Bio Process Additives Description The present invention relates to biosynthetic processes based on mammalian cell cultures using certain additives for improving the productivity, especially for the preparation of pharmaceutically active proteins such as antibodies and fragments of antibodies, as well as to cell culture media for mammalian cells containing said additive(s). In general, oxidative stress and other radical reactions can lead to damage at molecules such as proteins, nucleic acids or other small or large molecular weight molecules such as lipids, amino acids, sugars or the like, e.g. at double bonds, sulphur etc.

Natural antioxidants (Vitamins C & E, polyphenols, amino acids such as cysteine and methionine, peptides such as gluthatione and carnosine) scavenge reactive oxygen species (ROS) and are used in medicine, personal care and nutrition as well as in fermentation processes. There are also examples of synthetic antioxidants and radical scavengers being used in fermentation, however it has been reported that certain agents, such as radical scavengers of the nitroxide class, lead to programmed cell death (=apoptosis) especially of mammalian cells.

Sterically hindered and therefore metastable Nitroxide radicals, such as the 2,2,6,6- Tetramethyl-1-piperidinyloxy radical and 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxyl have already been examined in few single cases as additives to cell culture. US 2004/024025, for example, describes that TEMPO causes increased apoptosis in human cancer cells due to SAPK and p38 MAPK signal pathway modulation, leading to protein phosphorylation. Thus results available are rather disencouraging from adding sterically hindered nitroxyls to cell cultures. However N-oxyl (TEMPO) was also used in cell cultures [US2004/024025] in a concentration of 10mM to prevent apoptosis) In addition it was shown that N-OH Derivatives at relatively low concentration (5mM) show a low Cytoxicity for human dermal fibroplast cells (US-2008-305055, Baschong et al) US 5,462,946 mentions the use of various nitroxide compounds as antioxidants capable of protecting cells, tissues, organs and whole organisms against the deleterious effects of harmful oxygen-derived species generated during oxidative stress in phar- maceutical formulations. It does not specifically refer to the use in the manufacture of products in cell culture or by biosynthesis in cell free systems. Among many other possible uses, it is mentioned only that they can be used in stabilizing labile chemical compounds which undergo spontaneous degradation by generating free radicals in media which are already present in the media as such.

No disclosures have been made how to increase yields of protein products in manufacturing processes based on mammalian cells. There is a need for the development of improved and broadly applicable additives to enhance the productivity of mammalian cell cultures.

It has now been found that, surprisingly, protein yield in a fermentation process based on mammalian cells may nevertheless be greatly improved when certain nitroxides are added at defined levels during the process.

The invention thus primarily pertains to a biosynthetic process or method for the manufacture of a protein in and by a cell culture system comprising mammalian cells in a suitable medium, which process comprises keeping the presence of one or more radical scavenging and/or antioxidative agents selected from the group consisting of steri- cally hindered nitroxyls, sterically hindered hydroxylamines, or salts or adducts thereof, in the culture medium at a level from 1 to 5000, and especially 5 to 5000 micromol (sterically hindered nitrogen) per litre of the cell culture system through a part or all of the biosynthetic manufacturing process. Preferred concentrations of the sterically hindered nitroxyls, sterically hindered hydroxylamines, or salts thereof, are from the range 1 to 1500 micromol, especially 5 to 1500 micromol, of the agent (amount of sterically hindered nitrogen) per litre of the cell culture system. Also of importance is the dosage range of 1 to less than 1000 micromol, e.g. 1 to 800 micromol or especially 5 to 600 micromol of the agent (each based on sterically hindered nitrogen) per litre of the cell culture system. The sterically hindered nitroxyls and sterically hindered hydroxylamines are mainly selected from 2,2,6,6-tetraalkylpiperidine compounds, like those of the formula I

wherein the asterisk (*) denotes the sterically hindered nitrogen atom; R is hydrogen, C1-C8alkoxy, oxyl or hydroxy, especially oxyl or hydroxy, and R' is H, oxo, hydroxy or X-A, where X is O or NH and A is H or stands for a residue of a mono, di, tri or tetrava- lent carboxylic acid or hydroxycarboxylic acid, where further valences of the di, tri or tetravalent carboxylic acid or hydroxycarboxylic acid are free COOH, are anionic car- boxylate counterbalanced by cationic ammonium, alkaline or alkaline earth ions and/or carr further hindered nitroxyl or hydroxylamine moieties of the formula

, wherein R is as defined above. Oxo stands for a double bonded

oxygen (=0), oxyl denotes the oxygen radical (-0 ). Especially preferred species of the formula I are TEMPO, TEMPOL (see abbreviations explained further below), as well as salts or adducts thereof.

For example, it has been found in the present invention that the addition of a small amount (especially below 5mM) of sterically hindered nitroxides (such as TEMPO) and related compounds such as the corresponding hydroxylamines to a mammalian cellcul- ture media are effectively enhancing the yield of pharmaceutically relevant proteins. As an example, significant effects on DC-SIGN/lgG protein expression of both sterically hindered nitroxides TEMPO and TEMPOL in CHO-K1 cells at concentration below 5mM are observed.

