U.S. patent application number 17/752074 was filed with the patent office on 2022-09-08 for method for the production of a glycosylated immunoglobulin.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Chugai Seiyaku Kabushiki Kaisha, Hoffmann-La Roche Inc.. Invention is credited to Reinhard Franze, Chikashi Hirashima, Thomas Link, Yoshinori Takagi, Shinya Takuma, Yuriko Tsuda.
Application Number | 20220282298 17/752074 |
Document ID | / |
Family ID | 1000006359481 |
Filed Date | 2022-09-08 |
United States Patent
Application |
20220282298 |
Kind Code |
A1 |
Franze; Reinhard ; et
al. |
September 8, 2022 |
METHOD FOR THE PRODUCTION OF A GLYCOSYLATED IMMUNOGLOBULIN
Abstract
Herein is reported a method for the production of an
immunoglobulin comprising the following steps: a) providing a
eukaryotic cell comprising a nucleic acid encoding the
immunoglobulin, b) cultivating the eukaryotic cell in a cultivation
medium wherein the amount of glucose available in the cultivation
medium per time unit is kept constant and limited to less than 80%
of the amount that could maximally be utilized by the cells in the
cultivation medium per time unit, and c) recovering the
immunoglobulin from the culture.
Inventors: |
Franze; Reinhard; (Penzberg,
DE) ; Hirashima; Chikashi; (Tokyo, JP) ; Link;
Thomas; (Penzberg, DE) ; Takagi; Yoshinori;
(Tokyo, JP) ; Takuma; Shinya; (Tokyo, JP) ;
Tsuda; Yuriko; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc.
Chugai Seiyaku Kabushiki Kaisha |
Little Falls
Tokyo |
NJ |
US
JP |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
Chugai Seiyaku Kabushiki Kaisha
Tokyo
|
Family ID: |
1000006359481 |
Appl. No.: |
17/752074 |
Filed: |
May 24, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17407758 |
Aug 20, 2021 |
11377678 |
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17752074 |
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17243309 |
Apr 28, 2021 |
11136610 |
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17407758 |
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16790177 |
Feb 13, 2020 |
11021728 |
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17243309 |
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16682401 |
Nov 13, 2019 |
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16790177 |
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14844570 |
Sep 3, 2015 |
10501769 |
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16682401 |
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12911300 |
Oct 25, 2010 |
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14844570 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/14 20130101;
C07K 2317/24 20130101; C07K 2317/76 20130101; C07K 16/2866
20130101; C07K 2317/41 20130101; C12P 21/005 20130101 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C07K 16/28 20060101 C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 26, 2009 |
EP |
09013455.2 |
Claims
1.-35. (canceled)
36. A composition comprising Tocilizumab protein with mannose-5
glycostructure (M5) attached to Asn.sup.297 of the Tocilizumab
protein, wherein the fraction of M5 is in a range from 2.8% to 10%
of the sum comprising M5, G(0), G(1), and G(2) oligosaccharide
attached to Asn.sup.297 of the Tocilizumab protein, wherein the
fraction equals area % fraction determined in a liquid
chromatography method.
37. The composition according to claim 36, wherein the Tocilizumab
protein with M5 attached thereto has been produced by a recombinant
Chinese Hamster Ovary (CHO) cell.
38. The composition according to claim 37, wherein the CHO cell is
cultured in cell culture at an initial cell density of at least
10.sup.5 cells/ml.
39. The composition according to claim 38, wherein the initial cell
density comprises 8-12.times.10.sup.5 cells/ml.
40. The composition according to claim 37, wherein the relative
antibody concentration produced by the CHO cell is greater than
100% of the antibody concentration resulting from constant feeding
of CHO cells.
41. The composition according to claim 37, wherein the Tocilizumab
protein is produced by a cell culture of CHO cells at a cultivation
volume from 10,000 L-50,000 L.
42. The composition according to claim 36, wherein the M5 fraction
is in a range from 2.8 to 8%.
43. The composition according to claim 36, wherein the M5 fraction
is in a range from 2.8 to 6%.
Description
RELATED APPLICATION
[0001] This is a continuation application which claims priority
under 35 USC .sctn. 120 to non-provisional application Ser. No.
12/911,300, filed Oct. 25, 2010, which claims priority to European
application no. 09013455.2 filed on Oct. 26, 2009, the contents of
which are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Herein is reported a method in the field of immunoglobulin
production in cells, whereby the glycosylation pattern of the
produced immunoglobulin can be modified based on the cultivation
conditions.
[0003] In recent years the production of immunoglobulins has
steadily increased and it is likely that immunoglobulins will
become the biggest group of therapeutics available for the
treatment of various diseases in the near future. The impact of
immunoglobulins emerges from their specificity, which comprises
their specific target recognition and binding function as well as
the activation of specific effects concurrently with or after
antigen/Fc-receptor binding.
[0004] The specific target recognition and binding is mediated by
the variable region of the immunoglobulin. Other parts of the
immunoglobulin molecule, from which effects originate, are
posttranslational modifications, such as the glycosylation pattern.
The posttranslational modifications do have an influence on the
efficacy, stability, immunogenic potential, binding etc. of an
immunoglobulin. In connection therewith complement-dependent
cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC)
and induction of apoptosis have to be addressed.
[0005] It has been reported that the glycosylation pattern of
immunoglobulins, i.e. the saccharide composition and number of
attached glycostructures, has a strong influence on the biological
properties (see e.g. Jefferis, R., Biotechnol. Prog. 21 (2005)
11-16). Immunoglobulins produced by mammalian cells contain 2-3% by
mass carbohydrates (Taniguchi, T., et al., Biochem. 24 (1985)
5551-5557). This is equivalent e.g. in an immunoglobulin of class G
(IgG) to 2.3 oligosaccharide residues in an IgG of mouse origin
(Mizuochi, T., et al., Arch. Biochem. Biophys. 257 (1987) 387-394)
and to 2.8 oligosaccharide residues in an IgG of human origin
(Parekh, R. B., et al., Nature 316 (1985) 452-457), whereof
generally two are located in the Fc-region and the remaining in the
variable region (Saba, J. A., et al., Anal. Biochem. 305 (2002)
16-31).
[0006] In the Fc-region of an immunoglobulin of class G
oligosaccharide residues can be introduced via N-glycosylation at
amino acid residue 297, which is an asparagine residue (denoted as
Asn.sup.297). Youings et al. have shown that a further
N-glycosylation site exists in 15% to 20% of polyclonal IgG
molecules in the Fab-region (Youings, A., et al., Biochem. J., 314
(1996) 621-630; see e.g. also Endo, T., et al., Mol. Immunol. 32
(1995) 931-940). Due to inhomogeneous, i.e. asymmetric,
oligosaccharide processing, multiple isoforms of an immunoglobulin
with different glycosylation pattern exist (Patel, T. P., et al.,
Biochem. J. 285 (1992) 839-845; Ip, C. C., et al., Arch. Biochem.
Biophys. 308 (1994) 387-399; Lund, J., et al., Mol. Immunol. 30
(1993) 741-748). Concurrently the structure and distribution of the
oligosaccharides is both highly reproducible (i.e. non-random) and
site specific (Dwek, R. A., et al., J. Anat. 187 (1995)
279-292).
[0007] Some characteristics of an immunoglobulin are directly
linked to the glycosylation of the Fc-region (see e.g. Dwek, R. A.,
et al., J. Anat. 187 (1995) 279-292; Lund, J., et al., J. Immunol.
157 (1996) 4963-4969; Lund, J., FASEB J. 9 (1995) 115-119; Wright,
A. and Morrison, S. L., J. Immunol. 160 (1998) 3393-3402), such as
for example thermal stability and solubility (West, C. M., Mol.
Cell. Biochem. 72 (1986) 3-20), antigenicity (Turco, S. J., Arch.
Biochem. Biophys. 205 (1980) 330-339), immunogenicity (Bradshaw, J.
P., et al., Biochim. Biophys. Acta 847 (1985) 344-351; Feizi, T.
and Childs, R. A., Biochem. J. 245 (1987) 1-11; Schauer, R., Adv.
