FIELD OF THE INVENTION
The present invention relates to a method of producing soluble TNFR:Fc by mammalian cell cultures. The method comprises feeding the cells with a primary feed and a secondary feed, wherein the secondary feed includes galactose and mannose. As a result, the sialylation of TNFR:Fc is increased in comparison to the sialylation of TNFR:Fc obtained by standard procedures. The present invention also relates to the use of a specific composition for increasing the sialylation of TNFR:Fc, to a method of isolating soluble TNFR:Fc from cell supematants using a fixed sequence of purification steps and to recombinant TNFR:Fc obtained by such methods.
BACKGROUND OF THE INVENTION
In the past, various methods were used for the cell culture manufacturing of recombinant glycoproteins or monoclonal antibodies. The ever-increasing demand of monoclonal antibodies and other recombinant proteins in properly glycosylated forms have increased the prospects of cell culture process development. In general, protein expression levels in mammalian cell culture-based systems are considerably lower than in microbial expression systems, such as bacterial or yeast expression systems. However, bacterial and yeast cells are limited in their ability to optimally express high molecular weight proteins, to properly fold a protein having a complex tertiary structure, and/or to provide the post-translational modifications necessary for the maturation of an expressed glycoprotein, thereby effecting its properties and biological activity. The degree and completeness of the glycosylation structure of a polypeptide product is an important product quality parameter, with sialic acid content commonly used as a measure of glycoprotein quality.
Enbrel (Etanercept) is a recombinant fusion protein comprising the extracellular domain of the human tumor necrosis factor receptor (TNFR) superfamily, member IB (p75) and the Fc domain of human IgGl. Enbrel is an important scientific advance that in many people has been shown to reduce the signs and symptoms of rheumatoid arthritis, polyarticular-course juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and psoriasis. TNF is a naturally occurring cytokine that is involved in normal inflammatory and immune responses. It plays an important role in the inflammatory processes of rheumatoid arthritis (RA), polyarticular-course juvenile rheumatoid arthritis (JRA) and the resulting joint pathology. Elevated levels of TNF are found in the synovial fluid of RA patients.

OBJECTIVE OF THE INVENTION
It was an object of the present invention to provide a highly-efficient method of producing soluble TNFR:Fc by mammalian cell cultures, with TNFRrFc being characterized by an increased and enhanced sialic acid content. Furthermore, it was an object of the present invention to provide a method for isolating recombinant soluble TNFR:Fc fusion protein from cell culture supernatant, wherein the method is characterized by high yield and purity of the end product.
DESCRIPTION OF ACCOMPANYING FIGURES
Figure 1 shows the cell growth pattern of CHO cells without a primary and secondary feed (A), CHO cells fed with a primary feed consisting of Cell Boost 5® growth supplement (HyClone, Logan, UT, USA) and a secondary feed consisting of a 1.5 M solution of galactose/marmose (1:1) (B), and CHO cells fed with a primary feed consisting of Cell Boost 5® growth supplement and a secondary feed consisting of 1 M galactose/mannose (1:1) in Cell Boost 5® growth supplement (C);
Figure 2 shows the progression of nutrient consumption and lactate accumulation during a fermentation run according to the present invention;
Figure 3 shows the progression of cell growth and cell viability during a fermentation run according to the present invention;
Figure 4 shows the progression of protein expression during a fermentation run according to the present invention;
Figure 5 shows chromatograms of the affinity chromatography step used for isolating soluble TNFR:Fc from cell culture supematants;
Figure 6 shows the chromatograms of the cation exchange chromatography step (A) and the subsequent anion exchange chromatography step (B) used for the isolation/purification of soluble TNFR:Fc;

