KR20130102936A - Composition for culture media additives for enhancing productivity of recombinant protein - Google Patents

Abstract

PURPOSE: A culture medium containing a medium additive composition is provided to improve productivity of recombinant proteins and the growth and proliferation of cells which produce the recombinant proteins. CONSTITUTION: A medium additive composition for improving productivity of a recombinant protein contains 0.050-0.150 mM of alanine, 0.450-1.350 mM of arginine, 0.344-1.031 mM of asparagine, 0.118-0.353 mM of aspartic acid, 0.028-0.184 mM of cysteine, 0.155-0.465 mM of cystine, 0.395-1.184 mM of glutamic acid, 0.040-0.120 mM of glycine, 0.143-0.429 mM of histidine, 1.145-3.435 mM of isoleucine, 1.527-4.580 mM of leucine, 1.134-3.401 mM of lysine, 0.749-2.246 mM of methionine, 0.515-1.545 mM of phenylalanine, 1.305-3.914 mM of proline, 4.048-12.143 mM of serine, 0.883-2.648 mM of threonine, and 0.307-0.920 mM of tyrosine. The composition improves the growth or proliferation of an animal cell line.

Description

Composition for culture media additives for enhancing productivity of recombinant protein

The present invention relates to a composition for adding a culture medium for improving the productivity of a recombinant protein, and more particularly, the present invention to improve the productivity of the recombinant protein and to improve the growth and / or growth of host cells, alanine, arginine , Asparagine, aspartic acid, cysteine, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine and tyrosine, the composition for the addition of a culture medium, the composition comprising the composition It relates to a medium and a method for producing a recombinant protein using the culture medium.

Proteins and polypeptides are becoming increasingly important as therapeutic agents. Since antibodies have a well-defined mechanism of action and fewer side effects, they have been developed and marketed worldwide in the United States for the treatment of autoimmune and cancer diseases requiring long-term treatment.

For such commercial production, using a recombinant protein production technology to increase its productivity. Recombinant protein production technology is a technique for synthesizing a desired protein by changing the sequence of a specific gene or by combining with other genes to make a different combination of genes and insert it into bacterial yeast, plant, animal cells. Genetically modified protein drugs are produced by mass production of the bioactive proteins required by the human body in abnormal conditions using these techniques.

Initially, the recombinant protein was produced by Escherichia coli, which can be rapidly grown, easily cultured, and cultured at low cost. However, since the complex structure of the human protein cannot be properly implemented, it has been produced in yeast cells, which are eukaryotic cells. However, the production in yeast, such as the degradation of foreign human protein by the self-degrading enzyme or the exact sugar chain addition capacity, and had a disadvantage of low production yield. To improve this, human-derived cells produce human proteins. While animal cells add precise conformation and sugar chains to human-derived proteins, they have slow growth rates, difficult control of the culture environment, and high costs of incubators and culture media.

Currently, about 60 to 70% of the recombinant drugs on the market with FDA approval are produced using animal cell culture methods, especially since most of the high value added glycoproteins are produced by animal cell culture methods. Research on culture conditions for improving research and development and productivity of recombinant cell lines is progressing rapidly.

Cell culture at high concentrations is also an essential process for increasing protein production efficiency. In order for a cell to grow to a high concentration, it must meet the requirements of abundant components of the medium. However, there is a disadvantage that the osmotic pressure is increased when the type and concentration of the components in the medium increases. Ordinary media also contain animal serum containing abundant proteins and hormones, which are problematic for clinical use. Therefore, the culture medium for the production of recombinant protein for use as a drug should be a medium that does not have serum but has a component that can replace it. In addition, since the optimum conditions of the medium are all different according to the type of cells, it is important to find the optimal culture medium composition for the animal cell culture to increase the productivity of the target protein.

