pecification
Title of Invention: glycosylation of recombinant glycoproteins control method
Art
[1]
The present invention relates to a method for the control of glycosylation of recombinant glycoproteins (A method for controlling glycosylation of recombinant glycoprotein).
BACKGROUND
[2]
Etanercept (Etanercept) is human p75 TNF-α receptor a ligand binding portion of (TNFR, TNF-α receptor) to a TNFR-Fc fusion protein, coupled to the Fc fragment of human IgGⅠ, Enbrel in 2002 by Amgen (Amgen) ( was released as trade name Enbrel). Etanercept suppresses immune responses associated with TNF-α, by competitive inhibition in that the TNF-α receptor, and TNF-α on the cell surface binding in vivo. Thus etanercept has clinical studies are underway for application to the rheumatoid arthritis, psoriasis, ankylosing spondylitis etc. has been used, vasculitis, Crohn's disease and Alzheimer's disease as TNF-α inhibitors.
[3]
On the other hand, a gene recombinant products refers to medicines such as a peptide or protein that is produced using a genetic engineering technique, as an active ingredient. Using genetic engineering, but in it for a large amount of highly purified recombinant protein difficult to obtain in a natural state, because the gene for the desired protein to be inserted from the outside into the host cell expressing the structure itself may be unstable. As well as by expression, etc. microorganism, copper, plant cells, not human because of the production of the protein Recombinant proteins can day the structural, physical and chemical, immunochemical, biological properties or characteristics side native protein and the difference in the (outer gwonohseok, FDC legislation study No. 5 Issue No. 1, 2, 13-21, 2010).
[4]
In particular, there is a structure I in the form of if and oligosaccharide glycosylation (glycosylation) may vary depending on the type of host cell, the operation of the recombinant method, culture conditions, when the glycoprotein. That is, it produces a wide variety of oligosaccharide (glycoform) it is possible to present the non-uniformity (heterogeneity) in accordance with the difference between the production conditions according to the production process of component per volume of differences constituting the sugar chain structure or a sugar chain in the glycoprotein. Sugar chain structure to each other, if the glycoprotein other in vivo pharmacokinetics and tissue distribution of the wild-type and different, to cause adverse reactions to the antagonistic action against native also and may cause immunological problems by acting as an antigen for long term continuous administration is.
[5]
As described above oligosaccharide is not to regulate the sugar chain structure can be an important factor that can affect the pharmacological effects and pharmacokinetics is very important in the field of development and the development of mass production technology of recombinant glycoproteins for pharmaceutical products.
[6]
In Korea Patent 2011-0139292 disclose prior literature with respect to the increase in the N- glycosylation site occupancy on the protein and the substrate with respect to the compositions and methods related to the control and that of glycosylation, Korea Patent Publication 2012-0134116 is treated glycoprotein , it discloses a method for.
Detailed Description of the Invention
SUMMARY
[7]
Under this background, the present inventors have completed the present invention and confirmed that can adjust the sugar chain pattern of the glycoprotein recombinant When using a medium containing intensive research efforts result, the insulin to develop a method for adjusting a glycosylated protein per recombinant .
Problem solving means
[8]
The main object of the present invention to provide a method for adjusting the sugar chain pattern (glycosylation pattern) of a glycoprotein, a recombinant comprising the step of culturing a microorganism comprising a polynucleotide encoding the glycoprotein recombinant in a medium containing insulin .
[9]
Another object of the invention is the growth phase of growing by culturing a microorganism comprising a polynucleotide encoding the recombinant glycoprotein from (a) the culture medium; And (b) to provide a method for adjusting the sugar chain pattern of a glycoprotein, a recombinant comprising a production step of producing a glycoprotein in the culture by the addition of insulin to the culture medium.
Effects of the Invention
[10]
Sugar chain pattern control of the recombinant glycoprotein process according to the invention can control the activity of the recombinant glycoprotein, folding, secretion, stability, plasma half-life and immune responses and the like.
Brief Description of the Drawings
[11]
Figure 1 shows the cleavage map of pCUCBin-mSig-TNFcept.
[12]
Figure 2 is a cell culture of time in one embodiment of the invention: the living cell number (unit 10 in accordance with (in days) 5 illustrates the cells / ml), and the survival rate (%).
