HK1171237B - Method for the production of a glycosylated immunoglobulin - Google Patents

Description

Method for producing glycosylated immunoglobulins

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 culture conditions. .

Background

The production of immunoglobulins has increased steadily in recent years and in the near future immunoglobulins are likely to be the largest group of therapeutics available for the treatment of a variety of diseases. The impact of immunoglobulins derives from their specificity, which includes their specific target recognition and binding functions and activation of specific effects simultaneously with or following binding to antigen/Fc receptors.

Specific target recognition and binding is mediated by the variable regions of immunoglobulins. Other parts of the immunoglobulin molecule that give rise to effects are post-translational modifications, such as glycosylation patterns. Post-translational modifications do have an impact on the efficacy, stability, immunogenic potential, binding, etc. of immunoglobulins. Here, mention must be made of complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and induction of apoptosis.

The glycosylation pattern of immunoglobulins, i.e. the sugar composition and the number of attached glycosyl structures, has been reported to have a strong influence on biological properties (see e.g. Jefferis, r., biotechnol. prog.21(2005) 11-16). Immunoglobulins produced by mammalian cells contain from 2 to 3% by mass of carbohydrate (Taniguchi, T. et al, biochem.24(1985) 5551-. For example, in class G immunoglobulins (IgG), this is equivalent to 2.3 oligosaccharide residues in mouse-derived IgG (Mizuochi, T. et al, Arch. biochem. Biophys.257(1987)387-394) and 2.8 oligosaccharide residues in human-derived IgG (Parekh, R.B. et al, Nature 316(1985)452-457), two of which are generally located in the Fc region and the remainder in the variable region (Saba, J.A. et al, anal. biochem.305(2002) 16-31).

In the Fc region of class G immunoglobulins oligosaccharide residues can be introduced by N-glycosylation of the amino acid residue at position 297, which is an asparagine residue (designated Asn)²⁹⁷). Youdings et al show the presence of additional N-glycosylation sites in 15% to 20% of the Fab region of polyclonal IgG molecules (Youdings, A. et al, biochem. J., 314(1996) 621-940; see also, e.g., Endo, T. et al, mol. Immunol.32(1995) 931-940). Due to heterogeneous (i.e., asymmetric) oligosaccharide processing, there are multiple isotypes of immunoglobulins with different glycosylation patterns (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). Both the structure and the distribution of oligosaccharides are highly reproducible (i.e., non-random) and site-specific (Dwek, R.A. et al, J.Ant.187 (1995) 279-292).

Some characteristics of immunoglobulins are directly related to 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), e.g., thermal stability and solubility (West, C.M., cell.biochem.72(1986)3-20), antigenicity (Turco, S.J., Arch.biochem.205 (1980)330-339), immunogenicity (Bradshaw, J.P. et al, Biophys.Achy. 351; Arch.biochem. 127.205, 1980)330-339), immunogenicity (Bradshaw, J.P. et al, J.P. Biophys.351; 1985. J., 1987. 92. J., Aust. 1987; ChalJ. 382. Biophys.201, J. 127. 126; biochem. 126, J. 126, biochem; biochem. 121; biochem. 235. 35, Biophys.76, J., Aust. 344; Cheng.76, J., Aust. 382. 76, J., Aust. 76, J., Biol.76, J., 11-344; Biol.76, J., Biol.11-344; and J., biochem; Biol.11-344; Biol.11, J., biochem; Biol.11, J., biochem, 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-.

Factors which influence the glycosylation pattern have been investigated, for example the presence of fetal bovine serum (Gawlitzek, M. et al, J.Biotechnol.42(2) (1995)117-131), buffer conditions (Muting, 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), the location and conformation of oligosaccharides and the host cell type and cell growth status (Hahn, T.J. and ocoche, C.F., J.Biol.Chem.267(1992) 982; Jchen. 987; Biotechnol. Na. et al, Biotechnol. 2001, Biotechnol. 89-327, Biotechnol. 89-75; Biotechnol. Na. et al, Biotechnol.9, J.9, Biotechnol. Vol.32-32; Biotechnol. Vol.32; Biotechnol. Vol.310; Biotechnol. Vol.05, Biotechnol. Vol.32; Biotech, Biotech., Vol.32; Biotech, Vol. et al., Vol.32; Biotech, Vol. 350; Biotech, Vol. 350; Biotech, Vol. et al., Vol. Vo, H. et al, Cytotechnology 16(1994) 151-.

Increased oligomannose structures as well as truncated oligosaccharide structures have been observed by recombinant expression of immunoglobulins in, for example, 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). Glycosylation changes in CHO cells, murine 3T3 cells, rat hepatoma cells, rat kidney cells and murine myeloma cells, such as the attachment or complete absence of the oligosaccharide moiety of smaller precursor oligosaccharides, have been observed under conditions of glucose starvation (Rearick, J.I. et al, J.biol.Chem.256(1981) 6255-. Wong, D.C.F. et al, Biotechnol.Bioeng.89(2005)164-177 reported strategies based on low glutamine/glucose concentrations.

Japanese patent application JP 62-258252 reports a perfusion culture of mammalian cells, while U.S. Pat. No. 5,443,968 reports a fed-batch culture method for protein-secreting cells. In WO98/41611 efficient cell culture methods are reported to adapt cells to metabolic states characterized by low lactate production. A method of culturing cells for the production of a substance is reported in WO 2004/048556. Elbein, A.D., Ann.Rev.biochem.56(1987)497-534 reported that mammalian cells transferred mannose-5 containing structures to proteins instead of mannose-9 containing structures when incubated in the absence of glucose. Takuma, s. et al, reported in biotechnol. bioenng.97 (2007)1479-1488 the effect of pCO2 dependence on CHO cell growth, metabolism and IgG production during glucose limitation.

SUMMARY

It has been found that the amount of mannose-5-glycosyl structure 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 culture process. The change in the amount of mannose-5 glycosyl structure in the glycosylation pattern can be obtained by reducing the amount of available glucose, for example by changing the DGL value from 1.0 to a smaller value (e.g. 0.8, 0.6, 0.5, 0.4 or 0.2). The DGL value or the amount of glucose available per time unit must be kept constant and at a determined reduced value per time unit, respectively.

