U.S. patent application number 13/459743 was filed with the patent office on 2012-11-01 for method of producing recombinant vitamin k dependent proteins.
This patent application is currently assigned to Inspiration Biopharmaceuticals, Inc.. Invention is credited to Marian J. Drohan, William N. Drohan, Michael J. Griffith.
Application Number | 20120276079 13/459743 |
Document ID | / |
Family ID | 43922560 |
Filed Date | 2012-11-01 |
United States Patent
Application |
20120276079 |
Kind Code |
A1 |
Drohan; William N. ; et
al. |
November 1, 2012 |
METHOD OF PRODUCING RECOMBINANT VITAMIN K DEPENDENT PROTEINS
Abstract
Methods for producing cell lines with high levels of
biologically active recombinant vitamin K dependent proteins are
described. The transfected cell lines do not include heterologous
genes for processing enzymes and are not subject to selection
pressure such as methotrexate resistance. Cell lines producing
Factor VII/VIIa and Factor IX are described. These cell lines can
be used for isolation of Factor VII/VIIa and/or Factor IX for
treatment of Hemophilia.
Inventors: |
Drohan; William N.;
(Springfield, VA) ; Drohan; Marian J.;
(Springfield, VA) ; Griffith; Michael J.; (San
Juan Capistrano, CA) |
Assignee: |
Inspiration Biopharmaceuticals,
Inc.
Laguna Niguel
CA
|
Family ID: |
43922560 |
Appl. No.: |
13/459743 |
Filed: |
April 30, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2010/054581 |
Oct 28, 2010 |
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13459743 |
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61256802 |
Oct 30, 2009 |
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Current U.S.
Class: |
424/94.64 ;
435/214; 435/219; 435/358; 435/69.1; 435/7.4 |
Current CPC
Class: |
C07K 14/745 20130101;
C12P 21/02 20130101; C12N 9/64 20130101; A61K 38/36 20130101; A61P
7/04 20180101 |
Class at
Publication: |
424/94.64 ;
435/214; 435/219; 435/358; 435/7.4; 435/69.1 |
International
Class: |
C12N 9/50 20060101
C12N009/50; A61P 7/04 20060101 A61P007/04; C12P 21/00 20060101
C12P021/00; C12N 5/10 20060101 C12N005/10; G01N 33/573 20060101
G01N033/573; A61K 38/48 20060101 A61K038/48; C12N 9/74 20060101
C12N009/74 |
Claims
1. A cell line which produces recombinant vitamin K dependent
protein having at least 20% biological activity, wherein the cell
line does not contain heterologous genetic material encoding
proteins involved in the post-translation modification of vitamin K
dependent proteins.
2. The cell line of claim 1, wherein the cell line is a mammalian
cell line.
3. The cell line of claim 2, wherein the mammalian cell line is
Chinese Hamster Ovary.
4. The cell line of claim 1, which has not been subjected to
selection with methotrexate.
5. The cell line of claim 1, wherein the Vitamin K dependent
protein is selected from the group consisting of Factor II, Factor
VII, Factor IX, Factor X, Protein C and Protein S.
6. The cell line of claim 5, wherein the Vitamin K dependent
protein is Factor VII/VIIa or Factor IX.
7. The cell line of claims 1, comprising a gene encoding the
Vitamin K dependent protein which is operably linked to a Chinese
hamster elongation factor 1 (CHEF-1) promoter.
8. A method of producing a recombinant biologically active Vitamin
K dependent protein comprising the steps of: (a) transfecting a
population of mammalian cells with a gene encoding the Vitamin K
dependent protein operably linked to a promoter; (b) performing at
least one round of cloning and screening to identify cell clones
which produce at least 10 mg/L of the Vitamin K dependent protein
which is at least 10% biologically active; (c) optionally,
repeating the cell cloning of step (b) one or more times to
identify single cells producing more than 10 mg/L of the Vitamin K
dependent protein which is at least 10% biologically active; and
(d) harvesting the Vitamin K dependent protein.
9. The method of claim 8, wherein the Vitamin K dependent protein
is selected from the group consisting of Factor II, Factor VII,
Factor IX, Factor X, Protein C and Protein S.
10. The method of claim 9, wherein the Vitamin K dependent Protein
is Factor IX.
11. The method of claim 10, wherein the cloning is limit dilution
cloning.
12. The method of claim 9, wherein the Vitamin K dependent Protein
is Factor VII.
13. The method of claim 12, wherein the cloning is semi-solid
matrix cloning.
14. The method of claim 8, wherein the promoter is a Chinese
hamster elongation factor 1 (CHEF-1) promoter.
15. The method of claim 8, further comprising preselecting cells
producing at least 10 mg/L Vitamin K dependent protein antigen
before step (b).
16. The method of claim 8, wherein the cell line is cultured in a
media that includes vitamin K.
17. The method of claim 8, wherein the cell line is cultured in a
media that does not include vitamin K.
18. The method of claim 8, wherein at least 20% of the Vitamin K
dependent protein is biologically active.
19. The method of claim 8, wherein at least 30% of the Vitamin K
dependent protein is biologically active.
20. The method of claim 8, wherein at least 40% of the Vitamin K
dependent protein is biologically active.
21. The method of claim 8, wherein at least 58% of the Vitamin K
dependent protein is biologically active.
22. The method of claim 8, wherein at least 70% of the Vitamin K
dependent protein is biologically active.
23. The method of claim 8, wherein at least 85% of the Vitamin K
dependent protein is biologically active.
24. The method of claims 8, wherein at least 95% of the Vitamin K
dependent protein is biologically active.
25. The method of claims 8, wherein at least 99% of the Vitamin K
dependent protein is biologically active
26. The method of claim 8, wherein the vitamin K dependent protein
is produced in an amount of at least 20 mg/L.
27. The method of any of claim 8, wherein the vitamin K dependent
protein is produced in an amount of at least 30 mg/L.
28. The method of claim 8, wherein the vitamin K dependent protein
is produced in an amount of at least 40 mg/L.
29. The method of claim 8, wherein the mammalian cells are Chinese
Hamster Ovary Cells.
30. A recombinant Factor IX protein produced by the method of claim
8.
31. A recombinant Factor VII protein produced by the method of
claim 8.
32. A pharmaceutical composition comprising the Factor IX protein
of claim 30 or the Factor VII protein of claim 31.
33. A kit comprising the recombinant Factor IX protein of claim 30
or the Factor VII protein of claim 31.
34. A method of treating hemophilia which comprises administering
an effective amount of the pharmaceutical composition of claim 32
to a patient in need thereof.
35. A recombinant Factor IX protein having at least 10% biological
activity.
36. The recombinant Factor IX protein of claim 35, having at least
20% biological activity.
37. The recombinant Factor IX protein of claim 35, having at least
30% biological activity.
38. The recombinant Factor IX protein of claim 35, having at least
40% biological activity.
39. The recombinant Factor IX protein of claim 35, having at least
58% biological activity.
40. A recombinant Factor VII/VIIa protein having at least 70%
biological activity.
41. A recombinant Factor VII/VIIa protein having at least 85%
biological activity.
42. A recombinant Factor VII/VIIa protein having at least 99%
biological activity.
43. A cell line which produces recombinant vitamin K dependent
protein having at least 20% biological activity, wherein the cell
line is generated by: (a) transfecting a population of mammalian
cells with a gene encoding the Vitamin K dependent protein operably
linked to a Chinese hamster elongation factor 1 (CHEF-1) promoter;
and (b) performing at least one round of cloning and screening to
identify cell clones which produce at least 10 mg/L of the Vitamin
K dependent protein which is at least 10% biologically active.
44. The method of claim 43, wherein the cloning is limit dilution
cloning or semi-solid-matrix cloning.
45. A pharmaceutical composition comprising the Factor VII protein
of claim 31.
Description
RELATED APPLICATIONS
[0001] This application claims priority to provisional application
No. 61/256,802, filed Oct. 30, 2009, which is incorporated herein
by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention related to recombinant vitamin K dependent
proteins and methods of preparing the protein in a mammalian cell
without the use of heterologous post-translational modification
enzymes.
[0004] 2. Description of the Related Art
[0005] Bleeding disorders can result from a deficiency in the
functional levels of one or more of the blood proteins,
collectively known as blood coagulation factors, that are required
for normal hemostasis, i.e. blood coagulation. The severity of a
given bleeding disorder is dependent on the blood level of
functional coagulation factors. Mild bleeding disorders are
generally observed when the functional level of a given coagulation
factor reaches about 5% of normal, but if the functional level
falls below 1%, severe bleeding is likely to occur with any injury
to the vasculature.
[0006] Medical experience has shown that essentially normal
hemostasis can be temporarily restored by intravenous infusion of
biological preparations containing one or more of the blood
coagulation factors. So-called replacement therapy, whereby a
biological preparation containing the deficient blood coagulation
factor is infused when bleeding occurs (on demand) or to prevent
bleeding (prophylactically), has been shown to be effective in
managing patients with a wide variety of bleeding disorders. In
general, for replacement therapy to be effective, intravenous
infusions of the missing coagulation factor are targeted to achieve
levels that are well above 5% of normal over a two- to three-day
period.
