slightly
higher in the culture with a lower NaHCO.sub.3 concentration (0.83
mg/10.sup.6cells/d vs. 0.67 mg/10.sup.6 cells/d). Therefore, a
glucose concentration of 4.65 g/L was considered to be reasonable
and was retained in further medium preparation.
[0142] Investigation of the Synperonic F68 concentration in the
Furin medium. The regular concentration of Synperonic F68 in the
cell culture medium was set at 0.25 g/L. The purpose of Synperonic
F68 in the medium is to protect the cells from damage due to
submerged oxygenation. Therefore, one experiment was carried out in
2.times.10 L bioreactors, where an increased Synperonic F68
concentration of 1.0 g/L vs. the regular concentration of 0.25 g/L
was investigated. The fermentation was carried out at 35.8.degree.
C. at a pH of 7.30 and with a pO.sub.2 of 20%.
[0143] With increasing Pluronic concentration (Synperonic F68), a
slightly higher specific growth rate and cell density could be
achieved. Thus, due to increased specific productivity, a
proportionally greater volumetric productivity of 365 vs. 278
kU/L/d could be achieved.
[0144] Supernatants from these experiments were collected and
filtered with a depth filter (Cuno Cart.Z08P4A30SP 4 discs)
followed by a membrane filter (Pall Fluorodyne II KA2DFLP2). The
filtered supernatants were concentrated by ultrafiltration
(Sartorius 30S1463901E--SG PSU 10KD, 0.2 m.sup.2) and diafiltrated
against the Furin diafiltration buffer. The sterile filtered
concentrate (Sartorius Sartobran P 523130748-00 0.45/0.2.mu.) was
purified on the Capto MMC column. Results from this purification
experiments revealed that the increased Synperonic F68 has no
detrimental influence on quality and/or purity of the purified
rFurin. No elevated Synperonic F68 concentrations could be found in
the eluate of the Capto MMC column, which were <0.15 mg/mL in
both eluates.
[0145] Despite these results the Synperonic F68 concentration in
the rFurin medium was kept at the original 0.25 g/L. However, in
case of scale up problems due to submerged oxygenation, an
increasing Synperonic concentration of up to 1.0 g/L could be
considered, with the potential of increased yields without
detrimental influence of the final Furin product.
[0146] Final Medium Composition for rFurin Production in
Fermentation. Based on the medium optimization experiments, the
following medium composition is shown to be optimal for the
fermentation of CHO clone 488-3 in bioreactor cultures for the
production of rFurin.
TABLE-US-00017 TABLE 17 Components of the basic medium and
optimized fermentation medium Basic Medium Optimized Medium
Concentration Concentration Component [g/kg] [g/kg] DMEM/F12 (1:1)
11.76 11.76 L-Glutamine 0.6 0.9 D-Glucose -- 1.5 Ethanolamine
0.00153 0.00153 Synperonic 0.25 0.25 Putrescine.cndot.2HCl 0.0036
0.0036 Methionine -- 0.01 Leucine -- 0.04 Phenylalanine -- 0.01
FeSO4.cndot.7H2O 0.0006 0.0006 CuSO4.cndot.5H2O 0.00000125
0.00000125 NaHCO3 2.0 1.5
[0147] The influence of different pO.sub.2 set points on growth
rate and productivity was also investigated. Three different
pO.sub.2 set points were tested, i.e. 10%, 20% and 50%. The
experiments were performed in 1.5 L bioreactors with gassing via
head space. All fermentation runs were carried out at 35.1.degree.
C. at pH 7.20. Comparison of the fermentation runs at 10, 20 and
50% pO.sub.2 showed a slight increase of the growth rates (0.59,
0.62 and 0.65 d.sup.-1) with increasing pO.sub.2 set points.
Likewise, the volumetric productivity was greatest at 50% pO.sub.2.
The mean cell counts of the different fermentation runs were very
similar. Thus, the increase in the volumetric productivity was the
result of the increasing growth rate.
[0148] Consequently, a pO.sub.2 set point of 50% seemed to
influence the growth rate positively, and as a result the
volumetric productivity was approx. 4% higher than at standard
conditions (=20% pO.sub.2). No effect on viability was observed.
Therefore, a setpoint of pO2=20% and a range for regulation between
10%-50% could be confirmed to be suitable for fermentation of the
CHO clone #488-3.
[0149] Optimization of temperature and pH to maximize the
volumetric productivity. The influence of pH and temperature on the
performance of the CHO-Furin clone was investigated. By using a
`design of experiments method`, different temperatures were
combined with different pH values to ascertain the conditions which
resulted in maximum volumetric productivity. Five temperatures were
combined with three pH values according to the "Doehlert Matrix",
resulting in seven combinations of temperature and pH, as shown in
FIG. 5.
[0150] The combination of 36.5.degree. C. and pH 7.20 was chosen as
the center point, which was applied to two fermentation lots. Table
18 sets out the culture conditions used in the different
fermentation lots.
