Modifying blue color formation in bioprocesses

Abstract

The present invention relates to the field of cell culture and recombinant protein production in mammalian cells. It specifically relates to a method for reducing formation of blue-colored compounds in a fed batch medium by using a high cysteine concentration and a reduced tryptophane concentration in the feed medium.

Description

Modifying Blue Color Formation in BioprocessesFIELD OF THE INVENTION

[0001] The present invention relates to the field of cell culture and recombinant protein production in mammalian cells. It specifically relates to a method for reducing formation of blue-colored compounds in a fed batch process by using a high cysteine concentration and a reduced tryptophane concentration in the feed medium.BACKGROUND OF THE INVENTION

[0002] The majority of recombinant therapeutic proteins in the biopharmaceutical industry are produced by mammalian cell culture due to their capacity for accurate protein folding and post- translational modifications. Within mammalian culture systems, Chinese hamster ovary (CHO) cells are the host of choice in industrial production processes. Their major advantage is their human-like post-translational modification pattern. Furthermore, CHO cells have already proved to be safe hosts and are more likely to be approved for novel therapeutic manufacturing. While the development of stable CHO cell lines with high productivity yielding a high-quality product has been thoroughly done during the past years, there is a constant need for further improvement of cell culture performance. Most therapeutic proteins are produced in a fed-batch method.

[0003] Growth, productivity and product quality are directly influenced by the choice and composition of the used media. The optimization of cell culture medium to fulfill the cells’ demand on nutrients and minimize the accumulation of inhibitory substances, has a high impact on process performance. The metabolism of CHO cells and other mammalian cells is characterized by an inefficiently high uptake of substrates such as carbon and nitrogen sources, which leads to high concentrations of cytotoxic or inhibiting byproducts such as lactate, ammonia and various other growth-inhibitory metabolites. Cytotoxic or inhibiting byproducts are particularly problematic in fed-batch processes, because the medium is not exchanged and hence cytotoxic byproducts accumulate over time. This may affect product yield and product quality. CHO cell culture media for fed-batch processes for optimal yield or product quality are known in the art. For example, US 2018 / 0030495 A1 , and CN 113088480 A describe cell culture feed media for enhancing recombinant antibody purity or product quality. US 12,077,796 B2 describes supplementation as a percentage of the expected antibody amount to improve product quality.

[0004] However, also other substances than the intended recombinant therapeutic proteins and common metabolites may be formed during fed-batch culture and accumulate in the cellculture fluid (CCF) and the later harvested cell culture fluid (HCCF). Such other substances may impact downstream purification and may lead to break-through of these other substances into the drug substance and / or affect reused material during purification. Particularly reused material such as chromatography columns have to be regenerated after each use and insufficient regeneration leads to accumulation of such substances. More aggressive means for regeneration on the other hand impact the standard process and potentially the duration of such material, which negatively affects the costs, especially for expensive material such as affinity chromatography columns.

[0005] In case of the identification of unusual substances, such as by visual means, there is therefore a need to reduce or avoid the formation of such substances in order to streamline purification and to avoid any potential contamination of the drug substrate. Additionally, the quality of the intended recombinant therapeutic protein, like folding, intactness of side chains, glycosylation pattern and the like or overall productivity of the process ought not or at least not significantly to be affected by any means chosen for reducing or avoiding the formation of such undesired substances.SUMMARY OF THE INVENTION

[0006] The present invention relates to the observation that low molecular weight compounds are formed in the supernatant of CHO cultures in cell culture fluid during the growth and production phase of the culture and lead to blue coloration of membrane filters or even columns in downstream purification of produced proteins, especially if the harvested cell culture fluid (HCCF) is stored at room temperature for a prolonged time, such as for about 6 to 48 hours, typically 24-30 hours. It was surprisingly found that a low tryptophane concentration in the feed medium and / or a high cysteine concentration, preferably high cystine concentration, in the feed medium reduce the formation of the blue-colored compounds. The present invention therefore provides methods for reducing the formation of blue-colored compounds in a fed-batch culture of CHO cells, wherein a low tryptophane concentration and / or a high cysteine concentration, preferably cystine concentration, are used in the feed medium. At the same time the quality of the intended recombinant therapeutic protein, like folding, intactness of side chains, glycosylation pattern and the like or overall productivity of the process are not or at least not significantly affected by this means for reducing or avoiding the formation of such undesired substances.

[0007] The present invention relates to a method for reducing formation of one or more blue colored compounds in a fed-batch culture of CHO cells, wherein the method comprises:(a) inoculating CHO cells encoding a recombinant protein in a basal cell culture medium to form a cell culture;(b) cultivating the CHO cells in the cell culture under conditions that allow expression of the recombinant protein and secretion into the cell culture fluid (CCF), wherein the cell culture is regularly fed with nutrients by adding a feed medium and / or one or more feed supplement(s);(c) harvesting the CCF to obtain the harvested cell culture fluid (HCCF) comprising the recombinant protein and storing the HCCF until purification; and(d) optionally purifying the recombinant protein from the HCCF, wherein(i) the cell culture comprises until harvesting the CCF in step (c) a cumulative amount of• cysteine of about 5.5-7.5 mM, preferably about 6.0-7.0 mM;• tryptophane of about 2.5-3.8 mM, preferably of about 2.5-3.5 mM; and• a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about 1.7 to 3.0, more preferably about 2.0 to 2.8; and / or(ii) the feed medium and / or one or more feed supplement(s) together provide for at least 10 days of the culture an average daily addition of• cysteine of about 250-400 pM, preferably about 300-380 pM;• tryptophane of about 120-230 pM, preferably of about 130-200 pM; and• a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about 1.7 to 3.0, more preferably about 2.0 to 2.8; wherein the molar amount of cysteine refers to the molar cysteine equivalent provided by cysteine and / or cystine or a salt or hydrate thereof. In certain embodiments the method comprises step (d) purifying the recombinant protein from the HCCF, such as purifying the recombinant protein from the HCCF by pre-filtration and affinity chromatography (e.g., Protein A chromatography for antibodies and other Fc containing proteins).

[0008] In certain embodiments, the feed medium and / or one or more feed supplement(s) together provide cysteine and tryptophane per day (pM / L / day) and / or cumulative until harvest (pM / L) at a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about 1.7 to 3.0, more preferably about 2.0 to 2.8. The method according to the invention results in reduced formation of one or more blue colored compounds in the CCF and / or HCCF compared to cysteine and tryptophane provided by a feed medium and / or one or more feed supplement(s) together per day (pM / L / day) and / or cumulative until harvest (pM / L) at a molar ratio of cysteine / tryptophane of less than 1.6 and optionally tryptophane concentrations above the range according to the invention.