Best Mode for Carrying Out the Invention
[13]
In some embodiments for achieving the above object, the present invention provides a sugar chain pattern of a glycoprotein, a recombinant comprising the step of culturing a microorganism comprising a polynucleotide encoding the glycoprotein recombinant in a medium containing insulin (glycosylation pattern ) to provide a method for controlling.
[14]
[15]
The protein (glycoprotein) sugar can be a sugar means that refers to a protein coupled with specific amino acids of the polypeptide, wherein the sugar is for example made of a sugar chain is one or two or more linked monosaccharides. In one embodiment the oligosaccharide is the number of monosaccharide oligosaccharide attached to a glycoprotein, a Foucault Oz, N- acetylglucosamine, N- acetyl galactosamine, galactose, manno Oz, monosaccharides, glucose, xylene, such as sialic acid Oz, manno odds may include a 6-phosphate, or a branched form, such as.
[16]
[17]
In one embodiment, the recombinant glycoprotein may be one of an immunoglobulin fusion protein. Wherein the immunoglobulin is an immunoglobulin; variable region of the heavy chain and light chain of (immunoglobulin Ig), heavy chain constant region 1 (CH1) with the exception of the portion other than the light chain constant domain (CL1), a heavy chain constant region 2 (CH2), heavy chain constant region It may be one containing the Fc portion of an immunoglobulin comprising a (CH3) and a hinge (hinge) portion.
[18]
[19]
For example other, the recombinant glycoprotein may be one of TNFR-Fc fusion protein.
[20]
The TNFR (tumor necrosis factor receptor) refers to a receptor protein that binds to TNF-α. The TNFR proteins TNFRI (p55), and include all of the protein or TNFRII (p75) protein, preferably not proteins or TNFRII, limited to this. In addition, the TNFRII may be mixed with TNFRSF1B (Tumor necrosis factor receptor superfamily member 1B). TNFRII the protein is divided into four domains, and a transmembrane (transmembrane) domain. Examples TNFRII may be a protein comprising the four domain and transmembrane region, which consists of 235 amino acids, but is not limited thereto. The TNFRI protein and can be obtained from known databases such as GenBank National Institutes of Health has information on TNFRII proteins. Examples Accession number that may be a protein NP_001056, or P20333, but not limited thereto.
[21]
The TNFR proteins can be used in the treatment of diseases which are so Genie an activity of binding to TNF-α is known to cause various diseases when overexpressed in the human body, by using this, mediated by TNF-α, such as autoimmune diseases. By fusing the Fc region of an immunoglobulin to the TNFR protein it can be used to do this by making the form of a fusion protein which increases the half-life.
[22]
The TNFR (tumor necrosis factor receptor) -Fc fusion protein is TNFR protein or all or a portion of which is connected by the Fc region and the enzyme action of the immunoglobulin, or the expression of a single polypeptide of the two polypeptides by genetic engineering resulting It means. Wherein the TNFR-Fc fusion protein, but may be connected via a direct connection the Fc region of the TNFR protein and an immunoglobulin or a peptide linker (peptide linker), but is not limited thereto. Wherein the TNFR-Fc Non-limiting examples of fusion proteins may include etanercept (US patent 7,915,225; 5,605,690;. Re 36,755).
[23]
Wherein the TNFR-Fc fusion proteins can be produced by fusing all or a portion of the TNFR protein and the immunoglobulin Fc region, and examples thereof include an immunoglobulin Fc domain that one to 235 11 of TNFRⅡ protein comprising the amino acid region and the hinge (hinge) region the fusion to the 232 amino acids, but is not limited thereto. In addition, the TNFR-Fc fusion protein may be codon optimized (codon optimization) according to the host cell to be expressed. Examples may be an enemy codon optimized TNFR-Fc fusion protein specifically in CHO cells, but are not limited to, . Wherein the TNFR-Fc fusion proteins, as well as the amino acid sequence, wherein the sequence that is 70% or more, preferably 80%, more preferably at least 90%, still more preferably 95% or more, most preferably 98% and substantially includes both if the protein having an activity of binding to the TNF-α amino acid sequence shown as the least similarity. Furthermore, if a sequence having such a similarity amino acid sequence having a biological activity the same as or correspond to the TNFR-Fc fusion protein, within the scope of the present invention, protein variants, some sequence having a deletion, modified, substituted or added in the amino acid sequence included is obvious.