TABLE-US-00018 TABLE 18 Experimental setup of the Doehlert Matrix:
Culture conditions of the different fermentation lots Fermentation
lot Temp. [.degree. C.] pH FUR_06/43-B07 35.1 7.20 FUR_06/43-B05
35.8 7.10 FUR_06/40-B03 35.8 7.30 FUR_06/40-B01 36.5 7.20
FUR_06/43-B02 36.5 7.20 FUR_06/40-B08 37.2 7.10 FUR_06/40-B04 37.2
7.30 FUR_06/40-B06 37.9 7.20
TABLE-US-00019 TABLE 19 Mean values of the fermentation data of the
fermentations FUR_06/40-B01, -B03, -B04, -B06, -B08 and
FUR_06/43-B02, -B05, -B07 Fermentation Temp. CC .mu. Furin p.sup.1)
qP.sup.1) Viability lot [.degree. C.] pH [10.sup.6/mL] [1/d]
[IU/mL] [kIU/L/d] [U/10.sup.6/d] [%] FUR_06/43-B07 35.1 7.20 1.74
0.595 930 543 312 97.7 FUR_06/40-B03 35.8 7.30 1.64 0.632 621 379
232 97.6 FUR_06/43-B05 35.8 7.10 1.73 0.647 683 429 250 98.5
FUR_06/40-B01 36.5 7.20 1.78 0.696 439 309 174 97.9 FUR_06/43-B02
36.5 7.20 1.66 0.684 454 310 186 96.8 FUR_06/40-B04 37.2 7.30 1.71
0.642 284 184 108 96.8 FUR_06/40-B08 37.2 7.10 1.68 0.652 338 215
128 98.3 FUR_06/40-B06 37.9 7.20 1.52 0.473 143 65 43 96.1
.sup.1)the mean volumetric and the mean specific productivity over
5 culture days were calculated by forming the the mean value of the
singel productivities
[0151] The data were analyzed statistically with the Response
Surface Methodology (RSM), using the "Minitab" software. RSM
explores the relationships between several explanatory variables
and one or more response variables. The main idea of RSM is to use
a set of designed experiments to obtain an optimal response. The
analysis focused on the volumetric and specific productivity as
well as on the growth rate.
[0152] Analysis of the data in reference to the volumetric
productivity. As a first step, a surface plot was created (FIG. 6).
The coordinates of the data in FIG. 6 are marked as points. The
surface shows the assumed correlation of the single data.
[0153] The surface plot supposes a linear correlation between the
parameters temperature/pH and the responding volumetric
productivity. The chart indicates an increase of the volumetric
productivity with decreasing temperature. The influence of the pH
is considered to be weak. Subsequent calculations showed a linear
correlation of the data (calculation not shown). By variance
analysis, an equation was generated which describes the correlation
between pH, temperature and volumetric productivity:
P=7693.1-162.4*Temp-202.8*pH
[0154] Based on the mathematical model a contour plot was generated
(FIG. 7) which illustrates the influence of temperature and pH on
the volumetric productivity. The dots indicate the conditions
(pH/temp.) which had been tested experimentally.
[0155] The contour plot shows that the area, where maximum
volumetric productivity can be expected, is at 35.1.degree. C. and
a pH of approx. 7.10. Both values are at the edge of the
experimental design which means that the real maximum could be
found even below those values. Furthermore the Contour plot shows
that the influence of the pH on the volumetric productivity is
marginal and slightly higher at low pH values.
[0156] The surface plot (FIG. 8) which is a three-dimensional
illustration of the mathematical model, gives the same result as
the contour plot: the strong influence of the temperature and the
weak influence of the pH on the volumetric productivity.
[0157] Analysis of the data in reference to the growth rate .mu..
The correlation of the growth rate with temperature and pH is
described in a quadratic equation. Again, a strong influence of the
temperature and a weak influence of the pH can be found. Strong
variation of the growth rate hampers the design of a mathematical
model. Analysis of variance and regression gave following
equation:
P=-103.539+5.706*Temp-0.079*Temp.sup.2-0.233*pH-0.020*pH.sup.2
[0158] The influence of the pH on the growth rate is statistically
not ascertainable, as indicated by the high P-value for the
pH-terms which was calculated to be 0.99 (calculation not
shown).
[0159] The surface plot (FIG. 9) illustrates the modeled
correlation three-dimensionally, demonstrates the quadratic
relationship, and shows a maximum growth rate at 36.5.degree.
C.
[0160] Analysis of the data shows a similar correlation of the
specific productivity with temperature and pH as seen for the
volumetric productivity (FIG. 10). The correlation is described by
a linear equation. The influence of the pH is again low as proved
by variance analysis (calculation not shown):
qP=4261.40-93.35*temp-93.77*pH
[0161] In summary, the experiments for optimization of temperature
and pH and subsequent analysis of the data gave a quite clear
result. The greatest volumetric productivity, which is considered
as the most important parameter for process optimization, was
achieved by culture at the lowest temperature (35.1.degree. C.) and
lowest pH (7.10). The influence of pH is statistically hardly
significant, and a pH value between 7.10 and 7.30 would give very
similar results. By decreasing the temperature from 37.degree. C.
to 35.1.degree. C., the volumetric productivity could be raised
from approx. 200 kU/L/d to 540 kU/L/d which is 2.7 times greater
(FIG. 11). Based on these results, the new set points for
temperature and pH, for the culture of the CHO-Furin clone in the
chemostat mode, were determined as 35.1.degree. C. and 7.15.
[0162] Comparison of chemostat mode to batch refeed mode. The
culture of the CHO-Furin clone in the chemostat mode at a low
temperature (35.1.degree. C.) resulted in high yields in small
scale experiments (1.5 L, 2.5 L, 10 L and 32 L) and was considered
to be the appropriate culture method for the large scale culture
for production of rFurin. However, initial fermentation runs in the
large scale (1200 L bioreactor in the PP1 facility) showed a strong
decrease of the growth rate at the chemostat mode. As an
alternative, to get higher growth rates, the batch reefed mode was
set up in the large scale. Experiments in the 10 L bioreactor scale
were carried out to compare growth and productivity in the
chemostat mode with the batch reefed mode. The cells were cultured
at 36.5.degree. C. at a pH of 7.15 and a pO.sub.2 of 20%. The batch
was split to a cell count of 0.6-0.7.times.10.sup.6 cells/mL every
second culture day.
[0163] The chemostat- and the batch reefed-fermentations were run
in parallel, using the same cell culture as inoculum. The cells
were cultured in the chemostat mode until day 6. Then one
fermentation run was switched to the batch refeed mode
(FUR.sub.--06.sub.--50-F03) while the other one was continued in
the chemostat mode (FUR.sub.--06.sub.--51-F04). Culture in the
batch refeed mode resulted in a mean cell count of
2.22.times.10.sup.6 cells/mL at the end of the batch, with a growth
rate of 0.64 d.sup.-1 (Table 20). In the chemostat mode, a growth
rate of 0.50 d.sup.-1 was obtained with an average cell count of
1.67.times.10.sup.6 cells/mL. Due to the higher cell count and
growth rate in the batch refeed mode, the volumetric productivity
was greater as well (238 vs. 197 kU/L/d).