[0007] Historically, patients who suffer from hemophilia, a
genetically acquired bleeding disorder that results from a
deficiency in either blood coagulation Factor VIII (hemophilia A)
or Factor IX (hemophilia B), were successfully treated by periodic
infusion of whole blood or blood plasma fractions of varying
degrees of purity.
[0008] More recently, with the advent of biotechnology,
biologically active preparations of synthetic (recombinant) blood
coagulation factors have become commercially available for
treatment of blood coagulation disorders. Recombinant blood
coagulation proteins are essentially free of the risks of human
pathogen contamination that continue to be a concern that is
associated with even high purity commercial preparations that are
derived from human blood.
[0009] Several of the proteins required for normal blood
coagulation are very complex in terms of having multiple structural
domains each being associated with a very specific functional
property that is essential for the overall effectiveness of the
protein in controlling hemostasis and/or preventing thrombosis. In
particular, the so-called "vitamin K-dependent" blood coagulation
proteins, e.g. Factors II, VII, IX, X, Protein C and Protein S, are
very complex proteins and must undergo extensive post-translational
modification for normal function. Achieving high levels of
functional vitamin K-dependent proteins by recombinant technology
has been limited by the structural complexity of these proteins and
the inability to create genetically engineered cell systems that
overcome the inherent deficiencies in the enzymatic activities
required for efficient and complete post-translational modification
to occur.
[0010] Kaufman, et al. (Kaufman, et al. (1986) The Journal of
Biological Chemistry, vol. 261 no. 21: 9622-9628) report the
production of recombinant, biologically active Factor IX. However,
while upwards of 100 .mu.g/mL of Factor IX was produced, the level
of active material was only 1.5%.
[0011] Other vitamin K dependent proteins have been produced
recombinantly with limited success. Jorgensen, et al. (Jorgensen,
et al. (1987) The Journal of Biological Chemistry, vol. 262 (14):
6729-6734) report that human prothrombin was produced in CHO cells
at a level of up to 0.55 .mu.g/ml. At this level, the
prothrombin'was all biologically active, However, when levels were
increased 10-15 fold, biological activity dropped to 60%. They
hypothesized that the .gamma.-carboxylation system of CHO cells is
limited and that only a certain level of protein can be efficiently
processed.
[0012] Messier, et al. (Messier, et al. (1991) Gene vol. 99:
291-294) cloned and expressed human Factor X in COS-1 Monkey kidney
Cells. Both the level produced (0.25-0.27 .mu.g/ml) and the
biological activity (9-10%) were low.
[0013] Herlitschka, et al. (Herlitschka, et al. (1996) Protein
Expression and Purification vol. 8: 358-364) used human prothrombin
as a reporter with hygromycin phosphotransferase/dihyrofolate
reductase (DHFR) as a dominant selection/amplification fusion
marker. Levels of up to 200-250 mU/10.sup.6 cells/24 hours were
produced using 293 kidney cells and 5-15 mU/10.sup.6 cells/24 hours
using CHO cells. Taking 1 Unit as equivalent to about 100 .mu.g,
this translates to a maximum level of 25 .mu.g using 293 cells.
Although the relative biological activity was not determined, the
authors indicated that the 293 cells had been chosen because of its
high carboxylation potential.
[0014] Himmelspach, et al. (Himmelspach, et al. (2000) Thrombosis
Research vol. 97:51-67) obtained 120 .mu.g/mL/day of recombinant
human Factor X using DHFR deficient CHO cells with methotrexate
selection. Biological activity was up to 25%. The role of Furin in
processing of Factor X was investigated by these workers. While
Factor X, like Factor IX, also requires gamma carboxylation and
post-translational cleavage, it is not clear why higher levels of
Factor X having biological activity have been obtained compared to
Factor IX. Significant amounts of the recombinant Factor X produced
remained covalently attached to the propeptide and/or remained as a
single chain precursor. In the presence of recombinant Furin
(PACE), the amount of biologically active Factor X approximately
doubled from 22% to 43%). In Factor X, removal of the propeptide
appeared to rely upon an endopeptidase other than Furin, while
light/heavy chain processing was furin-dependent.
[0015] Sun, et al. (Sun, et al. (2005) Blood vol, 106 (12):
3811-3815) reported that the percentage of carboxylated Factor X
can be increased from 50% to 95% by coexpression of Vitamin K
epoxide reductase (VKOR).
[0016] Wasley, et al. (Wasley, et al. The Journal of Biological
Chemistry vol. 268 (12): 8458-8465, 1993) reported that Factor IX
is poorly processed in Chinese Hamster Ovary (CHO) cells but that
coexpression of PACE (Paired basic Amino acid Cleaving Enzyme)
improved processing and specific activity 2-3 fold.
[0017] Among the problems encountered in recombinant systems is
that in order to produce biologically active Factor IX and Factor
VII/VIIa, substantial gamma-carboxylation of glutamic acid residues
in the amino terminal region of the protein referred to as the
gla-domain, is needed. For example, FVII/VIIa has 10 gla residue
sites which should be carboxylated and Factor IX has 12. A majority
of these residues must be gamma-carboxylated in order to have
bioaetive protein. Additionally, pro-Factor IX, a form of Factor IX
that contains a propeptide domain that is required for the
efficient intracellular gamma-carboxylation of the protein, must be
processed properly prior to secretion, as must Factor VII be
processed prior to secretion.
[0018] One approach is to co-transfect with genes encoding enzymes
which function to post-translationally process Factor IX.
Appropriate enzymes include Vitamin K dependent .gamma.-glutamyl
carboxylase (VKGC), Vitamin K dependent epoxide reductase (VKOR),
and Paired basic amino acid converting enzyme (PACE). U.S.
application Ser. No. 11/643,563, filed Dec. 21, 2006 is directed to
this approach.
[0019] VKGC incorporates a carboxyl group into glutamic acid to
modify multiple residues within the vitamin K dependent protein
within about 40 residues of the propeptide, within the so-called
"gla domain", VKOR is important for vitamin K dependent proteins
because vitamin K is converted to vitamin K epoxide during
reactions in which it is a cofactor. The amount of vitamin K in the
human diet is limited. Therefore, vitamin K epoxide must be
converted back to vitamin K by VKOR to prevent depletion.
Consequently, co-transfection with VKOR enhances the appropriate
cycling of vitamin K inside the cell and provides sufficient
vitamin K for proper functioning of the vitamin K dependent enzymes
such as VKGC. The term "PACE" is an acronym for paired basic amino
acid converting (or cleaving) enzyme; PACE, is a subtilisin-like
endopeptidase, i.e., a propeptide-cleaving enzyme which exhibits
specificity for cleavage at basic residues of a polypeptide, e.g.,
-Lys-Arg-, -Arg-Arg, or -Lys-Lys-.
[0020] While the above mentioned enzymes may be incorporated into a
transgenic cell line for processing of vitamin K dependent
proteins, mammalian cells naturally produce certain levels of these
enzymes endogenously.
[0021] The approach taken here is a process of initial selection,
whereby a gene, such as a DNA sequence with introns or a cDNA
encoding a gene product for a vitamin K dependent protein such as
Factor VII or Factor IX is cloned into mammalian cells, followed by
selection for transfected clones. The high level expressers are
identified, isolated and optionally pooled and may be re-cloned. In
any case, the cloned cells are cultured to select even higher
expressing clones. The method selects for cell lines which express
high levels of vitamin K dependent proteins without requiring
co-transfection with multiple heterologous genes, such as genes
encoding enzymes necessary for the post-translational modification
of vitamin K dependent proteins.
Definitions
[0022] The term cloning as used herein refers to manipulations for
isolating and establishing clones. The term "clone" has its usual
and customary meaning and refers to a population of cells produced
generated from a single parent cell and which therefore should be
genetically identical. In some embodiments, limit dilution cloning
is used to produce high producing clones. "Limit dilution cloning"
has its usual and customary meaning and refers to a process of
obtaining a monoclonal cell population starting from a polyclonal
mass population of cells. The starting (polyclonal) culture is
serially diluted until a single cell is statistically isolated and
used to derive a monoclonal culture is obtained.
[0023] In alternate embodiments, "semi-solid matrix cloning"
methods are used. Semi-solid matrix cloning refers to seeding cells
at very low densities into into a semi-solid matrix, typically
although not necessarily following a the transfection and selection
process. Preferably, the cells are seeded into a semi-solid matrix
with as few population doublings as possible. The shortest number
of population doublings minimizes the risk of losing the highest
expressing cells in the original mixed population, since these
cells would carry the greatest metabolic burden and likely be
overgrown by the faster growing cells that express lower levels of
recombinant protein or none at all.