A first aspect as reported herein is a method for producing a polypeptide (in one embodiment an immunoglobulin) in a eukaryotic cell, said method comprising the steps of:

a) providing a eukaryotic cell comprising a nucleic acid encoding a polypeptide,

b) culturing the cell under conditions wherein the glucose limitation level (DGL) is maintained constant and wherein the DGL is less than 0.8, and

c) recovering the polypeptide from the culture medium,

wherein the polypeptide fraction having a mannose-5 glycosyl structure is 10% or less of the sum of the amount of polypeptide having a mannose-5 glycosyl structure, the amount of polypeptide G (0) isoform, the amount of polypeptide G (1) isoform and the amount of polypeptide G (2) isoform.

In one embodiment, the DGL is kept constant in the range from 0.8 to 0.2. In other embodiments, the DGL remains constant over a range from 0.6 to 0.4. In another embodiment, the polypeptide fraction having a mannose-5 glycosyl structure is 8% or less of the total comprising the polypeptide having a mannose-5 glycosyl structure, the polypeptide G (0) isoform, the polypeptide G (1) isoform and the polypeptide G (2) isoform. In yet another embodiment, the polypeptide is an immunoglobulin, in one embodiment an immunoglobulin of the class G or E.

Another aspect as reported herein is a method for producing an immunoglobulin, said method comprising the steps of:

a) providing a mammalian cell comprising a nucleic acid encoding an immunoglobulin,

b) culturing the cells in a culture medium, wherein the amount of glucose available in the culture medium per time unit is kept constant and limited to less than 80% of the maximum available amount of cells in the culture medium per time unit, and

c) recovering the immunoglobulin from the cells or the culture medium.

In one embodiment, the amount of glucose available per time unit in the medium is kept constant and limited to a value in the range from 80% to 20%. In other embodiments, the range is from 60% to 40%. In another embodiment, the cells in the culture medium are living cells in the culture medium.

In one embodiment of the aspects reported herein, the eukaryotic cell is selected from the group consisting of a CHO cell, an NS0 cell, a HEK cell, a BHK cell, a hybridoma cell, a,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 reported herein, the culturing is at a pH value in the range of from about pH 7.0 to about pH 7.2.

In yet another embodiment of the aspects reported herein, the culturing is a continuous or fed-batch culture. In another embodiment, the method may comprise a final step of purifying the polypeptide. In yet another embodiment, the cells are cultured for 6 to 20 days, or 6 to 15 days. In other embodiments, the cells are cultured for 6 to 8 days.

Another aspect as reported herein is a composition comprising an immunoglobulin, wherein the composition has been prepared by a method as reported herein.

In one embodiment, the immunoglobulin is an anti-IL-6R antibody. In other embodiments, the anti-IL-6R antibody comprises Tocillizumab. In another embodiment, the mannose-5 glycosyl structure attached to the anti-IL-6R antibody is 8% or less. In still other embodiments, the mannose-5 glycosyl structure is 6% or less. In another embodiment, the mannose-5 glycosyl structure is 4% or less. In other embodiments, the G (0) glycosyl structure attached to the anti-IL-6R antibody is in the range of from 40% to 46% and the G (2) glycosyl structure attached to the anti-IL-6R antibody is in the range of from 9% to 11%.

Detailed Description

Herein is reported a method for producing an immunoglobulin comprising the following steps:

a) culturing a mammalian cell comprising a nucleic acid encoding an immunoglobulin in a culture medium at a constant DGL of less than 0.8 (i.e., the amount of glucose available per time unit is constant and is 80% or less of the maximum amount of glucose available to the cell per time unit), and

b) recovering the immunoglobulin from the cells or the culture medium.

Using the method as reported herein it is possible to obtain immunoglobulins wherein the amount of immunoglobulin with mannose-5 glycosyl structure is dependent on the adjusted DGL value and wherein the amount is the fraction of the immunoglobulin with mannose-5 glycosyl structure and the sum of the amounts of immunoglobulin G (0) isotype, immunoglobulin G (1) isotype and immunoglobulin G (2) isotype. In one embodiment, the DGL is from 0.8 to 0.2. The fraction in this embodiment is 10% or less. In another embodiment, DGL is from 0.6 to 0.4. The fraction in this embodiment is 6% or less. Using the method as reported herein it is possible to obtain immunoglobulins wherein the fraction of immunoglobulins with mannose-5 glycosyl structure is 10% or less of the total comprising the amount of immunoglobulins with mannose-5 glycosyl structure, the amount of immunoglobulin G (0) isotype, the amount of immunoglobulin G (1) isotype and the amount of immunoglobulin G (2) isotype. In another embodiment, the fraction is an area-% fraction determined in a liquid chromatography method. In one embodiment, the DGL is maintained in the range of from 0.8 to 0.2. In another embodiment, the DGL is maintained in the range of from 0.6 to 0.2. In yet another embodiment, the DGL is maintained in the range of from 0.6 to 0.4. In one embodiment, the maximum amount of glucose available to the cell per time unit, determined on the basis of at least five cultures, is the average amount of glucose utilized in cultures where all compounds are available in excess (i.e., where no compound limits the growth of the cell). In one embodiment, the fractions are determined on day seven of culture.

Methods and techniques known to those skilled in the art useful for carrying out the present invention are described, for example, in Ausubel, F.M (eds.), Current Protocols in Molecular Biology, volumes I through III (1997), Wiley and Sons; sambrook, j, et al, Molecular Cloning: ALABORT MANUAL, third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); glover, n.d. (eds.), DNA Cloning: APractcal Appliach, volumes I through II (1985); freshney, r.i. (eds.), Animal cellcure (1986); miller, J.H. and Calos, M.P. (eds.), Gene Transfer vector 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. (eds.), 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).

The use of recombinant DNA technology enables the production of a variety of derivatives of polypeptides. Such derivatives can be modified, for example, by substitution, alteration or exchange at a single or several amino acid positions. Derivatization can be carried out, for example, by means of targeted mutagenesis. Such variations can be readily made by those skilled in the art (Sambrook, J. et al, Molecular Cloning: A Laboratory Manual, third edition, Cold Spring Harbor Laboratory Press, New York, USA (2001); Hames, B.D. and Higgins, S.G., Nucleic acid hybridization-a practical proproach (1985) IRL Press, Oxford, England).