[0009] In certain embodiments, in (i) more than 15% of the cysteine are provided as cystine, preferably more than 20%, even more preferably more than 30% are provided as cysteineand / or in (ii) more than 30% of the cysteine are provided as cystine, preferably more than 40%, even more preferably more than 45% are provided as cystine.

[0010] According to the invention, the feeding with nutrients by adding a feed medium and / or one or more feed supplement(s) may starts between day 0 and day 4, preferably at days 2-3. In certain embodiments cysteine and tryptophane are provided daily by the feed medium and / or one or more feed supplement(s) starting at least on days 2-4 at a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about 1.7 to 3.0, more preferably about 2.0 to 2.8.

[0011] Preferably, the feed medium and / or one or more feed supplement(s) together provide for at least 12 days of the culture an average daily addition of cysteine of about 250-400 pM, preferably about 300-380 pM; tryptophane of about 120-230 pM, preferably of about 130-200 pM, and a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about 1.7 to 3.0, more preferably about 2.0 to 2.8. The average daily addition may be provided per day or at a respective higher concentration at larger intervals. In certain embodiments, the basal medium comprises cysteine at about 2-2.5 mM and tryptophane at about 0.8-1.5 mM.

[0012] The one or more blue colored compounds are low molecular weight organic chemical compounds comprising 2-3 indole residues. According to the invention the one or more blue colored compounds comprise a compound of the following structure:In certain embodiments, the one or more blue colored compounds further comprise a compound comprising three indole residues and the sum formula C34H20N5O5S2.

[0013] The one or more blue colored compounds are typically present in the HCCF or in the CCF and the HCCF. If present, the one or more blue colored compounds in the HCCF result in blue coloration of filters and / or of one or more chromatography columns in step (d), specifically in blue coloration of an affinity chromatography column in step (d).

[0014] In another aspect, the invention relates to a method of manufacturing a recombinant protein of interest comprising the steps of a) inoculating CHO cells encoding a recombinant protein in a basal cell culture medium to form a cell culture; b) cultivating the CHO cells in the cell culture under conditions that allow expression of the recombinant protein and secretion into the cell culture fluid (CCF), wherein the cellculture is regularly fed with nutrients by adding a feed medium and / or one or more feed supplement(s); c) harvesting the CCF to obtain the harvested cell culture fluid (HCCF) comprising the recombinant protein and storing the HCCF until purification; and d) optionally purifying the recombinant protein from the HCCF, wherein(i) the cell culture comprises until harvesting the CCF in step (c) a cumulative amount of• cysteine of about 5.5-7.5 mM, preferably about 6.0-7.0 mM;• tryptophane of about 2.5-3.8 mM, preferably of about 2.5-3.5 mM; and• a molar ratio of cysteine / tryptophane of about 1 .6 to 3.0, preferably about 1.7 to 3.0, more preferably about 2.0 to 2.8; and / or(ii) the feed medium and / or one or more feed supplement(s) together provide for at least10 days of the culture an average daily addition of• cysteine of about 250-400 pM, preferably about 300-380 pM;• tryptophane of about 120-230 pM, preferably of about 130-200 pM; and• a molar ratio of cysteine / tryptophane of about 1 .6 to 3.0, preferably about 1.7 to 3.0, more preferably about 2.0 to 2.8; wherein the molar amount of cysteine refers to the molar cysteine equivalent provided by cysteine and / or cystine or a salt or hydrate thereof.SHORT DESCRIPTION OF FIGURES

[0015] Figure 1 : Loading HCCF after a prolonged HCCF hold time provided blue coloration on column pre-filters (A) and at the top of capture column resins (B; staining indicated by the right-hand bracket and arrow).

[0016] Figure 2: Hold time of HCCF leads to a blue coloration of filters. HCCF was stored for up to 28 h at room temperature (RT) prior to filtration using a Sartopore 2 filter. Hold times of HCCF are indicated at the left-hand side, the filters are shown in the middle and the coloring is indicated on the right-hand side. In the last row HCCF was filtered 3 h after harvest and the filtered HCCF was stored at RT for 23 h prior to a second filtration step using a Sartopore 2 filter. Hold times of HCCF up to 7 h did not lead to a blue coloration of the filters. After 21 h HCCF hold time at RT the filter turned blue. If the HCCF was filtered 3 h after harvest and the filtered HCCF was stored at RT for 23 h, the filter of a second filtration step also turned blue.

[0017] Figure 3: Column regeneration using 6 M urea in WFI. A MabSelect capture resin comprising staining was regenerated using 6 M urea in WFI in downflow mode. The colored band (indicated by the dotted line circle) moved downwards from the top of the resin bed (lefthand image) when it started to detach (second image from the left) and slowly moveddownwards (third and fourth image from the left) and finally moved out of the column (righthand image) at approximately 1 cm per CV of regeneration solution.

[0018] Figure 4: Structural identification study and formation during cell culture process. (A) Proposed structures of compounds “Blue 1” and “Blue 2” with structural unsolved middle part and (B) Blue 1 and Blue 2 formation during cell culture process using LC-MS / MS after 24 hours hold time at RT following harvest. (C) Blue staining indicated extraction of blue substances after basic separation with 0.1 % ammonium bicarbonate. (D) Formation of “Blue 1” and “Blue 2” during increasing hold time of HCCF after harvest using LC-MS / MS. HCCF samples of 3 L runs for three different recombinant proteins (labelled as protein 1 and protein 2) were harvested after 14 days and stored for up to 48 h at room temperature (RT), frozen and analyzed together after all samples were collected by LC-MS / MS measurements targeted against blueOl (solid line) and blue02 (dotted line). Color formation starts after about three hours of hold time and reaches a plateau after about 24 hours.

[0019] Figure 5: Analysis of formation kinetics in upstream intermediates using liquid- chromatography-mass spectrometry / mass spectrometry (LC-MS / MS). An LC-MS / MS method was developed that allows relative quantification of both identified blue substances in complex biological matrices, such as cell culture supernatants. (A) High sensitivity is ensured by three dimensions of targeted analysis (retention time, Q1 mass to Q3 massed of two fragmentation reactions per analyte). Polysorbate 20 introduced a noticeable background signal (ghost peaks). The method shows linear response in spiking studies and is thus suitable for relative quantification with acceptable reproducibility. This method was used to investigate formation of the blue substances in upstream intermediates and drug substance and drug product clearance; SRM: Selective Reaction Monitoring; LC: liquid chromatography; ESI: electronspray ionization. (B) Principle of tandem mass spectrometry using ESI (electro-spray ionization) for ion generation. The sample is ionized by the ESI method and precursor ions are generated. Those ions are separated by their m / z ratio (Q1) and subsequently fragmented (Q2), resulting in product ions that are again separated by their m / z ratio (Q3) and finally detected; CID: collision induced dissociation; Q1: first quadrupole; Q2: collision cell, Q3: second quadrupole; DP: declustering potential; CE: collision energy; CXP: collision cell exit potential.