[0024] Cells are seeded at very low densities (typically
1,000-4,000 but may be as high as 10,000 cells per ml) into a
mixture of media, media supplements, conditioned medium (usually
5-20% volume:volume), and a generally inert, biologically
compatible, semi-solid medium such as methylcellulose. After
seeding the low density cells into the mixture as a low density
single cell suspension and letting it "gel" for a few minutes into
a semi-solid, the culture plates are returned to an incubator
(37.degree. C., with humidity and carbon dioxide buffering
atmosphere) and allowed to sit undisturbed for anywhere from
.about.1week to .about.3weeks. During this time many of the
suspended single cells will grow slowly but eventually begin to
replicate and form "colonies" representing a cluster of daughter
cells all genetically identical to the single suspended cell from
which they were derived. About two weeks after initially seeding
the cells into the semisolid matrix one observes the culture plates
for colony formation (number of colonies, their size and how far
separated they are from one another in the gel) and then individual
colonies are picked and seeded into separate cluster plates, each
as a clonal population. They are expanded thru larger plates and
screened.
[0025] The term "bioactive" as used herein in reference to vitamin
K dependent proteins is a broad term which has its usual and
customary meaning of a substance which has an effect on living
matter. In the present context the meaning is expanded to include
zymogen forms which may not be bioactive per se but are capable of
activation. Activation may be by administration to a living body
(that is, activation occurs within the body after administration by
endogenous factors) or may be in vitro activated by treatment with
an appropriate enzyme or set of incubation conditions (e.g. pH,
concentration, temperature, etc.). For example, in the case of
Factor VII, proteolytic cleavage is needed to convert the zymogen,
Factor VII, to the active form Factor VIIa. Likewise, Factor IX is
a zymogen which requires proteolytic cleavage to Factor IXa. In the
context of the invention, Factor VIIa and Factor IXa are considered
"bioactive". However, Factor VII and Factor IX are also considered
to be "bioactive" if they have been appropriately
post-translationally modified, (with Gla residues for example), so
that they are capable of being converted to the bioactive form
either in vivo or in vitro.
[0026] The term "gene" as used herein has its usual and customary
meaning of a DNA sequence which may contain introns and exons and
also includes a cDNA encoding a gene product.
[0027] As used herein, the terms "treating," "treatment,"
"therapeutic," or "therapy" do not necessarily mean total cure or
abolition of the disease or condition. Any alleviation of any
undesired signs or symptoms of a disease or condition, to any
extent can be considered treatment and/or therapy. Furthermore,
treatment may include acts that may worsen the patient's overall
feeling of well-being or appearance.
SUMMARY OF THE INVENTION
[0028] Embodiments of the invention are directed to cell lines
which produce recombinant vitamin K dependent protein having at
least 20% biological activity. Preferably, the cell line does not
contain heterologous genetic material encoding proteins involved in
the post-translation modification of vitamin K dependent proteins.
Preferably, the cell line is a mammalian cell line, more
preferably, the mammalian cell line is a Chinese Hamster Ovary
(CHO) cell line. Preferably, the gene encoding the Vitamin K
dependent protein is operably linked to the Chinese hamster
elongation factor 1 (CHEF-1) promoter.
[0029] In some embodiments, the cell line is cultured in a media
that includes vitamin K. In some embodiments, the cell line is
cultured in a media that does not include vitamin K.
[0030] In some embodiments, the cell line does not contain
heterologous DHFR. In preferred embodiments, the cell line has not
been subjected to selection with methotrexate.
[0031] Preferably, Vitamin K dependent protein is Factor II, Factor
VII, Factor IX, Factor X, Protein C or Protein S, and more
preferably, Factor VII/VIIa or Factor IX.
[0032] Embodiments of the invention are directed to methods of
producing a recombinant biologically active Vitamin K dependent
protein which include one or more of the following steps: [0033]
(a) transfecting a population of mammalian cells with a gene
encoding the Vitamin K dependent protein operably linked to a
promoter; [0034] (b) performing at least one round of cloning and
screening to identify cell clones which produce at least 10 mg/L of
the Vitamin K dependent protein which is at least 10% biologically
active; [0035] (c) optionally, repeating the cell cloning of step
(b) one Or more times to identify single cells producing more than
10 mg/L of the Vitamin K dependent protein which is at least 10%
biologically active; and [0036] (d) harvesting the Vitamin K
dependent protein.
[0037] Preferably, the Vitamin K dependent protein of the method is
Factor II, Factor VII, Factor IX, Factor X, Protein C or Protein S,
more preferably Factor IX or Factor VII. In preferred embodiments,
the Vitamin K dependent protein is operably linked to the Chinese
hamster elongation factor 1 (CHEF-1) promoter. In preferred
embodiments, the mammalian cell line is a Chinese Hamster Ovary
Cell line. The media may optionally include Vitamin K.
[0038] Preferably, cloning is by limit dilution cloning or
semi-solid matrix cloning methods. More preferably, limit dilution
cloning is used for producing recombinant Factor IX and semi-solid
matrix cloning is used for producing recombinant Factor
VII/VIIa.
[0039] In preferred embodiments, the method may include
preselecting cells producing at leak 10 mg/L Vitamin K dependent
protein antigen, preferably Factor VII/VIIa or Factor IX antigen,
before step (b). In some embodiments, cells are selected which
produce at least 10 mg/L Vitamin K dependent protein antigen,
preferably Factor VII/VIIa or Factor IX antigen, after step
(b).
[0040] In preferred embodiments, the Vitamin K dependent protein is
Factor IX and at least 20%, more preferably at least 30%, and yet
more preferably at least 40% of the Vitamin K dependent protein is
biologically active. In a most preferred embodiment, at least 58%
of the Vitamin K dependent protein is biologically active.
[0041] In preferred embodiments, the Vitamin K dependent protein is
Factor VII/VIIa and at least 60%, more preferably 70%, more
preferably 80%, more preferably 90%, more preferably 95%, more
preferably 98%, more preferably 99% and most preferably 100% of the
Vitamin K dependent protein is biologically active.
[0042] In preferred embodiments, the vitamin K dependent protein is
produced in an amount of at least 20 mg/L, more preferably at least
30 mg/L, and yet more preferably at least 40 mg/L.
[0043] Embodiments of the invention are directed to recombinant
Vitamin K dependent proteins and pharmaceutical preparations
containing Vitamin K dependent proteins, in particular Factor
VII/VIIa protein and Factor IX protein, where the protein is
produced by any of the methods described above.
[0044] Embodiments of the invention also include kits which may
include one or more Vitamin K dependent proteins, preferably as a
pharmaceutical composition. Preferably the kit includes Factor
VII/VIIa or Factor IX protein in a pharmaceutically acceptable
carrier.
[0045] Embodiments of the invention are directed to methods of
treating hemophilia by administering an effective amount of a
pharmaceutical preparation of a Vitamin K dependent protein, in
particular where the Vitamin K dependent protein is Factor VII/VIIa
or Factor IX, to a patient in need of treatment for hemophilia or
uncontrollable hemorrhage.
[0046] Embodiments of the invention are directed to recombinant
Factor IX proteins having at least 10%, more preferably 20%, yet
more preferably 30%, yet more preferably 40% and yet more
preferably 58% biological activity.
[0047] Embodiments of the invention are directed to Factor VII/VIIa
protein having at least 60%, more preferably 70%, more preferably
80%, more preferably 90%, more preferably 95%, more preferably 98%,
more preferably 99% and most preferably 100% biological
activity.
[0048] Embodiments of the invention are directed to cell lines
which produce recombinant vitamin K dependent protein having at
least 20% biological activity. Preferably, the cell line is
generated by transfecting a population of mammalian cells with a
cDNA encoding the Vitamin K dependent protein operably linked to a
Chinese hamster elongation factor 1 (CHEF-1) promoter; and
performing at least one round of cloning and screening to identify
cell clones which produce at least 10 mg/L of the Vitamin K
dependent protein which is at least 10% biologically active.
Preferably, techniques of limit dilution cloning or semi-solid
matrix cloning are used.
[0049] Further aspects, features and advantages of this invention
will become apparent from the detailed description of the preferred
embodiments which follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] These and other feature of this invention will now be
described with reference to the drawings of preferred embodiments
which are intended to illustrate and not to limit the
invention.
[0051] FIG. 1 shows Factor IX ELISA total antigen results for CHO
cell first transfectant clones. There were 152 T335 clones and 171
T337 clones.
[0052] FIG. 2 shows Factor IX clones % activity versus titer.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0053] Kaufman, et al. (supra) teach a method of increasing the
expression level of a gene encoding Factor IX in CHO cells by
including a gene encoding DHFR on the plasmid containing the gene
for Factor IX and using the selective pressure of increasing levels
of methotrexate. As the cells having a high copy number of the
plasmid encoding DHFR are selected by increasing methotrexate
levels, increased levels of Factor IX are also produced. However,
the limitation of this method is that, while reasonably high levels
of Factor IX are produced, the level of the processing enzymes
remain low. So most of the Factor IX produced by this method is not
biologically active.