The term "nucleic acid" denotes a naturally occurring or partially or completely non-naturally occurring nucleic acid molecule encoding a polypeptide. Nucleic acids can be accumulations of DNA fragments isolated or synthesized by chemical means. The nucleic acid can be integrated into another nucleic acid, for example, in an expression plasmid or in the genome/chromosome of a eukaryotic cell. The term "plasmid" includes shuttle and expression plasmids. Typically, the plasmid will also comprise a prokaryotic proliferation unit comprising an origin of replication (e.g., ColE1 origin of replication) and a selectable marker (e.g., an ampicillin or tetracycline resistance gene) for replication and selection of the plasmid, respectively, in prokaryotic cells. Procedures and methods for converting amino acid sequences (e.g., of a polypeptide) into corresponding nucleic acids encoding the respective amino acid sequences are generally known to those skilled in the art. Thus, a nucleic acid is characterized by its nucleic acid sequence consisting of a single nucleotide and likewise by the amino acid sequence of the polypeptide encoded thereby.

The term "expression cassette" means a nucleic acid containing elements (e.g., a promoter, a polyadenylation site, and 3 '-and 5' -untranslated regions) necessary for the expression of at least a structural gene contained in/from a cell and for secretion.

The term "gene" denotes, for example, a segment on a chromosome or plasmid, which is necessary for the expression of a polypeptide. In addition to the coding region, a gene contains other functional elements, including a promoter, introns, and one or more transcription terminators. "structural gene" means the coding region of a gene that does not contain a signal sequence.

The term "expression" refers to the transcription and translation of structural genes within a cell. The transcription level of a structural gene in a cell can be determined based on the amount of the corresponding mRNA present in the cell. For example, mRNA transcribed from a selected nucleic acid can be quantified by PCR or by Northern hybridization (see, e.g., Sambrook et al (supra)). Polypeptides encoded by nucleic acids can be quantified by a variety of methods [ e.g., by ELISA, by assaying the biological activity of the polypeptide, or by employing methods that do not rely on such activity (e.g., Western blotting or radioimmunoassay) using antibodies that recognize and bind to the polypeptide (see, e.g., Sambrook et al (supra) ].

The term "cell" means a cell into which a nucleic acid encoding a polypeptide, which in one embodiment is a heterologous polypeptide, has been introduced. The term "cell" includes prokaryotic cells for plasmid/vector propagation, as well as eukaryotic cells for expression of structural genes. In one embodiment, the eukaryotic cell used to express the immunoglobulin is a mammalian cell. In another embodiment, the mammalian cell is selected from the group consisting of CHO cells, NS0 cells, Sp2/0 cells, COS cells, HEK cells, BHK cells, and,Cells and hybridoma cells. Furthermore, the eukaryotic cell can be selected from insect cells, such as Armoracia chinensis (Spodoptera frugiperda), sf cells, Drosophila melanogaster (Drosophila melanogaster), mosquito cells (Aedes aegypti, Aedes albopictus), and silkworm cells (Bombyx Mori).

The term "polypeptide" denotes a polymer of amino acid residues linked by peptide bonds, whether naturally or synthetically produced. 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 polymers comprising more than one polypeptide may be referred to as "proteins". The polypeptide may comprise components other than amino acids, such as carbohydrate groups. Non-amino acid components may be added to the polypeptide by the cell producing the polypeptide and may vary with the cell type. Polypeptides are defined herein in terms of their amino acid sequence in the N-to-C-terminal direction. The addition of which, for example carbohydrate groups, is generally not illustrated, but may still be present.

The term "heterologous DNA" or "heterologous polypeptide" refers to a DNA molecule or polypeptide, or a population of DNA molecules or a population of polypeptides, that does not naturally occur within a given cell. A DNA molecule heterologous to a particular cell may contain DNA from the species of the cell (i.e., endogenous DNA) so long as the DNA is combined with non-host DNA (i.e., exogenous DNA). For example, a DNA molecule comprising a non-cellular DNA segment (e.g., encoding a polypeptide) operably linked to a cellular 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 a foreign promoter. The polypeptide encoded by the heterologous DNA molecule is a "heterologous" polypeptide.

The term "expression plasmid" denotes a nucleic acid comprising at least one structural gene encoding a polypeptide to be expressed. Typically, the expression plasmid comprises a prokaryotic plasmid propagation unit (including an origin of replication and a selectable marker (e.g., for E.coli)), a eukaryotic selectable marker, one or more expression cassettes for expressing the structural gene of interest, each comprising, in turn, a promoter, at least one structural gene, and a transcription terminator comprising a polyadenylation signal. Gene expression is usually under the control of a promoter, and such structural genes are 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.

The term "isolated polypeptide" refers to a polypeptide that is substantially free of associated cellular components (e.g., carbohydrates, lipids, or other proteinaceous or non-proteinaceous impurities that are not covalently associated with the polypeptide). Generally, in certain embodiments, a preparation of an isolated polypeptide contains 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 isolated polypeptides is by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the preparation and the appearance of a single band after Coomassie Brilliant blue staining of the gel. However, the term "isolated" does not exclude the presence of the same polypeptide in an alternative physical form (e.g., disomic) or alternatively glycosylated or derivatized form.

Immunoglobulins are generally classified into five distinct classes: IgA (class A immunoglobulin), IgD, IgE, IgG and IgM. Between these classes, immunoglobulins differ in overall structure and/or amino acid sequence, but have identical building blocks. A complete immunoglobulin consists of two pairs of polypeptide chains, each pair comprising an immunoglobulin light polypeptide chain (abbreviated: light chain) and an immunoglobulin heavy polypeptide chain (abbreviated: heavy chain). The chain comprises in sequence a variable region and a constant region. In the light chain, both regions consist of one domain, whereas in the heavy chain, the variable region consists of one domain and the constant region comprises up to 5 domains (in the direction from the N to the C terminus): c_H1-domain, optional hinge region domain, C_H2-Domain, C_H3-domain and optionally C_H4-domain. Immunoglobulins can be broken down into Fab and Fc regions. Complete light chain, heavy chain variable domain and C_HThe 1-domain is called the Fab region (fragment antigen binding region). The Fc region comprises C_H2-、C_H3-and optionally C_H4-domain.