Amino acids are one of the major energy sources in CHO cell growth. In particular, CHO cells in culture actively absorb and metabolize 20 amino acids. Specifically, the glycosylation pathway from glucose is used to create energy and cell building blocks such as ATP (adenosine triphosphate) and NADPH (nicotinamide adenine dinucleotide phosphate), and absorb amino acids including glutamine to absorb energy and Create architectural elements and other derivatives. Numerically, the energy needed for growth is about 70% from glucose and about 30% from glutamine. On the other hand, in the assimilation of building elements for cell composition, the glucose-based pathway must be followed by several complex biochemical pathways through basic intermediate metabolites. However, when the amino acid is absorbed and used from outside the cell, only a relatively small amount of energy is consumed for absorption, and the substance can be used immediately after the cell is absorbed. In particular, given that most of the building elements in the cell are composed of proteins, it is reasonable that the direct absorption and use of amino acids is the first priority for the cell. Such biological characteristics mean that the addition of sufficient amino acids in the medium in the culture for recombinant protein production can increase the recombinant protein production rate and cell growth. However, the amino acid-related metabolism of all cells, including CHO cells, is so complex that metabolism of one amino acid affects the metabolism of another amino acid. In addition, the total ratio of proteins constituting each cell and the stoichiometry of each amino acid constituting the protein have different characteristics between species. In particular, when mass-producing a recombinant protein, the cell will additionally require as many amino acids as the stoichiometric coefficient of the amino acids constituting the recombinant protein. This metabolic process is so delicate that eventually unbalanced concentrations and absorption rates of each amino acid result in low cell growth or low recombinant protein productivity. Thus, optimization of the amount of amino acid components in the medium to increase cell growth and recombinant protein production is one of the most important parts in process development, particularly in medium optimization. In particular, such optimization is also a very popular research field in recent years.

Under this background, the present inventors add a composition for adding a medium consisting of a specific amount of amino acids to a conventional culture medium, and when producing a transformant that produces a recombinant protein using the culture medium to which the composition is added, the recombinant It was confirmed that the productivity of the protein as well as the growth and proliferation of the transformant can be improved, and completed the present invention.

One object of the present invention is to provide a composition for the addition of a culture medium that can improve the productivity of the recombinant protein and the growth and growth of the transformant.

Another object of the present invention is to provide a culture medium containing the composition.

Still another object of the present invention is to provide a method for producing a recombinant protein using the culture medium.

As one embodiment for achieving the above object, the present invention provides alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glycine, histidine which can improve the productivity of recombinant protein and the growth and proliferation of transformants. Provided is a composition for adding a culture medium consisting of isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine and tyrosine. At this time, the content of each amino acid included in the composition is not particularly limited, but 0.050 to 0.150mM alanine, 0.450 to 1.350mM arginine, 0.344 to 1.031mM asparagine, 0.118 to 0.353mM aspartic acid, 0.028 to 0.184 mM cysteine, 0.155 to 0.465 mM cystine, 0.395 to 1.184 mM glutamic acid, 0.040 to 0.120 mM glycine, 0.143 to 0.429 mM histidine, 1.145 to 3.435 mM isoleucine, 1.527 to 4.580 mM leucine, 1.134 to 3.401 Preference is given to mM lysine, 0.749-2.246 mM methionine, 0.515-1.545 mM phenylalanine, 1.305-3.914 mM proline, 4.048-12.143 mM serine, 0.883-2.648 mM threonine and 0.307-0.920 mM tyrosine.

As used herein, the term "recombinant protein" refers to a protein expressed in a mammalian host cell genetically engineered to express the protein of interest. The recombinant protein may be the same as or similar to a protein normally expressed in mammalian host cells. In addition, the recombinant protein may be exogenous to the host cell, that is, heterologous to the protein normally expressed in the host cell. Alternatively, the recombinant protein may be chimeric in that some contain the same or similar to a protein normally expressed in the host cell, while others contain an amino acid sequence that is exogenous to the host cell. For the purposes of the present invention, the recombinant protein is not particularly limited, but may be a monoclonal antibody or an Fc-containing fusion protein.

The term "amino acid" in the present invention means any amino acid of the 20 natural amino acids generally used for polypeptide formation. The form of the amino acid included in the composition of the present invention for the purposes of the present invention is not particularly limited, but may be included in the form of a powder for cell culture.

The cell line which can improve its growth or proliferation with the composition for adding a culture medium of the present invention may be an animal cell. Preferably it may be a CHO cell, more preferably may be a CHO-K1 cell line, but is not limited thereto.

According to one embodiment of the present invention, the productivity of the recombinant protein in the medium containing the composition for the addition of the culture medium containing the amino acid mixture of the present invention not only improved (Fig. 2), it was confirmed that the growth and proliferation of the cell line is improved Could be (FIG. 3).