Preferred sterically hindered nitroxyls and sterically hindered hydroxylamines are selected from compounds of the formula I wherein R is oxo or hydroxy, X is O and A is H or stands for a residue of acetic acid, glycolic acid, citric acid, lactic acid, oxalic acid, malonic acid, hydroxymalonic acid, succinic acid, maleic acid, glutaric acid, hydroxyglu- taric acid, sebacic acid.

Salts, and adducts, of the sterically hindered nitroxyls, sterically hindered hydroxylamines, 2,2,6,6-tetraalkylpiperidine compounds, are usually selected from with mineral or organic acids (such as hydrochloric acid, hydrobromic acid, acetic acid, glycolic acid, citric acid, lactic acid, oxalic acid, malonic acid, hydroxymalonic acid, succinic acid, maleic acid, glutaric acid, hydroxyglutaric acid, propane tricarboxylic acid, hydroxypro- pane tricarboxylic acid, ethylenediamine tetraacetic acid, sebacic acid, cinnamic acid, including free carboxylates and/or alkali carboxylates thereof especially in case of di-, tri- and tetracarboxylic acids whose acid functionalities are not fully counterbalanced by

The product obtainable according is one or more products selected from the group consisting of antibodies, hormones, vaccines, toxins, enzymes, proteins useful in cancer treatment other than those already mentioned, proteins useful in the treatment of bone disorders other than those already mentioned, batch proteins, interferons, inter- leukins, inerleukin receptor antagonists, blood factors, thrombolytic agents, hematopoietic growth factors, other growth factors, tumor necrosis factor, other proteins, nucleic acids, especially antisense therapeutics, ribozymes, gene therapeutics and siRNAs, and small molecules, especially sugars, amino acids, vitamins, steroids, antibiotics or antibiotics precursors, oligopeptides and lipids, and is obtained in free and/or in salt form; and wherein especially the protein prepared is selected from antibodies, antibody precursors and fragments of antibodies.

The process according to the invention is generally carried out using cell culture media known in the art of mammalian cell cultivation. The medium thus often contains one or more further active agents; agents may, for example, be selected from antibiotics, antioxidants and other radical scavengers, such as beta-lactames, glycopeptides, poly- ketides, aminoglycosides, polypeptides, chinolones, sulfonamides, vitamines, benzofu- ranone compounds, nitrones, phenolic antioxidants (see, for example, formula 1 of US- 2008-305055, especially compounds 7-33 of Table 1), natural antioxidants or radical scavengers such as tocopherols, coumestanes (such as compounds described in WO 08/1 10465); especially preferred as further components in the present cell culture media are antibiotics (such as penicillin, streptomycin), tocopherols (such as vitamin E, vitamin A). Further components, such as the above optional constituents, are often present in amounts ranging from 1 to about 10000 microgram / ml culture medium, for example 5 to 1000 microgram / ml culture medium.

Mammalian cells for cultivation according to the present process are generally selected from cell lines known in the art for biosynthetic processes; examples are ovary cells, myeloma cells, kidney cells, retina derived cells, B cells, hybridoma cells, more particularly Chinese hamster ovary cells (such as CHO-K1 cells), mouse myeloma cells, baby hamster kidney cells, green monkey kidney cells, human retina derived cells, human embryonic kidney cells, hybridoma cells; especially suitable are Chinese hamster ovary cells, and hybridoma cells (e.g. as known in the art as obtainable by the fusion of antibody-producing cells (B-cells) with myeloma cells).

The protein obtained according to the invention may conveniently be converted in a further step into a pharmaceutical or nutraceutical composition, e.g. by admixing it with one or more pharmaceutically and/or nutraceutically acceptable carrier materials. The conversion may be effected immediately from the culture medium, or preferably after workup such as isolation or concentration of the desired protein product by methods known in the art (chromatography, affinity chromatography, centrifugation, lyophiliza- tion etc.). The protein product obtainable according to the invention may be free and/or in form of a salt.

The invention includes the use of one or more of the antioxidative agents selected from the group consisting of sterically hindered nitroxyls, sterically hindered hydroxylamines, or salts or adducts thereof, as described above, for enhancing the quantity and/or the quality of the product, especially protein, obtainable in a biosynthetic process of manufacture based on mammalian cells, said use comprising adding said additive(s) into a medium in which a cell culture of mammalian cells is comprised. Most preferred mammalian cells are Chinese hamster ovary cells (such as CHO-K1 cells) and hybridoma cells. The invention further comprises using one or more of said additives within a medium in which a cell culture of said mammalian cells is comprised. Further included is a method of use, or the use, of one or more of the above additives for the reduction of detrimental effects of radicals in the cell fermentation process for the product. The invention further pertains to a mammalian cell culture, typically of cell lines known in the art for biosynthetic processes such as ovary cells, myeloma cells, kidney cells, retina derived cells, B cells, hybridoma cells, more particularly Chinese hamster ovary cells, mouse myeloma cells, baby hamster kidney cells, green monkey kidney cells, human retina derived cells, human embryonic kidney cells, hybridoma cells, in liquid or solid form, which cell culture comprises one or more sterically hindered nitroxyls, sterically hindered hydroxylamines, compounds of formula I , or salts or adducts thereof, without or with addition of one or more other components (such as antibiotics etc.) as described above. Most preferred mammalian cells in said culture are Chinese hamster ovary cells (such as CHO-K1 cells) or hybridoma cells. In consequence, the invention includes a cell culture medium for said mammalian cells, or a main component (like concentrate, kit, carbon source, energy source, or in case of a complex medium the extract, e.g. yeast extract etc.) of said mammalian cell culture medium, which com- prises one or more radical scavenging and/or antioxidative additives as defined above, especially of formula I like TEMPO and/or TEMPOL.