[0020] Figure 6: Product titer (normalized) in DoE optimization of blue coloration for cell line A as a function of (A) tryptophane, wherein (+1) represents 9.67 mM, (0) represents 7.25 mM [-25%] and (-1) represents 4.83 mM [-50%] tryptophane in Feed 1 or (B) cysteine, wherein (+1) represents 8.33 mM, (0) represents 4.17 mM [-50%] and (-1) represents 0 mM [-100%] cystine in Feed 2. The values of titer are normalized to the highest value of the DoE. Highest product titers could be obtained at high cystine feeding (R2=0.99).

[0021] Figure 7: Product titer (normalized) in DoE optimization of blue coloration for cell line A as a function of tryptophane and cysteine, wherein for tryptophan (+1) represents 9.67 mM, (0) represents 7.25 mM [-25%] and (-1) represents 4.83 mM [-50%] in Feed 1 and for cysteine (+1) represents 8.33 mM, (0) represents 4.17 mM [-50%] and (-1) represents 0 mM [-100%] in Feed 2. The values of titer are normalized to the highest value of the DoE. Highest product titers could be obtained at high cystine feeding (R2=0.99).

[0022] Figure 8: Harvest viability (%) in DoE optimization of blue coloration for cell line A at day 14 as a function of (A) tryptophane, wherein (+1) represents 9.67 mM, (0) represents 7.25 mM [-25%] and (-1) represents 4.83 mM [-50%] tryptophane in Feed 1 or (B) cysteine, wherein (+1) represents 8.33 mM, (0) represents 4.17 mM [-50%] and (-1) represents 0 mM [-100%] cystine in Feed 2. Highest harvest viability could be obtained at low tryptophane and cystine feeding with overall only minor impact (R2=0.91).

[0023] Figure 9 Harvest viability (%) in DoE optimization of blue coloration for cell line A at day 14 as a function of tryptophane and cysteine, wherein for tryptophan (+1) represents 9.67 mM, (0) represents 7.25 mM [-25%] and (-1) represents 4.83 mM [-50%] in Feed 1 and for cysteine (+1) represents 8.33 mM, (0) represents 4.17 mM [-50%] and (-1) represents 0 mM [-100%] in Feed 2 (R2=0.91).

[0024] Figure 10: Blue 1 formation for cell line A in DoE optimization of blue coloration as a function of (A) tryptophane, wherein (+1) represents 9.67 mM, (0) represents 7.25 mM [-25%] and (-1) represents 4.83 mM [-50%] tryptophane in Feed 1 or (B) cysteine, wherein (+1) represents 8.33 mM, (0) represents 4.17 mM [-50%] and (-1) represents 0 mM [-100%] cystine in Feed 2 (R2=0.97).

[0025] Figure 11 : Blue 1 formation for cell line A in DoE optimization of blue coloration at day 14 as a function of tryptophane and cysteine, wherein for tryptophan (+1) represents 9.67 mM, (0) represents 7.25 mM [-25%] and (-1) represents 4.83 mM [-50%] in Feed 1 and for cysteine (+1) represents 8.33 mM, (0) represents 4.17 mM [-50%] and (-1) represents 0 mM [-100%] in Feed 2 (R2=0.97).

[0026] Figure 12: Blue 2 formation for cell line A in DoE optimization of blue coloration as a function of (A) tryptophane, wherein (+1) represents 9.67 mM, (0) represents 7.25 mM [-25%] and (-1) represents 4.83 mM [-50%] tryptophane in Feed 1 or (B) cysteine, wherein (+1) represents 8.33 mM, (0) represents 4.17 mM [-50%] and (-1) represents 0 mM [-100%] cystine in Feed 2 (R2=0.96).

[0027] Figure 13: Blue 2 formation for cell line A in DoE optimization of blue coloration at day 14 as a function of tryptophane and cysteine, wherein for tryptophan (+1) represents 9.67 mM, (0) represents 7.25 mM [-25%] and (-1) represents 4.83 mM [-50%] in Feed 1 and for cysteine (+1) represents 8.33 mM, (0) represents 4.17 mM [-50%] and (-1) represents 0 mM [-100%] in Feed 2 (R2=0.96).

[0028] Figure 14: Viable cell density (VCD) [106cells / ml], total cell density (TCD) [106cells / ml], viability [%], product concentration [mg / L], glucose concentration [g / L], lactate [g / L], osmolarity [mOsmol / kg] and pH as indicated above the graphs for low tryptophane (-1) and high cysteine (+1) (grey circles) compared to standard medium (black circles) over the course of the process are shown.DETAILED DESCRIPTION OF THE INVENTION

[0029] The general embodiments “comprising” or “comprised” encompass the more specific embodiment “consisting of”. Furthermore, singular and plural forms are not used in a limiting way. As used herein, the singular forms “a”, “an” and “the” designate both the singular and the plural, unless expressly stated to designate the singular only.

[0030] The term “cell cultivation” or “cell culture” includes cell cultivation and fermentation processes in all scales (e.g. from micro titer plates to large-scale industrial bioreactors, i.e. from sub mL-scale to > 10.000 L scale), in all different process modes (e.g. batch, fed-batch, perfusion, continuous cultivation), in all process control modes (e.g. non-controlled, fully automated and controlled systems with control of e.g. pH, temperature, oxygen content), and in all kind of fermentation systems (e.g. single-use systems, stainless steel systems, glass ware systems). According to the invention the cell culture is a mammalian cell culture (preferably of CHO cells) and is a fed-batch culture. In a preferred embodiment the cell culture is a cell culture in a volume of > 1 L, preferably > 2L, > 10L, > 1.000L, > 5000L and more preferably > 10.000L.