[0164] The data indicate that the batch reefed mode is a preferable
culture method for the CHO-Furin clone in the 10 L scale, which
results in even slightly higher volumetric productivities than in
the chemostat mode (at 36.5.degree. C. and a pH of 7.15). However,
in the batch refeed mode, harvest- and further downstream processes
are restricted to certain intervals, while in the chemostat mode,
harvesting can be performed continuously. Thus, each method has its
advantages. No optimization experiments for the parameters of the
batch reefed mode were performed like for the optimal cell counts
at the end of a batch or for the best split ratio.
TABLE-US-00020 TABLE 20 Mean values of the fermentation data from
the fermentation runs FUR_06_51-F04 (Chemostat) and Fur_06_50-F03
(Batch Refeed) D .mu. CC Furin P Ferment. lot Vol. Mode [1/d] [1/d]
[1E6/mL] [IU/mL] [kIU/L/d] FUR_06_51-F04 10 L chemostat 0.494 0.497
1.67 401.4 198 FUR_06_50-F03 10 L batch -- 0.637 2.22 (end).sup.1)
539.8 238.sup.2) refeed .sup.1)Mean value of the cell counts at the
end of each batch (2 batches) .sup.2)Mean value of the
productivities at the end of each batch (2 batches)
[0165] Scale Up Investigations. Comparison of Rushton type-versus
ball type-impellers at different agitation rates. The influence of
the agitator type on growth rate and productivity was investigated.
The standard setting used in the bioreactors for experiments with
the CHO-Furin clone comprised a Rushton impeller and four baffle
plates. The 1,000 L bioreactor for rFurin production was equipped
with three pairs of ball impellers without any baffle plates.
Therefore, an experiment was carried out to test three different
settings in 10 L bioreactors: one reactor was equipped with the
standard setting, another reactor with two pairs of ball impellers
and no baffle plates, and a third reactor was assembled like the
standard one but the agitation rate was reduced from 170 rpm to 90
rpm.
[0166] Table 21 gives an overview of the bioreactor set-ups. The
agitation rate of 60 rpm with ball impellers gave a similar tip
speed as the rushton impeller at 90 rpm (see Table 21). However,
the tip speeds cannot be equated with each other due to the
different geometry of the impellers (shape, diameter).
TABLE-US-00021 TABLE 21 10 L Bioreactor Set-ups for
impeller/agitation rate experiment Impeller Agita- type/ Baffle
tion Fermentation lot diam. Levels plates rate Tip speed
FUR_06/37_F03 Rushton 2 4 170 0.712 m s.sup.-1 80 mm FUR_06/35_F01
Rushton 2 4 90 0.377 m s.sup.-1 80 mm FUR_06/38_F04 Ball 2 -- 60
0.440 m s.sup.-1 140 mm, 39.5 mm diameter
[0167] The fermentation conditions were as follows: 37.degree. C.,
pH 7.15, pO.sub.2 of 20% and pCO.sub.2 of 6-7%. (Medium: 4.65 g/L
Glc, 1.5 g/L NaHCO.sub.3). Comparison of the fermentation data
showed that the performance in the bioreactor with the standard
set-up, i.e. equipped with the rushton impeller and agitated at a
rate of 170 rpm, was higher than in the bioreactors with the other
set-ups (Table 22). The use of ball impellers at 60 rpm resulted in
a somewhat lower growth rate (0.57 vs. 0.61d-1), a lower volumetric
(197 vs. 227 kU/L/D) compared to the rushton impeller at 170 rpm.
The viability was hardly affected though. Using a rushton-impeller
stirred bioreactor at an agitation rate of 90 rpm instead of 170
rpm clearly diminished the growth rate (0.54 vs. 0.61d-1),
volumetric productivity (175 vs. 227 kU/L/D) and cell specific
productivity (115 vs. 138 U/10.sup.6 cells/day).
[0168] The data from this experiment showed that both, a ball
impeller at 60 rpm and Rushton impellers at a reduced agitation
rate, result in a declined performance of the CHO-Furin clone
compared to the Rushton impellers at 170 rpm. Nevertheless, the
results also revealed that it is possible to use a ball impeller
for culture of the CHO-Furin clone. Among the set-ups which were
tested, the set-up with the rushton impeller at 170 rpm plus four
baffle plates displayed the roughest conditions. Notably, however,
no decrease of cell viability was observed under these conditions,
which indicates a high mechanical resistance of the investigated
CHO-Furin clone.
TABLE-US-00022 TABLE 22 Mean values of the fermentation data of
three fermentation runs of the CHO-Furin clone in bioreactors with
different set-ups (impeller type, baffle plates) Fermentation CC D
Furin .mu. P qP Viability lot Set-up [10.sup.6/ml] [d.sup.-1]
[IU/mL] [d.sup.-1] [kU/L/d] [U/10.sup.6/d] [%] FUR_06/37_F03
Rushton 1.65 0.628 361.37 0.614 227 138 95.4 170 rpm FUR_06/38_F04
Ball 1.52 0.600 328.10 0.568 197 130 94.1 impeller 60 rpm
FUR_06/35_F01 Rushton 1.53 0.574 305.31 0.544 175 115 94.3 90
rpm
[0169] Comparison of different agitation rates with pitched blade
impellers in 32 L bioreactors. For the GMP run campaign
ORFURFB07002 in the PP1 the bioreactor (working volume 950 L) was
equipped with a pitched blade impeller type (2 pcs., d=700 mm)
instead of the previously used ball type impellers.