[0054] While one approach to address this problem is to add
heterologous processing enzymes, processing of Vitamin K dependent
proteins is complex. Other factors may be involved which then
become limiting in the presence of heterologous factors.
[0055] In contrast, the present selection method selects for cell
lines which produce high levels of biologically active vitamin K
dependent proteins. The nucleic acid encoding the vitamin K
dependent protein of interest is cloned into a vector where it is
operably linked to a strong promoter, preferably CHEF-1. This
approach does not rely upon methotrexate selection and selects for
optimal levels of all of the necessary post-translational enzymes,
as well as vitamin K dependent protein. Vitamin K dependent
proteins include Factors II, VII, IX, X, Protein C and Protein S.
In particular, the present inventors have found that for production
of both recombinant Factor VII/VIIa and Factor IX protein, use of
the CHEF-1 promoter operably linked to a gene coding for Factor
VII/VIIa or Factor IX in the present method produces very high
levels of biologically active Factor VII/VIIa or Factor IX, even
without introduction of genes for processing factors and/or
addition of processing factors to provide means for
post-translational processing of the recombinant Factor VII/VIIa
and Factor IX proteins.
[0056] Any appropriate cell line may be used including, but not
limited to insect cells, plant cells and mammalian cells. Mammalian
cell lines include Chinese Hamster Ovary (CHO) cells and HEK 293
cells. The cell may be selected from a variety of sources, but is
otherwise a cell that may be transfected with an expression vector
containing a nucleic acid, preferably a cDNA of a vitamin
K-dependent protein.
[0057] In some embodiments, from a pool of transfected cells,
clones are selected that produce quantities of the vitamin
K-dependent protein over a range(Target Range) that extends from
the highest level to the lowest level that is minimally acceptable
for the production of a commercial product. Cell clones that
produce quantities of the vitamin K-dependent protein within the
Target Range may be combined to obtain a single pool or multiple
sub-pools that divide the clones into populations of clones that
produce high, medium or low levels of the vitamin K-dependent
protein within the Target Range. Alternatively, the clones are not
pooled but are maintained as monoclonal cultures. It is considered
to be within the scope of the present invention that transfected
cells that produce a vitamin K-dependent protein within the Target
Range may be analyzed to determine the extent to which fully
functional protein is produced Levels of vitamin K dependent
protein antigen may be determined by conventional ELISA. Commercial
kits are available such as VisuLize.RTM. (Factor IX Antigen ELISA
kit from Affinity biologicals (Ancaster, Ontario, Canada).
[0058] In preferred embodiments of the method of the present
invention, the selected transfectant pool is cloned to determine
the optimal level of production of fully functional vitamin
K-dependent protein. It is contemplated that higher percentages of
fully functional vitamin K-dependent protein will be produced by
cell clones that produce lower total amounts of the vitamin
K-dependent protein within the Target Range. In preferred
embodiments, the optimal level of production will be the highest
level of functional vitamin K-dependent protein.
[0059] Means to assay bioactivity of vitamin K dependent proteins
are well known and include one- and two-stage clotting assays as
well as chromogenic assays. For example, assay of Factor IX may use
a Universal Coagulation Reference Plasma (UCRP) as a standard for
Factor IX activity and Factor IX-deficient plasma for dilution of
calibration standards and unknown samples. The assay involves
mixing plasma with activator and calcium chloride to initiate the
clotting cascade, with formation of the fibrin clot measured by
absorbance. The clotting time measured in this assay is the aPTT
(activated partial thromboplastin time), the time required for the
absorbance to cross a pre-determined threshold value. Accurate
determination of Factor IX activity is achieved by comparing the
signal of the unknowns to Factor IX Reference Standard (UCRP)
assayed simultaneously. In preferred embodiments, the assay is
conducted on an automated coagulation analyzer. Potency in units/mL
is obtained by use of an international WHO standard for blood
coagulation factor IX.
[0060] clotting assays are also known for FVII/FVIIa (U.S. Pat. No.
5,750,358 which is incorporated herein by reference).
[0061] Chromogenic assays include conventional chromogenic FVIIa
Bioactivity assays such as BIOPHEN FVII.RTM. (Ref No. 221304)
available from HYPHEN BioMed. A cleavage product produced during
the assay is measured spectrophotometrically.
Genetic Engineering Techniques
[0062] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, e.g., Sambrook, et al., "Molecular Cloning; A
Laboratory Manual", 2nd ed (1989); "DNA Cloning", Vols. I and II
(D. N Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gait ed.
1984); "Nucleic Acid Hybridization" (B. D. Hames & S. J.
Higgins eds. 1984); "Transcription and Translation" (B. D. Hames
& S. J. Higgins eds. 1984); "Animal Cell Culture" (R. I.
Freshney ed. 1986); "Immobilized Cells and Enzymes" (IRL Press,
1986); B. Perbal, "A Practical Guide to Molecular Cloning" (1984);
the series, Methods in Enzymology (Academic Press, Inc.),
particularly Vols. 154 and 155 (Wu and Grossman, and Wu, eds.,
respectively); "Gene Transfer Vectors for Mammalian Cells" (J. H.
Miller and M. P. Calos eds. 1987, Cold Spring Harbor Laboratory);
"Immunochemical Methods in Cell and Molecular Biology", Mayer and
Walker, eds. (Academic Press, London, 1987); Scopes, "Protein
Purification: Principles and Practice", 2nd ed. 1987
(Springer-Verlag, N.Y.); and "Handbook of Experimental Immunology"
Vols I-IV (D. M. Weir and C. C. Blackwell eds 1986). All patents,
patent applications, and publications cited in the background and
specification are incorporated herein by reference.
[0063] The production of cloned genes, recombinant DNA, vectors,
transformed host cells, proteins and protein fragments by genetic
engineering is well known. See, e.g., U.S. Pat. No. 4,761,371 to
Bell et al. at Col. 6 line 3 to Col. 9 line 65; U.S. Pat. No.
4,877,729 to Clark et al. at Col. 4 line 38 to Col. 7 line 6; U.S.
Pat. No. 4,912,038 to Schilling at Col. 3 line 26 to Col. 14 line
12; and U.S. Pat. No. 4,879,224 to Wallner at Col. 6 line 8 to Col.
8 line 59.
Expression Vectors
[0064] A vector is a replicable DNA construct. Many transfection
methods to create genetically engineered cells that express large
quantities of recombinant proteins are well known. Embodiments of
the invention are not dependent on the use of any specific
expression vector. In preferred embodiments, cells are transfected
with an expression vector that contains the cDNA encoding the
protein.
[0065] Vectors are used herein either to amplify DNA encoding
Vitamin K Dependent Proteins and/or to express DNA which encodes
Vitamin K Dependent Proteins. An expression vector is a replicable
DNA construct in which a DNA sequence encoding a Vitamin K
dependent protein is operably linked to suitable control sequences
capable of effecting the expression of a Vitamin K dependent
protein in a suitable host. The need for such control sequences
will vary depending upon the host selected and the transformation
method chosen. Generally, control sequences include a
transcriptional promoter, an optional operator sequence to control
transcription, a sequence encoding suitable mRNA ribosomal binding
sites, and sequences which control the termination of transcription
and translation.
[0066] Amplification vectors do not require expression control
domains. All that is needed is the ability to replicate in a host,
usually conferred by an origin of replication, and a selection gene
to facilitate recognition of transformants.
[0067] Vectors comprise plasmids, viruses (e.g., adenovirus,
cytomegalovirus), phage, and integratable DNA fragments (i.e.,
fragments integratable into the host genome by recombination). The
vector replicates and functions independently of the host genome,
or may, in some instances, integrate into the genome itself.
Expression vectors should contain a promoter and RNA binding sites
which are operably linked to the gene to be expressed and are
operable in the host organism.
[0068] DNA regions are operably linked or operably associated when
they are functionally related to each other. For example, a
promoter is operably linked to a coding sequence if it controls the
transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
permit translation.
[0069] Transformed host cells are cells which have been transformed
or transfected with one or more Vitamin K dependent protein
vector(s) constructed using recombinant DNA techniques.
[0070] In a preferred embodiment, a promoter for the elongation
factor-1.alpha. from Chinese hamster is used (CHEF1) to provide
high level expression of a vitamin K dependent coagulation factor
and/or processing factor(s). The CHEF1 vector is used as described
in Deer, et al. (2004) "High-level expression of proteins in
mammalian cells using transcription regulatory sequences from the
Chinese Hamster EF-1.alpha. gene" Biotechnol Prog. 20: 880-889 and
in U.S. Pat. No. 5,888,809 which is incorporated herein by
reference. The CHEF1 vector utilizes the 5' and 3' flanking
sequences from the Chinese hamster EF-1.alpha.. The CHEF1 promoter
sequence includes approximately 3.7 kb DNA extending from a SpeI
restriction site to the initiating methionine (ATG) codon of the
EF-1.alpha. protein. The DNA sequence is set forth in SEQ ID NO: 1
of U.S. Pat. No. 5,888,809.