[0031] The term “fed-batch” as used herein relates to a cell culture in which the cells are fed continuously or periodically with a feed medium containing nutrients and optionally one or more feed supplement(s). The feeding may start shortly after starting the cell culture on day 0 or more typically one, two or three days after starting the culture. Feeding may follow a preset schedule, such as every day, every two days, every three days etc. Alternatively, the culture may be monitored for cell growth, nutrients or toxic by-products and feeding may be adjusted accordingly. In general, the following parameters are often determined on a daily basis and cover the viable cell concentration, product concentration (titer) and several metabolites such as glucose, pH, lactate, osmolarity (a measure for salt content), and ammonium (growth inhibitor that negatively affects the growth rate and reduces viable biomass). Compared to batch cultures (cultures without feeding), higher product titers can be achieved in the fed-batch mode. Typically, a fed-batch culture is stopped at some point and the cells and / or the medium is harvested and the product of interest, such as a heterologous protein or a recombinant virus, is isolated and / or purified. A fed-batch process is typically maintained about 2-3 weeks, e.g., about 10-24 days, about 12 to 21 days, about 12 to 18 days, preferably about 12 to 16days. Particularly, a fed-batch process for the production of a heterologous protein is typically maintained about 2-3 weeks, e.g., about 10-24 days, about 12 to 21 days, about 12 to 18 days, preferably about 12 to 16 days.

[0032] The term “harvest” as used herein refers to cell culture fluid (CCF) from which the cells have been separated, such as by centrifugation and / or filtration, preferably centrifugation, to form the harvested cell culture fluid (HCCF). After harvest the HCCF is typically stored until purification. The term “storing the HCCF until purification”, as used herein refers to a holding time before the HCCF is subjected to further purification, such as affinity chromatography (e.g., Protein A chromatography) in a storage tank. The terms “storage” or “stored” and “hold time” are used synonymously herein. Storage is typically performed at room temperature (RT) and, if not otherwise indicated, storage or holding is performed at RT (e.g., 15-25°C), preferably within 18-22°C. The term “room temperature” as used herein refers to a temperature of 15°C to 25°C, as defined in the European Pharmacopoeia 11.0 (htps: / / pheur.edqm.eu, excerpt of 19 May 2025, 1. General notice, 1.2.3 Temperature).

[0033] By definition any nucleic acid, sequences or genes introduced into a host cell are called “heterologous nucleic acid” “heterologous sequences”, “heterologous genes”, “heterologous RNAs” or “transgenes” or “recombinant gene” with respect to the host cell, even if the introduced sequence is identical to an endogenous nucleic acid, sequence or gene in the host cell. A “heterologous” or “recombinant” protein or RNA is thus a protein or RNA expressed from a heterologous nucleic acid, sequence or gene, preferably a DNA. In a preferred embodiment, the introduced nucleic acid, sequence or gene is not identical to an endogenous nucleic acid sequence or gene of the host cell in question.

[0034] The term “encodes” and “codes for” as used herein refers broadly to any process whereby the information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first. The second molecule may have a chemical structure that is different from the chemical nature of the first molecule. For example, in some aspects, the term “encode” describes the process of semi-conservative DNA replication, where one strand of a double-stranded DNA molecule is used as a template to encode a newly synthesized complementary sister strand by a DNA-dependent DNA polymerase. In other aspects, a DNA molecule can encode an RNA molecule (e.g., by the process of transcription that uses a DNA-dependent RNA polymerase enzyme). Also, an RNA molecule can encode a polypeptide, as in the process of translation. When used to describe the process of translation, the term “encode” also extends to the triplet codon that encodes an amino acid. In some aspects, an RNA molecule can encode a DNA molecule, e.g., by the process of reverse transcription incorporating an RNA-dependent DNA polymerase. In another aspect, a DNA molecule can encode a polypeptide, where it is understood that “encode” as used in that case incorporates both the processes of transcription and translation.

[0035] The terms “polypeptide” or “protein” are used interchangeably. These terms refer to polymers of amino acids of any length. These terms also include proteins that are post- translationally modified through reactions that include, but are not limited to glycosylation, glycation, acetylation, phosphorylation, oxidation, amidation or protein processing. Modifications and changes, for example fusions to other proteins, amino acid sequence substitutions, deletions or insertions, can be made in the structure of a polypeptide while the molecule maintains its biological functional activity. For example, certain amino acid sequence substitutions can be made in a polypeptide or its underlying nucleic acid coding sequence and a protein can be obtained with similar or modified properties. Amino acid modifications can be prepared for example by performing site-specific mutagenesis or polymerase chain reaction mediated mutagenesis on its underlying nucleic acid sequence. The terms “polypeptide” and “protein” thus also include, for example, fusion proteins consisting of an immunoglobulin component (e.g. the Fc component) and a growth factor (e.g. an interleukin), antibodies or any antibody derived molecule formats or antibody fragments.

[0036] The term “recombinant protein” or “recombinant protein of interest” as used herein refers to any protein produced in a CHO cell that is encoded by a heterologous nucleic acid that has been introduced into the cell, particularly to a therapeutic protein.

[0037] The term “cell culture medium” as used herein is a medium to culture mammalian cells comprising a minimum of essential nutrients and components such as vitamins, trace elements, salts, bulk salts, amino acids, lipids, carbohydrates in a preferably buffered medium. Typically a cell culture medium for mammalian cells has an about neutral pH, such as a pH of about 6.5 to about 7.5, preferably about 6.8 to about 7.3, more preferably about 7. Non limiting examples for such cell culture media include commercially available media like Ham's F12 (Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma), Minimal Essential Medium (MEM; Sigma), Iscove's Modified Dulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), CHO-S-lnvitrogen), serum-free CHO Medium (Sigma), and protein-free CHO Medium (Sigma) etc. as well as proprietary media from various sources. The cell culture medium may be a basal cell culture medium. The cell culture medium may also be a basal cell culture medium to which the feed medium and / or one or more feed supplements (additives) have been added. The cell culture medium may also be referred to as fermentation broth, if the cells are cultured in a fermenter or bioreactor.

[0038] The term “basal medium” or “basal cell culture medium” as used herein is a cell culture medium to culture mammalian cells as defined below. It refers to the medium in which the cells are cultured from the start of a cell culture run and is typically not used as an additive to another medium, although various components may be added to the basal medium. The basal medium serves as the base to which optionally further additives (or supplements) and / or a feed medium may be added during cultivation, i.e. , a cell culture run, resulting in a cell culturemedium. The basal cell culture medium is provided from the beginning of a cell cultivation process, i.e., for inoculation of the cells. In general, the basal cell culture medium provides nutrients such as carbon sources, amino acids, vitamins, bulk salts (e.g. sodium chloride or potassium chloride), various trace elements (e.g. manganese sulfate), pH buffer, lipids and glucose. Major bulk salts are usually provided only in the basal medium and should not exceed a final osmolarity in the cell culture of about 280-350 mOsmo / kg, so that the cell culture is able to grow and proliferate at a reasonable osmotic stress.