As used herein, the term "immunoglobulin" refers to a protein composed of one or more polypeptides. The immunoglobulin-encoding genes include various constant region genes as well as a number of immunoglobulin variable region genes. In one embodiment, the term "immunoglobulin" encompasses monoclonal antibodies and fragments thereof, such as isolated heavy chains, or heavy chain constant regions, and includes at least immunoglobulin heavy chain C_H2-domain fusion polypeptide. In one embodiment of the method as reported herein the immunoglobulin is an intact immunoglobulin, in another embodiment the immunoglobulin is the Fc region of an intact immunoglobulin. In another embodiment, the immunoglobulin is an immunoglobulin, or an immunoglobulin fragment, or an immunoglobulin conjugate.

The term "immunoglobulin fragment" means a C comprising at least an immunoglobulin, or alpha heavy chain_H2-domain, and/or C of an immunoglobulin or heavy chain_H3-domain polypeptide. Also encompassed are derivatives and variants thereof, wherein C_H2-or C_HThe N-glycosylation motif Asn-Xaa-Ser/Thr in the 3-domain was not altered.

The term "immunoglobulin conjugate" denotes a C comprising at least an immunoglobulin, or alpha heavy chain, fused to a non-immunoglobulin polypeptide_H2-domain, and/or C of an immunoglobulin or heavy chain_H3-domain polypeptide. Wherein C is_H2-or C_HThe N-glycosylation motif Asn-Xaa-Ser/Thr in the 3-domain was not altered.

C attached to immunoglobulin heavy chain_HAsn of 2-Domain²⁹⁷(IgG, IgE) or Asn²⁶³(IgA) and/or C_HAsn of 3-Domain³⁹⁴、Asn⁴⁴⁵Or Asn⁴⁹⁶The oligosaccharides of (IgE, IgD) have a bifurcated structure (Mizuochi, T. et al, Arch. biochem. Biophys.257(1987)387-394), i.e.they are formed by a core structure with an optional Fuc (. alpha.1-6) linkage at the terminal GlcNAc residue

Man(α1-4)GlcNAc(β1-4)GlcNAc→Asn

And (4) forming. The two outer arms are linked to the terminal mannose of a core structure having the general formula:

gal (. beta.1-4) GlcNAc (. beta.1-2) Man (. alpha.1-6) → Man, and

Gal(β1-4)GlcNAc(β1-2)Man(α1-3)→Man，

wherein the terminal galactose residue is optional (Man ═ mannose, GlcNAc ═ N-acetylglucose, Gal ═ galactose, Fuc ═ fructose).

Table 1: glycosylation sites of immunoglobulins

Immunoglobulins	Residues capable of attaching glycosyl structures
		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

The terms "amount of immunoglobulin G (0) isotype, amount of immunoglobulin G (1) isotype, and amount of immunoglobulin G (2) isotype" denote the sum of the amounts of different, heterologous, di-branched oligosaccharides N-linked to the asparagine (Asn) of the immunoglobulin. The G (2) isoforms have terminal galactose residues on each outer arm of the oligosaccharide structure, the G (1) isoforms have galactose residues only on the (α 1-6) or (α 1-3) linked outer arms, and the G (0) isoforms do not have galactose residues on both outer arms.

The term "mannose-5-glycosyl structure" refers to an oligomannose structure linked to an Asn residue of a polypeptide, comprising or consisting of 5 mannose residues and two N-acetylglucose core residues, forming a three-branched structure.

One aspect as reported herein is a method for producing an immunoglobulin, said method comprising the steps of:

a) culturing a eukaryotic cell, preferably a mammalian cell, comprising one or more nucleic acids encoding an immunoglobulin in a culture medium, wherein the amount of glucose available per time unit in the culture medium is kept constant and limited to a value of less than 80% of the maximum amount of eukaryotic cells cultured per time unit, and

b) recovering the immunoglobulin from the cell or the culture medium and thereby producing the immunoglobulin.

The method is used to obtain an immunoglobulin comprising up to 10% of immunoglobulins having a mannose-5 glycosyl structure. 10% is calculated based on the sum of the amount of immunoglobulin having a mannose-5 glycosyl structure, the amount of immunoglobulin G (0) isotype, the amount of immunoglobulin G (1) isotype, and the amount of immunoglobulin G (2) isotype.

The term "glucose limitation" and its abbreviation "DGL" are used interchangeably herein to denote the ratio of the current specific glucose consumption rate of an individual cell in culture to the known maximum specific glucose consumption rate of an individual cell or individual cells of the same species. The degree of glucose limitation is defined as

Wherein qGlc is the current specific glucose consumption rate of a single cell;

qGlc_{maximum of}The known maximum specific glucose consumption rate for that single cell or for a single cell of the same species.

DGL can be at DGL_{Maintenance of}And 1, and DGL_{Maintenance of}(< 1 and > 0) means complete growth restriction and 1 means no restriction or completeGlucose excess.

Introduction of a glycosyl structure into a polypeptide (e.g., an immunoglobulin) is a post-translational modification. Due to the incompleteness of the glycosylation process of the respective cell, each of the expressed polypeptides obtained has a glycosylation pattern comprising a different glycosyl structure. Thus, a polypeptide is obtained from a cell expressing it in the form of a composition comprising different glycosylated forms of the same polypeptide (i.e., having the same amino acid sequence). The sum of the individual glycosyl structures is expressed as a glycosylation pattern, including, for example, polypeptides with complete deletion of the glycosyl structure, differentially processed glycosyl structures, and/or different compositions of glycosyl structures.

One glycosyl structure is the mannose-5 glycosyl structure (also known as high mannose, Man5, M5, or oligomannose). It has been reported that the fraction of recombinantly produced polypeptides having a mannose-5-glycosyl structure increases with prolonged culture time or under conditions of glucose starvation (Robinson, D.K. et al, Biotechnol. Bioeng.44(1994) 727-.

It has been found that the amount of mannose-5-glycosyl structure 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 during culture. It has been found that by reducing the amount of glucose, i.e. by changing the DGL value from 1.0 to a smaller value, e.g. 0.8, 0.6, 0.5, 0.4 or 0.2, a modification of the amount of mannose-5 glycosyl structure in the glycosylation pattern can be obtained. In one embodiment, the DGL value is held constant at a range of values (e.g., from 0.8 to 0.2, or from 0.6 to 0.4). In other words, the production of a polypeptide (in one embodiment, an immunoglobulin) can be performed under conditions in which the cultured cells can obtain a limited amount of glucose in order to obtain a polypeptide with a defined amount of mannose-5-glycosyl structure in the glycosylation pattern. It has been found that culturing with an amount of glucose available per time unit that is 80% or less of the maximum amount of glucose available per time unit cell (in one embodiment log grown cells), i.e., a DGL of 0.8 or less, produces a polypeptide having a glycosylation pattern wherein the amount of mannose-5 glycosyl structure is altered compared to culturing with a DGL of 1.0. In one embodiment, the cell density is viable cell density. In addition, the yield of the obtained polypeptide is increased.