Exp. Med. Biol. 228 (1988) 47-72), clearance rate/circulatory
half-life (Ashwell, G. and Harford, J., Ann. Rev. Biochem. 51
(1982) 531-554; McFarlane, I. G., Clin. Sci. 64 (1983) 127-135;
Baenziger, J. U., Am. J. Path. 121 (1985) 382-391; Chan, V. T. and
Wolf, G., Biochem. J. 247 (1987) 53-62; Wright, A., et al.,
Glycobiology 10 (2000) 1347-1355; Rifai, A., et al., J. Exp. Med.
191 (2000) 2171-2182; Zukier, L. S., et al., Cancer Res. 58 (1998)
3905-3908), and biological specific activity (Jefferis, R. and
Lund, J., in Antibody Engineering, ed. by Capra, J. D., Chem.
Immunol. Basel, Karger, 65 (1997) 111-128).
[0008] Factors influencing the glycosylation pattern have been
investigated, such as for example presence of fetal calf serum in
the fermentation medium (Gawlitzek, M., et al., J. Biotechnol.
42(2) (1995) 117-131), buffering conditions (Muthing, J., et al.,
Biotechnol. Bioeng. 83 (2003) 321-334), dissolved oxygen
concentration (Saba, J. A., et al., Anal. Biochem. 305 (2002)
16-31; Kunkel, J. P., et al., J. Biotechnol. 62 (1998) 55-71; Lin,
A. A., et al., Biotechnol. Bioeng. 42 (1993) 339-350), position and
conformation of the oligosaccharide as well as host cell type and
cellular growth state (Hahn, T. J. and Goochee, C. F., J. Biol.
Chem. 267 (1992) 23982-23987; Jenkins, N., et al., Nat. Biotechnol.
14 (1996) 975-981), cellular nucleotide-sugar metabolism (Hills, A.
E., et al., Biotechnol. Bioeng. 75 (2001) 239-251), nutrient
limitations (Gawlitzek, M., et al., Biotechnol. Bioeng. 46 (1995)
536-544; Hayter, P. M., et al., Biotechnol. Bioeng. 39 (1992)
327-335), especially glucose restriction (Tachibana, H., et al.,
Cytotechnology 16 (1994) 151-157), and extracellular pH (Borys, M.
C., et al., Bio/Technology 11 (1993) 720-724).
[0009] Increased oligomannose structures as well as truncated
oligosaccharide structures have been observed by the recombinant
expression of immunoglobulins e.g. in NS0 myeloma cells (Ip, C. C.,
et al., Arch. Biochem. Biophys. 308 (1994) 387-399; Robinson, D.
K., et al., Biotechnol. Bioeng. 44 (1994) 727-735). Under glucose
starvation conditions variations in glycosylation, such as
attachment of smaller precursor oligosaccharides or complete
absence of oligosaccharide moieties, have been observed in CHO
cells, Murine 3T3 cells, rat hepatoma cells, rat kidney cells and
Murine myeloma cells (Rearick, J. I., et al., J. Biol. Chem. 256
(1981) 6255-6261; Davidson, S. K. and Hunt, L. A., J. Gen. Virol.
66 (1985) 1457-1468; Gershman, H. and Robbins, P. W., J. Biol.
Chem. 256 (1981) 7774-7780; Baumann, H. and Jahreis, G. P., J.
Biol. Chem. 258 (1983) 3942-3949; Strube, K.-H., et al., J. Biol.
Chem. 263 (1988) 3762-3771; Stark, N. J. and Heath, E. C., Arch.
Biochem. Biophys. 192 (1979) 599-609). A strategy based on low
glutamine/glucose concentrations was reported by Wong, D. C. F., et
al., Biotechnol. Bioeng. 89 (2005) 164-177.
[0010] The Japanese Patent Application JP 62-258252 reports a
perfusion culture of mammalian cells, whereas U.S. Pat. No.
5,443,968 reports a fed-batch culture method for protein secreting
cells. In WO 98/41611 a method for cultivating cells is reported
effective to adapt the cells to a metabolic state characterized by
low lactate production. A method for culturing cells in order to
produce substances is reported in WO 2004/048556. Elbein, A. D.,
Ann. Rev. Biochem. 56 (1987) 497-534, reports that mammalian cells
when incubated in the absence of glucose transfer mannose-5
containing structures instead of mannose-9 containing structures to
proteins. The dependence of pCO2 influences during glucose
limitation on CHO cell growth, metabolism and IgG production is
reported by Takuma, S., et al. in Biotechnol. Bioeng. 97 (2007)
1479-1488.
SUMMARY OF THE INVENTION
[0011] It has been found that the amount of the mannose-5
glycostructure in the glycosylation pattern of a polypeptide
produced by a eukaryotic cell can be modified based on the amount
of glucose provided to the cell in the cultivation process. By
reducing the amount of glucose available, e.g. by changing the DGL
value from 1.0 to smaller values of e.g. 0.8, 0.6, 0.5, 0.4, or
0.2, a modification in the mannose-5 glycostructure amount in the
glycosylation pattern can be obtained. The DGL value or
respectively the amount of glucose available per time unit has to
be kept constant and at a defined reduced value per time unit.
[0012] A first aspect as reported herein is a method for the
production of a polypeptide, in one embodiment of an
immunoglobulin, in a eukaryotic cell, comprising the following
steps [0013] a) providing a eukaryotic cell comprising a nucleic
acid encoding the polypeptide, [0014] b) cultivating the cell under
conditions wherein the degree of glucose limitation (DGL) is kept
constant and wherein the DGL is less than 0.8, and [0015] c)
recovering the polypeptide from the culture, wherein the fraction
of the polypeptide with a mannose-5 glycostructure is 10% or less
of the sum comprising the amount of the polypeptide with a
mannose-5 glycostructure, the amount of the polypeptide G(0)
isoform, the amount of the polypeptide G(1) isoform, and the amount
of the polypeptide G(2) isoform.
[0016] In one embodiment the DGL is kept constant in the range from
0.8 to 0.2. In a further embodiment the DGL is kept constant in the
range from 0.6 to 0.4. In another embodiment the fraction of the
polypeptide with a mannose-5 glycostructure is 8% or less of the
sum comprising the polypeptide with a mannose-5 glycostructure, the
polypeptide G(0) isoform, the polypeptide G(1) isoform, and the
polypeptide G(2) isoform. In still another embodiment the
polypeptide is an immunoglobulin, in one embodiment an
immunoglobulin of class G or E.
[0017] Another aspect as reported herein is a method for the
production of an immunoglobulin comprising the following steps:
[0018] a) providing a mammalian cell comprising a nucleic acid
encoding the immunoglobulin, [0019] b) cultivating the cell in a
cultivation medium wherein the amount of glucose available in the
cultivation medium per time unit is kept constant and limited to
less than 80% of the amount that could maximally be utilized by the
cells in the cultivation medium per time unit, and [0020] c)
recovering the immunoglobulin from the cells or the cultivation
medium.
[0021] In one embodiment the amount of glucose available in the
cultivation medium per time unit is kept constant and limited to a
value in the range from 80% to 20%. In a further embodiment the
range is from 60% to 40%. In another embodiment the cells in the
cultivation medium are the viable cells in the cultivation
medium.
[0022] In one embodiment of the aspects as reported herein the
eukaryotic cell is selected from CHO cells, NS0 cells, HEK cells,
BHK cells, hybridoma cells, PER.C6.RTM. cells, insect cells, or
Sp2/0 cells. In one embodiment the eukaryotic cell is a Chinese
Hamster Ovary (CHO) cell. In another embodiment of the aspects as
reported herein the cultivating is at a pH value in the range from
about pH 7.0 to about pH 7.2.
[0023] In still another embodiment of the aspects as reported
herein the cultivating is a continuous or a fed-batch cultivating.
The methods may comprise in another embodiment a final step of
purifying the polypeptide. In still another embodiment the cell is
cultivated for six to twenty days or for six to fifteen days. In a
further embodiment the cell is cultivated for six to eight
days.