[0039] The term “feed” or “feed medium” as used herein relates to a concentrate of nutrients / a concentrated nutrient composition used as a feed in a culture of mammalian cells. Thus, it is provided as a concentrate that is added into the cell culture. It is provided as a “concentrated feed medium” to minimize dilution of the cell culture, typically a feed medium is provided at 10-50 ml / L / day, preferably at 15-45 ml / L / day, more preferably at 20-40 ml / L / day and even more preferably at 30 ml / L / day based on the culture starting volume (CSV, meaning the start volume on day 0) in the vessel. This corresponds to a daily addition of about 1-5%, preferably about 1.5-4.5%, more preferably about 2-4% and even more preferably about 3% of the culture starting volume. For cultures using high density seeding or ultra-high density seeding higher feeding rates may be beneficial such as 10-50 ml / L / day, 15-45 ml / L / day or 25-45 ml / L / day. This corresponds to a daily addition of about 1-5%, about 1.5-4.5%, or about 2.5-4.5 % of the culture starting volume. The feeding rate is to be understood as an average feeding rate over the feeding period. A feed medium typically has higher concentrations of most, but not all, components of the basal cell culture medium. Generally, the feed medium substitutes nutrients that are consumed during cell culture, such as amino acids and carbohydrates, while salts and buffers are of less importance and are commonly provided with the basal medium. The feed medium is typically added to the (basal) cell culture medium / fermentation broth in fed-batch mode. The feed medium added (repeatedly or continuously) to the basal medium results in the cell culture medium. The feed may be added in different modes like continuous or bolus addition or via perfusion related techniques (chemostat or hybrid-perfused system). Preferably, the feed medium is added daily, but may also be added more frequently, such as twice daily or less frequently, such as every second day or every third day. More preferably the feed medium is added continuously. The addition of nutrients is commonly performed during cultivation (i.e., after day 0). In contrast to the basal medium, the feed medium typically consists of a highly concentrated nutrient solution (e.g. > 6x) that provides all the components similar to the basal medium except for ‘high-osmolarity-active compounds’ such as major bulk salts (e.g., NaCI, KCI, NaHCOs, MgSC>4, Ca(NOa)2). Typically, a 6x-fold concentrate or higher of the basal medium without or with reduced bulk salts maintains good solubility of compounds and sufficiently low osmolarity (e.g. 270-1500 mOsmo / kg, preferably 310-800 mOsmo / kg) in order to maintain osmolarity in the cell culture at about 270-550 mOsmo / kg, preferably at about280-450 mOsmo / kg, more preferably at about 280-350 mOsmo / kg. The feed medium may be added as one complete feed medium or the feeding may comprise a feed medium and additionally one or more feed supplement(s) for separate addition to the cell culture. Thus, in a fed-batch the cell culture is regularly fed with nutrients by adding a feed medium and / or one or more feed supplement(s) (preferably a feed medium and optionally one or more feed supplement(s)). The use of one or more feed supplements may be necessary due to different feeding schedules, such as regular feeding and feeding on demand as often performed for glucose addition, which is therefore typically at least also provided as a separate feed. The use of one or more feed supplements may also be necessary due to low solubility of certain compounds, solubility at different pH of certain compounds and / or interactions of compounds in the feed medium at high concentrations. The feed medium is preferably chemically defined (optionally comprising a recombinant protein, such as insulin or IGF). It does not contain cells, has not been in contact with cells in culture or does not contain cell derived metabolic waste products. Thus, as used herein, the term “feed medium” excludes a pre-conditioned medium derived from a cell culture or a culture medium in cell culture, i.e., in the presence of cells (also referred to as cell culture medium herein).

[0040] The term “feed supplement” as used herein relates to a concentrate of a nutrient (one or more than one nutrient), which might be added to the feed medium before use or may be added separately from the feed medium to the basal medium and / or the cell culture medium. Thus, a compound may be provided with the feed medium or the one or more feed supplement(s) or a compound may be provided with the feed medium and the one or more feed supplement(s). For example, cysteine may be added in a two-feed strategy with the feed medium and a feed supplement. As the feed medium, the “feed supplement” is provided as a concentrate in order to avoid dilution of the cell culture. The term “feed medium and / or one or more feed supplement(s)” as used herein refers to feeding with nutrients by adding a feed medium and optionally one or more feed supplement(s). A compound (e.g., cysteine or tryptophane) may be added with the feed medium or the one or more feed supplement(s) or with the feed medium and one or more feed supplement(s). Typically and preferably, compounds such as cysteine and tryptophane, are added with the feed medium and optionally one or more feed supplement(s).

[0041] The cell culture medium, both basal medium and feed medium, is preferably serum- free and chemically defined. The basal medium and / or the feed medium may further be protein-free. A “serum-free medium” as used herein refers to a cell culture medium for in vitro cell culture, which does not contain serum from animal origin. This is preferred as serum may contain contaminants from said animal, such as viruses, and because serum is ill-defined and varies from batch to batch. The basal medium and the feed medium according to the invention are serum-free.

[0042] A “chemically defined medium” as used herein refers to a cell culture medium suitable for in vitro cell culture, in which all components are known. More specifically it does not comprise any supplements such as animal serum or plant, yeast or animal hydrolysates. A chemically defined medium is therefore also serum-free. The basal medium and the feed medium according to the invention are preferably chemically defined. In one embodiment the basal medium and / or the feed medium are serum-free and chemically-defined and optionally comprises a recombinant growth factor such as insulin or insulin-like growth factor (IGF). The basal medium and / or the feed medium as referred to herein comprise no further proteins, except for, once in cell culture to provide the cell culture medium, proteins produced by the mammalian cell to be cultured.

[0043] A “protein-free medium” as used herein refers to a cell culture medium for in vitro cell culture comprising no proteins (except for proteins produced by the cell to be cultured once in cell culture), wherein protein refers to polypeptides of any length, but excludes single amino acids, dipeptides or tripeptides. Specifically, growth factors such as insulin and insulin-like growth factor (IGF) are not present in the medium. Preferably, the basal medium and feed medium according to the present invention are chemically defined and protein-free.

[0044] The term “viability” as used herein refers to the % viable cells in a cell culture as determined by methods known in the art, e.g., trypan blue exclusion with a Cedex device based on an automated-microscopic cell count (Innovatis AG, Bielefeld) or ViCell from Beckman-Coulter. However, there exist of number of other methods for the determination of the viability such as fluorometric (such as based on propidium iodide), calorimetric or enzymatic methods that are used to reflect the energy metabolism of a living cell e.g. methods that use LDH lactate-dehydrogenase or certain tetrazolium salts such as alamar blue, MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) or TTC (tetrazolium chloride).