[24]
Wherein the Fc is an immunoglobulin; variable region of the heavy chain and light chain of (immunoglobulin Ig), heavy chain constant region 1 (CH1) with the exception of the portion other than the light chain constant domain (CL1), a heavy chain constant region 2 (CH2), heavy chain constant region ( It refers to CH3) and a hinge (hinge) portion of an immunoglobulin comprising part. In addition, Fc regions of the present invention includes derivatives thereof as well as the sequence of natural-type amino acid sequence. It means that the amino acid sequence derivative is one or more amino acid residues in the native amino acid sequence that has a different sequence by a deletion, insertion, or entirely vivo conservative substitutions, or a combination thereof. In addition, the immunoglobulin Fc region may be an Fc region of the IgG, IgM, IgE, IgA or IgD-derived or a combination of both (combination) or a mixed (hybrid). Preferably, but may lose obtained from the combination is known to be derived from IgG that enhance the half-life of the protein, more preferably IgG1 or derived from, any subclass (i.e. IgG1, IgG2, IgG3 and IgG4).
[25]
The Fc region may be obtained by cutting by using a recombinant vector, a gene encoding the Fc region from the genetic engineering manufacturing or purified polyclonal antibody or a single polyclonal antibody to a suitable protein cleavage enzyme, such as papain, pepsin.
[26]
Wherein the TNFR-Fc fusion proteins can be obtained by expression by introducing the expression vector into a host cell comprising a polynucleotide encoding the fusion protein. In one embodiment of the present invention was used pCUCBin-mSig-TNFcept vector as an expression vector comprising a polynucleotide encoding a TNFR-Fc fusion protein was transduced them in CHO cells expressing TNFR-Fc fusion protein.
[27]
[28]
The microorganisms in the present invention can be used in the same sense as the host cell or transformant. Non-limiting examples can be animal cells, plant or yeast host. In an embodiment of the present invention but using CHO cells (Chinese Hamster Ovarian ovary cell) into the microorganism is not limited to one that can be mutated to a polynucleotide encoding the recombinant glycoprotein.
[29]
If the polynucleotide is as long as can be expressed in the microorganism, it may be located is inserted into the chromosome of the microorganism, or in addition to chromosomal location or include all of them, regardless. The polynucleotide includes DNA and RNA encoding the target protein. A method of containing the polynucleotide can be used if the method used in the art without limitation. For example, may be included in the form of a microorganism is expressed in its own expression cassette (cassette expression) gene construct containing all the elements required. In one embodiment the other may be used a method of mutation by expression vector containing the nucleotide sequence of the polynucleotide encoding the desired protein operably linked to suitable control sequences so as to express the desired protein in a suitable host. The control sequences include any operator sequence, sequences that control the termination of the sequence, and a transcription and translation encoding a suitable mRNA ribosome-binding site for regulating the promoter, such that transcription can initiate transcription. Vector may then be transformed into a suitable host cell, replicate independently of the host genome, or functions, may be integrated into the genome itself. The expression vector as long as it can replicate in a host cell is not particularly limited, it is possible to use any vector known in the art.
[30]
[31]
Sugar pattern (glycosylation pattern) of the recombinant glycoprotein is meant the expression of sugar chain represented by the glycosylation (glycosylation) of the glycoprotein. For example, glycosylation or without binding to the protein, sugar types, glycosylation type, sugar content, monosaccharides containing a molar ratio (sex per minute) of the composition, the structure of the position of the sugar chain, the sugar chain containing the sequence, glycosylation place, Saccharin Chemistry share, may be to include a number of oligosaccharide, or structure-specific relative content or the like. Depending on the sugar chain pattern of the recombinant glycoprotein can lead to differences in the biological activity and in vivo stability.
[32]
Insulin in the present invention may be to adjust the N- linked glycosylated (N-linked glycosylation) of the recombinant glycoprotein. The N- linked glycosylated in the present invention can be used in the same sense and the N- glycosylation. For example, the insulin may be to reduce the content of the N- glycans of recombinant glycoproteins. May be used in the present invention to the N- glycans, it is defined as N- linked sugar chains, and per this can mean a case bonded to the asparagine amino acids in the protein.
[33]
Insulin in the present invention may be to control the connection of each O- glycosylated recombinant proteins (O-linked glycosylation). O- glycosylated at the connection with the present invention it can be used in the same sense and O- glycosylation. For example other, the insulin may be to reduce the content of O- glycans of recombinant glycoproteins. May be used in the present invention as the O- glycan are as defined and O- linked sugar chains, the sugar can represent a case bound to a serine or threonine amino acid of the protein.