Abbreviations

FCS fetal calf serum

M mol/litre

TEMPO 1-oxyl-4-hydroxy-2,2,6,6-tetramethylpiperidine

TEMPOL 1 ,4-dihydroxy-2,2,6,6-tetramethylpiperidine

TEMPOL adduct is the salt comprising 3 molecular units of TEMPOL and 1 molecular unit of 2-hydroxy-propane-1 ,2,3-tricarboxylic acid

Tris Tris-hydroxymethyl-aminomethane

Tween20 polysorbate 20 (polyoxyethylene-sorbitan monolaurate of 20 ethylene units)

Example 1

CHO-K1 cells producing DC-SIGN/lgG are cultivated in F-12 nutrient mixture medium (cat. 21765, Gibco) supplemented with 10% FCS (cat. 10500, Lot. 07Q2396K, Gibco), optionally with further addition of penicillin (100 U)/streptomycin (100 μg/ml) (cat.

P4333, Sigma). The cultures are performed using TC-flasks in a HeraCell incubator (Heraeus) at 37°C, 5% C02 and 80% humidity. At 80% confluence, TEMPO or TEMPOL suspended in the culture medium (75 mM) and sterile filtrated are added to the cells at 10 μΜ, 100 μΜ and 1 mM concentrations. TEMPOL adduct is added in 10 mg/L, 100 mg/L, 250 mg/L and 500 mg/L concentrations (corresponding to 42 μΜ, 421 μΜ, 1.05 mM and 2.1 mM with respect to TEMPOL).

The cells are inspected daily by microscopy. The medium is collected after 1 week of cell culture, filtrated (0.22 μΜ), mixed (1 :1) with loading buffer (50 mM tris-HCI pH 7.6, 150 mM NaCI and 0.05% Tween20) and loaded on a protein A column for affinity purification. Two washing steps are performed, the first with 10 bed volumes of loading buffer and the second with 2 bed volumes of 5 mM NH40Ac (pH 5.0). The protein is eluted with 10 mL of 500 mM NH40Ac (pH 3.4). The collected fractions are immediately neutralized with 2.5 M Tris-HCI solution and concentrated by ultrafiltration (Ami- con, YM 50). Results of microscopic inspections:

No difference between cells growing in the presence or in the absence of antibiotics is observed. Cells grow normally during the first week in presence of TEMPO or TEM- POL. In the presence of TEMPOL adduct, cells grow normally during the first 5 days; after one week, cells start to detach from the TC-flask forming aggregates of dead cells.

Protein Quantification:

The protein expression in the presence of 10, 100 and 1000 μΜ concentrations of TEMPO, TEMPOL with or without antibiotics (penicillin/streptomycin) and in the presence of TEMPOL adduct in 10 mg/L, 100 mg/L, 250 mg/L and 500 mg/L concentrations is determined by HPLC.

The result shows a significant effect of TEMPO, TEMPOL and TEMPOL adduct on the protein expression in CHO-K1 cells. In the presence of penicillin/streptomycin, TEMPO improves the protein expression especially at 100 μΜ and 1 mM concentrations; with addition of TEMPOL, the protein expression is increased at 10 and 100 μΜ (Figure 1). The same effect of TEMPOL on the protein expression is observed in the absence of antibiotics in the culture medium (Figure 2).

The presence of TEMPOL adduct improves the protein expression especially at 100 and 250 mg/L concentrations (Fig. 3). Example 2: The procedure of example 1 is repeated using Hybridoma cells instead of CHO-K1 cells. The results show improved antibody production.

Brief description of Figures: Figure 1 : Evaluation of DC-SIGN expression in CHO-K1 cells in the presence of

TEMPO and TEMPOL. K = positive control; T = TEMPO; TL = TEMPOL; 10,100 and 1000 μΜ; (+) presence of penicillin/streptomycin in the culture medium.

Figure 2: Evaluation of DC-SIGN expression in CHO-K1 cells in the presence of TEMPO and TEMPOL. K = positive control; T = TEMPO; TL = TEMPOL; 10,100 and 1000 μΜ; (-) absence of penicillin/streptomycin in the culture medium. Figure 3: Evaluation of DC-SIGN expression in CHO-K1 cells in the presence of TEM- POL adduct at 10, 100, 250 and 500 mg/L, absence of penicillin/streptomycin in the culture medium.