[0045] The term “producing” or “highly producing”, “production”, “production and / or secretion”, “producing”, “production cell” or “producing at high yield” as used herein relates to the production of a (recombinant) protein of interest, such as a therapeutic protein encoded by a nucleic acid. An “increased production and / or secretion” or “production at high yield” relates to the expression of the protein of interest and means in the context of the recombinant protein an increase in cell specific productivity, increased titer, increased overall productivity of the cell culture or a combination thereof. Increased titer as used herein relates to an increased concentration in the same volume, i.e. , an increase in total yield of a recombinant protein.

[0046] The term “reduced”, as used herein, generally means a reduction by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 75%, or at least about 80%, or at least about 90%, or 100% as compared to a control cell culture compared to the same method wherein cysteine and tryptophane are provided by afeed medium and / or one or more feed supplement(s) together per day (pM / L / day) and / or cumulative until harvest (pM / L) at a molar ratio of cysteine / tryptophane of less than 1.6. As used herein, a “control cell culture” or “control mammalian cell culture” is a cell culture using the same cell (same CHO cell clone) producing the same product using the same method according to the invention, wherein cysteine and tryptophane are provided at a molar ratio of cysteine / tryptophane of less than 1.6 and optionally tryptophane concentrations above the range according to the invention.

[0047] The present invention provides a method for reducing formation of one or more blue colored compounds in a fed-batch culture of CHO cells, wherein the method comprises:(a) inoculating CHO cells encoding a recombinant protein in a basal cell culture medium to form a cell culture;(b) cultivating the CHO cells in the cell culture under conditions that allow expression of the recombinant protein and secretion into the cell culture fluid (CCF), wherein the cell culture is regularly fed with nutrients by adding a feed medium and / or one or more feed supplement(s);(c) harvesting the CCF to obtain the harvested cell culture fluid (HCCF) comprising the recombinant protein and storing the HCCF until purification; and(d) optionally purifying the recombinant protein from the HCCF, wherein(i) the cell culture comprises until harvesting the CCF in step (c) a cumulative amount of• cysteine of about 5.5-7.5 mM, preferably about 6.0-7.0 mM;• tryptophane of about 2.5-3.8 mM, preferably of about 2.5-3.5 mM ; and• a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about1.7 to 3.0, more preferably about 2.0 to 2.8; and / or(ii) the feed medium and / or one or more feed supplement(s) together provide for at least 10 days of the culture an average daily addition of• cysteine of about 250-400 pM, preferably about 300-380 pM;• tryptophane of about 120-230 pM, preferably of about 130-200 pM; and• a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about1.7 to 3.0, more preferably about 2.0 to 2.8; wherein the molar amount of cysteine refers to the molar cysteine equivalent provided by cysteine and / or cystine or a salt or hydrate thereof.

[0048] In another aspect the invention provides method for manufacturing a recombinant protein of interest comprising the steps of:(a) inoculating CHO cells encoding a recombinant protein in a basal cell culture medium to form a cell culture;(b) cultivating the CHO cells in the cell culture under conditions that allow expression of the recombinant protein and secretion into the cell culture fluid (CCF), wherein the cell culture is regularly fed with nutrients by adding a feed medium and / or one or more feed supplement(s);(c) harvesting the CCF to obtain the harvested cell culture fluid (HCCF) comprising the recombinant protein and storing the HCCF until purification; and(d) optionally purifying the recombinant protein from the HCCF, wherein(i) the cell culture comprises until harvesting the CCF in step (c) a cumulative amount of• cysteine of about 5.5-7.5 mM, preferably about 6.0-7.0 mM;• tryptophane of about 2.5-3.8 mM, preferably of about 2.5-3.5 mM ; and• a molar ratio of cysteine / tryptophane of about 1 .6 to 3.0, preferably about1 .7 to 3.0, more preferably about 2.0 to 2.8; and / or(ii) the feed medium and / or one or more feed supplement(s) together provide for at least 10 days of the culture an average daily addition of• cysteine of about 250-400 pM, preferably about 300-380 pM;• tryptophane of about 120-230 pM, preferably of about 130-200 pM; and• a molar ratio of cysteine / tryptophane of about 1 .6 to 3.0, preferably about1 .7 to 3.0, more preferably about 2.0 to 2.8; wherein the molar amount of cysteine refers to the molar cysteine equivalent provided by cysteine and / or cystine or a salt or hydrate thereof.

[0049] In certain embodiments of the methods of the invention (i) the cell culture comprises until harvesting the CCF in step (c) a cumulative amount of cysteine of about 5.5-7.5 mM, preferably about 6.0-7.0 mM; of tryptophane of about 2.5-3.8 mM, preferably about 2.5-3.5 mM; and a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about 1 .7 to 3.0, more preferably about 2.0 to 2.8; and / or (ii) the feed medium and / or one or more feed supplement(s) together provide for at least 12 days of the culture an average daily addition of cysteine of about 250-400 pM, preferably about 300-380 pM; of tryptophane of about 120-230 pM, preferably about 130-200 pM, and at a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about 1.7 to 3.0, more preferably about 2.0 to 2.8. The cumulative amount as referred to herein means the amount provided in the basal medium and added with the feed medium and / or one or more feed supplement(s) together over the course of the cell culture until harvest. The person skilled in the art would understand that the cysteine and thetryptophane can be added with the feed medium or the one or more feed supplement(s) or with the feed medium and the one or more feed supplement(s), preferably with the feed medium and optionally the one or more feed supplement(s). Typically, the course of the cell culture until harvest lasts for 12-16 days, preferably for 14 days. The basal medium and the feed medium are typically adopted to each other to provide the required nutrients and the person skilled in the art would understand that a lower concentration in the basal medium may be compensated to a certain extent by the addition of a higher concentration in the feed medium (and / or one or more feed supplement(s)) and vice versa. Without being limited thereto a suitable concentration of cysteine in the basal medium is about 2-2.5 mM and a suitable concentration of tryptophane is about 0.8-1.5 mM. Preferably, cysteine has a concentration of 2.1-2.4 mM cysteine and 1.0 to 1.3 mM tryptophane in the basal medium. The basal medium may comprise cysteine or cystine or a salt or a hydrate thereof as cysteine source, such as L- cysteine HCI H2O or L-cystine 2 HCI. Preferably it comprises cysteine or a salt thereof.