[34]
Insulin in the present invention may be to adjust the N- linked glycosylated and O- glycosylated connection of each recombinant protein.
[35]
In one embodiment of the present invention, showed that the addition of insulin that affect sugar chain pattern of the glycoprotein (Table 2). In particular, it is shown to the content of the N- glycans and / or O- glycans changes with the addition of insulin. Specifically, that the N- glycans and / or O- be adjusted so as to decrease the content of the glycan was identified in accordance with the addition of insulin. During the step of culturing the cells capable of producing glycoproteins in particular, it was confirmed that the addition of insulin in the sugar chain pattern important adjustment in the production stage.
[36]
[37]
The concentration of the insulin may be of 0.0001 mg / L to 1g / L, based on the total volume of medium. It was confirmed that in one embodiment of the invention, be adjusted to the amount of the N- glycans and / or O- glycans decreases with increasing concentrations of insulin (Table 2).
[38]
[39]
The medium may be used without limitation so long as it is used to culture the microorganism or host cell comprising a polynucleotide encoding the glycoprotein in the art. In one embodiment the medium is a glutamic acid (L-Glutamine), thymidine (Thymidine), alanine (Alanine), Arginine (Arginine, monohydrochloride), asparaginase Nin (Asparagine, monohydrate), aspartic acid (Aspartic acid), Cysteine ​​(Cysteine) , glycine (glycine), histidine (histidine), isoleucine (isoleucine), leucine (leucine), lysine (lysine, monohydrochloride), methionine (methionine), phenylalanine (phenylalanine), proline (proline), serine (serine), threonine ( Threonine), tryptophan (tryptophan), tyrosine (tyrosine, disodium salt, dehydrate), Val (valine) may include amino acids such as. Examples other day, the medium was glucose (Glucose), sodium chloride, sodium hydrogen carbonate (Sodium bicarbonate), (Sodium chloride), calcium chloride (Calcium chloride, ananhydrous), sulfate, cupric (Cupric sulfate, pentahydrate), nitric acid (II), iron ( Ferric nitrate, nonahydrate), sulfuric acid (I) iron (ferrous sulfate, heptahydrate), potassium (potassium chloride), magnesium sulfate (magnesium sulfate, ananhydrous), magnesium chloride (magnesium chloride, ananhydrous), sodium phosphate (sodium phosphate, monobasic or dibasic, monohydrate), zinc sulphate (zinc sulfate, heptahydrate), hypoxanthine (hypoxanthine), Fu Trail new dihydrochloride (Putrescine, dihydrochloride), sodium pyruvate (pyruvate sodium), biotin (biotin), calcium pantothenate (D- Calcium pantothenate), choline chloride (choline chloride), cyanocobalamin (cyanocobalamin), folic acid (folic acid), inositol (i-inositol), nicotinamide (nicotinamide), pyridoxal (pyridoxal, monohydrochloride), pyridoxine (Pyrid oxine, monohydrochloride), riboflavin (Riboflavin), Thiamin (Thiamine, monohydrochloride), glucose (glucose, anhydrous), potassium chloride (Potassium Chloride), sodium phosphate (NaH2PO4 · H2O), sodium bicarbonate (NaHCO3), free acid (HEPES) , dextran sulfate (dextran sulfate), NaCl (sodium chloride), ascorbic acid (ascorbic acid), D- biotin (D-biotin), choline chloride (choline chloride), folic acid (folic acid), Hypep 1510, or of its 2 may include more thereof. Initial species may further comprise a MTX to increase the expression levels during culture.
[40]
The culture can be a perfusion culture (perfusion culture). The culture may be a culture solution means that the culture sloppy around microorganism. Depending on the sugar chains in a desired pattern, the concentration of insulin throughout the perfusion culturing can be easily adjusted.
[41]
[42]
In another aspect, the growing step of the present invention are grown by culturing a microorganism comprising a polynucleotide encoding the recombinant glycoprotein from (a) the culture medium; And (b) it provides a method for adjusting the sugar chain pattern of a glycoprotein, a recombinant comprising a production step of producing a glycoprotein in the culture by the addition of insulin to the culture medium.
[43]
In one embodiment, the recombinant glycoprotein may be one of an immunoglobulin fusion protein. For example other, the recombinant glycoprotein may be one of TNFR-Fc fusion protein. This is as described above.
[44]
The step (a) the growth phase may be one further comprising a seed culture stage.