[0024] Another aspect as reported herein is a composition
comprising an immunoglobulin, wherein the composition has been
prepared with a method as reported herein.
[0025] In one embodiment the immunoglobulin is an anti-IL-6R
antibody. In a further embodiment the anti-IL-6R antibody comprises
Tocilizumab. In another embodiment the mannose-5 glycostructure
attached to the anti-IL-6R antibody is 8% or less. In still a
further embodiment the mannose-5 glycostructure is 6% or less. In
another embodiment the mannose-5 glycostructure is 4% or less.
[0026] The invention also concerns a composition comprising an
antibody that binds human interleukin 6 receptor (anti-IL-6R
antibody) with oligosaccharide attached thereto, wherein mannose-5
glycostructure (M5) content in the composition is 8% or less, e.g.
less than 5%, for example, 4% or less. In one embodiment, the
anti-IL-6R antibody is Tocilizumab and/or has been produced by a
recombinant Chinese Hamster Ovary (CHO) cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIGS. 1A and 1B Viable cell density (FIG. 1A) and cell
viability profiles (FIG. 1B) in the fed-batch mode using the DGL
control; open circle: initial cell density of 8.times.10.sup.5
cells/ml; filled triangle: initial cell density of
10.times.10.sup.5 cells/ml; open square: initial cell density of
12.times.10.sup.5 cells/ml.
[0028] FIG. 2 Time courses of DGL in the fed-batch mode in
immunoglobulin production; circle: initial cell density of
8.times.10.sup.5 cells/ml; triangle: initial cell density of
10.times.10.sup.5 cells/ml; square: initial cell density of
12.times.10.sup.5 cells/ml.
[0029] FIG. 3 Feeding profiles based on DGL by the fed-batch mode
in immunoglobulin production; circles: initial cell density of
8.times.10.sup.5 cells/ml; triangle: initial cell density of
10.times.10.sup.5 cells/ml; square: initial cell density of
12.times.10.sup.5 cells/ml.
[0030] FIG. 4 Immunoglobulin production profiles by the fed-batch
mode in the DGL control; open circles: initial cell density of
8.times.10.sup.5 cells/ml; filled triangle: initial cell density of
10.times.10.sup.5 cells/ml; open square: initial cell density of
12.times.10.sup.5 cells/ml; filled small circle: constant feeding
method: FR=0.02 g glucose/h (control)
[0031] FIG. 5 Time curse of DGL during a fed-batch cultivation of a
cell: diamond: single feed daily feeding, square: dual feed daily
feeding; triangle: single feed profile feeding; X: dual feed
profile feeding.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Herein is reported a method for the production of an
immunoglobulin comprising the following steps: [0033] a)
cultivating a mammalian cell comprising a nucleic acid encoding the
immunoglobulin in a cultivation medium at a constant DGL of less
than 0.8 (i.e. the amount of glucose available per time unit is
constant and 80% or less of the amount of glucose that can
maximally be utilized by the cell per time unit), and [0034] b)
recovering the immunoglobulin from the cells or the culture
medium.
[0035] With the method as reported herein an immunoglobulin can be
obtained wherein the amount of the immunoglobulin with a mannose-5
glycostructure depends on the adjusted DGL value, and wherein the
amount is the fraction of the sum of the amount of the
immunoglobulin with a mannose-5 glycostructure, and of the
immunoglobulin G(0) isoform, and of the immunoglobulin G(1)
isoform, and of the immunoglobulin G(2) isoform. In one embodiment
the DGL is from 0.8 to 0.2. In this embodiment the fraction is 10%
or less. In another embodiment the DGL is from 0.6 to 0.4. In this
embodiment the fraction is 6% or less. With the method as reported
herein an immunoglobulin can be obtained wherein the fraction of
the immunoglobulin having a mannose-5 glycostructure is 10% or less
of the sum comprising the amount of the immunoglobulin with a
mannose-5 glycostructure, the amount of the immunoglobulin G(0)
isoform, the amount of the immunoglobulin G(1) isoform, and the
amount of the immunoglobulin G(2) isoform. In another embodiment
the fraction is the area-% fraction determined in a liquid
chromatography method. In one embodiment the DGL is maintained in
the range from 0.8 to 0.2. In another embodiment the DGL is
maintained in the range from 0.6 to 0.2. In still another
embodiment the DGL is maintained in the range from 0.6 to 0.4. In
one embodiment the amount of glucose that can maximally be utilized
by the cell per time unit is the average amount of glucose that is
utilized in a cultivation in which all compounds are available in
excess, i.e. no compound is limiting the growth of the cell,
determined based on at least five cultivations. In one embodiment
the fraction is determined on day seven of the cultivation.
[0036] Methods and techniques known to a person skilled in the art,
which are useful for carrying out the current invention, are
described e.g. in Ausubel, F. M. (ed.), Current Protocols in
Molecular Biology, Volumes I to III (1997), Wiley and Sons;
Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, Third
Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (2001); Glover, N. D. (ed.), DNA Cloning: A Practical
Approach, Volumes I and II (1985); Freshney, R. I. (ed.), Animal
Cell Culture (1986); Miller, J. H. and Calos, M. P. (eds.), Gene
Transfer Vectors for Mammalian Cells, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1987); Watson, J. D., et al.,
Recombinant DNA, Second Edition, N.Y., W. H. Freeman and Co (1992);
Winnacker, E. L., From Genes to Clones, N.Y., VCH Publishers
(1987); Celis, J. (ed.), Cell Biology, Second Edition, Academic
Press (1998); Freshney, R. I., Culture of Animal Cells: A Manual of
Basic Techniques, Second Edition, Alan R. Liss, Inc., N.Y.
(1987).
[0037] The use of recombinant DNA technology enables the production
of numerous derivatives of a polypeptide. Such derivatives can, for
example, be modified in individual or several amino acid positions
by substitution, alteration or exchange. The derivatization can,
for example, be carried out by means of site directed mutagenesis.
Such variations can easily be carried out by a person skilled in
the art (Sambrook, J., et al., Molecular Cloning: A laboratory
manual, Third Edition (2001) Cold Spring Harbor Laboratory Press,
New York, USA; Hames, B. D. and Higgins, S. G., Nucleic acid
hybridization--a practical approach (1985) IRL Press, Oxford,
England).
[0038] The term "nucleic acid" denotes a naturally occurring or
partially or fully non-naturally occurring nucleic acid molecule
encoding a polypeptide. The nucleic acid can be build up of
DNA-fragments which are either isolated or synthesized by chemical
means. The nucleic acid can be integrated into another nucleic
acid, e.g. in an expression plasmid or the genome/chromosome of a
eukaryotic cell. The term "plasmid" includes shuttle and expression
plasmids. Typically, the plasmid will also comprise a prokaryotic
propagation unit comprising an origin of replication (e.g. the
ColE1 origin of replication) and a selectable marker (e.g.
ampicillin or tetracycline resistance gene), for replication and
selection, respectively, of the plasmid in prokaryotic cells. To a
person skilled in the art procedures and methods are well known to
convert an amino acid sequence, e.g. of a polypeptide, into a
corresponding nucleic acid encoding the respective amino acid
sequence. Therefore, a nucleic acid is characterized by its nucleic
acid sequence consisting of individual nucleotides and likewise by
the amino acid sequence of a polypeptide encoded thereby.
[0039] The term "expression cassette" denotes a nucleic acid that
contains the elements necessary for expression and optionally for
secretion of at least the contained structural gene in/from a cell,
such as a promoter, polyadenylation site, and 3'-and
5'-untranslated regions.
[0040] The term "gene" denotes e.g. a segment on a chromosome or on
a plasmid, which is necessary for the expression of a polypeptide.
Beside the coding region a gene comprises other functional elements
including a promoter, introns, and one or more transcription
terminators. A "structural gene" denotes the coding region of a
gene without a signal sequence.