[0050] In certain embodiments of the methods of the invention (i) the cell culture comprises until harvesting the CCF in step (c) a cumulative amount of cysteine of about 6.0-7.0 mM; of tryptophane of about 2.5-3.5 mM; and a molar ratio of cysteine / tryptophane of about 1.7 to 3.0, more preferably about 2.0 to 2.8; and / or (ii) the feed medium and / or one or more feed supplement(s) together provide for at least 10 days, preferably for at least 12 days, of the culture an average daily addition of cysteine of about 300-380 pM; of tryptophane of about 130-200 pM; and at a molar ratio of cysteine / tryptophane of about 1.7 to 3.0, more preferably about 2.0 to 2.8. Preferably, (i) the cell culture comprises until harvesting the CCF in step (c) a cumulative amount of cysteine of about 6.0-7.0 mM; of tryptophane of about 2.5-3.5 mM; and a molar ratio of cysteine / tryptophane of about 1.7 to 3.0, more preferably about 2.0 to 2.8; and / or (ii) the feed medium and / or one or more feed supplement(s) together provide for at least 12 days of the culture an average daily addition of cysteine of about 300-380 pM; tryptophane of about 130-200 pM; and at a molar ratio of cysteine / tryptophane of about 1.7 to 3.0, more preferably about 2.0 to 2.8. Also, the feed medium and / or the one or more feed supplement(s) may comprise cysteine or cystine or a salt or a hydrate thereof as cysteine source, such as L-cysteine HCI H2O or L-cystine 2 HCI.

[0051] In a preferred embodiment in (i) (cumulative amounts) more than 15% of the cysteine are provided as cystine, preferably more than 20%, even more preferably more than 30% are provided as cystine and / or in (ii) (average daily addition) more than 30% of the cysteine are provided as cystine, preferably more than 40%, even more preferably more than 45% are provided as cystine. In a more preferred embodiment in (i) more than 20% of the cysteine are provided as cystine, and / or in (ii) more than 40% are provided as cystine; or in (i) more than 30% are provided as cystine and / or in (ii) more than 45% are provided as cystine.

[0052] The term “average daily addition” refers to a daily addition, an addition every two days, an addition every three days or a combination thereof, wherein the concentrations are provided at a respective higher concentration when provided at larger intervals to provide the same average concentration per day. Thus, in certain embodiments, the average daily addition is provided per day or at a respective higher concentrations at larger intervals, i.e. , twice the concentration when provided every second day. The addition of the feed medium and / or one or more feed supplement(s) may be as bolus additions daily or every second or third day or the addition of the feed medium and / or one or more feed supplement(s) may be continuous.

[0053] In certain embodiments of the methods of the invention the feed medium and / or the one or more feed supplement(s) together provide cysteine and tryptophane per day (pM / L / day) and / or cumulative until harvest (pM / L) at a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about 1.7 to 3.0, more preferably about 2.0 to 2.8, even more preferably about 2.0 to 2.6. Preferably, more than 30% of the cysteine equivalents are provided as cystine, more preferably more than 40% or even more than 45% of the cysteine equivalents are provided as cystine. The feeding with nutrients by adding a feed medium and / or one or more feed supplement(s) typically starts between day 0 and day 4, preferably between days 2-4, preferably between days 2-3. In specific embodiments feeding starts at das 2.

[0054] In certain embodiments cysteine and tryptophane are provided daily by the feed medium and / or one or more feed supplement(s) starting at least on days 2-4 at a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about 1.7 to 3.0, more preferably about 2.0 to 2.8. In certain embodiments cysteine and tryptophane are provided daily by the feed medium and / or one or more feed supplement(s) starting at least on days 2-4 at a molar ratio of cysteine / tryptophane of about 2.0 to 2.8 (or 2.0 to 2.6). Preferably, more than 45% of the cysteine equivalents are provided as cystine.

[0055] The methods of the invention result in reduced formation of one or more blue colored compounds in the CCF and / or HCCF compared to cysteine and tryptophane provided by a feed medium and / or one or more feed supplement(s) together per day (pM / L / day) and / or cumulative until harvest (pM / L) at a molar ratio of cysteine / tryptophane of less than 1.6.

[0056] The methods according to the invention further comprises storing the HCCF until purification in step (c). Typically, the HCCF is stored for > 6 h to up to 48 h, preferably for about > 6 h to > 24 h. In certain embodiments the method comprises storing the HCCF for at least about 6 h until purification in step (c), such as from about 6 to 48 hours, preferably from about 24 to 48 hours, more preferably from about 24 to 30 hours (at RT).

[0057] The term “blue colored compounds” as used herein refers to a compound that has at least at neutral pH or higher a blue color (e.g., at neutral and basic pH), such as at least at a pH of > 6.5, preferably at least at a pH of > 6.5 - 8. The one or more blue colored compounds are low molecular weight organic chemical compounds. The term “low molecular weight organic chemical compound” as used herein refers to a compound of a molecular weight of less than 1000 Da, preferably less than 750 Da. The one or more blue colored compounds further comprise at least 2-3 indole residues, preferably the one or more blue colored compounds further comprises at least 2-3 indole residues.

[0058] In certain embodiments, the one or more blue colored compounds comprise a compound of the following structure:This compound is also referred to as “Blue 1” herein.

[0059] In certain embodiments the one or more blue colored compounds further comprise a compound comprising three indole residues and the sum formula C34H20N5O5S2. Thus, the one or more blue colored compound may comprise a compound of the following structure:and / or a compound comprising three indole residues and the sum formula C34H20N5O5S2 (also referred to as “Blue 2”).

[0060] In a specific embodiment the one or more blue colored compounds are a compound of the following structure:and a compound comprising three indole residues and the sum formula C34H20N5O5S2.

[0061] The one or more blue colored compounds are present in the harvested cell culture fluid (HCCF) or in the cell culture fluid (CCF) and the HCCF. The term “harvesting the CCF” comprises isolating the cell culture medium comprising the product of interest from the CHO cells, e.g., by centrifugation and / or filtration. The term “purifying the recombinant protein from the HCCF” or “isolating the recombinant protein from the HCCF” as used herein includes purifying the product of interest from the cell culture medium following harvest of the cell culture medium comprising the product of interest. Preferably the HCCF is prepared by separation from the cells. Methods for purifying recombinant (heterologous) proteins, including antibodies, or recombinant virus are known in the art.