Figure 7 shows the electrophoretic pattern of formulated TNFR:Fc produced and isolated according to the present invention (Lane 3) in comparison to the reference medicinal product (RMP) Enbrel® (API-Etanercept) (Lane 2) and a molecular weight marker (Lane 1) on a 12% reducing silver stained gel (A) and a 8% non-reducing silver stained gel (B);
Figure 8 shows western blotting of formulated TNFR:Fc produced and isolated according to the present invention (Lane 3) in comparison to the reference medicinal product (RMP) Enbrel® (API-Etanercept) (Lane 2) and a molecular weight marker (Lane 1) imder reducing (left panel) and non-reducing (right panel) conditions;
Figure 9 shows isoelectric focusing of formulated TNFR:Fc produced and isolated according to the present invention (Lane 2) and of the reference medicinal product (RMP) Enbrel® (API-Etanercept) (Lane 1);
Figure 10 shows 2D gel electrophoresis of formulated TNFR:Fc produced and isolated according to the present invention (B), referred to as drug substance (DS), and of the reference medicinal product (RMP) Enbrel® (API-Etanercept) (A);
Figure 11 shows the HPLC profiles of formulated TNFR:Fc produced and isolated according to the present invention (B) and of the reference medicinal product (RMP) Enbrel® (API-Etanercept) (A);
Figure 12 shows the hydrophobicity profiles (reverse phase HPLC) of formulated TNFR:Fc produced and isolated according to the present invention (B) and of the reference medicinal product (RMP) Enbrel® (API-Etanercept) (A);
Figure 13 shows the size exclusion profiles of formulated TNFR:Fc produced and isolated according to the present invention (B) and of the reference medicinal product (RMP) Enbrel (API-Etanercept) (A);
Figure 14 shows MALDI-TOF analysis of formulated TNFR:Fc produced and isolated according to the present invention (B) and of the reference medicinal product (RMP) Enbrel® (API-Etanercept) (A);

Figure 15 shows the deconvoluted mass spectral profiles obtained by MALDI-TOF analysis of the tryptic digests of formulated TNFR:Fc produced and isolated according to the present invention (B) and of the reference medicinal product (RMP) Enbrel® (API-Etanercept) (A);
Figure 16 shows HPLC-based tryptic peptide mapping analysis of formulated TNFR:Fc produced and isolated according to the present invention (B) and of the reference medicinal product (RMP) Enbrel® (API-Etanercept) (A);
Figure 17 shows HPLC-based glycan analysis of formulated TNFR;Fc produced and isolated according to the present invention (B) and of the reference medicinal product (RMP) Enbrel® (API-Etanercept) (A);
Figure 18 shows UV spectroscopy analysis of formulated TNFR:Fc produced and isolated according to the present invention (B) and of the reference medicinal product (RMP) Enbrel® (API-Etanercept) (A);
Figure 19 shows CD spectroscopy analysis of the tertiary structure of formulated TNFR:Fc produced and isolated according to the present invention (B) and of the reference medicinal product (RMP) Enbrel® (API-Etanercept) (A); and
Figure 20 shows a cytotoxicity rescue assay using the reporter cell line L929 with formulated TNFR:Fc produced and isolated according to the present invention (DS) and the reference medicinal product (RMP) Enbrel® (API-Etanercept).
DETAILED DESCRIPTION OF THE INVENTION
The objects of the present invention are solved by a method for producing soluble TNFR:Fc,
comprising:
culturing mammalian cells which produce soluble TNFR;Fc,
feeding the cells with a primary feed over a first time period, during which first time period
the cells are cultured at a first temperature, followed by
feeding the cells with a secondary feed, which is different fi-om the primary feed, over a
second time period, during which second time period the cells are cultured at a second
temperature.

wherein the primary feed comprises glucose, lipids, amino acids, vitamins, trace elements, cholesterol and growth factors in an aqueous solvent, and
wherein the secondary feed comprises glucose, lipids, amino acids, vitamins, trace elements, cholesterol, growth factors, galactose and mannose in an aqueous solvent.
In one embodiment of the present invention, the galactose and mannose in the secondary feed are each present at a concentration of about 0,2 to 1 mol/L, preferably 0,4 to 0,8 mol/L, more preferably about 0,4 to 0,6 mol/L.
In one embodiment, the initial seeding density of the culture of mammalian cells which produce soluble TNFR:Fc is about 0,4 to 0,6 x 106 cells/mL of culture.
In one embodiment, the first time period starts immediately after inoculation of the culture and ends 40 to 180 hours, preferably 60 to 160 hours, more preferably 80 to 160 hours after inoculation of the culture.
In one embodiment, the cell density is > 3,5 x 106 cells/mL of culture at the start of the second time period.
In one embodiment, the second time period starts 40 to 180 hours, preferably 60 to 160 hours, more preferably 80 to 160 hours after inoculation of the culture.
In one embodiment, the second time period ends, i.e. the cell culture is stopped, when cell viability drops below 70%. Preferably, the culture is stopped at a cell viability of 60 to 70%.
In one embodiment, the cells are fed with the primary feed or secondary feed about every 22 to 26 hours, preferably about every 24 hours over the first time period and second time period.
In one embodiment, the first temperature is about 36 to 37 °C.
In one embodiment, the second temperature is about 30 to 33 °C.
In one embodiment, the second temperature is 32 +/- 0.5 °C and is fiirther reduced to a third temperature of 31 +/- 0.5 °C after 260 to 290 hours after inoculation of the culture.