The term "the amount of glucose maximally available to a cell per time unit" means the amount of glucose maximally consumed or utilized or metabolized per time unit by an individual cell under optimal growth conditions in the logarithmic growth phase in culture without any nutrient limitation. Thus, the amount of glucose maximally utilized by the cells per time unit can be determined by determining the amount of glucose metabolized by the cells per time unit under optimal growth conditions in the logarithmic growth phase in culture without any nutrient limitation. Further increasing the amount of glucose available will not increase (i.e., change) the maximum amount of glucose available to the cell per unit of time. This amount defines the maximum level of glucose consumption by an individual cell. This does not mean that the genetically modified cell type does not have an even higher maximum level of glucose consumption. Alternatively, the maximum amount of glucose available to the cells per time unit can be determined based on previous culture and monitoring data.

The method reported herein is particularly simple to perform, involves minimal measurement and control effort, and is particularly economical.

Cultured cells grow and consume nutrients at a maximum rate in an uneconomical manner without limiting, for example, inadequate nutrient supply. One of the media nutrients consumed is glucose, which is metabolized by the cultured cells to produce energy and building blocks for cellular metabolism. In the presence of excess glucose, cellular metabolism proceeds with maximum glucose conversion. For example, the maximum amount of glucose available to a cell per time unit can be determined from the glucose consumption of a log grown cell in the presence of excess glucose that is used or will be cultured under the same culture conditions (i.e., with an amount of glucose available per time unit that is less than the amount that the cell can utilize)This maximum amount is calculated. This value is generally from 0.006 to 190 mmol/h/10⁹In the range of cells (Baker, K.N. et al, Biotechnol.Bioeng.73(2001) 188-. In one embodiment, qGlc is performed under standard process conditions of pH 7.0_{Most preferably Big (a)}Is about 0.142 mmol/hr/10⁹A cell.

In one embodiment, the method as reported herein is carried out under conditions wherein the amount of available glucose per time unit is kept constant and is at 80% or less (0.8. gtoreq. DGL > 0) of the maximum amount of glucose available to the cell per time unit, in one embodiment the amount of available glucose is kept constant and is at 60% or less (0.6. gtoreq. DGL > 0), in another embodiment at 50% or less (0.5. gtoreq. DGL > 0), in yet another embodiment at about 40%. The term "about" is used in this application to indicate that a value is not an exact value, it is merely the middle point of a range where a value can vary by up to 10%, i.e., the term "about 40%" indicates a range from 44% to 36% (DGL ═ 0.44-0.36).

In one embodiment, the culture uses an amount of glucose available per time unit that remains constant in a range between 80% and 10% of the maximum amount of glucose available per time unit of the cell (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 other embodiments, 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 another embodiment, the amount of glucose available is maintained between 80% and 60% (0.8. gtoreq. DGL. gtoreq.0.6).

In one embodiment, the method comprises the step of culturing the cell under conditions in which the DGL is kept constant and at a value of about 0.4, whereas culturing comprises reducing the DLG to a value of about 0.4 starting with a DGL of between 1.0 and 0.5, and thereafter keeping the DGL constant. In one embodiment, the DGL is reduced over a period of 100 hours. The term "keeping the DGL constant" and grammatical equivalents thereof means maintaining the DGL value over a period of time, i.e., the variation of the DGL value is within 10% of the value (see, e.g., fig. 2).

The immunoglobulin is recovered directly after production or after cell lysis. In one embodiment, the recovered immunoglobulin is purified using methods known to those skilled in the art. Different methods are well established and widely used for protein purification, such as affinity chromatography using microbial proteins (e.g., protein a or protein G affinity chromatography), ion exchange chromatography (e.g., cation exchange (carboxymethyl resin), anion exchange (aminoethyl resin) and mixed exchange), thiophilic adsorption (e.g., β -mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g., using phenyl-agarose, aza-arenophilic resin or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g., using ni (ii) -and cu (ii) -affinity materials), size exclusion chromatography and electrophoresis methods (e.g., gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, m.a., appl.biochem.biotech.75(1998) 93-102).

For example, purification methods for immunoglobulins typically include multi-step chromatographic portions. In the first step, the non-immunoglobulin polypeptides and immunoglobulin fractions are separated by affinity chromatography (e.g., with protein a or G). Thereafter, ion exchange chromatography, for example, can be performed to separate the individual immunoglobulin classes and remove traces of protein a co-eluted from the first column. Finally, a chromatography step is used to separate immunoglobulin monomers and multimers and fragments of the same class.

General chromatographic methods and their use are known to those skilled in the art. See, e.g., Heftmann, e. (eds), Chromatography, 5 th edition, Part a: fundamentalsand technologies, Elsevier Science Publishing Company, New York, (1992); deyl, Z. (eds.), Advanced chromatography and Electromigration methods in Biosciences, Elsevier Science BV, Amsterdam, The Netherlands (1998); poole, c.f. and Poole, s.k., Chromatography Today, elsevier science Publishing Company, New York (1991); scopes, r.k., ProteinPurification: principles and Practice (1982); sambrook, j, et al (eds.), Molecular Cloning: a Laboratory Manual, third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); or Ausubel, F.M. et al (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1990).

In one embodiment, the recovered immunoglobulin is characterized by the amount of immunoglobulin having a mannose-5 glycosyl structure relative to the amount of the population that is the sum of the amounts of immunoglobulin having a mannose-5 glycosyl structure, immunoglobulin G (0) isotype, immunoglobulin G (1) isotype, and immunoglobulin G (2) isotype. Using the methods reported herein, the amount of immunoglobulin having a mannose-5 glycosyl structure is 10% or less of the population in one embodiment, 8% or less of the population in another embodiment, and 6% or less of the population in another embodiment.