[0041] The term "expression" denotes the transcription and
translation of a structural gene within a cell. The level of
transcription of a structural gene in a cell can be determined on
the basis of the amount of corresponding mRNA that is present in
the cell. For example, mRNA transcribed from a selected nucleic
acid can be quantitated by PCR or by Northern hybridization (see
e.g. Sambrook et al. (supra)). A polypeptide encoded by a nucleic
acid can be quantitated by various methods, e.g. by ELISA, by
determining the biological activity of the polypeptide, or by
employing methods that are independent of such activity, such as
Western blotting or radioimmunoassay, using antibodies that
recognize and bind to the polypeptide (see e.g. Sambrook et al.
(supra)).
[0042] The term "cell" denotes a cell into which a nucleic acid
encoding a polypeptide, in one embodiment a heterologous
polypeptide, has been introduced. The term "cell" includes both
prokaryotic cells used for propagation of plasmids/vectors as well
as eukaryotic cells used for expression of the structural gene. In
one embodiment a eukaryotic cell for the expression of an
immunoglobulin is a mammalian cell. In another embodiment the
mammalian cell is selected from CHO cells, NS0 cells, Sp2/0 cells,
COS cells, HEK cells, BHK cells, PER.C6.RTM. cells, and hybridoma
cells. A eukaryotic cell can be selected in addition from insect
cells, such as caterpillar cells (Spodoptera frugiperda, sf cells),
fruit fly cells (Drosophila melanogaster), mosquito cells (Aedes
aegypti, Aedes albopictus), and silkworm cells (Bombyx Mori), and
the like.
[0043] The term "polypeptide" denotes a polymer of amino acid
residues joined by peptide bonds, whether produced naturally or
synthetically. Polypeptides of less than about 20 amino acid
residues may be referred to as "peptides". Polypeptides of more
than 100 amino acid residues or covalent and non-covalent
aggregates comprising more than one polypeptide may be referred to
as "proteins". Polypeptides may comprise non-amino acid components,
such as carbohydrate groups. The non-amino acid components may be
added to the polypeptide by the cell in which the polypeptide is
produced, and may vary with the type of cell. Polypeptides are
defined herein in terms of their amino acid sequence in N- to
C-terminal direction. Additions thereto, such as carbohydrate
groups, are generally not specified, but may be present
nonetheless.
[0044] The term "heterologous DNA" or "heterologous polypeptide"
denotes a DNA molecule or a polypeptide, or a population of DNA
molecules or a population of polypeptides, which do not exist
naturally within a given cell. DNA molecules heterologous to a
particular cell may contain DNA derived from the cell's species
(i.e. endogenous DNA) so long as that DNA is combined with non-host
DNA (i.e. exogenous DNA). For example, a DNA molecule containing a
non-cell's DNA segment, e.g. encoding a polypeptide, operably
linked to a cell's DNA segment, e.g. comprising a promoter, is
considered to be a heterologous DNA molecule. Likewise, a
heterologous DNA molecule can comprise an endogenous structural
gene operably linked to an exogenous promoter. A polypeptide
encoded by a heterologous DNA molecule is a "heterologous"
polypeptide.
[0045] The term "expression plasmid" denotes a nucleic acid
comprising at least one structural gene encoding a polypeptide to
be expressed. Typically, an expression plasmid comprises a
prokaryotic plasmid propagation unit, including an origin of
replication and a selection marker, e.g. for E.coli, an eukaryotic
selection marker, and one or more expression cassettes for the
expression of the structural gene(s) of interest each in turn
comprising a promoter, at least one structural gene, and a
transcription terminator including a polyadenylation signal. Gene
expression is usually placed under the control of a promoter, and
such a structural gene is to be "operably linked to" the promoter.
Similarly, a regulatory element and a core promoter are operably
linked if the regulatory element modulates the activity of the core
promoter.
[0046] The term "isolated polypeptide" denotes a polypeptide that
is essentially free from associated cellular components, such as
carbohydrate, lipid, or other proteinaceous or non-proteinaceous
impurities, which are not covalently associated with the
polypeptide. Typically, a preparation of an isolated polypeptide
contains in certain embodiments the polypeptide in a highly
purified form, i.e. at least about 80% pure, at least about 90%
pure, at least about 95% pure, greater than 95% pure, or greater
than 99% pure. One way to show that a particular protein
preparation contains an isolated polypeptide is by the appearance
of a single band following sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-page) of the preparation and Coomassie
Brilliant Blue staining of the gel. However, the term "isolated"
does not exclude the presence of the same polypeptide in
alternative physical forms, such as dimers, or alternatively
glycosylated or derivatized forms.
[0047] Immunoglobulins in general are assigned into five different
classes: IgA (immunoglobulin of class A), IgD, IgE, IgG and IgM.
Between these classes the immunoglobulins differ in their overall
structure and/or amino acid sequence but have the same building
blocks. Complete immunoglobulins are built up of two pairs of
polypeptide chains, each comprising an immunoglobulin light
polypeptide chain (short: light chain) and an immunoglobulin heavy
polypeptide chain (short: heavy chain). In turn the chains comprise
a variable region and a constant region. In a light chain both
regions consist of one domain, whereas in a heavy chain the
variable region consists of one domain and the constant region
comprises up to five domains (in N- to C-terminal direction): the
C.sub.H1-domain, optionally the hinge region domain, the
C.sub.H2-domain, the C.sub.H3-domain, and optionally the
C.sub.H4-domain. An immunoglobulin can be dissected in a Fab- and
an Fc-region. The entire light chain, the heavy chain variable
domain and the C.sub.H1 domain are referred to as Fab-region
(fragment antigen binding-region). The Fc-region comprises the
C.sub.H2-, C.sub.H3-, and optionally the C.sub.H4-domain.
[0048] As used herein, the term "immunoglobulin" denotes a protein
consisting of one or more polypeptides. The encoding immunoglobulin
genes include the different constant region genes as well as the
myriad immunoglobulin variable region genes. The term
"immunoglobulin" comprise in one embodiment monoclonal antibodies
and fragments thereof, such as an isolated heavy chain, or a heavy
chain constant region, as well as fusion polypeptides comprising at
least an immunoglobulin heavy chain C.sub.H2-domain. In one
embodiment of the method as reported herein the immunoglobulin is a
complete immunoglobulin, in another embodiment the immunoglobulin
is an Fc-region of a complete immunoglobulin. In another embodiment
the immunoglobulin is an immunoglobulin, or an immunoglobulin
fragment, or an immunoglobulin conjugate.
[0049] The term "immunoglobulin fragment" denotes a polypeptide
comprising at least the C.sub.H2-domain of an immunoglobulin delta,
epsilon, or alpha heavy chain, and/or the C.sub.H3-domain of an
immunoglobulin epsilon or delta heavy chain. Encompassed are also
derivatives and variants thereof wherein the N-glycosylation motif
Asn-Xaa-Ser/Thr in the C.sub.H2- or C.sub.H3-domain is not
changed.
[0050] The term "immunoglobulin conjugate" denotes a polypeptide
comprising at least the C.sub.H2-domain of an immunoglobulin delta,
epsilon, or alpha heavy chain, and/or the C.sub.H3-domain of an
immunoglobulin epsilon or delta heavy chain fused to a
non-immunoglobulin polypeptide. Therein the N-glycosylation motif
Asn-Xaa-Ser/Thr in the C.sub.H2- or C.sub.H3-domain is not
changed.
[0051] The oligosaccharides attached to Asn.sup.297 (IgG, IgE) or
Asn.sup.263 (IgA) of a C.sub.H2-domain and/or to Asn.sup.394,
Asn.sup.445, or Asn.sup.496 (IgE, IgD) of a C.sub.H3-domain of an
immunoglobulin heavy chain have a biantennary structure (Mizuochi,
T., et al., Arch. Biochem. Biophys. 257 (1987) 387-394), i.e. they
consist of a core structure of
Man(.alpha.1-4)GlcNAc(.beta.1-4)GlcNAc.fwdarw.Asn
with an optional Fuc(.alpha.1-6) linkage at the terminal GlcNAc
residue. Two outer-arms are connected to the terminal mannose of
the core structure having the formula
Gal(.beta.1-4)GlcNAc(.beta.1-2)Man(.alpha.1-6).fwdarw.Man, and
Gal(.beta.1-4)GlcNAc(.beta.1-2)Man(.alpha.1-3).fwdarw.Man,
wherein the terminal galactose residues are optional (Man=mannose,
GlcNAc=N-acetyl glucose, Gal=galactose; Fuc=fucose).