[0170] 2 day batch reefed cycles were carried out during this
campaign. In order to assess this change compared to the previous
campaigns, a 32 L bioreactor was set up with a similar type of
pitched blade impellers (1 piece, d=140 mm), and fermentations were
carried out using cell suspension from the PP1 facility and medium
to ensure comparable materials to be used.
[0171] A similar batch reefed process was carried out, where a 2-3
consecutive two day batch cycles were started with a starting cell
density of 0.5.times.10E06 cells/mL. The experiment was carried out
at 2 different agitation rates, i.e. 55 rpm and 120 rpm). Culture
conditions were identical with the culture conditions used in the
PP1: pH-SP 7.15, Temp-SP 35.5.degree. C., pO2-SP 20%. Data from the
last batch were compared after adaptation to the respective
agitation conditions.
TABLE-US-00023 TABLE 23 Growth rate, cell count and vol.
productivity of the batches of run FUR_07/06-F07 with pitched blade
impellers in the 32 L bioreactor FUR_07/06- Agitation F07I rate
CC.sup.1) Split.sup.2) Furin .mu. P.sup.3) Intervall [rpm]
[10.sup.6/ml] 1:x [IU/mL] [d.sup.-1] [kU/L/d] Batch K02- 120 1.95
3.89 1197.8 0.680 445 K04 K10-K12 50 1.50 3.13 812.0 0.570 276
.sup.1)cell count at the end of each batch .sup.2)theoretical split
ratio to be applied in a 2 batch reefed process: split ratio =
e{circumflex over ( )}(2 .times. .mu.) .sup.3)calculation of
volumetric productivity: P = (1 - (1/split ratio)) .times. product
titer/2 days
[0172] The experiment demonstrated again the effect of agitation
conditions on specific growth rates of the CHO clone #488-3. The
increasing growth rates led to greater final cell densities at the
end of the batch, when applying the same starting cell densities.
Volumetric productivities increased from 276 kU/L/D to 445 kU/L/D
(+61%), whereas the final cell density increased by 30% (1.95 vs.
1.5.times.110E06 cells/mL). These results indicate that the
increased agitation rate had a positive impact on cell-specific
productivity.
[0173] It also might be concluded that fermentation runs, which
resulted in average productivities of around 200 kU/L/D were mainly
a result of the applied low agitation rates of 20 rpm, and not due
to the impeller design itself. Here it could be demonstrated, that
pitched blade impellers can result in yields >400 kU/L/D, if the
agitation rates are adjusted accordingly.
Example 4
Purification of Recombinant Furin (rFURIN)
[0174] This example provides methods for the filtering and
purifying rFurin. The collected cell culture supernatants were
first filtered on depth filters (Cuno Zetaplus filters) to get them
cell-free and particle-free, followed by membrane filtration at
0.45 .mu.m PVDF filters (PALL Fluorodyne II). The filtered cell
culture supernatant containing the rFurin was then concentrated by
ultrafiltration on 10 K PES UF cassettes from Sartorius (Sartocon
PESU 10 kDa) with concentration factors ranging from 10-50. The
furin concentrates (with furin activity ranging from 290-1700
Units/ml) were then stored in aliquots at <-60.degree. C.
[0175] In an effort to get a more homogeneous rFurin preparation
for use in the VWF maturation process, experiments were conducted
to partially purify the rFurin from the cell culture supernatant. A
partially purified rFurin reagent is easier to use for
characterization and release testing. It also results in a reduced
presence of CHO host cell proteins in the VWF maturation
process.
[0176] A purification procedure on an anion exchange resin was
developed that required a loading conductivity of <5 mS/cm (RT)
for efficient binding of rFurin. The elution was then performed as
a step procedure at an ionic strength of approximately 500-300 mM
NaCl and during the screening phase, a gradient elution up to 300
mM NaCl was applied (see overview in Table 24). The original pH of
the buffers of 6.7 (RT) was increased to 7.5 to improve binding of
rFurin during loading, in particular at high protein loading and
high liner flow rates (see summary of relevant buffers in Table
25). The purification experiments were performed on EMD TMAE
(Merck) and CaptoQ (GE Healthcare) anion exchange resins that
differed in the stability of the packed and the maximum flow rate
to operate. The analytical data summarized in Table 26 show that
rFurin can be concentrated from the cell culture supernatant up to
362 fold with yields ranging from 20-71%. The rFurin activities in
the eluate pools was between 639 Units/ml and 27651 Units/ml
depending on the load applied. The CHO impurity level in the eluate
was found in a range between 10-134 ng CHO protein/Unit rFurin and
reduction rates up to 12.3 with a slightly better performance for
CHO reduction found for the CaptoQ resin.
[0177] In summary, purification of rFurin by anion exchange
chromatography proved to be an option for concentration, which is
important for storage of the rFurin reagent at <-60.degree. C.
In addition, a slight CHO reduction by a factor from 3-12 is
important in reducing the concentration of CHO protein in the
preparation of VWF.
TABLE-US-00024 TABLE 24 Approximate procedure for the purification
of rFurin on anion exchange resin. For EMD TMAE (Merck) the flow
rate applied was 150/75 cm/h, for CaptoQ (GE Healthcare) the flow
rate was 600/300. Flow rate Step Buffer CV [cm/h] comment
Equilibration FEQ buffer 10 150-600 Load CCS diluted with EQ1 up to
1200 Conductivity of the load .ltoreq.5 mS/cm; Wash 1 EQ buffer up
to 50 Optional; at high column Wash 2 Mix of EQ buffer and Approx.