In one embodiment, the mammalian cells are selected from the group comprising CHO, Hybridoma, NSO, HEK 293, MDCK, BHK cells, mouse L-cells and myeloma cell lines, such as J558L and SP2/0. In a preferred embodiment, the mammalian cells are CHO cells.
In one embodiment, the sialylation of TNFR:Fc is increased compared to sialylation of TNFR:Fc produced without feeding the cells with a primary feed and with a secondary feed. Preferably, the sialylation of TNFR:Fc is increased by at least 20 %, more preferably at least 30 %.
The objects of the present invention are also solved by a composition comprising glucose, lipids, amino acids, vitamins, trace elements, cholesterol, growth factors, galactose and mannose in an aqueous solvent, wherein galactose and mannose are each present at a concentration of about 0,2 to 1 mol/L, preferably 0,4 to 0,8 mol/L, more preferably about 0,4 to 0,6 mol/L.
The objects of the present invention are also solved by the use of a composition comprising
glucose, lipids, amino acids, vitamins, trace elements, cholesterol, growth factors, galactose
and marmose in an aqueous solvent for increasing the sialylation of TNFR:Fc produced in
mammalian cells,
wherein, in the composition, galactose and mannose are each present at a concentration of
about 0,2 to 1 mol/L, preferably 0,4 to 0,8 mol/L, more preferably about 0,4 to 0,6 mol/L,
wherem the composition is added about every 22 to 26 hours, preferably about every 24 hours
to a culture of mammalian cells which produce soluble TNFR:Fc,
wherein the culture has an initial seeding density of about 0,4 to 0,6 x 106cells/mL of culture,
and
wherein the composition is added to the cells, when the cells have a cell density of > 3,5 x
106 cells/mL of culture.
In a preferred embodiment, the composition contains 60 to 75%, preferably 65%, of a 1.5 M solution of galactose/mannose (1:1) and 25 to 40 %, preferably 35%, of 3x Cell Boost 5®.
The objects of the present invention are further solved by a method for isolating soluble TNFR:Fc, said method comprising:

performing the method as described above;
filtering the cell culture of mammalian cells which produce soluble TNFR:Fc, resultant from
performing the method as described above, and
subjecting the filtrate to a fixed sequence of purification steps of affinity chromatography,
followed by cation exchange chromatography, followed by anion exchange chromatography.
In a preferred embodiment, affinity chromatography is performed with protein A resin. Culture supematants/filtrates are preferably concentrated and clarified before chromatography.
The objects of the present invention are also solved by a recombinant soluble TNFR:Fc produced according to the method as described above and isolated according to the method as described above.
The term "soluble TNFR:Fc", as used herein, is meant to refer to the soluble fusion protein consisting of tumor necrosis factor alpha receptor (TNFR) and human IgG Fc, which is secreted from the cells into the cell culture medium. "Mammalian cells which produce soluble TNFR:Fc" refers to mammalian cells, which contain a plasmid or expression vector comprising a nucleotide sequence coding for the TNFR:Fc fusion protein. Alternatively, a nucleotide sequence coding for the TNFR:Fc fusion protein can be incorporated into the genome of a host cell. A variety of mammalian cell expression systems are available for either transient expression or stable expression of recombinant genes. These systems are known to a person skilled in the art. Chinese hamster ovary (CHO) cell stable expression systems (CHO SES) are used for the expression of the TNFR:Fc fusion protein according to the present invention. Moreover, baby hamster kidney (BHK) cells, human embryonic kidney (HEK) 293 cells, mouse L-cells, and myeloma cell lines like J558L and Sp2/0, etc., can also be employed as hosts for the establishment of stable transfectants.
The phrase "culturing mammalian cells", as used herein, refers to the culturing of mammalian cells under conditions that allow for protein production. For example, cells can be cultured by batch, semi fed-batch, fed-batch, perfusion and continuous fermentation. In a preferred embodiment, the cells are cultured by a fed-batch process.