Host Cells
[0071] Suitable host cells include prokaryote, yeast or higher
eukaryotic cells such as mammalian cells and insect cells. Cells
derived from multicellular organisms are a particularly suitable
host for recombinant Vitamin K Dependent protein synthesis, and
mammalian cells are particularly preferred. Propagation of such
cells in cell culture has become a routine procedure (Tissue
Culture, Academic Press, Kruse and Patterson, editors (1973)),
Examples of useful host cell lines are VERO and HeLa cells, Chinese
hamster ovary (CHO) cell lines, and WI138, HEK 293, BHK, COS-7, CV,
and MDCK cell lines. Expression vectors for such cells ordinarily
include (if necessary) an origin of replication, a promoter located
upstream from the DNA encoding vitamin K dependent protein(s) to be
expressed and operatively associated therewith, along with a
ribosome binding site, an RNA splice site (if intron-containing
genomic DNA is used), a polyadenylation site, and a transcriptional
termination sequence. In a preferred embodiment, expression is
carried out in Chinese Hamster Ovary (CHO) cells using the
expression system of U.S. Pat. No. 5,888,809, which is incorporated
herein by reference.
[0072] The transcriptional and translational control sequences in
expression vectors to be used in transforming vertebrate cells are
often provided by viral sources. For example, commonly used
promoters are derived from Cauliflower mosaic virus (cmv), polyoma,
Adenovirus 2, and Simian Virus 40 (SV40). See. e.g., U.S. Pat. No.
4,599,308.
[0073] An origin of replication may be provided either by
construction of the vector to include an exogenous origin, such as
may be derived from SV 40 or other viral (e.g. Polyoma, Adenovirus,
VSV, or BPV) source, or may be provided by the host cell
chromosomal replication mechanism. If the vector is integrated into
the host cell chromosome, the latter is often sufficient.
[0074] Rather than using vectors which contain viral origins of
replication, one can transform mammalian cells by the method of
cotransformation with a selectable marker and the DNA for the
Vitamin K Dependent protein(s). Examples of suitable selectable
markers are dihydrofolate reductase (DHFR) or thymidine kinase.
This method is further described in U.S. Pat. No. 4,399,216 which
is incorporated by reference.
[0075] Other methods suitable for adaptation to the synthesis of
Vitamin K Dependent protein(s) in recombinant vertebrate cell
culture include those described in M-J. Gething et al., Nature 293,
620 (1981); N. Mantel et al., Nature 281, 40; A. Levinson et al.,
EPO Application Nos. 117,060A and 117,058A.
[0076] Host cells such as insect cells (e.g., cultured Spodoptera
frugiperda cells) and expression vectors such as the baculovirus
expression vector (e.g., vectors derived from Autographa
californica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or
Galleria ou MNPV) may be employed in carrying out the present
invention, as described in U.S. Pat. Nos. 4,745,051 and 4,879,236
to Smith et al. In general, a baculovirus expression vector
comprises a baculovirus genome containing the gene to be expressed
inserted into the polyhedrin gene at a position ranging from the
polyhedrin transcriptional start signal to the ATG start site and
under the transcriptional control of a baculovirus polyhedrin
promoter.
[0077] Prokaryote host cells include gram negative or gram positive
organisms, for example Escherichia coli (E. coli) or Bacilli.
Higher eukaryotic cells include established cell lines of mammalian
origin as described below. Exemplary host cells are E. coli W3110
(ATCC 27,325), E. coli B, E. coli X1776 (ATCC 31,537), E. coli 294
(ATCC 31,446). A broad variety of suitable prokaryotic and
microbial vectors are available. E. coli is typically transformed
using pBR322. Promoters most commonly used in recombinant microbial
expression vectors include the betalactamase (penicillinase) and
lactose promoter systems (Chang et al., Nature 275, 615 (1978); and
Goeddel et al., Nature 281, 544 (1979)), a tryptophan (trp)
promoter system (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980)
and EPO App. Publ. No. 36,776) and the tae promoter (H. De Boer et
al., Proc. Natl. Acad. Sci. USA 80, 21 (1983)). The promoter and
Shine-Dalgarno sequence (for prokaryotic host expression) are
operably linked to the DNA encoding the Vitamin K Dependent
protein(s), i.e., they are positioned so as to promote
transcription of Vitamin K Dependent Protein(s) messenger RNA from
the DNA.
[0078] Eukaryotic microbes such as yeast cultures may also be
transformed with Vitamin K Dependent Protein-encoding vectors. see,
e.g., U.S. Pat. No. 4,745,057. Saccharomyces cerevisiae is the most
commonly used among lower eukaryotic host microorganisms, although
a number of other strains are commonly available. Yeast vectors may
contain an origin of replication from the 2 micron yeast plasmid or
an autonomously replicating sequence (ARS), a promoter, DNA
encoding one or more Vitamin K Dependent proteins, sequences for
polyadenylation and transcription termination, and a selection
gene. An exemplary plasmid is YRp7, (Stinchcomb et al., Nature 282,
39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschemper et al.,
Gene 10, 157 (1980)). Suitable promoting sequences in yeast vectors
include the promoters for metallothionein, 3-phosphoglycerate
kinase (Hitzeman et al., J. Biol. Chem. 255, 2073 (1980) or other
glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7, 149 (1968);
and Holland et al., Biochemistry 17, 4900 (1978)). Suitable vectors
and promoters for use in yeast expression are further described in
R. Hitzeman et al., EPO Publn. No. 73,657.
[0079] Cloned genes of the present invention may code for any
species of origin, including mouse, rat, rabbit, cat, porcine, and
human, but preferably code for Vitamin K dependent proteins of
human origin. DNA encoding Vitamin K dependent proteins that is
hybridizable with DNA encoding for proteins disclosed herein is
also encompassed. Hybridization of such sequences may be carried
out under conditions of reduced stringency or even stringent
conditions (e.g., conditions represented by a wash stringency of
0.3M NaCl, 0.03M sodium citrate, 0.1% SDS at 60.degree. C. or even
70.degree. C. DNA encoding the Vitamin K dependent protein
disclosed herein in a standard in situ hybridization assay. See J.
Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed.
1989)(Cold Spring Harbor Laboratory)).
[0080] As noted above, the present invention provides a method of
providing a functional Vitamin K dependent proteins. In general,
the method comprises culturing a host cell which expresses a
vitamin K dependent protein; and then harvesting the proteins from
the culture. The culture can be carried out in any suitable
fermentation vessel, with a growth media and under conditions
appropriate for the expression of the vitamin K dependent
protein(s) by the particular host cell chosen. Vitamin K dependent
protein can be collected directly from the culture media, or the
host cells lysed and the vitamin K dependent protein collected
therefrom. Vitamin K dependent protein can then be further purified
in accordance with known techniques.
[0081] As a general proposition, the purity of the recombinant
protein produced according to the present invention will preferably
be an appropriate purity known to the skilled art worker to lead to
the optimal activity and stability of the protein. The vitamin K
dependent protein, such as Factor IX or Factor VII/VIIa is
preferably of ultrahigh purity. Preferably, the recombinant protein
has been subjected to multiple chromatographic purification steps,
such as affinity chromatography, ion-exchange chromatography and
preferably immunoaffinity chromatography to remove substances which
cause fragmentation, activation and/or degradation of the
recombinant protein during manufacture, storage and/or use.
Illustrative examples of such substances that are preferably
removed by purification include thrombin and Factor IXa; other
protein contaminants; proteins, such as hamster proteins, which are
released into the tissue culture media from the production cells
during recombinant protein production; non-protein contaminants,
such as lipids; and mixtures of protein and non-protein
contaminants, such as lipoproteins. The purification of the
recombinant vitamin K dependent protein may also include in vitro
activation of the zymogen into the active protease form. For
example, in the case of Factor VII, purification may optionally
include an in vitro activation step to Factor VIIa.
[0082] Purification procedures for vitamin K dependent proteins are
known in the art. For example, see U.S. Pat. No. 5,714,583, which
is incorporated herein by reference. A method commonly used in
purification of vitamin K dependent protein is pseudochromatography
which involves metal ion elution from a positively charged resin
such as Q-Sepharose HP (See U.S. Pat No. 4,981,952 which is
incorporated herein by reference) In the case of vitamin K
dependent proteins, the method relies upon the ability of the gla
domain to bind metal ions such as calcium.
[0083] Factor IX DNA coding sequences, along with vectors and host
cells for the expression thereof, are disclosed in European Patent
App. 373012, European Patent App. 251874, PCT Patent Appl. 8505376,
PCT Patent Appln. 8505125, European Patent Appln. 162782, and PCT
Patent Appln. 8400560. Genes for other coagulation factors are also
known and available, for example, Factor II (Accession No.