According to another embodiment for achieving the above object, the present invention provides a culture medium comprising a composition for culture medium addition.

As used herein, the term "culture medium" means a nutrient-containing solution for nourishing proliferative mammalian cells. Such solutions generally provide the essential and non-essential amino acids, vitamins, energy sources, lipids and trace elements necessary for cells to minimally proliferate and / or survive. In addition, the solution may further comprise components that enhance proliferation and / or survival above the minimum rate, including hormones and growth factors. This solution is preferably prepared at a pH and salt concentration that is optimal for cell survival and proliferation. The medium may also be a defined medium, ie a serum-free medium that is free of components, proteins or hydrolysates of the unknown composition. A "limiting medium" is a composition that is free of components derived from animals and all components have a known chemical structure. For the purposes of the present invention, the culture medium is not particularly limited thereto, but may be a medium for transformant culture capable of producing monoclonal antibodies or Fc-containing fusion proteins.

Preferably the culture medium of the present invention can exclude glutamine. Glutamine is commonly used with glucose as a major carbon source for cells to make energy and building blocks, but too high concentrations of glutamine are bad because they produce byproducts such as ammonia and induce unbalanced amino acid metabolism. This results in poor culture results.

Also preferably, the culture medium of the present invention may not include animal serum. Normal animal serum contains large amounts of proteins and hormones, which can improve cell growth and protein productivity, but the use of these animal proteins is limited in their clinical use. Therefore, it is preferable that the culture medium for producing recombinant protein for use in medicine does not contain serum.

According to another embodiment for achieving the above object, the present invention provides a method for producing a recombinant protein using the culture medium. Specifically, the method for producing a recombinant protein using the culture medium of the present invention comprises the steps of culturing the transformant to produce a recombinant protein of interest using the culture medium and the desired recombinant protein from the culture Recovering the same.

The recombinant protein of the present invention may be a monoclonal antibody or an Fc-containing fusion protein. Preferably, the Fc-containing fusion protein may be a TNFR: Fc fusion protein. More preferably, the TNFR: Fc fusion protein may be etanercept. The recombinant protein is a protein produced by a host cell transformed with a recombinant expression vector in which a TNFR: Fc gene is inserted into a target gene. The transformed host cell is a mammalian host cell, and a CHO (chinese hamster ovary) cell line is most widely used, but mouse-derived NS0 (myeloma) for the production of baby hamster kidney (BHK), human embryo kidney (HEK) and therapeutic antibodies cell line). Preferably it may be a CHO cell, more preferably may be a CHO-K1 cell line, but is not limited thereto.

The term "etanercept" of the present invention is used as a therapeutic agent for autoimmune diseases, acting as a TNF inhibitor, as a therapeutic agent for rheumatoid, juvenile rheumatoid, psoriatic arthritis, ankylosing spondylitis and the like. Etanercept is a fusion protein produced by the expression of recombinant DNA. That is, it is the product of DNA produced by binding the human gene of the water-soluble TNF receptor 2 to the Fc region gene of human immunoglobulin G1 (IgG1).

By culturing the cells producing the desired recombinant protein using the culture medium containing the composition for adding a culture medium of the present invention, not only can the productivity of the desired recombinant protein be improved, but also the desired recombinant protein is produced. Because it can improve the growth and proliferation of cells to be able to be widely used for the effective production of the recombinant protein of interest.

1 is a graph showing the consumption and production rate of amino acids in recombinant CHO-K1 according to an embodiment of the present invention (-: consumption, +: production, ASX: Asn + Asp, GLX: Gln + Glu).
Figure 2 is a graph showing the effect of CDHFA on cell growth and titer under temperature shift conditions in accordance with an embodiment of the present invention.
Figure 3 is a graph showing the effect of CDHFA and nucleosides in cell growth according to an embodiment of the present invention.
Figure 4 is a graph showing the results of measuring the viable cell number, survival rate, titer and specific productivity (q _p ) in NBS culture according to an embodiment of the present invention.
Figure 5 is a graph showing the results of measuring the concentration of glucose, lactate, ammonia and the weight molar osmolality in NBS culture according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these examples.