[45]
Medium of step (a) may be one which does not contain insulin.
[46]
The step (b) may be to a different concentration of insulin in the sugar chain is added according to the desired pattern. In one embodiment of the present invention, it was found in the growth step of the content of the N- glycans and / or O- glycans changes with the addition of insulin. Specifically, that the N- glycans and / or O- be adjusted so as to decrease the content of the glycan was identified in accordance with the addition of insulin. During the step of culturing the cells capable of producing glycoproteins in particular, it was confirmed that the addition of insulin in the sugar chain pattern important adjustment in the production stage.
[47]
The concentration of the insulin may be one of 0.0001mg / L to 1g / L, based on the total volume of medium. It was confirmed that can adjust to the amount of the N- glycans and / or O- glycans decreases with increasing concentrations of insulin (Table 2).
[48]
Insulin may be to control the connection of N- glycoprotein recombinant glycosylated (N-linked glycosylation) and O- glycosylated connect (O-linked glycosylation). For example, the insulin may be to reduce the content of the N- glycans of recombinant glycoproteins. For example other, the insulin may be to reduce the content of O- glycans of recombinant glycoproteins.
[49]
[50]
In another aspect, the invention provides a sugar chain pattern for adjusting the medium composition of the glycoprotein, a recombinant containing the insulin. The insulin can be included in 0.0001mg / L to 1g / L, based on the total medium.
[51]
For example, the medium may be those that are used only in the production step of the step of culturing the microorganism.
[52]
[53]
Mode for the Invention
[54]
It will be described in detail by the following Examples to the present invention. However, the following examples are not intended to limit the scope of the present invention by way of example to be only, to illustrate the present invention.
[55]
[56]
Example 1: Preparation of cell lines for the production of glycoproteins
[57]
[58]
1-1. Preparation of vector
[59]
[60]
Restriction method commonly used in molecular biology such as the treatment and purification of plasmid DNA, DNA fragments bonded and transformation of E. coli in the (Restriction enzyme) are at least to the methods introduced in Molecular Cloning (2 plate), such as Sambrook It was added and modified.
[61]
[62]
By using the cDNA library as a template a mRNA isolated from HUVEC cell line clone the human p75 TNF receptor (TNFR) gene and to fuse it with the Fc Region of Human IgG1 to give the TNFR-IgG1. This use and pTOP-BA-RL-pA vector; by the (Republic of Korea Patent Publication No. 10-2012-0059222 No. 'CMVe' and 'CB', and that has a 'Beta-actin Intron') of the mold, as shown in FIG. 1 as cleavage map was prepared pCUCBin-mSig-TNFcept vector.
[63]
[64]
1-2. Cell cultures
[65]
[66]
The CHO / dhfr- (CHO DXB11) was used as a cell. CHO / dhfr- the Dihydrofolate Reductase (DHFR) is a gene-deficient cells, over a mutation after the separation from the ovarian cells of Chinese Hamster.
[67]
[68]
1-3. Transfected cell lines selected for conversion and production
[69]
[70]
Using a P75 TNF receptor (TNFR) the pCUCBin-mSig-TNFcept vector and CHO / dhfr- (CHO DXB11) containing the gene to create a cell transformed with the gene was amplified using MTX concentrations. Picking the candidates that meet the criteria of this cell line was selected as for the production and then evaluate whether monoclonal. Then into the cell line in a glass bottle and stored in liquid nitrogen.
[71]
[72]
Example 2: Recovery of protein and culturing the cell line for protein production per
[73]
[74]
The medium used in the culture was used differently depending on the cultivation stage. Media X011SB (Merck Millipore, Cat. No. 102443) at a basal medium added with insulin in 5.8g / l, seed culture phase (Phase Seed Cultivation) In anhydrous glucose (Sigma) 10g / l, and L-Glutamine, Glycine, Serine (Sigma) for each of 0.584 g / l was used as the medium (media EC-SI) was added, the growth phase (growth phase) in a were added 0.584 g / l of anhydrous glucose, 5g / l and l-Glutamine, Glycine, Serine was used as culture medium (EC-GM), was used for production (production phase) in anhydrous glucose 15g / l and respectively added 0.584 g / l of l-Glutamine, Glycine, Serine medium (EC-PM).