The term "basal medium", as used herein, refers to starting medium to which cells are added to begin the culture (t = 0 hours). Non-limiting examples for suitable basal media are MAM-PF®2 (BioConcept, Allschwil, Switzerland), Power CH02® and Power CH03® (Lonza Biologies, Basel, Switzerland). The basal medium can be supplemented with one or more supplements, such as L-glutamine and Pluronic F68. In a preferred embodiment, the basal medium is MAM-PF®2 supplemented with 2 to 4 mM stable L-glutamine and 0.15% Pluronic F68.
The term "inoculation" refers to the addition of cells to a basal medium (with our without supplements) to begin the culture (t = 0 hours).
The phrase "feeding the cells", as used herein, refers to adding a substance to the cell culture after inoculation. The term "feed", as used herein, thus refers to any addition of any substance made to a cell culture after inoculation. Preferably, feeding the cells with the primary feed is started immediately after inoculation, i.e. the first time period starts immediately after inoculation of the culture (t = 0 hours). The term "immediately after inoculation", as used herein, means within 30 min, preferably within 15 min, after inoculation of the culture. For the primary feed, the preferred ratio between feed and total culture volume is 1:150 to 1:200. For the secondary feed, the preferred ratio between feed and total culture volume is 1:80 to 1:120.
The term "cell density", as used herein, refers to the number of cells per unit volume. There are numerous tests and methods for measuring cell density, which are well known to a person skilled in the art.
The term "cell viability", as used herein, is meant to refer to the number of living cells in relation to the total number cells in a sample. There are numerous tests and methods for measuring cell viability, which are well known to a person skilled in the art. Testing for cell viability usually involves looking at a sample cell population and staining the cells or applying chemicals to show which are living and which are dead. When a sample is stained with various dyes or treated with chemicals, it is then subject to microscopic examination to evaluate cell viability.

The primary and secondary feeds according to the present invention comprise glucose, lipids (e.g. linoleic acid, oleic acid, arachidonic acid), amino acids (e.g. all essential amino acids, and generally all 20 amino acids), vitamins (e.g. ascorbic acid, biotin), trace elements (e.g. inorganic compounds or naturally occurring elements, that are typically required at very low concentrations, typically in the micromolar range), cholesterol and growth factors (e.g. insulin, transferring, epidermal growth factor) in an aqueous solvent. The primary feed can be a commercially available cell growth supplement, such as Cell Boost 5®, Cell Boost 2®, Cell Boost 6® (HyClone, Logan, UT, USA) and CHO feed supplement (Sigma Aldrich, USA). Preferably, the primary feed does not contain any galactose and mannose. The secondary feed, which contains galactose and mannose at a concentration of about 0,2 to 1 mol/L, preferably 0,4 to 0,8 mol/L, more preferably about 0,4 to 0,6 mol/L, can be based on a conmiercially available cell growth supplement, such as Cell Boost 5® (HyClone, Logan, UT, USA). For example, a concentrate of the growth supplement, such as 3x Cell Boost 5®, can be mixed with a concentrated 1:1 solution of galactose/mannose. In a particular preferred embodiment of the present invention, the primary feed is Cell Boost 5®, and the secondary feed contains 60 to 75%, preferably 65%, of a 1.5 M solution of galactose/mannose (1:1) and 25 to 40 %, preferably 35%, of 3x Cell Boost 5®.
The present invention provides an improved process for the manufacture of recombinant TNFR:Fc by mammalian cell cultures. In particular, the invention provides a method that allows high cell density cell culture processes and maintenance of high cell viability for a longer culture period. In addition, the invention helps in achieving proper glycosylation of recombinant TNFR:Fc, i.e. the addition of the required number of sialic acid residues with the correct linkages, which is essential for its biological activity . The cell culture manufacturing process starts with seeding the bioreactor at a predefined cell density in a chemically defined medium. The culture is fed in two stages: primary feeding, which is designed to achieve high cell growth, and secondary feeding, which is designed to maintain high cell viability and to achieve hyperglycosylation of the recombinant TNFR:Fc. With the at least two temperature shifts, a high protein yield with a concomitant high sialic acid content can be achieved. Furthermore, the invention provides a bioreactor operation procedure for the manufacture of recombinant TNFR:Fc. The present invention also provides a method for the rapid and efficient recovery of recombinant TNFR:Fc from cell culture supernatant or cell culture medium by means of affinity chromatography, followed by cation and anion exchange chromatography. The fixed sequence of purification steps (affinity chromatography - cation