10 loading this step was FEL buffer omitted Step Elution 30%
FEL/70% FEQ 14 75-300 Gradient 100% FEQ - 14 75-300 Optional: used
for initial elution 30% FEL/70% FEQ screening experiments Post
Various buffers (FEL, 15 n.d. conditioning NaCl buffer, CIP
buffer)
TABLE-US-00025 TABLE 25 Buffers for the purification of furin on
anion exchange resin Buffer formulation Equilibration 50 mM HEPES,
1 mM CaCl.sub.2, At high loadings buffer (FEQ) pH = 6.7-7.5 at RT
the pH of the buffers Elution buffer 50 mM HEPES, 1 mM CaCl.sub.2,
1 were increased to (FEL) M NaCl, pH = 6.7-7.5 at RT improve
binding NaCl buffer 2 M NaCl CIP buffer 0.5 M NaOH
TABLE-US-00026 TABLE 26 Summary of relevant purification
experiments of rFurin on anion exchange resin. The total protein
content was determined using a Bradford assay. The CHO reduction
rate is calculated as CHO protein loaded divided by total CHO
protein found in the eluate pool. Load Eluate Column Conc. load
Factor U Furin Vol yield CHO Run Furin/ml U/mg load/Vol % Furin
ng/U ID resin U/ml protein eluate activity Furin reduction comment
011 2705 639 n.d. 32 36 134 3.6 TMAE; RT, elution 500 mM NaCl 013
4016 2259 n.d. 33 57 51 3.0 TMAE; RT, elution 400 mM NaCl 015 4403
2007 n.d. 41 46 50 3.8 TMAE; RT, elution 300 mM NaCl 018 10635 2584
n.d. 65 37 41 6.5 TMAE; RT, elution 300 mM NaCl 021 8684 3550 n.d.
126 20 59 5.5 TMAE; RT, elution 300 mM NaCl 024 4151 903 n.d. 19 54
38 6.3 CaptoQ; RT; elution 300 mM NaCl 025 E1 15696 6185 n.d. 45 10
12.3 CaptoQ; RT; gradient E2 1068 n.d. 12 n.d. n.d. elution 026 E1
31316 10237 14138 51 8 10.1 CaptoQ; 4.degree. C.; gradient E2 1868
n.d. 17 n.d. n.d. elution 028 32635 27651 10011 288 68 19 3.6 TMAE;
4.degree. C.; elution 300 mM NaCl 029C 23682 12642 7789 176 71 14.4
6.9 CaptoQ; RT; elution 300 mM NaCl 029T 23806 14922 2671 362 30
65.1 3.3 TMAE; RT, elution 300 mM NaCl 033 20625 6843 4568 n.d. 35
n.d. 4.6 CaptoQ; RT; elution 300 mM NaCl; load UF/DF conc. 035
26244 13209 6967 n.d. 78 n.d. 1.9 TMAE; RT, elution 300 mM NaCl;
load UF/DF conc.
Example 5
Concentration, Purification, and Analysis of rFurin
[0178] This example describes other methods used in the
concentration and purification (i.e., downstream processing) of
large-scale rFurin. Such processing methods include
ultrafiltration, diafiltration, and capto-MMC chromatography, that
was carried out in the production of substantially animal
protein-free rFurin. It also describes methods of analyzing protein
concentration, specific activity, and contamination by host cell
protein and DNA.
[0179] Ultrafiltration. The supernatant (approx. 800-1200 kg in the
Chemostat campaigns, and approx. 550-700 kg in the RFB-campaigns)
was separated from the cells and concentrated to a final volume of
3545 L by ultrafiltration. The parameters and setpoints of the
Ultrafiltration/Diafiltration System (UFS) during the concentration
step are listed in Table 27.
TABLE-US-00027 TABLE 27 Operating Parameters and Setpoints for the
Concentration step Parameter Setpoint Temperature of the filtered
10-15.degree. C. harvest Filter Area 14 m.sup.2 Feed Pressure
0.9-1.5 bar Retentate Pressure 0.7-1.2 bar Permeate Pressure 0-0.1
bar Transmembrane Pressure 0.7-1.0 bar Specific Cross Flow Rate
300-600 L/h/m.sup.2 (Feed flow rate) Specific Permeate Flow Rate
15-40 L/h/m.sup.2 Concentration factor 20-30 Processing Time 3-4
h
[0180] Diafiltration. Immediately after finishing the concentration
step, diafiltration of the retentate was initiated. The parameters
and setpoints of the UFS during the diafiltration step are listed
in Table 28.
TABLE-US-00028 TABLE 28 Operating Parameters and Setpoints for the
Diafiltration Step Parameter Setpoint Filter Area 14 m.sup.2 Feed
Pressure 0.9-1.5 bar Retentate Pressure 0.7-1.2 bar Permeate
Pressure 0-0.1 bar Transmembrane Pressure 0.7-1.1 bar Specific
Cross Flow Rate 300-600 L/h/m.sup.2 (Feed flow rate) Specific
Permeate Flow Rate 7-20 L/h/m.sup.2 Target Conductivity <2 mS/cm
Diafiltration rate 4-5.5 (v.sub.permeate/v.sub.retentate)
Processing Time.sup.1) 0.75-1.25 h
[0181] When the conductivity of the retentate has fallen below 2
mS/cm (determined with the inline conductivity probe of the UFS),
this process step was finished. This low conductivity is required
to ensure a quantitative binding of the rFurin to the
chromatographic gel in the subsequent purification step. The
diafiltered product was transferred and the pH-value of the
diafiltered product was adjusted to 6.0 by adding a 1 M acetic acid
solution. The product was stored at room temperature (RT) before
applying to the Capto-MMC column.
[0182] Capto-MMC Chromatography. The Capto-MMC gel, a multimodal
cation exchanger, was used to bind rFurin and to eliminate the vast
majority of contaminants from the diafiltrated product. After
equilibration of the chromatography gel, the diafiltered product is
loaded to the column. A 0.22 .mu.m filter capsule was installed to
perform an online filtration of the diafiltered product. The
further chromatographic steps are listed and detailed in Table
29.