[75]
[76]
Carried out after the quick thawing the vial containing the cell line prepared in Example 1 in a water bath, the cells were transferred to Falcon Tube containing 10mL of medium. After the supernatant was removed primarily by centrifugation. This inoculated in Erlenmeyer Flask final volume was resuspended in Media EC-SI 10mL to 50mL, and the cells were cultured in 2L based on the amount of work (Working Volume) using a 5L CelliGen310 incubator. 5 via the conference seed culture cell number x 10 2 6 reaches cells / mL by the perfusion culture was started in EC-GM medium exchange. As the cell number increases by increasing the speed at which the medium was replaced so that the cells can be better differentiated. The number of cells (viable cell number) is 1.5 x 10 7 when it reaches the cells / mL by exchanging (Fig. 2) to the EC-medium PM medium and allowed to transition from growth phase to production phase. Proceed to recovery (harvest) over a total of four times, followed by purifying the recovered protein. The result is calculated as four times the average value.
[77]
[78]
Example 3. Quantitative Analysis of glycan (glycan)
[79]
[80]
3-1. O-glycan content analysis
[81]
[82]
Embodiment the sample purified in Example 2 was prepared such that 100 μl with 1.0 mg / mL concentration by diluting with 25 mM Sodium Phosphate, pH 6.3 buffer. Each sample N- glycosidase F (Glycosidase F; 1U / μl, Roche) is 4 μl and New lamina kinase (Neuraminidase; 1U / 100 μl, Roche) 1 μl, trypsin (Trypsin; 1mg / mL, Promega) 2 μl was added to react at 37 ℃ for 18 hours and was conducted LC-MS analysis.
[83]
Were analyzed; (Vydac, Cat No. 218TP54 4.6 mm x 250 mm, 5 μm, 300 Å.) Tryptic peptide degradation (Tryptic Peptide) by using said sample, and a 80 μl injection, C18 RP column. Mobile phase A was 0.1% TFA in Water, mobile phase B was used a 0.1% TFA in 80% Cold ACN, it was analyzed for 150 minutes Gradient conditions. Were detected for the peptides at 215 nm using a UV detector (detector), material that has already been run through the LC is mass spectrometry (Mass spectrometry; LTQ XL, Thermo) to connect to the by performing MS analysis O-glycopeptide relative area ratio (Relative of was calculated Area,%).
[84]
[85]
3-2. N-glycan content analysis
[86]
[87]
Example 2 The purified samples and reference standard (etanercept, Pfizer) was prepared by diluting each with 3.0mg / mL The sample dilution buffer (25 mM Sodium Phosphate, pH 6.3 buffer), so that in the. A mixture of 100 μl of each sample and N-glycosidase F 6 μl solution was reacted for 20 hours at 37 ℃. Was vortexed reaction solution was added to 400 μl of ethanol, and the ball end. It only takes a supernatant after centrifugation contained transferred to Eppendorf Tube, a solution using a Speed-Vac Concentrator and completely dried. The 2-AA label on the dried sample was mixed into 10 μl. After reacting at 45 ℃ it cooled at room temperature.
[88]
It was poured a disposable culture tube, and then, purified water, 30% acetic acid and acetonitrile (Acetonitrile) placed the GlycoClean S cartridge (Cartridge) above in turn. Then after loading the sample is cooled in the center of the membrane cartridge it was flushed with acetonitrile. Purified water was added to the cartridge to gather in Eppendorf Tube so as to elute the N-glycan. The resulting solution was freeze-dried and stored until Glycan analysis.
[89]
A 0.5mM ammonium acetate (pH 5.6) as mobile phase A, 250mM ammonium acetate (pH5.6) was analyzed for 130 minutes Gradient conditions in HPLC column (AsahiPak NH2P-50 4E, 4.6x250 mm) with a mobile phase B. It was detected using a fluorescence detector, and calculating a sum of area for peaks by the number of sialic acid (Sialic acid) present at the terminal of the N-glycan to the analysis result. If the absence of sialic acid if neutral, 1 gae (monosialyl) -1 charge, 2 gae (disialyl) be expressed as -2 charge.
[90]
[91]
Experimental Example 1. Cells cultured in the medium without addition of insulin from production
[92]
[93]
After a second embodiment cultured and the cell line in the same medium except for insulin in the production steps, to analyze the relative area ratio (%) of the content (%) of the N-glycan-specific culture temperature and the O-glycopeptide Table 1 It is shown.