TABLE-US-00001 TABLE 1 Glycosylation sites of immunoglobulins.
immunoglobulin residue to which a glycostructure class can be
attached IgG Asn 297 IgE Asn 255, Asn 297, Asn 361, Asn 371, Asn
394 IgA Asn 263, Asn 459 IgD Asn 445, Asn 496 IgM Asn 395
[0052] The term "the amount of the immunoglobulin G(0) isoform, the
amount of the immunoglobulin G(1) isoform, and the amount of the
immunoglobulin G(2) isoform" denotes the sum of the amounts of the
different, heterogeneous, biantennary oligosaccharides N-linked to
an asparagine (Asn) of an immunoglobulin. The G(2) isoform has a
terminal galactose residue on each of the outer-arms of the
oligosaccharide structure, the G(1) isoform bears only a galactose
residue on either the (.alpha.1-6) or (.alpha.1-3) linked
outer-arm, and the G(0) isoform bears no galactose residue on both
outer-arms.
[0053] The term "mannose-5 glycostructure" denotes an
oligomannose-structure linked to an Asn residue of a polypeptide
comprising or consisting of five mannose residues and two N-acetyl
glucose core residues, forming a triantennary structure.
[0054] One aspect as reported herein is a method for the production
of an immunoglobulin comprising the following steps: [0055] a)
cultivating a eukaryotic cell, preferably a mammalian cell,
comprising one or more nucleic acid(s) encoding the immunoglobulin
in a cultivation medium wherein the amount of glucose available in
the cultivation medium per time unit is kept constant and limited
to a value of less than 80% of the amount that could maximally be
utilized by the eukaryotic cells in the cultivating per time unit,
and [0056] b) recovering the immunoglobulin from the cell or the
culture medium and thereby producing an immunoglobulin.
[0057] With this method an immunoglobulin is obtained comprising at
most 10% of an immunoglobulin with a mannose-5 glycostructure. The
10% are calculated based on the sum of the amount of the
immunoglobulin with a mannose-5 glycostructure, the amount of the
immunoglobulin G(0) isoform, the amount of the immunoglobulin G(1)
isoform, and the amount of the immunoglobulin G(2) isoform.
[0058] The terms "degree of glucose limitation" and its
abbreviation "DGL", which can be used interchangeably herein,
denote the ratio of the current specific glucose consumption rate
of a single cell in a cultivation to the maximum known specific
glucose consumption rate of the single cell or a single cell of the
same kind. The degree of glucose limitation is defined as
DGL = qGlc qGlc m .times. ax ##EQU00001##
with qGlc=current specific glucose consumption rate of a single
cell;
[0059] qGlc.sub.max=maximum known specific glucose consumption rate
for this single cell or a single cell of the same kind.
[0060] The DGL can vary between DGL.sub.maintenance and 1 whereby
DGL.sub.maintenance (<1 and >0) denotes complete growth
limitation and 1 denotes no limitation or complete glucose
excess.
[0061] The introduction of glycostructures to polypeptides, e.g.
immunoglobulins, is a post-translational modification. Due to
incompleteness of the glycosylation procedure of the respective
cell every expressed polypeptide is obtained with a glycosylation
pattern comprising different glycostructures. Thus, a polypeptide
is obtained from a cell expressing it in form of a composition
comprising differently glycosylated forms of the same polypeptide,
i.e. with the same amino acid sequence. The sum of the individual
glycostructures is denoted as glycosylation pattern, comprising
e.g. polypeptides with completely missing glycostructures,
differently processed glycostructures, and/or differently composed
glycostructures.
[0062] One glycostructure is the mannose-5 glycostructure (also
denoted as high-mannose, Man5, M5, or oligo-mannose). It has been
reported, that the fraction of recombinantly produced polypeptides
with the mannose-5 glycostructure is increased with prolonged
cultivation time or under glucose starvation conditions (Robinson,
D. K., et al., Biotechnol. Bioeng. 44 (1994) 727-735; Elbein, A.
D., Ann. Rev. Biochem. 56 (1987) 497-534).
[0063] It has been found that the amount of the mannose-5
glycostructure in the glycosylation pattern of a polypeptide
produced by a eukaryotic cell can be modified based on the amount
of glucose provided to the cell in the cultivation process. It has
been found that by reducing the amount of glucose, i.e. by changing
the DGL value from 1.0 to smaller values of e.g. 0.8, 0.6, 0.5,
0.4, or 0.2, a modification in the mannose-5 glycostructure amount
in the glycosylation pattern can be achieved. In one embodiment the
DGL value is kept constant at a value within a range, such as from
0.8 to 0.2, or from 0.6 to 0.4. That is, the production of a
polypeptide, in one embodiment of an immunoglobulin, can be
performed under conditions wherein a restricted amount of glucose
is available to the cultivated cell in order to obtain the
polypeptide with a defined amount of the mannose-5 glycostructure
in the glycosylation pattern. It has been found that a cultivation
with an amount of glucose available per time unit of 80% or less of
the amount of glucose that can maximally be utilized by the cells
per time unit, in one embodiment by exponentially growing cells,
i.e with a DGL of 0.8 or less, yields a polypeptide with a
glycosylation pattern in which the amount of the mannose-5
glycostructure is changed compared to a cultivation with a DGL of
1.0. In one embodiment the cell density is the viable cell density.
Additionally the obtained polypeptide yield is increased.
[0064] The term "the amount of glucose that can maximally be
utilized by the cell per time unit" denotes the amount of glucose
that is maximally consumed or utilized or metabolized per time unit
by a single cell under optimum growth conditions in the exponential
growth phase in a cultivation without any nutrient limitation.
Thus, the amount of glucose that can maximally be utilized by the
cell per time unit can be determined by determining the amount of
glucose that is metabolized per time unit by a cell under optimum
growth conditions in the exponential growth phase in a cultivation
without any nutrient limitation. A further increase of the
available amount of glucose will not further increase, i.e. change,
the amount of glucose that can maximally be utilized by the cell
per time unit. This amount defines the maximum level of glucose
consumption of a single cell. This does not denote that a
genetically modified version of the cell might not have an even
higher maximum level of glucose consumption. Alternatively the
amount of glucose that can be maximally be utilized by the cell per
time unit can be determined based on previous cultivations and the
monitored data.
[0065] The process as reported herein is particularly simple to
carry out, associated with a minimum effort for measuring and
control, and particularly economic.
[0066] Without restrictions, e.g. insufficient nutrient supply,
cultivated cells grow and consume nutrients at maximum rates in an
uneconomic manner. One of the consumed culture medium nutrients is
glucose, which is metabolized by the cultivated cells in order to
produce energy and building blocks for the cell's metabolism. In
the presence of excess glucose the cell's metabolism is running at
the maximum turnover rate for glucose. The amount of glucose that
can maximally be utilized by the cell per time unit can for example
be determined from the glucose consumption of exponentially growing
cells in the presence of excess glucose cultivated with or under
the same cultivation conditions that will also be used in the
cultivation with restricted glucose, i.e. with an amount of glucose
available per time unit that is smaller than that which can be
utilized by the cell. This maximum amount can be calculated easily
by determining the cell density and glucose concentration at the
beginning and end of a fixed time range. The value is normally in a
range from 0.006 to 190 mmol/hour/10.sup.9 cells (Baker, K. N., et
al., Biotechnol. Bioeng. 73 (2001) 188-202; WO 98/41611; Muthing,
J., et al., Biotechnol. Bioeng. 83 (2003) 321-334; WO 2004/048556).
In one embodiment the qGlc.sub.max is about 0.142
mmol/hour/10.sup.9 cells under standard process conditions at pH
7.0.