TABLE-US-00029 TABLE 29 Capto-MMC Chromatography Steps Linear
Chromatography flow rate Quantity step Buffer/Product cm/h
CV.sup.1) 1 Acid wash ES4 online diluted to 300 1 0.2M acetic acid
2 Equilibration EP2 150 37 3 Absorption 35-45 L diafiltered 150
product 4 Equilibration EP2 150 30 5 Washing WPF 150 10 6 Gradient
Elution EL1/MMC eluate 150 15 7 Elution II EL2/MMC eluate II 150 10
8 Regeneration ES4 150 10 9 Regeneration SHD 150 10 .sup.1)CV =
column volume: volume of the packed resin in the chromatography
column
[0183] In the downstream processing, no issues were observed. The
downstream steps (Filtration, UF, DF) could be carried out at the
same conditions used for the harvests from the Chemostat culture.
Mean total rFurin activities yields of 91% (Campaign ORFU06002),
66% (Campaign ORFU07001) and 84% (Campaign ORFU07002) could be
achieved. The only minor change is given by the fact, that the
starting volume for the UF-step is somewhat lower compared to
continuous culture. But this change has no effect on the quality of
the purified product. (See data on total activity, Host Cell DNA,
Host Cell Protein; see Table 30).
[0184] The mean values of all three campaigns for the Host Cell
Protein and Host Cell DNA content reveal rather low mean maximum
values of 8.55 .mu.g/ml (ranging from 2.2-10.4 .mu.g/ml) and 13.48
ng/ml (ranging from 0.0-23.9 ng/ml), respectively (see Table 30).
The chromatographic step was able to reduce the specific
contamination to low mean maximum values of 0.35 ng CHO Protein/U
rFurin Activity (ranging from 0.13-0.52 ng CHO Protein/U) and 0.148
pg Host Cell DNA/U rFurin Activity (ranging from 0.0-0.365 pg DNA/U
rFurin Activity).
TABLE-US-00030 TABLE 30 Capto-MMC Chromatography impurity results -
Campaign comparison Host Cell Host Cell CHO-Protein CHO-Protein/
DNA DNA/rFurin Content rFurin Activity Content Activity campaign
.mu.g/mL ng/U ng/mL pg/U ORFU06002 8.55 0.22 1.66 0.059 ORFU07001
3.50 0.35 2.54 0.031 ORFU07002 2.36 0.026 13.48 0.148
[0185] Furin Characterization. The quality and functionality of
rFurin was assessed by following biochemical/biophysical methods
(Table 31).
TABLE-US-00031 TABLE 31 Analytical methods for rFurin Method Aim
Comment Fluorescent Enzyme activity Low molecular weight substrate
cleavage substrate Furin use test Enzyme activity, rVWF substrate
maturation efficacy SDS-PAGE Protein composition Western blot assay
IEF Protein composition Coomassie staining, Western blot assay
RP-HPLC Protein composition Protein fingerprint
[0186] Furin Activity Assay. The purified rFurin batches (Capto-MMC
eluate pools) were tested for enzymatic activity of Furin. The
substrate is a short synthetic peptide containing the dibasic
recognition sequence attached to a fluorescent amino-methyl
coumarin (AMC) group, that is released after cleavage
(BOC-RVRR-AMC). The released fluorogenic group can be detected by
excitation at 380 nm and subsequent measurement of the emitted
light at 435 nm. One activity unit is defined as the release of 1
pMol of AMC per minute at 30.degree. C.
[0187] Depending on the fermentation and purification efficacy the
measured values of rFurin activity were in the range of about 10000
U/ml up to more than about 100000 U/ml, with a mean value of
approx. 69000 U/ml (Table 32, Table 32, and Table 34). An increase
of rFurin activity for the RFB mode campaigns was noticed,
especially when comparing the mean values of the Chemostat campaign
ORFU06002 (47737 U/ml) with the mean values of RFB campaigns
ORFU07001 (77653 U/ml) and ORFU07002 (93178 U/ml). (Overall, rFurin
activity ranged from about 10000 to greater than 100000 U/ml; all
data not shown).
[0188] The specific activity of Furin is expressed as the activity
U/.mu.g protein (see Tables 32-34). The mean specific activity for
campaign ORFU06002 was 269 U/.mu.g protein, increasing to 500
U/.mu.g for ORFU07001 and 563 U/.mu.g for ORFU07002, respectively.
(Overall, specific activity ranged from 124-620 U/.mu.g protein;
data not shown). Thus, specific activity doubled for the two RFB
campaigns, a result of the higher enzymatic activity of the RFB
rFurin compared to the batches produced in Chemostat mode.
TABLE-US-00032 TABLE 32 Campaign ORFU06002 Capto-MMC Eluate Pool
Furin Specific rFurin Activity Protein Content Activity Furin-Lot
Chemostat Mode [U/ml] [.mu.g/ml] [U/.mu.g] ORFUCHR06002MMC01 28231
148 190.8 ORFUCHR06002MMC02 16307 162 124.3 ORFUCHR06002MMC03 44854
174 257.8 ORFUCHR06002MMC04 39102 148 264.2 ORFUCHR06002MMC05 71871
236 304.5 ORFUCHR06002MMC06 44292 142 311.9 ORFUCHR06002MMC07 68728
155 443.4 ORFUCHR06002MMC08 68510 271 252.8 Mean Value 47737 180
269
TABLE-US-00033 TABLE 33 Campaign ORFU07001 Capto-MMC Eluate Pool
Protein Specific rFurin Furin Activity Content Activity Furin-Lot
Repeated Fed-Batch [U/ml] [.mu.g/ml] [U/.mu.g] ORFUCHR07001MMC01
77090 151 510.5 ORFUCHR07001MMC02 68450 112 611.2 ORFUCHR07001MMC03
87420 231 378.4 Mean Value 77653 165 500
TABLE-US-00034 TABLE 34 Campaign ORFU07002 Capto-MMC Eluate Pool
Protein Specific rFurin Furin Activity Content Activity Furin-Lot
Repeated Fed-Batch [U/ml] [.mu.g/ml] [U/.mu.g] ORFUCHR07002MMC01
91200 162 563.0 ORFUCHR07002MMC02 96400 160 602.5 ORFUCHR07002MMC03
95020 173 549.2 ORFUCHR07002MMC04 85690 190 451.0 ORFUCHR07002MMC05
102670 174 590.1 ORFUCHR07002MMC06 88090 142 620.4 Mean Value 93178
167 563
[0189] Furin-Use-Test Activity. The Furin-Use-Test is designed to
quantify the efficacy of rFurin to process pro-VWF to mature rVWF.