Example  One: CHO - K1  Consumption and Production of Amino Acids by Cell Lines

In the production of etanercept using the transformed CHO-K1 cell line, the present inventors improve the productivity of the etanercept and at the same time improve the cell growth of the transformed CHO-K1 cell line producing the etanercept. , A composition for adding a culture medium consisting of amino acids was devised.

The content of each amino acid was obtained through the stoichiometric calculation of the desired incubation period to obtain the desired etanercept production in consideration of the amino acid content ratio of the total protein constituting CHO cells and the amino acid content ratio etanercept, Through this, the basic requirement for amino acid of CHO cells producing etanercept was obtained. In order to determine the major amino acid consumption of the recombinant CHO-K1 cell line producing etanercept, the recombinant CHO-K1 cell line was cultured using chemical restriction medium. The amino acid composition of the chemical restriction medium used at this time is shown in Table 1.

Composition of Amino Acids in Chemical Synthesis Medium amino acid Concentration (mM) amino acid Concentration (mM) Alanine
Arginine
Asparagine
Aspartic acid
Cystine
Cysteine
Glutamic acid
Glycine
Histidine
Isoleucine 2086.4
696.6
125.0
126.1
100.0
123.0
2588.3
1628.4
389.6
1301.9 Leucine
Lee Sin
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Balin 1799.8
1442.7
1837.2
944.9
740.9
2420.3
2240.5
161.3
472.4
1568.8

The concentration of amino acids remaining in each medium was measured through 11 feeding fed-batch cycles through three feedings, and the consumption and production rate of major amino acids were calculated according to each cell growth cycle. . This is shown in FIG.

Each production / consumption rate thus obtained was compared with the stoichiometric coefficient according to the theoretical production / consumption rate of recombinant CHO-K1. For the theoretical stoichiometric calculation, the amino acid composition ratio of CHO-K1 is shown in Table 2 below, and the etanercept production capacity of recombinant CHO-K1 was set to 40% of the total protein. The calculated experimental and theoretical production / consumption rates are shown in Table 3.

Amino acid fraction of constituent proteins in CHO cells (Biotechnol. Prog., 2001, 17 (6), pp1032-1041) Stoichiometric Equilibrium of CHO Cells DNA
RNA
lipids
carbohydrate
protein 1.9%
5.8%
7.7%
7.0%
70.6% amino acid Mole fraction amino acid Mole fraction Alanine
Arginine
Asparagine
Aspartic acid
Cysteine
Glutamine
Glutamic acid
Glycine
Histidine
Isoleucine, 0.095
0.063
0.048
0.039
0.028
0.052
0.064
0.078
0.022
0.052 Leucine
Lee Sin
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Balin 0.088
0.089
0.020
0.021
0.028
0.057
0.061
0.006
0.020
0.059

Looking at the results, it can be seen that there is a large difference in the theoretical expected value and the experimental production / consumption rate. This is because the theoretical values represent simple mole fractions, while the experimental values represent the consumption of each amino acid under specific environmental conditions. In other words, the actual metabolism is a complex biochemical pathway is linked to each other, which means that the theoretical values in static conditions alone cannot fully explain the experimental values.

Standardized experimental and theoretical consumption / production rates amino acid Experimental value Theoretical value amino acid Experimental value Theoretical value Asparagine or aspartic acid
Serine
Glutamine or glutamic acid
Glycine
Histidine
Arginine
Threonine
Alanine
Proline 0.15
35.02
1.82
-7.03
1.00
1.69
5.65
1.76
2.72 3.63
3.29
4.93
2.98
1.00
2.39
3.21
3.43
2.60 Cysteine
Tyrosine
Balin
Methionine
Lee Sin
Isoleucine
Leucine
Phenylalanine 9.02
1.96
4.61
4.44
4.56
4.52
10.11
3.35 1.84
1.06
2.93
0.80
3.38
1.68
3.36
1.01

Therefore, in order to overcome this, metabolic flux analysis (MFA) and fluxomics techniques linked thereto should be introduced, and considering that existing medium compositions are mainly close to theoretical values, this means that each of the amino acids required in the culture medium in culture is required. Since the concentration is not properly distributed, it was analyzed that the concentration of amino acid must be properly reestablished in order to increase the growth of recombinant CHO-K1 and the productivity of the recombinant protein.