[0067] The method as reported herein is performed in one embodiment
under conditions wherein the amount of glucose available per time
unit is kept constant and at 80% or less of the amount of glucose
that can maximally be utilized by the cell per time unit
(0.8.gtoreq.DGL>0), in one embodiment the amount of glucose
available is kept constant and at 60% or less (0.6>DGL>0), in
another embodiment at 50% or less (0.5.gtoreq.DGL>0), and in
still another embodiment at about 40%. The term "about" as used
within this application denotes that the value is no exact value it
is merely the central point of a range wherein the value can vary
up to 10%, i.e. the term "about 40%" denotes a range from 44% to
36% (DGL=0.44-0.36).
[0068] In one embodiment the cultivating is with an amount of
glucose available per time unit that is kept constant in a range
between 80% and 10% of the amount of glucose that can maximally be
utilized by the cell per time unit (0.8.gtoreq.DGL.gtoreq.0.1). In
another embodiment the amount of glucose available is kept constant
in a range between 60% and 10% (0.6.gtoreq.DGL.gtoreq.0.1). In a
further embodiment the amount of glucose available is kept constant
in a range between 50% and 10% (0.5.gtoreq.DGL.gtoreq.0.1). In
another embodiment the amount of glucose available is kept constant
in a range between 45% and 20% (0.45.gtoreq.DGL.gtoreq.0.2). In
also an embodiment the amount of glucose available is kept between
80% and 60% (0.8.gtoreq.DGL.gtoreq.0.6).
[0069] In one embodiment the method comprises the step of
cultivating the cell under conditions wherein the DGL is kept
constant and at a value of about 0.4, whereby the cultivating
comprises starting with a DGL between 1.0 and 0.5, lowering the DGL
to a value of about 0.4, and keeping the DGL constant thereafter.
In one embodiment the lowering of the DGL is within a time period
of 100 hours. The term "keeping the DGL constant" and grammatical
equivalents thereof denote that the DGL value is maintained during
a time period, i.e. the variation of the DGL value is within 10% of
the value (see e.g. FIG. 2).
[0070] The immunoglobulin is recovered after production, either
directly or after disintegration of the cell. The recovered
immunoglobulin is in one embodiment purified with a method known to
a person skilled in the art. Different methods are well established
and widespread used for protein purification, such as affinity
chromatography with microbial proteins (e.g. protein A or protein G
affinity chromatography), ion exchange chromatography (e.g. cation
exchange (carboxymethyl resins), anion exchange (amino ethyl
resins) and mixed-mode exchange), thiophilic adsorption (e.g. with
beta-mercaptoethanol and other SH ligands), hydrophobic interaction
or aromatic adsorption chromatography (e.g. with phenyl-sepharose,
aza-arenophilic resins, or m-aminophenylboronic acid), metal
chelate affinity chromatography (e.g. with Ni(II)- and
Cu(II)-affinity material), size exclusion chromatography, and
electrophoretical methods (such as gel electrophoresis, capillary
electrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75
(1998) 93-102).
[0071] For example, a purification process for immunoglobulins in
general comprises a multistep chromatographic part. In the first
step non-immunoglobulin polypeptides are separated from the
immunoglobulin fraction by an affinity chromatography, e.g. with
protein A or G. Afterwards, e.g., ion exchange chromatography can
be performed to disunite the individual immunoglobulin classes and
to remove traces of protein A, which has been coeluted from the
first column. Finally a chromatographic step is employed to
separate immunoglobulin monomers from multimers and fragments of
the same class.
[0072] General chromatographic methods and their use are known to a
person skilled in the art. See for example, Chromatography,
5.sup.th edition, Part A: Fundamentals and Techniques, Heftmann, E.
(ed.), Elsevier Science Publishing Company, New York, (1992);
Advanced Chromatographic and Electromigration Methods in
Biosciences, Deyl, Z. (ed.), Elsevier Science BV, Amsterdam, The
Netherlands, (1998); Chromatography Today, Poole, C. F. and Poole,
S. K., Elsevier Science Publishing Company, New York, (1991);
Scopes, R. K., Protein Purification: Principles and Practice
(1982); Sambrook, J., et al. (ed.), Molecular Cloning: A Laboratory
Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 2001; or Current Protocols in Molecular
Biology, Ausubel, F. M., et al. (eds), John Wiley & Sons, Inc.,
New York (1990).
[0073] In one embodiment the recovered immunoglobulin is
characterized by the amount of the immunoglobulin having a
mannose-5 glycostructure with respect to the amount of a
population, which is the sum of the amount of the immunoglobulin
with a mannose-5 glycostructure, the immunoglobulin G(0) isoform,
the immunoglobulin G(1) isoform, and the immunoglobulin G(2)
isoform. With the method as reported herein the amount of the
immunoglobulin with a mannose-5 glycostructure is in one embodiment
10% or less of the population, in another embodiment 8% or less of
the population, and in a further embodiment 6% or less of the
population.
[0074] The method as reported herein can be performed in certain
embodiments as continuous cultivation, as fed-batch cultivation, or
as combination thereof, e.g. starting as fed-batch cultivation with
subsequent crossover to a continuous cultivation. Additionally, the
method as reported herein can be performed in different ways. For
example, in one embodiment prior to the cultivating under
conditions with a DGL value below 1.0, i.e. for example under
conditions wherein the available amount of glucose is 80% or less
of the amount of glucose that can maximally be utilized by the cell
in the culture per time unit, the cultivating is with an excess of
glucose, i.e. a DGL value of 1.0. In another embodiment the
cultivating is started with an amount of glucose as contained in
standard culture media, e.g. between 1 and 10 g/l culture medium,
e.g. in order to obtain a predefined cell density, e.g. in one
embodiment of 10.sup.5 cell/ml. In a further embodiment the
starting of the cultivating is in the presence of an excess amount
of glucose, i.e. a DGL of 1.0, and adding an amount of glucose per
time unit, which is 80% or less of the amount of glucose that can
maximally be utilized per time unit by the cells in the
cultivation. In another embodiment the feeding is started once the
amount of glucose present in the culture medium has dropped to or
below a preset value in the cultivation. In the last two cases the
amount of glucose available in the culture is reduced by the
metabolism of the cells in the cultivation.
[0075] In one embodiment the amount of glucose, which is available
or added per time unit and which is less than the amount of glucose
that can maximally be utilized, is kept at the same value, i.e.
constant, in the method as reported herein. For example, if an
amount of 50% of the amount of glucose that can maximally be
utilized per time unit is available, this amount is available in
all time units of the method in which a restricted glucose feeding
is performed. It has to be pointed out that this value is a
relative value. Though, as the viable cell density changes during
the cultivation (i.e. it increases in the beginning, reaches a
maximum, and drops thereafter again) the absolute amount of
available glucose changes accordingly as it is a relative value
depending on the absolute viable cell density. As the relative
value is kept constant (i.e. at e.g. 80%) but the absolute
reference value changes (i.e. e.g. increasing viable cell density)
also the relative absolute value changes (i.e. 80% of an increasing
value are also increasing).
[0076] The term "per time unit" denotes a fixed time range, such as
1 minute, 1 hour, 6 hours, 12 hours, or 24 hours. In one embodiment
the time unit is 12 hours or 24 hours. The term "amount of glucose
available per time unit" as used within this application denotes
the sum of 1) the amount of glucose contained in the cultivation
medium of a cultivation at the beginning of a fixed time range and
2) the amount of glucose added, i.e. fed, during the time unit.
Thus, an amount of glucose is added to the cell cultivation medium,
e.g. to the cultivation vessel, which increases the amount of
glucose in the cultivation medium at the beginning of the fixed
time range to the predetermined amount. This amount of glucose can
be added, e.g., as solid, dissolved in water, dissolved in a
buffer, or dissolved in a nutrient medium, whereby water and buffer
shall not contain glucose. The amount of glucose to be added
corresponds to the amount of glucose to be available reduced by the
amount of glucose present in the medium in the cultivation vessel.