In certain embodiments, the method as reported herein may be performed as a continuous culture, a fed-batch culture or a combination thereof, e.g. starting with a fed-batch culture, before switching to a continuous culture. Furthermore, the methods reported herein may be carried out in different ways. For example, in one embodiment, the culture uses an excess of glucose, i.e., a DGL value of 1.0, prior to culture with a DGL value of less than 1.0, i.e., under conditions such that the amount of glucose available is 80% or less of the maximum amount of glucose available to the cultured cells per unit time. In another embodiment, the culture is started with the amount of glucose contained in a standard culture medium, for example between 1 and 10g/l of culture medium, in order to obtain, for example, a predetermined cell density, such as 10 in one embodiment⁵Cells/ml. In other embodiments, the culturing is initiated by the presence of an excess of grapesSugar conditions, i.e., a DGL of 1.0, and the amount of glucose added per time unit is 80% or less of the maximum amount of glucose available per time unit for the cultured cells. In another embodiment, feeding is initiated once the amount of glucose present in the medium drops to or below a predetermined value in the culture. In the latter two cases, the amount of glucose available in the culture is reduced by the metabolism of the cells in culture.

In one embodiment, in the method as reported herein, the amount of glucose available or added per time unit and less than the amount of glucose maximally available remains the same value, i.e. remains constant. For example, if an amount of 50% of the maximum available amount of glucose per time unit is available, this amount is available in all time units of the process for carrying out the limited glucose feeding. It should be noted that this value is a relative value. However, as the viable cell density changes during the culture (i.e., it increases initially, reaches a maximum, and then decreases again), the absolute amount of glucose available changes accordingly, as it is a relative value that depends on the absolute viable cell density. Since the relative value remains constant (i.e., at, for example, 80%) while the absolute reference value changes (i.e., for example, increased viable cell density), the absolute value of the relative also changes (i.e., 80% of the increase is also increased).

The term "per time unit" denotes a fixed time range, e.g. 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" is used in the present application to denote the sum of 1) the amount of glucose contained in the culture medium at the beginning of the fixed time frame and 2) the amount of glucose added (i.e.fed) during the time unit. Thus, an amount of glucose is added to the cell culture medium (e.g., in the culture vessel) that increases the amount of glucose in the medium at the beginning of the fixed time range to a predetermined amount. This amount of glucose can be added as a solid, for example, dissolved in water, dissolved in a buffer, or dissolved in a nutrient matrix, whereas water and buffers will not contain glucose. The amount of glucose added corresponds to the amount of glucose to be utilized minus the amount of glucose present in the medium of the culture vessel. The process of adding the amount of glucose can be carried out as a single addition, as multiple small, equal-fraction additions, or as a continuous addition during the time unit as previously described.

The methods reported herein are suitable for any kind of culture and any culture scale. For example, in one embodiment, the process is used in a continuous or fed-batch process; in another embodiment, the culture volume is from 100ml up to 50,000l, in another embodiment from 100l to 10,000 l. The method as reported herein is useful for the production of immunoglobulins comprising 10% or less, or 8% or less, or 6% or less of immunoglobulins with a mannose-5 glycosyl structure. In one embodiment, the immunoglobulin is immunoglobulin G or E. The methods reported herein comprise eukaryotic cells, wherein the cells comprise in sequence a nucleic acid encoding a heavy chain of an immunoglobulin or a fragment thereof and a nucleic acid encoding a light chain of an immunoglobulin or a fragment thereof. In one embodiment, the eukaryotic cell is selected from the group consisting of a CHO cell, a NS0 cell, a BHK cell, a hybridoma cell, a,Cells, Sp2/0 cells, HEK cells and insect cells.

The composition and components of the media and concentrations of nutrients required for optimal growth of different cells, in addition to the amount of glucose, are well known to those skilled in the art, and the appropriate media can be selected for cell culture (see, e.g., Mather, J.P. et al, in Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioclassification, Vol.2 (1999)777- > 785).

In one embodiment, the amount of glucose that must be available to the cells in culture is calculated according to the method reported herein by multiplying the viable cell density that can normally be reached in the culture vessel at a certain point in time of the culture by the volume of the culture vessel and the maximum amount of glucose available to the cells grown logarithmically per time unit and by the predicted DGL. In more detail, the future progression of the glucose concentration and the cell density is predicted from the progression of the glucose concentration in the culture and the progression of the cell density in the culture before the actual time point. Using this prediction, the amount of glucose that should be added to the culture to achieve the predicted DGL is calculated using the following equation:

(glucose to be added (pg glucose/ml/h) ═

(Current cell Density (cells/ml) x

(maximum glucose consumption rate of cells (pg glucose/cell/h)) x

(DGL value) -

The amount of glucose present in the medium in the culture vessel.

In one embodiment, the pH of the culture is between pH 6.5 and pH 7.8. In another embodiment, the pH is between pH 6.9 and pH 7.3. In other embodiments, the pH is between pH 7.0 and pH 7.2. As outlined in example 1, it was found that in a constant feeding process, a limited glucose feeding in combination with a pH of 7.0 was able to effectively adjust the M5 content to a defined value, i.e. below 8%, compared to a pH of 7.2. In the cultivation in the fed-batch process at a pH of 7.0 or 7.2, respectively, it was found that the M5 content could be adjusted to less than 5.5% using the DGL control method. It was found that as the pH value of the culture was decreased, the increase in the amount of M5 due to the decrease in DGL value could be prevented.

In one embodiment, the cultivation is carried out at a temperature between 27 ℃ and 39 ℃ and in another embodiment between 35 ℃ and 37.5 ℃.

Using the methods reported herein, any polypeptide containing a glycosyl structure can be produced, e.g. immunoglobulins, interferons, cytokines, growth factors, hormones, plasminogen activators, erythropoietin, etc.

The culturing in the method as reported herein can be carried out using any agitation or shaking culture apparatus for mammalian cell culture, such as a fermenter-type culture apparatus, an air-lift-type culture apparatus, a culture flask-type culture apparatus, a spinner flask-type culture apparatus, a microcarrier-type culture apparatus, a fluidized bed-type culture apparatus, a hollow fiber-type culture apparatus, a roller flask-type culture apparatus, or a packed bed-type culture apparatus.

In one embodiment, the method as reported herein is carried out for up to 15 days. In another embodiment, the culturing is for 6 to 15 days. In one embodiment, the immunoglobulin is an anti-IL-6R antibody.