[94]
[95]
TABLE 1
The culture temperature (production) Harvest (Recurrence) N-glycan -2 charge (%, mean) Relative ratio of O-glycopeptide (%, mean)
30 o C H1 12.5 56.13
H2
H3
H4
32 o The the C H1 16.1 54.38
H2
H3
H4

[96]
[97]
Experimental Example 2 Comparison of sugar chain pattern change in insulin was added in the production step
[98]
[99]
As compared to a sugar chain pattern of the glycoprotein according to whether the addition of insulin from production steps of Example 2 are shown in Table 2 below.
[100]
[101]
TABLE 2
Incubation temperature Insulin concentrations medium Harvest (Recurrence) N-glycan -2 charge (%, mean) Relative ratio of O-glycopeptide (%, mean)
30 o C 0mg / L H1 12.5 56.13
H2
H3
H4
0.003mg / L H1 10.4 54.68
H2
H3
H4
0.009mg/L H1 10.9 52.68
H2
H3
H4
0.03mg/L H1 9.7 49.5
H2
H3
H4

[102]
[103]
As a result, during the step of culturing the cell to produce the glycoprotein, it showed that affect sugar chain pattern of the addition of insulin glycoprotein in the production phase. In particular, it is shown to the content of the N- glycans and / or O- glycans changes with the addition of insulin. Specifically, that the N- glycans and / or O- be adjusted so as to decrease the content of the glycan was identified in accordance with the addition of insulin.
[104]
[105]
From the above description, those skilled in the art will appreciate that may be embodied in other specific forms of the present invention without changing the technical spirit or essential characteristics. The embodiments described above In this regard, the examples should be understood as illustrative and not be limiting in all aspects. The scope of the invention should be construed as the meaning and scope, and all such modifications as derived from the equivalent concepts of the claims to be described later, rather than the description above within the scope of the invention.
Industrial Applicability
[106]
Sugar chain pattern control of the recombinant glycoprotein process according to the invention in particular, since in the growth phase may change the content of the N- glycans and / or O- glycans in accordance with the addition of insulin, in particular the uniformity of the bonding of a sugar molecule It may be useful in important medicines for recombinant protein production per.

Claims
[Claim 1]
Method of adjusting the sugar chain pattern (glycosylation pattern) of a glycoprotein, a recombinant comprising the step of culturing a microorganism comprising a polynucleotide encoding the recombinant glycoprotein from the culture medium containing insulin.
[Claim 2]
According to claim 1, wherein, wherein the recombinant glycoprotein is an immunoglobulin fusion protein.
[Claim 3]
According to claim 1, wherein, wherein the recombinant glycoprotein is a TNFR-Fc fusion protein.
[Claim 4]
According to claim 1, wherein the concentration of the insulin is to a 0.0001mg / L to 1g / L, based on the total volume of the medium, the method.
[Claim 5]
The method of claim 1, wherein the insulin is a method to control the connection of N- glycoprotein recombinant glycosylated (N-linked glycosylation) and O- glycosylated connect (O-linked glycosylation).
[Claim 6]
According to claim 1, wherein the, method of the insulin reducing the content of N- glycans of recombinant glycoproteins.
[Claim 7]
According to claim 1, wherein, the method to reduce the content of insulin is O- glycans of recombinant glycoproteins.
[Claim 8]
The method of claim 1, wherein the culture method is a perfusion culture.
[Claim 9]
(A) growing step of growing by culturing a microorganism comprising a polynucleotide encoding the recombinant glycoprotein from the culture medium; And (b) a method of adjusting the sugar chain pattern of a glycoprotein, a recombinant comprising a production step of producing a glycoprotein in the culture by the addition of insulin to the culture medium.
[Claim 10]
The method of claim 9 wherein the (b) step is the production, method to different insulin concentrations was added according to the sugar chain in a desired pattern.
[Claim 11]
11. The method of claim 10, wherein the, method of the insulin reducing the content of N- glycans or O- glycans of recombinant glycoproteins.
[Claim 12]
The method of claim 9, wherein, wherein the recombinant glycoprotein is an immunoglobulin fusion protein.
[Claim 13]
The method of claim 9, wherein, wherein the recombinant glycoprotein is a TNFR-Fc fusion protein.
[Claim 14]
10. The method of claim 9, wherein the concentration of the insulin is to a 0.0001mg / L to 1g / L, based on the total volume of the medium, the method.
[Claim 15]
10. The method of claim 9, wherein the culture method is a perfusion culture.