The maturation efficacy is expressed as the amount of Furin units
required for the maturation of 1 VWF Antigen unit (U Furin/U VWF).
The substrate is a proVWF/VWF preparation that has been purified at
Pilot scale according to the current manufacturing procedure but
omitting the Furin maturation and the final purification step on
Superose 6. The rVWF substrate concentration was 100 U Ag/ml
(F8HL.sub.--24.sub.--01UF02-R).
[0190] Four dilutions of the sample were tested in a 1 ml Eppendorf
Tube to cover specific Furin concentrations of, e.g., 0.25-2.0 U/U
VWF. The reaction and dilution buffer was 100 mM HEPES, 1 mM
CaCl.sub.2, pH7.0 and the maturation experiment was performed for
16 hours at 37.degree. C. under slight agitation. After the
incubation the enzymatic reaction is stopped by addition of
SDS-sample buffer and heating the samples for 5 minutes at
>90.degree. C. The samples are then analysed by SDS-PAGE on 8%
gels using silver staining of the separated polypeptides. The
specific Furin activity required to get a maturation efficacy of
>95% as evaluated by visual inspection of the gels (the proVWF
band should represent less than 5% compared to the mature VWF band)
is then reported and used as a quality attribute of the rFurin
tested.
TABLE-US-00035 TABLE 35 Campaign ORFU06002 Capto-MMC Eluate Pool
Use-test Maturation Furin Activity Activity Degree Furin-Lot
Chemostat Mode [U/ml] [U/U] [%] ORFUCHR06002MMC01 28231 0.7 >95
ORFUCHR06002MMC02 16307 0.4 >95 ORFUCHR06002MMC03 44854 1.1
>95 ORFUCHR06002MMC04 39102 1.3 >95 ORFUCHR06002MMC05 71871
1.0 >95 ORFUCHR06002MMC06 44292 1.0 >95 ORFUCHR06002MMC07
68728 1.0 >95 ORFUCHR06002MMC08 68510 1.0 >95 Mean Value
47737 0.58 >95
TABLE-US-00036 TABLE 36 Campaign ORFU07001 Capto-MMC Eluate Pool
Use-test Maturation Furin Activity Activity Degree Furin-Lot
Repeated Fed-Batch [U/ml] [U/U] [%] ORFUCHR07001MMC01 77090 0.5
>95 ORFUCHR07001MMC02 68450 0.5 >95 ORFUCHR07001MMC03 87420
0.5 >95 Mean Value 77653 0.5 >95
TABLE-US-00037 TABLE 37 Campaign ORFU07002 Capto-MMC Eluate Pool
Use-test Maturation Furin Activity Activity Degree Furin-Lot
Repeated Fed-Batch [U/ml] [U/U] [%] ORFUCHR07002MMC01 91200 0.5
>95 ORFUCHR07002MMC02 96400 0.5 >95 ORFUCHR07002MMC03 95020
0.5 >95 ORFUCHR07002MMC04 85690 0.3 >95 ORFUCHR07002MMC05
102670 0.6 >95 ORFUCHR07002MMC06 88090 1.0 >95 Mean Value
93178 0.6 >95
[0191] Good and consistent maturation activity for proVWF was found
for all tested rFurin batches. The mean values of the campaigns are
below 1.0 U Furin/U VWF:Ag and the maturation degree exceeds 95% in
all cases. (Tables 35-37). Maturation activities of all rFurin
batches are comparable and, referring to the calculated mean values
of the campaigns ORFU06002, ORFU07001 and ORFU07002, almost no
differences can be found between rFurin produced in Chemostat mode
and RFB-mode.
[0192] Furin SDS-PAGE and Silver Stain. 8% SDS-PAGE with silver
staining and Western blot analysis was performed for all rFurin
batches to ensure consistent quality and visualize the degree of
impurity. As seen in FIG. 12, the band patterns of the Capto-MMC
eluates of campaign ORFU06002 and ORFU07002 correlate to a high
degree; all samples show a prominent Furin band at approx. 60 kDa.
A trend to slightly lower molecular weight of the Furin bands is
visible in samples of campaign ORFU06002 from batches MMC01 to
MMC08 (FIG. 12, lanes 1-8). Samples of those batches were
deglycosylated with the effect that this trend was not visible
anymore in subsequent SDS-PAGE with silvers staining (data not
shown), supporting the assumption that during campaign ORFU06002,
rFurin was glycosylated slightly different or to a lesser degree in
course of ongoing fermentation. This trend was not noticed in RFB
campaign ORFU07002, suggesting constant glycosylation of the rFurin
during the whole campaign. The impurities in batches from ORFU06002
and ORFU07002 are almost completely the same; however, samples from
the RFB-mode campaign ORFU07002 show less intense bands in the 40
kDa region, polypeptides that were particularly strong enriched in
samples of campaign ORFU06002.
[0193] SDS-PAGE Western Blot. Western blot analysis for all samples
was performed using a monoclonal anti-Furin antibody (FIG. 13). The
prominent band at .about.60 kDa can be identified as the Furin band
and is found in all samples. Comparability of the samples is very
high. Slight variations in band intensity are due to the different
Furin concentration in the samples. Overall, SDS-PAGE analysis
underlines the comparability of rFurin produced in Chemostat and
RFB mode.