exchange chromatography - anion exchange chromatography) resuhs in high yield and homogeneity of the end product.
The invention is now further described by means of the following examples, which are supposed to illustrate, but not to limit the present invention:
Example 1: Small-scale cell cultures of CHO cells producing soluble TNFR:Fc
All the experiments were carried out in 125 mL shake flasks with an initial total culture volume of 15 mL in each flask. MAM-PF®2 (BioConcept, AUschwil, Switzerland) with 2mM stable L-glutamine was used as the basal culture medium. The flasks were seeded at a seeding density of 0.5 x 106 cells/mL. Prior to seeding, the cells were harvested from a starting culture at log phase and centrifuged at 1000 rpm for 5 min. The cell pellet was resuspended in 3mL MAM-PF®2 supplemented with 2mM stable glutamine and 0.15% Pluronic F68. 1 mL of the resuspended cell suspension was added to each of the pre-labeled shake flasks. After inoculation, all the three flasks were transferred to a CO2 incubator shaker at temperature of 37°C, 5% CO2 and 100 rpm. Primary and secondary feeds as shown in Table 1 were added to the cultures of experiments # 2 and # 3 according to the regimen shown in Tables 2 to 4 (right column). After preparation, the feeds were stored at 2-4 °C. The culture flasks were sampled every 24/48 hours and the samples were assessed for cell count/cell viability (Tables 2 to 4). The experiment was terminated at 192 hrs of culture age, and the samples were analyzed for sialic acid content using the resorcinol method (Table 5). In this method, addition of HCl releases the bound sialic acid present in the molecule, which in turn reacts with resorcinol-copper sulphate to form a blue coloured complex. The degree of staining is proportional to the concentration of the sialic acid present in a sample. Figure 1 shows that the use of a feeding regimen according to the present invention led to a significantly increased cell count and cell viability at later culture stages. Furthermore, as shown in Table 5, a feeding regimen according to the present invention used in experiment # 3 significantly increased the glycosylation of recombinant TNFR:Fc.

Example 2: Fermentation of CHO cells producing soluble TNFR:Fc
Before seeding, the bioreactor was assembled and sterilized by autoclaving at 121 C for 60 min. After sterilization, the bioreactor was charged with 7000 mL of commercially available animal component free, chemically defined media (MAM-PF®2 supplemented with 2mM stable L-glutamine and 0.15% Pluronic F68). Afterwards, the bioreactor was kept under positive pressure with an air flow rate of 0.2 L per min. The bioreactor was aerated over night for 100% air saturation. The d02 electrode was calibrated after stabilization of the dissolved oxygen value. A sterile connection between the seed bottle and the seed port on the bioreactor head plate was created. The seed was then aseptically transferred to the bioreactor using a peristaltic pump. The bioreactor was seeded with a seeding density of 0.5 x 106 cells/mL. After seeding, the bioreactor was allowed to run with the following pre-set parameters: pH: 6.9-7.2;
dOi: 30 - 60% of air saturation; Temperature: 30 - 38°C; Stir speed: 70-80 rpm.

The bioreactor was sampled every 24/48 hours for in-process quality control analysis. The bioreactor process was a fed-batch process with feeding of different nutrients at definite culture stages. During the first 120 hrs of culture time, the bioreactor was daily fed with 60 mL of primary feed that comprised glucose, lipids, amino acids, vitamins, trace elements, cholesterol and growth factors (Cell Boost 5®). Starting from 144 hrs of culture age, the bioreactor was daily fed with 100 mL of secondary feed with about 0.5 mol/L galactose and about 0.5 mol/L mannose in addition to glucose, lipids, amino acids, vitamins, trace elements, cholesterol and growth factors (Cell Boost 5®). During first 120 hrs of culture age the bioreactor was operated with the following pre-set and controlled parameters: pH: 7.1 ±0.1;
dOi: 30 - 60% of air saturation; Temperature: 36 - 37°C; Stir speed: 70-80 rpm.
From 120 to 288 hrs, the bioreactor was operated with the following parameters:
pH: 6.9 ±0.05;
d02:30 - 60% of air saturation;
Temperature: 32 ± 0.5 °C;
Stir speed: 70-80 rpm.
From 288 hrs till the harvest, the bioreactor was operated with the following parameters:
pH: 6.9 ±0.05;
dOa: 30 - 60% of air saturation;
Temperature: 31 ±0.5°C;
Stir speed: 70-80 rpm.
The cells were harvested at a cell viability of 60 - 70%. The growth pattern, protein expression profile and nutrient consumptions are depicted in Figures 2,3 and 4.