The methods reported herein are exemplified with antibodies against the human interleukin-6 receptor as reported, for example, in EP 0409607, EP 0628639, US 5,670,373 or US 5,795,965 (incorporated by reference in their entirety), since in the present invention the laboratory has access to sufficient quantities of the antibody and cell lines expressing the antibody. And are not intended to limit the scope of the present invention.

The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It will be appreciated that modifications can be made to the method described without departing from the spirit of the invention.

Drawings

FIG. 1 viable cell density (a) and cell viability profile (b) in fed-batch mode using DGL control; hollow circle: 8x 10⁵Initial cell density of cells/ml; solid triangle: 10x10⁵Initial cell density of cells/ml; hollow blocks: 12x 10⁵Initial cell density of cells/ml.

FIG. 2 time course of DGL in fed-batch mode of immunoglobulin production; circle: 8x 10⁵Initial cell density of cells/ml; triangular: 10x10⁵Initial cell density of cells/ml; and (4) square block: 12x 10⁵Initial cell density of cells/mlAnd (4) degree.

FIG. 3 DGL-based feed profile in fed-batch mode of immunoglobulin production; circle: 8x 10⁵Initial cell density of cells/ml; triangular: 10x10⁵Initial cell density of cells/ml; and (4) square block: 12x 10⁵Initial cell density of cells/ml.

FIG. 4 immunoglobulin production profile in fed-batch mode under DGL control; hollow circle: 8x 10⁵Initial cell density of cells/ml; solid triangle: 10x10⁵Initial cell density of cells/ml; hollow blocks: 12x 10⁵Initial cell density of cells/ml; solid small circle: the constant feeding method comprises the following steps: FR ═ 0.02g glucose/h (control)

FIG. 5 time course of DGL in fed-batch culture of cells: diamond shape: feeding every day during single feeding; and (4) square block: feeding materials for two times every day; triangular: supplementing by a single supplementing spectrum; x: double-time supplement spectrum supplement

Examples

Materials and methods

Cell line:

an exemplary CHO cell line, in which the amount of mannose-5 glycosyl structure of recombinantly produced immunoglobulin can be modified, is a CHO cell line comprising nucleic acid encoding an anti-IL-6 receptor antibody according to EP 0409607 and US 5,795,965. For culturing the recombinant CHO cells, any medium can be used as long as glucose supplementation according to the method of the present invention can be carried out. Exemplary media are IMDM, DMEM or Ham's F12 matrix or combinations thereof, which have been adapted to the mass ratio of media components to glucose employed in the methods reported herein. Glucose can also be excluded from the culture medium and added separately to the culture.

Culturing:

CHO cells expressing anti-IL-6R antibody were cultured in 1l or 2l fermentation vessels. The feed medium contains 15 to 40g/l glucose. Glucose can be fed with a separate concentrated solution containing e.g. 400g/l glucose. The cultivation is carried out at a pH value ranging from pH 7.0 to pH 7.2.

Determination of the glycosyl structure:

for the analysis of the IgG glycosylation pattern, a method according to Kondo et al (Kondo, A. et al, Agric. biol. chem.54(1990)2169-2170) was used. IgG was purified from the centrifuged supernatant of the medium using a small-scale protein a column. The oligosaccharides of the purified IgG were released using N-glycosidase F (Roche diagnostics GmbH, Mannheim, Germany) and labeled with 2-aminopyridine at the reducing end. The labeled oligosaccharides were analyzed by reverse phase chromatography (HPLC). Each peak was assigned to the oligosaccharide by mass spectrometry and standard.

And (3) glucose determination:

use of YSI 2700SELECT^TMThe glucose concentration was determined using an analyzer (YSI, Yellow Springs, OH, USA) according to the manufacturer's manual.

Viable cell density determination:

using an automated image processing and analysis system ((ii) a Innovatis, germany) and trypan blue dye exclusion methods, viable cell density was determined.

Example 1: DGL control and the effect of pH on antibody production and mannose-5 glycosyl Structure (M5) content

Humanized anti-human IL-6 receptor antibodies were used (Tocilizumab,) The antibody was tested according to Japanese unexamined patent publication No.99902/1996 by using the human elongation factor I alpha promoter as reported in example 10 of International patent application publication No. WO 92/19759 (corresponding to US 5,795,965, US 5,817,790 and US7,479,543).

In the constant absolute feeding method, an effect of pH control on immunoglobulin production was observed. Table 2 shows the effect of pH control on antibody oligosaccharide production and M5 content in the constant feed mode.

Table 2: effect of pH control in constant Absolute Fed mode

At pH 7.0, the amount of mannose-5 glycosyl structure (M5) was adjusted to less than 5.5%. The DGL value decreased from 0.80 to 0.21 due to the change in cell density. On the other hand, at pH 7.2, the amount of M5 fluctuated between 8.7% and 25.2%, and was higher than that at pH 7.0. The DGL value at pH 7.2 was changed from 0.73 to 0.25. In addition, in this case, the immunoglobulin production at pH 7.2 was greater than 120% (relative value compared to that at pH 7.0). Higher immunoglobulin production induced higher M5 levels of more than 8% in the constant absolute feed method. Thus, in the constant absolute amount feeding method, the use of pH 7.0 control enables the M5 content to be effectively adjusted to a lower value, i.e., less than 8%, than the pH 7.2 control method.

The DGL control method (constant relative volume feeding method) was also used for fed-batch mode immunoglobulin production at various pH values and analyzed for M5 content. Table 3 shows the effect of DGL control and pH on immunoglobulin production and M5 content after the start of feeding on days 2-3.

Table 3: effect of DGL and pH control in fed-batch mode

No.	Day (d)]Sample (2)	pH set point	DGL	Relative antibody concentration [% ]]	Content of M5 [% ]]
						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

At pH 7.0, the DGL control method is applied over a DGL range from 0.2 to 0.8. As a result, the content of M5 was adjusted to 4.0% or less. On the other hand, at pH 7.2, the DGL value was manipulated to be in the range from 0.4 to 0.6. At this time, the content of M5 can be controlled to be less than 5.5%.

Example 2: cultivation with different DGL values

Culture of CHO cells containing nucleic acids encoding anti-IL-6R antibodies was performed with different DGL values. The results are summarized in table 4 below.

Table 4: effect of DGL control values on immunoglobulin production and M5 content

Controlled DGL strategies showing a DGL value of 0.4 to 0.6 have a reduced mannose-5 content compared to constant feeding.