In the present invention, hypotheses were set for several conditions, and a medium additive was prepared by predicting metabolism based on the hypotheses set above, which was named "CDHFA" and its specific composition is shown in Table 4.

The CDHFA used in the experiment was prepared in powder form to contain the component contents as shown in Table 4, and the prepared CDHFA was directly dissolved in a medium, and 3.99 g of CDHFA was added per 1 L of the basic culture medium. The pH of the medium to which CDHFA was added was 7.33, and the osmotic pressure was found to be 307 mOsm / kg.

Composition of CDHFA amino acid Optimal concentration
(mM) Minimum concentration
(mM) Concentration
(mM) amino acid Optimal concentration
(mM) Minimum concentration
(mM) Concentration
(mM) Alanine
Arginine
Asparagine
Aspartic acid
Cysteine
Cystine
Glutamic acid
Glycine
Histidine 0.100
0.900
0.687
0.235
0.056
0.310
0.789
0.080
0.286 0.050
0.450
0.344
0.118
0.028
0.155
0.395
0.040
0.143 0.150
1.350
1.031
0.353
0.084
0.465
1.184
0.120
0.429 Isoleucine
Leucine
Lee Sin
Methionine
Phenylalanine
Proline
Serine
Threonine
Tyrosine 2.290
3.053
2.267
1.497
1.030
2.609
8.095
1.765
0.613 1.145
1.527
1.134
0.749
0.515
1.305
4.048
0.883
0.307 3.435
4.580
3.401
2.246
1.545
3.914
12.143
2.642
0.920

Example  2: CDHFA Check the effect of

Example  2-1: Effect on Recombinant Protein Productivity

It was intended to confirm the effect on the recombinant protein productivity of the CDHFA prepared in Example 1.

Specifically, using a recombinant CHO-K1 cell line producing etanercept used in Example 1 in a 250 ml Erlenmeyer flask containing about 30 to 50 ml of CDHFA-added medium at 37 ° C., 8% carbon dioxide, and 100 rpm. The culture was carried out and cultured by changing the temperature to 30 ° C. on the 4th day of culture at the time when the cell concentration reached about 0.8 to 1.0 × 10 ⁷ cells / ml, and the proliferation rate of the cells and the etanercept produced from the cultured cells. Productivity was measured and compared (FIG. 2). In this case, as a control, a culture cultured using a medium without CDHFA was used, and all experiments were performed twice.

CHO-K1 used in the present invention is a cell line produced by the present inventors, which synthesizes recombinant DNA using the amino acid sequence of etanercept and clones it into a vector system developed in-house, and then uses a general method known in the art. Introduced stable cell lines (Maniatis, T. et al . Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, New York, 1982).

2 is a graph showing the effect of CDHFA on cell growth and titer under temperature shift conditions. As shown in the graph shown in the upper part of FIG. 2, the control group showed little cell proliferation after temperature change, whereas in the experimental group to which the CDHFA was added, the growth of the cells was confirmed until the 6th day of culture. In addition, as shown in the graph shown in the lower part of Figure 2, the analysis of the expression level of etaneremteum, while the control group showed a yield of about 600 mg / L, while in the experimental group all the high yield of about 1 g / L or more I could see it.

Therefore, it was confirmed that the productivity of etanercept can be greatly improved by the addition of the CDHFA.

Example  2-2: Effects on Cell Growth

In the present invention, the cells were inoculated at a constant concentration (1.0 × 10 ⁷ cells / ml) in a basic culture medium to simulate the existing process, and the cells were cultured by changing the culture temperature. In this case, the time point for changing the temperature was set based on a late exponential growth phase of the cell growth cycle. However, some cell proliferation was observed even after the temperature change, and because of the possibility of change of cell growth cycle due to the change of medium composition, nucleosides (NS, 0.01 g / L adenosine, guanosine, cytidine, uridine, thymidine, 0.001) g / L hypoxanthin) was added to confirm the effect.

Specifically, in a 250 ml Erlenmeyer flask containing about 30 to 50 ml of medium containing CDHFA, fed-batch cultivation was carried out under conditions of 37 ° C., 8% carbon dioxide, and 100 rpm. , Cell proliferation was measured and compared (Fig. 3). In this case, as a control, a culture cultured using a medium to which CDHFA was not added was used.