Example 3: Isolation of soluble TNFR:Fc
Clarification of the cell culture harvest was carried out by using a cellulose disposable filter with 650-1000 cm effective filtration area and with an operating pressure of not more than 30 psi. The filtrate was checked for turbidity and target protein content. The clarified harvest was diafiltered and buffer exchanged with Tris buffer pH 7.2 - 7.6 using a 30 kDa-TFF membrane at a Trans Membrane Pressure of 5-10 psi. Protein A affinity chromatography was performed with a column of 30 mm in diameter, with Tris buffer pH 7.2 - 7.6 as equilibration buffer. After the sample was loaded on to the column, it was washed with equilibration buffer, followed by 50 mM Tris-HCl, 250 mM NaCl pH 7.4 buffer solution. The protein of interest was eluted with citrate buffer (Figure 5). The eluate was held for 45 -60 min at acidic pH at room temperature for virus inactivation and then neutralized. The protein A eluate fi-action of Runl and Run 2 was pooled and buffer exchanged with Tris buffer pH 6.8 - 7.2 using a 30 kDa-TFF membrane at a Trans Membrane Pressure of 5-10 psi. Cation exchange chromatography in negative binding mode was carried out with a column at an operational flow rate of 10 mL/min. The column was equilibrated with Tris buffer pH 6.8-7.2. The protein of interest was collected in the flow through. This step was used for the removal of process related impurities like leachate protein A, host cell DNA and host cell protein. (Figure 6 A). Thereafter, the flow through was filtered for virus removal using a viral removal filter having an effective filtration area of 0.01 m2. Anion exchange chromatography was carried out with the nanofiltrate after equilibrating the column with Tris buffer pH 6.8-7.2. The protein of interest was eluted with elution buffer using a NaCl salt gradient. This step was used for the removal of process related impurities like host cell DNA and host cell protein (Figure 6 B). The eluate was buffer exchanged and concentrated using a 30 kDa-TFF membrane at a Trans Membrane Pressure (TMP) of 5 - 10 psi . The buffer exchanged protein solution was filtered using 0.2 μm filter. Isolated TNFR:Fc was formulated using formulation buffer containing 10 mg/mL sucrose, 5.8 mg/mL of sodium chloride, 5.3 mg/mL of L-arginine hydrochloride, 2.6 mg/mL sodium phosphate monobasic monohydrate and 0.9 mg/mL sodium phosphate dibasic anhydrous) and adjusted to pH 6.3±0.2. Formulated TNFR:Fc that was produced and isolated according to the present invention is herein also referred to as drug substance (DS) or active pharmaceutical ingredient.

Example 4: Characterization of isolated/formulated TNFR:Fc
Silver staining of a 12% reducing SDS PAGE gel revealed a single band at -75 kDa, matching the band of the reference medicinal product (RMP) Enbrel® (AFI-Etanercept) (Figure 7 A). An 8% non-reducing SDS-PAGE showed a single band corresponding to approximately 150 kDa, which was consistent with the RMP (Figure 7 B). Western blot analysis showed an immune-specificity comparable to that of the RMP under both reducing and non-reducing conditions (Figure 8). Isoelectric focusing showed that the isoelectric point of the formulated TNFR:Fc was consistent with that of the RMP (Figure 9). The 2-dimensional gel electrophoresis profile of the purified drug substance also corresponded to that of the RMP (Figure 10). Protein A HPLC revealed a retention time of 6.3 min for the drug substance, which matched the retention time of the RMP (Figure 11). Reverse phase HPLC also revealed an identical retention time for the purified drug substance and the RMP (Figure 12). Size exclusion chromatography for determination of the oligomeric state revealed a retention time of 6,9 min (% of main peak is 100) for the drug substance, which was identical to that of the RMP (Figure 13). Intact molecular mass estimation performed by high-sensitivity MALDI-TOF MS analysis revealed a molecular mass of ~ 145 kDa for both the isolated/formulated TNFR:Fc and the RMP (Figure 14). Peptide mass fingerprinting by MALDI-TOF MS revealed a high degree of similarity between the drug substance and the RMP (Figure 15). Peptide mapping using HPLC showed corresponding profiles for the drug substance and the RMP (Figure 16). Identification of cleaved glycans by HPLC revealed comparable glycosylation profiles for the purified drug substance and the RMP (Figure 17). UV spectral analysis showed an essentially identical primary structure profile for the purified drug substance and the RMP (Figure 18). CD spectral analysis demonstrated an essentially identical tertiary structure for the purified drug substance and the RMP (Figure 19 A and B). A cytotoxicity rescue assay with the reporter cell line L929 derived from a murine sarcosoma showed that the degree of the observed TNF alpha induced cytotoxicity was inversely proportional to the amount of the RMP and the drug substance (Figure 20). The potency value of the sample was calculated using CFR 21/part 11 compliance with parallel line assay (PLA) software.