Example 3: cultivation with different feeding strategies

Culture of CHO cells containing nucleic acid encoding anti-IL-6R antibody was performed with one DGL value, but with a different feeding strategy. The results are summarized in table 5 below.

Table 5: effect of the feeding strategy on viability and viable cell Density

In a single feed experiment, a single feed containing all nutrients and glucose was used. In a double feed experiment, two feeds were used: the first feed contained total nutrients and low glucose concentrations of 15g/l, while the second feed contained high glucose concentrations. These different feeding experiments were carried out in the following manner: the feed rate was adjusted daily in one group and followed in the other group by a predetermined profile based on the record of viable cell density development in earlier cultures. As can be seen from table 5, viability and viable cell density can be comparatively independent of the feeding strategy used.

Example 4: glucose limiting System (DGL) control of immunoglobulin production in fed-batch mode

CHO cells (8.0-12X 10) were seeded in serum-free medium as described previously⁵Cells/ml). At 37 deg.C, 98% relative humidity and 10% CO₂Cells were grown at atmospheric pressure. In fed-batch culture, the feed medium containing glucose was fed to the main fermentor from day 2 or 3 from the start of culture. According to U.S. patent application publication No. US 2006/0127975 a1, the feeding strategy follows a method of controlling the Degree of Glucose Limitation (DGL). DGL can be defined as the ratio of the observed specific glucose consumption rate to the known maximum specific glucose consumption rate at which these cells are free to utilize glucose (DGL Q (glc)/Q (glc)_{Maximum of}Wherein q (glc) is the current observed specific glucose consumption rate; q (glc)_{Maximum of}The maximum known specific glucose consumption rate for these cells).

Figure 1 shows the viable cell density and cell viability profile of the culture. In various cell densities shown in FIG. 2, the DGL was controlled to a value of 0.4-0.5. Depending on the cell density at that time, the feed rate was changed once or twice per day. FIG. 3 shows the DGL-based feed profile in fed-batch mode. The feed rate was varied between 0.8 and 1.6ml/h depending on the cell density. With this feeding strategy applied, an immunoglobulin production profile as shown in fig. 4 was obtained. As shown in Table 6 (feed rate 0.02g glucose/h), 10X10 was used⁵Cells/ml and 12X 10⁵At the inoculation scale of cells/ml, immunoglobulin production was almost identical and more than 120% of immunoglobulin production at day seven in the constant feeding method. Despite the 20% difference in initial cell density, it is possible to achieve nearly equal immunoglobulin titers using the DGL control method. In addition, when the inoculation scale was set to 8.0X 10⁵At cell/ml, more than 110% (relative) of immunoglobulin was obtained on day 7, despite a 20 hour delay from the start of feeding. Among these results, the DGL control method was able to achieve stable immunoglobulin production at various vaccination scales.

Example 5: DGL controls the effects on mannose-5 glycosyl structure and galactosylation of oligosaccharides

The glycosylation pattern of immunoglobulins produced using DGL-controlled fed-batch culture was analyzed. Table 6 shows the results of the oligosaccharide analysis of immunoglobulins obtained from fed-batch cultures with controlled DGL compared to the constant feed method (feed rate: 0.02g glucose/h). At 8.0x 10⁵At the seeded scale of cells/ml, the content of mannose-5 glycosyl structure (M5) was 2.8%. At 10x10⁵Cells/ml and 12X 10⁵At the seeded scale of cells/ml, the content of M5 was 4.1% and 3.8%, respectively. Under all culture conditions, the DGL control method was able to adjust the content of M5 to less than 5.0%.

Meanwhile, under each condition, the immunoglobulin G (0) isotype and the immunoglobulin G (2) isotype were controlled within a range from 40% to 46% and from 9.0% to 11%, respectively.

Table 6: effect of DGL control values on immunoglobulin production and glycosylation patterns

Claims (14)

1. Use of a method comprising:

a) culturing a eukaryotic cell comprising a nucleic acid encoding an immunoglobulin in a culture medium, wherein the ratio of the current specific glucose consumption rate of individual cells in the culture medium to the maximum known specific glucose consumption rate of such cells is kept constant, characterized in that the culture is at a pH value from pH 6.5 to 7.5, and

b) recovering the immunoglobulin from the culture,

for reducing the amount of immunoglobulins having a mannose-5 glycosyl structure in an immunoglobulin preparation.

2. Use according to claim 1, characterized in that the ratio is from 0.8 to 0.2.

3. Use according to claim 2, characterized in that the ratio is from 0.6 to 0.4.

4. Use of a method comprising:

a) culturing a eukaryotic cell comprising a nucleic acid encoding an immunoglobulin in a culture medium, wherein the amount of glucose available per time unit of the culture medium is kept constant and limited to a constant value of less than 80% of the maximum available amount of the cell per time unit of the culture medium, characterized in that the culturing is at a pH value from pH 6.5 to 7.5, and

b) recovering the immunoglobulin from the culture,

for reducing the amount of immunoglobulins having a mannose-5 glycosyl structure in an immunoglobulin preparation.

5. Use according to claim 4, characterized in that the constant value is less than 80% and more than 20%.

6. Use according to any one of claims 1 to 5, characterized in that the culture is a fed-batch culture.

7. Use according to claim 6, characterized in that the culture is a fed-batch culture, wherein the feeding is started on day 2 or 3 of the culture.

8. Use according to claim 1, characterized in that the cultivation is at a pH value from pH 6.9 to 7.3.

9. Use according to claim 8, characterized in that the cultivation is at a pH value from pH 6.95 to pH 7.05 or at a pH value from pH 7.15 to pH 7.25.

10. Use according to any one of claims 1 or 4, characterized in that the immunoglobulin is a class G or class E immunoglobulin.

11. Use according to any one of claims 1 or 4, characterized in that the eukaryotic host cell is selected from the group consisting of CHO cells, NS0 cells, HEK cells, BHK cells, hybridoma cells, DNA fragments, DNA,Cells, insect cells and Sp2/0 cells.

12. Use according to claim 11, characterized in that the eukaryotic cell is a Chinese Hamster Ovary (CHO) cell.

13. Use according to any one of claims 1 or 4, characterized in that the cultivation of the host cell is carried out for 6 to 20 days.

14. Use according to any one of claims 1 or 4, characterized in that the immunoglobulin is an anti-IL-6R antibody.