[0194] To support conventional SDS-PAGE, Isoelectric Focusing (IEF)
was performed on samples of campaigns ORFU06002 and ORFU07002. IEF
was performed between pH 7.0 and pH 3.0, using vertical IEF. The
polypeptides were visualized with Coomassie staining and Western
blot analysis. IEF and subsequent Western blotting of rFurin
samples of campaign ORFU06002 provided specific band patterns for
Furin (see FIG. 14). Up to seven separate bands in the region of pH
4.5 to pH 5.5 can be identified, with at least five bands present
in all samples.
[0195] IEF of samples of campaign ORFU07002 was carried out using
Coomassie staining and Western blot analysis for visualization.
Coomassie staining reveals the specific band pattern with all
samples showing at minimum five separate bands in the range of pH
4.5 to pH 5.5, and up to eight bands can be identified (see FIG.
15, Lane 3).
[0196] Western blot analysis (see FIG. 16) corroborates these
results, with some samples showing not all the bands visible in the
Coomassie gel (see FIG. 17), probably due to incomplete transfer of
the proteins from the gel to the blotting membrane.
[0197] Furin Reverse Phase HPLC. Samples from campaigns ORFU06002
and ORFU07003 (Capto-MMC eluates) were tested with C4 RP-HPLC in
order to establish a fingerprint pattern for rFurin. The peak
patterns of the samples of the two campaigns were compared (see
FIG. 17 and FIG. 18).
[0198] Chromatograms of all tested samples show a characteristic
main peak at a retention time of approx. 13 min., which can be
assigned to Furin. The peak heights correlate well with the Furin
concentration in the samples. Other protein impurities can be seen
as minor peaks in the range of 8 min to 17 min. All samples from
campaign ORFU07002 show significantly less and smaller peaks from
impurities than those from ORFU06002. This fact is well in
accordance with the results of SDS-PAGE, as in the RFB mode
campaign ORFU07002 a smaller number and decreased amount of
impurities were found.
[0199] Analytical data obtained from the above discussed
characterization methods prove very good comparability of the
rFurin produced in both Chemostat mode and RFB mode. No major
differences in rFurin quality could be detected from the data
available, however rFurin batches produced in RFB mode showed
higher specific activity and less impurities compared to production
in Chemostat mode. The chromatographic step accounts for the very
low Host Cell Protein and Host Cell DNA content in the Bulk Drug
Substance (BDS). At least a complete production process consisting
of a RFB-fermentation and the whole down-stream processing
(Filtration, UF/DF, Capto-MMC chromatography) is maybe easier to be
implemented for the commercial production of rFurin, e.g. in Single
Use Bioreactor (SUB) systems.
Example 6
Safety, Sterility, and Stability Testing
[0200] This example describes the safety, sterility, and stability
testing that is performed to determine and maintain the quality of
the CHO cell bank. Testing on sterility/mycoplasma has to be
performed in accordance to requests of the ICH-Guideline Q5D. The
quality of the cell bank has to be checked by determination of
average viability and cell density of the thawed cells and
subsequent growth rate of the cultures.
[0201] Cells were tested for viral safety (Table 38), genetic
stability (Table 39), and identity (Table 40). Cells were found to
be sterile, free of mycoplasma, free from extraneous agents, free
from retroviruses, negative for MVM virus, negative for
adventitious viruses, negative for rodent viruses, free from
porcine and bovine viruses, and free from Cache Valley virus
(CVV).
[0202] Cell banks were examined with regard to the Furin
producer/non-producer ratio by FACS analysis. They are also tested
for their long term stability (ability to produce Furin over a
period of time). Further, the secreted Furin produced under
serum-free conditions has to be investigated with respect to
generated isoforms. All data should indicate that stable growth and
Furin production can be achieved using these cell banks.
[0203] Cells from the MCB/WCB should show stable growth and Furin
expression over the entire production process.
TABLE-US-00038 TABLE 38 Viral Safety Program Test MCB WCB MEPC
Adventitious viruses in vitro x x x (CHO, A9, Vero, MRC-5 Cells)
Adventitious viruses in vivo x (x) x (in suckling mice, adult mice,
guinea pigs, and embryonated eggs) Rodent viruses x -- -- MAP test
with LCMV challenge Rodent viruses x -- -- HAP Rodent viruses x --
x MMV (in vitro assay) Retroviruses x -- x EM (Transmission
electron microscopic examination) Retroviruses x -- x Retroviral
Infectitvity Assay with Pert Endpoint Retroviruses x -- x Ex S +
L-(in vitro detection of xenotropic retroviruses by Mink S +
L-Focus) Retroviruses/Co cultivation -- -- x Detection of
infectious retroviruses by co-cultivation with HEK 293 cells (5
passages) Bovine virus: x -- -- Detection of viral contaminants in
Bovine serum according CMPC & US 9CFR requirements Porcine
virus: x -- -- In vitro assay, detection of porcine viral
contaminants using PPK indicator cells according to 9 CFR PCR
Bovine polyomavirus x -- x Real time PCR of Cache Valley x -- x
Virus (CVV) (x) viral testing has not to be performed on the WCB
from which the MEPC have been prepared
TABLE-US-00039 TABLE 39 Genetic Stability Program Test MCB WCB MEPC
Productivity: x x x Furin activity assay Sequencing x -- x
quantitative PCR (gene copy x -- x number) Southern Blot x -- x
Northern Blot x -- x
TABLE-US-00040 TABLE 40 Identity Program Test MCB WCB MEPC
Isoenzyme analysis x x x
[0204] The present invention has been described in terms of
particular embodiments found or proposed to comprise preferred
modes for the practice of then invention. It will be appreciated by
those of ordinary skill in the art that, in light of the present
disclosure, numerous modifications and changes can be made in the
particular embodiments exemplified without departing from the
intended scope of the invention. Therefore, it is intended that the
appended claims cover all such equivalent variations which come
within the scope of the invention as claimed.
* * * * *