NM.sub.--000506), Factor VII (Accession No. NM.sub.--019616, and
Factor X (Accession No. NM.sub.--000504).
[0084] It will be understood by those of skill in the art that
numerous and various modifications can be Made without departing
from the spirit of the present invention. Therefore, it should be
clearly understood that the forms of the present invention are
illustrative only and are not intended to limit the scope of the
present invention.
Pharmaceutical Compositions
[0085] The blood clotting Factor formulations may be formed by
methods well known in the art. Vitamin K dependent protein
compositions may be blended with conventional excipients such as
binders, including gelatin, pre-gelatinized starch, and the like;
lubricants, such as hydrogenated vegetable oil, stearic acid and
the like; diluents, such as lactose, mannose, and sucrose;
disintegrants, such as carboxymethyl cellulose and sodium starch
glycolate; suspending agents, such as povidone, polyvinyl alcohol,
and the like; absorbents, such as silicon dioxide; preservative,
such as methylparaben, propylparaben, and sodium benzoate;
surfactants, such as sodium lauryl sulfate, polysorbate 80, and the
like; and colorants, such as F.D & C. dyes and the like.
[0086] Liquid form preparations include solutions, suspensions, and
emulsions. Aqueous solutions suitable for oral use are prepared by
dissolving the active component in water or other suitable liquid
and adding suitable colorants, flavors, stabilizing agents, and
thickening agents as desired. Aqueous solutions suitable for oral
use may also be made by dispersing the finely divided active
component in water or other suitable liquid with viscous material,
such as natural or synthetic gums, resins, methylcellulose, sodium
carboxymethylcellulose, and other suspending agents known in the
art.
[0087] Also included are solid form preparations Which are intended
to be converted, shortly before use, to liquid form preparations
for either oral or parental administration. Such liquid forms
include solutions, suspensions, and emulsions. These particular
solid form preparations are provided in unit dose form and as such
are used to provide a single liquid dosage unit. Alternatively,
sufficient solid preparation may be provided so that the after
conversion to liquid form, multiple individual liquid doses may be
obtained by measuring predetermined volumes of the liquid form
preparation as with a syringe, teaspoon, or other volumetric
measuring device.
[0088] Pharmaceutical compositions of Vitamin K dependent protein
for injection or intravenous administration comprise
therapeutically effective amounts of Vitamin K dependent protein
and an appropriate physiologically acceptable carrier. A variety of
aqueous carriers may be used, e.g., buffered water, saline, 0.3%
glycine and the like. Stabilizers such as plant-derived
glycoproteins, albumin, free amino acids, small peptides,
lipoprotein, and/or globulin may also be added. Other components of
the pharmaceutical compositions of the invention can include
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions, such as pH adjusting and
buffering agents, tonicity adjusting agents and the like, for
example, sodium acetate, sodium lactate, sodium chloride, potassium
chloride, calcium chloride, etc.
[0089] The solid and liquid forms may contain, in addition to the
active material, flavorants, colorants, stabilizers, buffers,
artificial and natural sweeteners, dispersants, thickeners,
solubilizing agents, and the like. The liquid utilized for
preparing the liquid form preparation is suitably water, isotonic
water, ethanol, glycerin, propylene glycol, and the like, as well
as combinations thereof. The liquid utilized will be chosen with
regard to the route of administration.
[0090] Preferably, the preparations are unit dosage form. In such
form, the preparation is subdivided into unit doses containing
appropriate quantities of the active components. The unit dosage
form can be a packaged preparation, such as packaged tablets or
capsules. The unit dosage can be a capsule, cachet, or tablet
itself or it can be the appropriate number of any of these in
packaged form.
[0091] The quantity of active material in a unit dose of
preparation is varied according to the particular application and
potency of the active ingredients.
EXAMPLES
Example 1
Determination of Factor IX Antigen Level
[0092] A gene for Factor IX was synthesized, operably linked to the
CHEF-1 promoter, and transfected into CHO cells. The primary
transfectants were grown up in a E-well microtiter plate and were
assayed in triplicate to give an average level of antigen
expression for the cells in the well. As presented in Table 1, the
cells were designated primary transfectant line T-335. It should be
kept in mind that this is an average production for the primary
transfectants. Some of the cells produce no Factor IX, some produce
a moderate level and some cells produce high levels of the protein:
The primary transfectants were followed by measuring the amount of
Factor IX antigen produced. Biological activity was not measured at
this early stage of expression. As a control, a Factor IX gene
construct in which the wild type Factor IX Propeptide sequence was
replaced with the Propeptide sequence from another Vitamin K
dependent protein, Protein C, was transfected into CHO cells and
designated primary transfectant cell line T-337.
TABLE-US-00001 TABLE 1 Expression of wild type Factor IX in
transfected CHO cells. Replicate (6-well model assay) A C Primary
Factor IX (mg/ B (mg/ AVG Transfectant Promoter Construct L) (mg/L)
L) (mg/L) T-335 CHEF-1 Wild Type 11.8 12.0 10.7 11.5 Factor IX
T-337 CHEF-1 Protein C 10.0 11.8 12.3 11.3 Propeptide - Factor
IX
[0093] All transfectants were assayed for Factor IX antigen by a
commercially available ELISA test which used a commercially
available Factor IX preparation as a standard (BeneFix, Wyeth
Laboratories).
[0094] The cloning and growth of CHO cells transfected with the
Factor IX gene are conducted by one of three methods. As one
skilled in the art appreciates, cell cultures are set up to produce
the amount of cells or tissue culture fluid needed for the
experiments which are performed with these materials. The smallest
size system is growth of cells in 96-well microtiter plates. The
cells on these plates were grown for 14 days but since they have
the smallest surface area for cells to multiply on, they produce
the fewest cells and in the least amount of tissue culture media
and lowest amounts of Factor IX. The second system is a 6-well
model assay as shown in Table 1 above. Cells were grown on a 6 well
tissue culture plates for 9 days. Cells grown in 6 well plates
generally produce an intermediate amount of cells in a medium
amount of tissue culture media and an intermediate amount of Factor
IX. The highest concentration (and actual number) of cells was
produced in a 1.5 liter shake flask incubated for 18 days.
[0095] Due to our extensive experience using all three of systems,
it is possible to extrapolate from the amount of Factor IX produced
in one system to that produced in another system. For example, the
amount of Factor IX produce in the shake Flask system is
essentially 6 times greater than produced in the 6-well model
system. In this way, when it is necessary for comparison, one can
extrapolate the quantity of Factor IX that will be produced in a
Shake Flask system from the amount measured in a smaller system
like the 6-well model or from the 96-well microtiter plates. Table
1 above indicates that CHO cells can be transfected with a wild
type Factor IX gene and produce an average of 11.5 mg/L in a 6-well
model assay system. By extrapolation, as described above, this
equates to 69 mg/L in a 1.5 L shake flask.
Example 2
[0096] For comparison, the wild type Factor IX gene of Example 1
was cloned into a construct using the CMV promoter and transfected
into HEK293 cells. As in Example 1 above, the primary transfectants
were grown up in a 6-well microliter plate. The titer for these
samples was less than 0.2 mg/L (data not shown). Further
experiments were conducted using the CHEF-1 promoter.
Example 3
[0097] In order to identify and characterize clones with high
levels of Factor IX expression, primary transfectants were cloned
by limit dilution into 96-well plates. As a consequence of the low
surface area for cells to grow the level of expression was less
that that reported in Example 1 where cells were grown on 6-well
microliter plates. One hundred and fifty two single cell clones
were identified from the T-335 primary transfectant experiment. The
Factor IX antigen expression level for each clone was measured by
ELISA analysis. The distribution of Factor IX expression levels
from the clones are shown in FIG. 1.
[0098] The T-335 clones were separated into clones that expressed
Factor IX at a level of greater than 0.4 mg/L and those that
produced Factor IX at lower levels. Clones expressing Factor IX at
levels higher than 0.4 mg/L were considered high expresser clones.
Clones with expression levels lower than 0.4 mg/L were considered
low expressers. The range of expression for Factor IX can be seen
to be distributed in a wide range between no expression to above
1.6 mg/L with an average of 0.87 mg/L of Factor IX. In order to
determine if other genes transfected into CHO Cells would have
essentially the same broad range of distribution when recloned,
cells form the primary T-337 transfection were also cloned by limit
dilution in the 96-well microtiter plate assay. As can be seen in
FIG. 1 above and Table 2 below, of the 171 clones evaluated the
range of antigen expression was again quite broad being between
none and above 1.8 mg/L with an average of 0.73 mg/L.