The process of adding the amount of glucose can be performed either
as single addition, as multiple addition of small, equal fractions,
or as continuous addition during a time unit as described
above.
[0077] The method as reported herein is suitable for any kind of
cultivation and any cultivation scale. For example, in one
embodiment the method is used for continuous or fed-batch
processes; in another embodiment the cultivation volume is from 100
ml up to 50,000 1, in another embodiment from 100 1 to 10,000 1.
The method as reported herein is useful for the production of
immunoglobulins with 10% or less, or 8% or less, or 6% or less of
the immunoglobulin having a mannose-5 glycostructure. In one
embodiment the immunoglobulin is an immunoglobulin G or E. The
method as reported herein comprises a eukaryotic cell, wherein the
cell in turn comprises a nucleic acid encoding the heavy chain of
an immunoglobulin or a fragment thereof and a nucleic acid encoding
the light chain of an immunoglobulin or a fragment thereof. The
eukaryotic cell is in one embodiment selected from CHO cells, NS0
cells, BHK cells, hybridoma cells, PER.C6.RTM. cells, Sp2/0 cells,
HEK cells, and insect cells.
[0078] A person skilled in that art is familiar with medium
compositions and components as well as nutrient concentrations
required by different cells for optimal growth in addition to the
amount of glucose and will choose an appropriate medium for the
cultivation of the cell (see e.g. Mather, J. P., et al. in
Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis,
and Bioseparation, Vol. 2 (1999) 777-785).
[0079] In one embodiment the amount of glucose that has to be
available to the cells in a cultivation according to the method as
reported herein is calculated by multiplying the viable cell
density, which can be achieved normally in the culture vessel at a
certain point of time of the cultivation, with the volume of the
culture vessel and the amount of glucose that can maximally be
utilized by the exponentially growing cells per time unit and by
the intended DGL. In more detail, from the course of the glucose
concentration in the cultivation and the course of the cell density
in the cultivation prior to the actual point of time the future
course of the glucose concentration and the cell density are
predicted. With this prediction the amount of glucose that has to
be added to the cultivation to achieve the intended DGL is
calculated with the following formula:
(glucose to be added [pg glucose/ml/h])=(current cell density
[cells/ml]).times.(maximum glucose consumption rate of the cell [pg
glucose/cell/h]).times.(DGL value)-amount of glucose present in the
medium in the cultivation vessel.
[0080] In one embodiment the pH value of the cultivation is between
pH 6.5 and pH 7.8. In another embodiment the pH value is between pH
6.9 and pH 7.3. In a further embodiment the pH value is between pH
7.0 and 7.2. It has been found as outlined in Example 1 that in
combination with a restricted glucose feeding with a pH value of
7.0 in the constant feeding method the M5 content can efficiently
be regulated to defined values, i.e. below 8%, compared to a pH
value of 7.2. In the cultivations in the fed-batch method at pH
values of 7.0 or 7.2, respectively, it was found that with the DGL
control method the M5 content could be regulated to be less than
5.5%. It has been found that with a reduction of the pH value of
the cultivation an increase of the M5 amount due to the lowering of
the DGL value can be traversed.
[0081] The cultivation is in one embodiment performed at a
temperature between 27.degree. C. and 39.degree. C., in another
embodiment between 35.degree. C. and 37.5.degree. C.
[0082] With the method as reported herein any polypeptide
containing a glycostructure can be produced, such as
immunoglobulins, interferons, cytokines, growth factors, hormones,
plasminogen activator, erythropoietin and the like.
[0083] The cultivating in the method as reported herein can be
performed using any stirred or shaken culture devices for mammalian
cell cultivation, for example, a fermenter type tank cultivation
device, an air lift type cultivation device, a culture flask type
cultivation device, a spinner flask type cultivation device, a
microcarrier type cultivation device, a fluidized bed type
cultivation device, a hollow fiber type cultivation device, a
roller bottle type cultivation device, or a packed bed type
cultivation device.
[0084] The method as reported herein is performed in one embodiment
for up to 15 days. In another embodiment the cultivating is for 6
to 15 days. In one embodiment the immunoglobulin is an anti-IL-6R
antibody.
[0085] The method as reported herein is exemplified with an
antibody to human interleukin-6 receptor as reported e.g. in EP 0
409 607, EP 0 628 639, U.S. Pat. Nos. 5,670,373, or 5,795,965
(herewith incorporated by reference in their entirety) as this
antibody and the cell line expressing it were available at
sufficient quantity in our laboratory at the time of the invention.
This is not intended to restrict the scope of the invention.
[0086] The following examples and figures are available to aid the
understanding of the present invention, the true scope of which is
set forth in the appended claims. It is understood that
modifications can be made in the procedures set forth without
departing from the spirit of the invention.
EXAMPLES
Materials and Methods
Cell Line
[0087] An exemplary CHO cell line in which the amount of the
mannose-5 glycostructure of a recombinantly produced immunoglobulin
can be modified is a CHO cell line comprising a nucleic acid
encoding an anti-IL-6 receptor antibody according to EP 0 409 607
and U.S. Pat. No. 5,795,965. For the cultivation of the recombinant
CHO cell any culture medium can be used as long a glucose
supplementation according to the method of the invention can be
performed. Exemplary culture media are IMDM, DMEM or Ham's F12
medium or combinations thereof, which have been adapted to the
method as reported herein in as much as the mass ratios of the
culture medium components to glucose are adopted. It is likewise
possible to exclude glucose from the cultivation medium and add it
to the cultivation separately.
Cultivation
[0088] CHO cells expressing an anti-IL-6R antibody were cultivated
in a 11 or 21 fermentation vessel. The feeding medium contained 15
to 40 g/l glucose. Glucose could be fed with a separate
concentrated solution containing of e.g. 400 g/l glucose. The
cultivation was performed at a pH value of in the range from pH 7.0
to pH 7.2.
Determination of the Glycostructure
[0089] For the analysis of IgG glycosylation pattern a method
according to Kondo et al. (Kondo, A., et al., Agric. Biol. Chem. 54
(1990) 2169-2170) was used. The IgG was purified from the
centrifuged supernatant of the cultivation medium using a small
scale protein A column. The oligosaccharide of the purified IgG was
released using N-glycosidase F (Roche Diagnostics GmbH, Mannheim,
Germany) and labeled with 2-amino pyridine at the reducing
terminus. The labeled oligosaccharide was analyzed by reverse-phase
chromatography (HPLC). Each peak was assigned by both mass
spectrometry and standards for the oligosaccharides.
Glucose Determination
[0090] The glucose concentration was determined using an YSI 2700
SELECT.TM. analyzer (YSI, Yellow Springs, Ohio, USA) with a method
according to the manufacturer's manual.
Viable Cell Density Determination
[0091] Viable Cell density was determined using an automatic image
processing and analysis system (CEDEX.RTM.; Innovatis, Germany) and
the trypan blue dye-exclusion method.
Example 1
Effects of the DGL Control and pH on Antibody Production and
Mannose-5 glycostructure (M5) Content
[0092] A test was conducted using a CHO cell strain producing
humanized anti-human IL-6 receptor antibody (Tocilizumab,
RoACTEMRA.RTM.), which was prepared in accordance with the method
described in Referential Example 2 of Japanese Unexamined Patent
Publication No. 99902/1996 by use of human elongation factor
I.alpha. promotor as reported in Example 10 of International Patent
Application Publication No. WO 92/19759 (corresponding to U.S. Pat.
Nos. 5,795,965, 5,817,790, and 7,479,543).
[0093] In the constant absolute amount feeding method, effects of
pH control on immunoglobulin production were observed. Table 2
shows the effects of pH control on antibody oligosaccharides
production and M5 content in constant feeding mode.