3 is a graph showing the effects of CDHFA and nucleosides on cell growth. As shown in FIG. 3, the control group entered the stationary phase after 4 days of culture at which temperature was changed, whereas the experimental group to which CDHFA was added had active cell proliferation until the 6th day of culture. About 2 × 10 ⁷ cells / ml were reached. However, the effect by NS added with CDHFA was insignificant.

Therefore, it can be seen that when the culture medium to which CDHFA is added is used, it has an effect of promoting cell growth as well as the amount of recombinant protein produced.

Example  3: Development of mass production process

To develop a mass production process using a 5L NBS bioreactor (New Brunswick Scientific, NJ, USA), a total of 17 days of incubation was performed based on a survival rate of 80% (FIGS. 4 and 5).

Figure 4 is a graph showing the results of measuring the live cell number, survival rate, titer and specific productivity (q _p ) in NBS culture according to an embodiment of the present invention, Figure 5 is NBS culture according to an embodiment of the present invention It is a graph showing the results of measuring the concentration of glucose, lactate, ammonia and the weight molar osmolality at.

As shown in Figure 4, in terms of cell growth profile (cell growth profile), on the second day of culture reached a cell concentration of about 9 × 10 ⁶ cells / ml, at this time by changing the pH and temperature production phase (production phase) Switched to The maximum cell density was about 1.3 × 10 ⁷ cells / ml, and the cell concentration tended to decrease from day 6. In addition, the survival rate tended to decrease slightly from the 8th day of culture, and the survival rate was about 80% on the 17th day of the final culture. The expression level was about 1,200 mg / L on day 14 and about 1,400 mg / L on day 17.

In addition, as shown in Figure 5, the glucose concentration was controlled by the addition of the feeding medium (feeding media), the culture was maintained without the increase of lactate and ammonia showed a tendency to increase gradually with increasing the incubation period. The osmotic pressure was about 400 mOsm / kg at the end of the culture. This proved in the 5 L process study that the new process has a high productivity of more than 1,400 mg / L. This is a double expression level compared to the existing process without the addition of CDHFA.

This indicates that high cell growth and proliferation rate and high yield of protein production are possible even in the mass production of recombinant protein using the medium containing the amino acid additive CDHFA of the present invention.

Claims (11)

0.050 to 0.150 mM alanine, 0.450 to 1.350 mM arginine, 0.344 to 1.031 mM asparagine, 0.118 to 0.353 mM aspartic acid, 0.028 to 0.184 mM cysteine, 0.155 to 0.465 mM cystine, 0.395 to 1.184 mM glutamic acid, 0.040 to 0.120mM glycine, 0.143 to 0.429mM histidine, 1.145 to 3.435mM isoleucine, 1.527 to 4.580mM leucine, 1.134 to 3.401mM lysine, 0.749 to 2.246mM methionine, 0.515 to 1.545mM phenylalanine, A composition for addition of a culture medium for improving the productivity of a recombinant protein, consisting of 1.305 to 3.914 mM proline, 4.048 to 12.143 mM serine, 0.883 to 2.648 mM threonine, and 0.307 to 0.920 mM tyrosine.
The method of claim 1,
A composition that exhibits an effect of enhancing the growth or proliferation of an animal cell line.
The method of claim 2,
Wherein said animal cell line is a CHO cell line.
The method of claim 3,
Wherein said CHO cell line is a CHO-K1 cell line.
A culture medium comprising a composition for adding a culture medium according to any one of claims 1 to 4.
The method of claim 5,
Culture medium is characterized in that the glutamine is excluded.
The method of claim 5,
The culture medium is a medium characterized by serum free.
(Iii) culturing the transformant to produce the desired recombinant protein using the culture medium of claim 5, to obtain a culture; And
(Ii) recovering the desired recombinant protein from the culture obtained.
9. The method of claim 8,
Wherein said recombinant protein is a monoclonal antibody or an Fc containing fusion protein.
10. The method of claim 9,
Wherein said Fc-containing fusion protein is a TNFR: Fc fusion protein.
The method of claim 10,
Wherein said TNFR: Fc fusion protein is Etanercept.

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