TABLE-US-00002 TABLE 2 Limit dilution cloning of Primary
Transfectants Primary Transfectant Cloned Factor IX Antigen Level #
of All Clones High Clones mg/L Expressors mg/L T335 Wild Type
Factor IX 152 0.46 0.87 T337 Protein C Propeptide- 171 0.29 0.73
Factor IX
[0099] The results of FIG. 1 and Table 2 demonstrate that when CHO
cells are transfected with a gene under the control of the CHEF-1
promoter, all cells are not transfected in a way that they can
produce detectable Factor IX and that those cells that are
transfected in a productive way produce varying amounts of Factor
IX. The reason that these cells produce varying levels of Factor IX
is not understood. Without intending to be limited by theory,
reasons may include that (1) some cells simply do not receive the
transfected gene, (2) others may receive a gene copy but it may not
integrate into the chromosome, (3) other cells may have the gene
integrated in a non-functional region of the chromosome, (4) yet
other cells may receive multiple copies of the gene but only some
are integrated in the correct position of the genome for expression
and (5) some cells-may receive multiple copies of the gene but only
some of the copies are integrated in a functional way at the time
the ELISA is performed on individual clones. The last possibility
predicts that as these cells divide and grow, more copies of
unintegrated DNA may become integrated with time, (6) Duplication
of integrated plasmid may occur and number of duplications may
increase upon further cloning or culturing. In the last two cases
(5) and (6), one predicts that upon further cloning or culturing,
(essentially repeating the above experiment), cell lines would be
isolated which would express higher levels of Factor IX (or other
protein, e.g., Factor VII/VIIa).
[0100] To determine if this were the case, high level expressors of
Wild Type Factor IX, as described in Table 2, were pooled and
expanded in a shake flask culture. Individual clones were then
isolated by limit dilution cloning. The clones producing the
highest level of Factor IX were isolated. The twenty highest
producers are presented in Table 3.
TABLE-US-00003 TABLE 3 Second limit dilution of pooled high
expressing clones of T-335 and selection for high Factor IX antigen
expression. Estimated Titer FIX Specific Active % 6-Well Sample
(.mu.g/ Clot Activity Activity FIX Active Titer # mL) Time (U/mL)
(U/mg) (.mu.g/mL) FIX (.mu.g/mL) P1B10 4.14 65.95 0.066 16 0.264 6%
104 P2B11 3.05 76.54 0.028 9 0.112 4% 76 P1G12 2.98 66.26 0.077 26
0.308 10% 75 P2C2 2.47 67.11 0.062 25 0.248 10% 62 P1H2 2.44 61.66
0.100 41 0.400 16% 61 P1A6 2.31 65.00 0.071 31 0.284 12% 58 P1B4
2.16 64.65 0.074 34 0.296 14% 54 P1G3 2.12 65.87 0.079 37 0.316 15%
53 P1A12 2.03 70.04 0.046 23 0.184 9% 51 P1A2 2.02 75.28 0.030 15
0.120 6% 50 P1H10 1.98 65.60 0.069 35 0.276 14% 50 P2D7 1.97 62.34
0.095 48 0.380 19% 49 P2D6 1.95 63.41 0.086 44 0.344 18% 49 P1D11
1.94 66.37 0.076 39 0.304 16% 49 P1D9 1.92 62.32 0.110 57 0.440 23%
48 P2D11 1.85 58.42 0.138 74 0.552 30% 46 P1H3 1.85 66.61 0.063 34
0.252 14% 46 P1G4 1.83 68.11 0.065 35 0.260 14% 46 P1B5 1.83 68.66
0.052 28 0.208 11% 46 P1E6 1.79 69.77 0.057 32 0.228 13% 45 The
"estimated 6-well titers" is based on our experience that about
25-fold more antigen is produced in 6-well plates when compared to
96-well plates.
[0101] As shown in Table 3, in the 96 well microliter model, the
range of production for Factor IX antigen from the highest yielding
20 clones is between 1.79 and 4.14 mg/L. As presented in the last
column of Table 3, one can estimate the concentration of Factor IX
that would be expected in 6-well microtiter plates. It is estimated
that the level of these clones in 6-microtitre plates would be
between 46 and 104 mg/L. This is a substantial increase from the
level of expression of the primary transfectants seen in Table 2 (a
range of 0-1.65 mg/L was reported). The biological activity of
Factor IX produced from this selected group of clones is between 4
and 30% (Table 3). It appears, that, in general, the higher the
level of Factor IX antigen production the lower the level of
biologically active Factor IX produced. This is shown graphically
in FIG. 2. This observation has been made by others attempting to
overproduce Vitamin K dependent coagulation factors.
[0102] It has always proven difficult to produce Factor IX in
tissue culture systems with more than 1.5-5% biologically activity.
For example, Kaufman (Kaufman, et al. The Journal of Biological
Chemistry vol. 261 (21):9622-9628, Jul. 25, 1986) report CHO cell
lines secreting more than 100 .mu.g/ml Factor IX by methotrexate
amplification in dihyrofolate reductase deficient cell lines. Yet,
biologically active Factor IX was never produced at a level higher
than 1.5 .mu.g/ml. It was suggested that the CHO cell line was
deficient in processing factors needed to make biologically active
Factor IX. It is generally accepted that the presence of processing
cofactors is necessary to produce biologically active vitamin K
dependent proteins in recombinant systems. We evaluated the
potential of our selection system to also identify clones producing
a high percentage of biologically active Factor IX. Table 4
presents the 20 Factor IX producing clones with the highest
percentage of biologically active Factor IX.
TABLE-US-00004 TABLE 4 Second limit dilution of high expresser
clones of T-335 and selection for high percentage of biologically
active Factor IX. Estimated Titer FIX Specific Active % 6-Well
Sample (.mu.g/ Clot Activity Activity FIX Active Titer # mL) Time
(U/mL) (U/mg) (.mu.g/mL) FIX (.mu.g/mL) P2A7 0.24 76.52 0.028 119
0.112 48% 6 P1B9 0.61 65.52 0.088 112 0.272 45% 15 P2D5 0.47 69.04
0.052 112 0.208 45% 12 P2A10 0.34 73.28 0.036 106 0.144 42% 8 P2B9
0.51 68.62 0.053 103 0.212 41% 13 P1B11 0.38 71.82 0.039 103 0.156
41% 10 P1D10 0.70 67.28 0.070 99 0.280 40% 18 P2D2 0.88 63.53 0.085
96 0.340 39% 22 P2C5 0.37 74.30 0.034 92 0.136 37% 9 P2D3 0.67
68.06 0.057 88 0.228 34% 17 P2A9 0.37 76.33 0.028 76 0.112 30% 9
P2D11 1.85 58.42 0.138 74 0.552 30% 46 P1H11 0.61 70.83 0.044 72
0.176 29% 15 P1E8 0.65 72.25 0.046 71 0.184 28% 16 P1E9 0.64 72.55
0.045 70 0.180 28% 16 P1E3 1.45 83.08 0.102 70 0.408 28% 36 P1E10
0.98 67.76 0.067 69 0.268 27% 24 P2E8 0.30 81.39 0.020 67 0.080 27%
7 P1C9 0.54 72.96 0.036 66 0.144 27% 14 P2D10 0.79 69.59 0.050 64
0.200 25% 20
[0103] As can be seen in Table 4, all of the 20 clones producing
Factor IX, which have been selected for high content of
biologically active material produce above 25% biologically active
Factor IX. In this experiment, the highest level of functional
Factor IX reported is for clone P2A7 at 48%. Specific activity
ranges from 64-119 U/mg. In contrast, Kaufman (ibid) reported
specific activity of 35-75 U/mg, up to half the specific activity
of plasma derived Factor IX (150 U/mg) using the adenovirus major
late promoter. By use of the CHEF-1 promoter in CHO cells,
biologically active recombinant Factor IX was produced at levels
much closer to the levels obtained with plasma-derived Factor IX.
It was unexpected that such high levels of biologically active
Factor IX protein could be produced recombinantly without addition
of processing factors.
[0104] In summary, limit dilution selection of clones which produce
Factor IX in a tissue culture system in conjunction with a high
level promoter system produced more Factor IX antigen and a higher
level of biologically active Factor IX protein than has been
possible in the past.
Example 4
[0105] In order to determine if our extrapolation of Factor IX
production from 96-well plates to 6-well microtiter plates was
accurate in both antigen produced and percentage biologically
active protein recovered, we expanded a group of eleven clones from
the second limited dilution experiment in 6 well microtiter plates.
As can be seen in Table 5, the results were similar than those
reported in 96 well micro titer plates. The level of Factor IX
antigen production was between 2 and 40 mg/L and the percentage of
biologically active Factor IX recovered was between 10 and 58%. The
level of biologically active Factor IX was significantly higher
than reported previously for any other Vitamin K dependent protein
prepared in the absence of vitamin K or processing factors involved
in post-translational processing of blood factor proteins.
TABLE-US-00005 TABLE 5 Factor IX antigen production and percentage
of biologically active Factor IX in 6-well microtiter plates.