TABLE-US-00002 TABLE 2 Effects of pH control in constant absolute
amount feeding mode. Relative antibody M5 Sample on pH set-
concentration content No. [day] point DGL [%] [%] 1 7 7.0 0.80-0.45
90.1 3.6 2 7 7.0 0.49-0.21 100 5.4 3 7 7.2 0.73-0.35 135.1 11.7 4 7
7.2 0.69-0.30 120 10.8 5 7 7.2 0.35-0.29 127 25.2 6 7 7.2 0.64-0.25
122.5 8.7
[0094] At pH 7.0 the amount of the mannose-5 glycostructure (M5)
was regulated to less than 5.5%. The DGL value declined from 0.80
to 0.21 due to the change of cell density. On the other hand, at pH
7.2, the M5 amount fluctuated between 8.7% and 25.2% and was higher
than that at pH 7.0. The DGL value at pH 7.2 varied from 0.73 to
0.25. Moreover, in this case, immunoglobulin production at pH 7.2
was more than 120% (relative value compared to pH 7.0). Higher
immunoglobulin production in the constant absolute amount feeding
method induces a higher M5 content of more than 8%. Therefore, with
a pH 7.0 control in the constant absolute amount feeding method the
M5 content could efficiently be regulated to lower values, i.e.
below 8%, compared to pH 7.2 control method.
[0095] The DGL control method (=constant relative amount feeding
method) was also used for the immunoglobulin production by
fed-batch mode at various pH values, and the M5 content was
analyzed. Table 3 shows the effects of DGL control after the start
of feeding at day 2-3 and pH on immunoglobulin production and M5
content.
TABLE-US-00003 TABLE 3 Effects of DGL and pH control in fed-batch
mode. Relative antibody M5 Sample on pH set- concentration content
No. [day] point DGL [%] [%] 1 7 7.0 0.8 102.7 2.9 2 7 7.0 0.6 96.2
2.7 3 7 7.0 0.4 100.0 3.3 4 7 7.0 0.3 91.1 3.9 5 7 7.0 0.2 83.0 4.0
6 7 7.2 0.6 100.9 4.4 7 7 7.2 0.4 90.1 5.3
[0096] At pH 7.0 the DGL control method was applied in the range of
a DGL from 0.2 to 0.8. As a result, the M5 content was regulated to
be equal or less than 4.0%. On the other hand, at pH 7.2, the DGL
value was operated in the range from 0.4 to 0.6. Here the M5
content could be controlled to be less than 5.5%.
Example 2
Cultivating With Different DGL Values
[0097] The cultivating of a CHO cell comprising a nucleic acid
encoding an anti-IL-6R antibody was performed with different DGL
values. The results are summarized in the following Table 4.
TABLE-US-00004 TABLE 4 Effects of DGL control value on
immunoglobulin production and M5 content. Relative Sample antibody
M5 G(0) G(1) G(2) on concentration content content content content
No [day] DGL [%] [%] [%] [%] [%] 1 7 0.6-0.5 107.3 3.5 38.4 46.7
11.4 2 7 0.4 111.0 3.5 38.8 46.9 10.8 3 7 0.2 111.5 4.5 40.1 45.2
10.1 4 8 const. 100.0 5.9 43.8 42.0 8.3 feeding
[0098] Compared to a constant feeding shows the controlled DGL
strategy with a DGL value of 0.4 to 0.6 a reduced mannose-5
content.
Example 3
Cultivating With Different Feeding Strategies
[0099] The cultivating of a CHO cell comprising a nucleic acid
encoding an anti-IL-6R antibody was performed with one DGL value
but with different feeding strategies. The results are summarized
in the following Table 5.
TABLE-US-00005 TABLE 5 Effects of feed strategy on viability and
viable cell density. viable cell Sample viability density
[.times.10.sup.6 No. after [h] DGL feeding adjustment [%] cells/ml]
1 112 0.4 single daily 71 5.1 2 115 0.4 dual daily 75 5.8 3 115 0.4
single profile 73 4.9 4 115 0.4 dual profile 70 5.1
[0100] In the single feed experiments a single feed was used
containing all nutrients and glucose. In the dual feed experiments
two feeds were used: the first feed contains all nutrients and
glucose at a low concentration of 15 g/l and the second feed
contains a high concentration of glucose. These different feed
experiments were performed in one set with a daily adjustment of
the feeding rate and in another set following a predetermined
profile based on the viable cell density development recorder in
earlier cultivations. As can be seen from Table 5 viability and
viable cell density are comparable independently of the employed
feeding strategy.
Example 4
[0101] Degree of Glucose Limitation (DGL) Control for
Immunoglobulin Production By the Fed-Batch Mode
[0102] CHO cells (8.0-12.times.10.sup.5 cells/ml) were inoculated
in serum free culture media as described above. The cells were
grown at 37.degree. C., 98% relative humidity, and 10% CO.sub.2
atmosphere. In the fed-batch cultivation the feeding medium
containing glucose was started to be fed to the main fermenter on
the 2.sup.nd or 3.sup.rd day from the beginning of the cultivation.
The feeding strategy followed the method to control the degree of
glucose limitation (DGL) according to U.S. Patent Application
Publication No. US 2006/0127975 A1. The DGL can be defined as the
ratio of the observed specific glucose consumption rate to the
maximum known specific glucose consumption rate when glucose is
freely available for these cells (DGL=Q(glc)/Q(glc).sub.max, where
Q(glc)=currently observed specific glucose consumption rate;
Q(glc).sub.max=maximum known specific glucose consumption rate for
these cells).
[0103] FIG. 1 shows the viable cell density and cell viability
profiles of the cultivation. The DGL was controlled to be at a
value of 0.4-0.5 in various cell densities as shown in FIG. 2. The
feeding rates were changed once or twice a day depending on the
cell density at that time. FIG. 3 shows the feeding profiles based
on DGL by the fed-batch mode. The feeding rate was changed between
0.8 and 1.6 ml/h depending on the cell density. With this feeding
strategy applied, an immunoglobulin production profile was obtained
as shown in FIG. 4. Using the inoculation size of 10.times.10.sup.5
cells/ml and 12.times.10.sup.5 cells/ml, the immunoglobulin
production was almost the same and more than 120% of the
immunoglobulin production in constant feeding method at day seven
as shown in Table 6 (feeding rate of 0.02 g glucose/h). In spite of
the 20% difference in the initial cell densities, it was possible
with the DGL control method to obtain approximately equivalent
immunoglobulin titer. Moreover, when the inoculation size was set
at 8.0.times.10.sup.5 cells/ml, despite the 20 hour delay of the
feeding start point, the immunoglobulin obtained was more than 110%
(relative value) at day seven. In these results, the DGL control
method could achieve a stable immunoglobulin production at various
inoculation sizes.
Example 5
The Effects of the DGL Control on the mannose-5 glycostructure and
Galactosylation of Oligosaccharides
[0104] Of the immunoglobulin produced by fed-batch cultivation
using the DGL control the glycosylation pattern was analyzed. Table
6 shows the result of the oligosaccharide analysis for the
immunoglobulin obtained from the DGL controlled fed-batch
cultivation in comparison with the constant feeding method (feeding
rate: 0.02 g of glucose/h). At the inoculation size of
8.0.times.10.sup.5 cells/ml, the content of mannose-5
glycostructure (M5) was 2.8%. At the inoculation size of
10.times.10.sup.5 cells/ml and 12.times.10.sup.5 cells/ml, the M5
content was 4.1% and 3.8%, respectively. At all cultivation
conditions, the DGL control method was able to regulate the M5
content to less than 5.0%.
[0105] Meanwhile, in each condition, immunoglobulin G(0) isoform
and immunoglobulin G(2) isoform were controlled at the range from
40% to 46% and from 9.0% to 11%, respectively.
TABLE-US-00006 TABLE 6 Effects of DGL control value on
immunoglobulin production and glycosylation pattern. inoculation
relative cell density antibody M5 G(0) G(1) G(2) Sample
[.times.10.sup.5 concentration content content content content No
on [day] DGL cells/ml] [%] [%] [%] [%] [%] 1 7 constant 10 100.0
3.5 45.7 41.5 9.2 feeding 2 7 0.4 8 112.5 2.8 41.7 44.7 10.8 3 7
0.4 10 122.6 4.1 42.9 43.1 9.8 4 7 0.4 12 127.1 3.8 45.5 41.5
9.1
* * * * *