1. Method for producing soluble TNFR:Fc, comprising:
culturing mammalian cells which produce soluble TNFR:Fc,
feeding the cells with a primary feed over a first time period, during which first time
period the cells are cultured at a first temperature, followed by
feeding the cells with a secondary feed, which is different from the primary feed, over
a second time period, during which second time period the cells are cultured at a
second temperature,
wherein the primary feed comprises glucose, lipids, amino acids, vitamins, trace
elements, cholesterol and growth factors in an aqueous solvent, and
wherein the secondary feed comprises glucose, lipids, amino acids, vitamins, trace
elements, cholesterol, growth factors, galactose and marmose in an aqueous solvent.
2. Method according to claim 1, wherein the galactose and mannose in the secondary feed are each present at a concentration of about 0,2 to 1 mol/L, preferably 0,4 to 0,8 mol/L.
3. Method according to claim 1 or 2, wherein the initial seeding density of the culture of mammalian cells which produce soluble TNFR:Fc is about 0,4 to 0,6 x 106 cells/mL of culture.
4. Method according to any of the foregoing claims, wherein the first time period starts immediately after inoculation of the culture and ends 40 to 180 hours, preferably 60 to 160 hours, more preferably 80 to 160 hours after inoculation of the culture.
5. Method according to any of the foregoing claims, wherein the cell density is > 3,5 x 106 cells/mL of culture at the start of the second time period.
6. Method according to any of the foregoing claims, wherein the second time period ends when cell viability drops below 70%.
7. Method according to any of the foregoing claims, wherein the cells are fed with the primary feed or secondary feed about every 22 to 26 hours, preferably about every 24 hours over the first time period and second time period.

8. Method according to any of the foregoing claims, wherein the first temperature is about 36 to 37 °C.
9. Method according to any of the foregoing claims, wherein the second temperature is about 30 to 33 °C.
10. Method according to any of the foregoing claims, wherein the mammalian cells are selected from the group comprising CHO, Hybridoma, NSO, HEK 293, MDCK, BHK cells, mouse L-cells and myeloma cell lines, such as J558L and SP2/0.
11. Method according to any of the foregoing claims, wherein the sialylation of TNFR:Fc is increased compared to sialylation of TNFR:Fc produced without feeding the cells with a primary feed and with a secondary feed.
12. Method to claim 11, wherein the sialylation of TNFR:Fc is increased by at least 20 %, more preferably at least 30 %.
13. Use of a composition comprising glucose, lipids, amino acids, vitamins, trace elements, cholesterol, growth factors, galactose and mannose in an aqueous solvent for increasing the sialylation of TNFR:Fc produced in mammalian cells,
wherein, in the composition, galactose and mannose are each present at a
concentration of about 0,2 to 1 mol/L, preferably 0,4 to 0,8 mol/L,
wherein the composition is added about every 22 to 26 hours, preferably about every
24 hours to a culture of mammalian cells which produce soluble TNFR:Fc,
wherein the culture has an initial seeding density of about 0,4 to 0,6 x 106 cells/mL of
culture, and
wherein the composition is added to the cells, when the cells have a cell density of >
3,5x106 cells/mL of culture.
14. Method for isolating soluble TNFR:Fc, said method comprising:
a) performing the method according to any of claims 1 to 12;
b) filtering the cell culture of mammalian cells which produce soluble TNFR:Fc,
resultant from step a), and

c) subjecting the filtrate to a fixed sequence of purification steps of affinity chromatography, followed by cation exchange chromatography, followed by anion exchange chromatography.
15, Recombinant soluble TNFR:Fc produced according to any of claims 1 to 12 and isolated according to claim 14.