Factor IX Specific Activity Activity Active FIX % Active Sample #
Titer (mg/L) (U/mL) (U/mg) (mg/L) FIX 117 37.1 2.7 74 11.0 30 143
40.1 2.2 56 9.0 22 23 16.3 1.6 101 6.6 40 125 10.3 1.5 142 5.9 57
46 10.3 1.5 145 5.9 58 21 10.4 1.3 121 5.1 49 137 9.9 1.1 116 4.6
46 106 9.2 1.1 124 4.6 50 135 7.8 0.5 66 2.1 27 105 18.4 0.4 24 1.8
10 103 2.7 0.3 104 1.1 42
[0106] As shown by Table 5, extrapolating production and Factor IX
antigen and percentage of biologically active Factor IX produced
from 96-well microtiter plates to production in 6 well-microtiter
plates is confirmed by experimental data.
Example 5
Recombinant Factor VII Produced by Semi-Solid Matrix Cloning
[0107] A gene for Factor VII was synthesized, operably linked to
the CHEF-1 promoter, and transfected into CHO cells using a vector
which included the DHFR marker for selection in Hypoxanthine
Thymidine (HT)-minus medium. The primary transfectants were grown
up in a microtiter plate and were assayed to give an average level
of antigen expression for the cells in the well. The pool of
primary transfectants were then subjected to cell cloning by a
semi-solid matrix cloning method.
[0108] The cells were seeded into a semi-solid matrix at very low
densities (typically 1,000-4,000 cells per ml) into a mixture of
media (Cloning medium A.RTM., Invitrogen), media supplements (4 mM
L-glutamine, 10 .mu.g/mL vitamin K, 1 mM CaCl.sub.2), conditioned
medium (5-20% of 7 day old culture of parental CHO cells), and a
generally inert, biologically compatible, semi-solid medium such as
methylcellulose (CloneMatrix.RTM.). After seeding the low density
cells into the mixture as a single cell suspension and letting it
"gel" for a few minutes, into a semi-solid, the culture plates are
returned to an incubator (37.degree. C., with humidity and carbon
dioxide buffering atmosphere) and allowed to sit undisturbed for
anywhere from .about.1 week to .about.3 weeks.
[0109] A fluorescent antibody against human FVII was included in
the semi-solid medium formulation such that the FVII-antibody that
was impregnated in the gel allowed detection of high expressing
colonies (clones) when the culture plates containing semi-solid
matrix with colonies were viewed under a fluorescence microscope.
About two weeks after initially seeding the cells into the
semisolid matrix the culture plates were observed for colony
formation (number of colonies, their size and how far separated
they are from one another in the gel) and then individual colonies
were picked and seeded into separate cluster plates, each as a
clonal population. They were expanded through larger plates and
screened for production of FVII in expansion medium (OptiCHO.RTM.,
L-glutamine, 200 mM, Vitamin K1 (2% in ethanol) and 1 M
CaCl.sub.2). The cells were split and grown in fresh medium.
Periodically, FVII/FVIIa levels and bioactivity were assessed.
[0110] FVII/FVIIa levels were determined by conventional FVII ELISA
assay.
[0111] Bioactivity was determined by a conventional chromogenic
FVIIa Bioactivity assay such as BIOPHEN FVII.RTM. (Ref No. 221304)
available from HYPHEN BioMed. The basis of the assay relies upon
the ability of Factor VII to activate Factor X to Factor Xa. After
forming an enzymatic complex with Tissue Factor, provided by rabbit
Thromboplastin, the FVII complex activates Factor X to Factor Xa
which activity is measured by cleavage of a chromogenic substrate
(SXa-11). Factor Xa cleaves the substrate and generates pNA. The
amount of pNA generated is directly proportional to the Factor Xa
activity. Finally, there is a direct relationship between the
amount of Factor VII in the assayed sample and the Factor Xa
activity generated, measured by the amount of pNA released,
determined by color development at 405 nm. Alternatively, a
two-stage coagulation (clotting) assay may be used to determine
FVII/FVIIa activity.
[0112] Calibration is performed with a normal pooled citrated
plasma, with the assigned value of 100% Factor VII. The assay kit
includes a standard plasma dilution of 1:1000. By definition, this
latter dilution of the pool represents the 100% Factor VII
activity, The dynamic range is from 0 to 200% FactorVII. The 200%
Factor VII activity is the 1:500 dilution of the plasma pool. A
standard curve was generated.
[0113] Samples were diluted in order to get a final Factor VII
concentration in the tested dilution range of 0.1 to 1 ng/ml. The
samples were placed in a microplate well or plastic tube and
incubated. The SXa-11 substrate was introduced followed by further
incubation. The reaction, was stopped by adding 60 .mu.L/well or
200 .mu.L/tube citric acid (20 g/L), or 20% acetic acid. The yellow
color is stable for 2 hours. The sample blank was prepared by
mixing the reagents in the opposite order from the test (i.e.,
Citric Acid (20 g/L), SXa-11 substrate, diluted plasma, Factor X,
and Thromboplastin-Ca). The concentration of bioactive FVII/FVIIa
was determined for the unknown samples from the standard curve
generated with plasma samples from the kit discussed above. The
concentration of bioactive FVII represents the amount in .mu.g/ml
of FVII/FVIIa in the supernatant that was sufficiently carboxylated
to be measured as "active FVIIa".
[0114] Table 6 shows the titers of FVII/VIIa antigen in the
supernatants of 15 different clones transfected with a FVII
encoding plasmid containing the DI-IFR selectable marker and a
CHEF-1 promoter and how much of this FVII/VIIa protein in the
supernatants was biologically active as measured by the chromogenic
assay described above. The various clones produce reasonably high
levels of FVII/VIIa antigen into their supernatant (e.g. up to 7
ug/ml) even though they do not contain plasmid for expressing any
exogenous (human) VKOR or VKGC. More importantly, for these
representative clones the proportion of FVII/VIIa antigen in the
supernatant which is bioactive is very high, ranging from 66% to
100% of the secreted FVII.
TABLE-US-00006 TABLE 6 Titers of FVII/FVIIa antigen and Bioactivity
in supernatants of 15 different clones. Clone # Ag (ng/ml)
Bioactivity (ng/ml) % Bioactive 1 7152 6848 96 2 5524 5214 94 3
5160 4194 81 4 5042 4766 95 5 4017 3109 77 6 4013 3009 75 7 3924
2587 66 8 3651 3062 84 9 3531 3937 100* 10 3377 3289 97 11 3286
2836 86 12 3064 3520 100* 13 2891 2488 86 14 2642 2514 95 15 2307
2341 100* *calculated value was greater than 100%
[0115] Table 7 shows similar kind of data as Table 6 except that
these results were obtained from the clones at a later stage, after
the individual clones had undergone more cumulative population
doublings. The clones have increased their volumetric
productivities (i.e. amount of FVII/VIIa antigen per unit volume of
culture supernatant) the longer they've been cultured. By comparing
the results in Table 7 for Clone #10 with that in Table 6, one sees
that this clone's expression levels increased from .about.3.3 ug/ml
in the secondary screening stage to .about.29ug/ml after time in
culture. The secondary screen results are from static cultures
grown in cluster plates whereas the later assays are derived from
cultures grown under more optimal culture conditions, i.e., in a
shaking flask where the cultures are better aerated and with better
availability of nutrients before the supernatant sample was
harvested for assaying.
TABLE-US-00007 TABLE 7 Titers of FVII/FVIIa antigen and Bioactivity
in supernatants of 5 selected clones after repeated culturing.
Clone # Ag (ng/ml) Bioactivity (ng/ml) % Bioactive 1 23000 15000 65
2 26000 19000 73 3 28000 20000 71 6 26000 18000 69 10 29000 21000
72
[0116] This increase in cell specific productivity (Qp) is shown in
the following Table for a particular clone. An increase of 0.4
pg/cell/day at initial assessment increases to 0.56 pg/cell/day as
a function of time in culture, without recourse to
supertransfection with processing factors. Typical values for
FVII/VIIa production before initiation of the cell stability
studies was 11-12 .mu.g/mL with 60-70% bioactivity. By the end of
productivity assessment #3, these numbers typically increased to
35-45 .mu.g/mL with somewhat higher bioactivity (65-70%).
TABLE-US-00008 TABLE 8 Increase in cell specific productivity (Qp)
as a function of time in culture Experiment Qp (pg/cell/day) Cell
Stability flasks: Prod. Assess #1 0.4 (23)* Prod Assess #2 0.4 (48)
Prod Assess #3 0.56 (73) *Numbers in parentheses refers to number
of total cumulative population doublings from the time the cells
recovered from transfection and selection until seeding in the
productivity assessment experiment indicated.
[0117] These results show that by cell cloning methods such as
limit dilution cloning and semi-solid matrix cloning, and with
repeated culturing, high levels of bioactive vitamin K dependent
proteins can be achieved without co-transfection of processing
factors such as VKOR, VKGC, and/or PACE.
[0118] It will be understood by those of skill in the art that
numerous and various modifications can be made without departing
from the spirit of the present invention. Therefore, it should be
clearly understood that the forms of the present invention are
illustrative only and are not intended to limit the scope of the
present invention.
* * * * *