[0062] The one or more blue colored compounds, if present in the HCCF, result in blue coloration of filters and / or of one or more chromatography columns in step (d). Specifically, the one or more blue colored compounds, if present in the HCCF, result in blue coloration of an affinity chromatography column in step (d). Although it has been demonstrated that the one or more blue colored compounds are completely removed during downstream purification, such compounds are still undesired. For example, such compounds are disadvantageous, because the coloration and / or contamination may required more stringent conditions for reused materials such as chromatography columns, specifically affinity chromatography columns of the capture step, which may reduce the lifetime of the resin, complicate the process and / or increase costs. Thus, the method of the invention is for increasing the lifetime of an affinity chromatography used in purifying the recombinant protein from the HCCF in step (d) of the present invention. The method of the present invention reduces the presence of the one or more blue colored compounds in the HCCF or the CCF and the HCCF, preferably the method of the present invention prevents the formation or accumulation of the one or more blue colored compounds in the HCCF or the CCF and the HCCF. In certain embodiments, the method results in reduced formation of one or more blue colored compounds in the CCF and / or HCCF compared to cysteine and tryptophane provided by a feed medium and / or one or more feed supplement(s) together per day (pM / L / day) and / or cumulative until harvest (pM / L) at a molar ratio of cysteine / tryptophane of less than 1.6.

[0063] The feed medium used in the methods according to the invention is added daily, preferably continuously during the feeding period of the fed-batch process. In one embodiment the feed medium is added starting from days 0 to 4. The person skilled in the art will understand that this may also depend on the seeding density. For a normal seeding density (0.7 to 1 x 106cells / ml) feeding is typically started at days 1-4, preferably days 2-3. Feeding is typically continued until at least 5 days before the end of the fed-batch process, until at least 4 days before the end of the fed-batch process, until at least 3 days before the end of the fed- batch process, until at least 2 days before the end of the fed-batch process and preferablyuntil the end of the process. More preferably feeding is started at days 2-3 and is continued at least until 2 days before the end of the fed-batch process, more preferably until the end of the cell fed-batch process. For high seeding densities (> 1 to 4 x 106cells / ml) feeding is typically started at days 0-4, preferably days 0-2.

[0064] The term “inoculating” as used herein refers to collecting a sample of CHO cells and placing them into a basal medium that contains the nutrients needed for growth. This step may also be referred to as seeding. The mammalian cells may be inoculated into the basal medium at different seeding densities. As referred to herein the terms “seeding” or “normal seeding” refer to a standard seeding density of about 0.3 x 106cells / ml to about 1 x 106cells / ml, the term “high seeding” refers to a seeding density of greater 1 x 106cells / ml to about 4 x 106cells / ml and the term “ultrahigh seeding” refers to a seeding density of greater 4 x 106cells / ml to about 20 x 106cells / ml or even higher, preferably of about 6 x 106cells / ml to about 15 x 106cells / ml, more preferably of 8 x 106cells / ml to about 12 x 106cells / ml.

[0065] The cysteine may be provided as cysteine or a salt and / or a hydrate thereof, as cystine or a salt thereof or as a dipeptide or tripeptide comprising cysteine. The cysteine salt and / or hydrate or the cystine or a salt thereof or the dipeptide or tripeptide comprising cysteine is provided at an equimolar concentration to the cysteine concentrations provided herein. The terms “cysteine” and “cystine” as used herein refer to L-cysteine and L-cystine.

[0066] The methods according to the invention are in vitro methods of culturing CHO cells used for high expression of a product of interest, such as a heterologous protein or a RNA products. Examples of CHO cells commonly used for heterologous protein production are CHO, CHO-K1 , CHO-DXB11 (also referred to as CHO-DUKX or DuxB11), CHO-S cells and CHO-DG44 cells or the derivatives / progenies of any of such cell line. Particularly preferred are CHO cells, such as CHO-DG44 and CHO-K1 cells. Most preferred are CHO-DG44 cells. Glutamine synthetase (GS)-deficient derivatives of the mammalian cell, particularly of the CHO-DG44 and CHO-K1 cell are also encompassed. These cells are particularly suitable for GS-based selection (such as methionine sulfoximine (MSX) selection) of clones stably expressing the heterologous protein. In one embodiment of the invention the mammalian cell is a Chinese hamster ovary (CHO) cell, preferably a CHO-DG44 cell, a CHO-K1 cell, a CHO DXB11 cell, a CHO-S cell, a CHO GS deficient cell or a derivative thereof. The CHO cell may further comprise one or more expression cassette(s) encoding a heterologous or recombinant protein, such as a therapeutic protein, preferably a recombinant secreted therapeutic protein. Exemplary CHO cells are also listed in Table 1 below.Table 1 : Suitable CHO production cell lines

[0067] CHO cells are most preferred, when being established, adapted, and completely cultivated under serum free conditions, and optionally in media, which are free of any protein / peptide of animal origin. Commercially available media such as Ham's F12 (Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma), Minimal Essential Medium (MEM; Sigma), Iscove's Modified Dulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), CHO-S-lnvitrogen), serum-free CHO Medium (Sigma), and protein-free CHO Medium (Sigma) are exemplary appropriate nutrient solutions. Any of the media may be supplemented as necessary with a variety of compounds, non-limiting examples of which are recombinant hormones and / or other recombinant growth factors (such as insulin, transferrin, epidermal growth factor, insulin like growth factor), salts (such as sodium chloride, calcium, magnesium, phosphate), buffers (such as HEPES), nucleosides (such as adenosine, thymidine), glutamine, glucose or other equivalent energy sources, antibiotics and trace elements. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. For the growth and selection of genetically modified cells expressing a selectable gene a suitable selection agent is added to the culture medium.

[0068] The recombinant protein may be any therapeutically relevant protein. Examples for therapeutic proteins are without being limited thereto antibodies, fusion proteins, cytokines and growth factor. The recombinant protein produced in the CHO cells according to the methods of the invention includes but is not limited to an antibody or a fusion protein, such as an Fc-fusion protein. Other recombinant proteins can be for example enzymes, cytokines, lymphokines, adhesion molecules, receptors and derivatives or fragments thereof, and any other polypeptides and scaffolds that can serve as agonists or antagonists and / or have therapeutic or diagnostic use.

[0069] A preferred recombinant protein is an antibody or a fragment or derivative thereof or a fusion protein. Thus, the method according to the invention can be advantageously used forproduction of antibodies, preferably monoclonal antibodies. Typically, an antibody is mono- specific, but an antibody may also be multi-specific. Thus, the method according to the invention may be used for the production of mono-specific antibodies, multi-specific antibodies, or fragments thereof, preferably of antibodies (mono-specific), bispecific antibodies, trispecific antibodies or fragments thereof, preferably antigen-binding fragments thereof. Unless specifically mentioned, the term “antibody” refers to a mono-specific antibody. Exemplary antibodies within the scope of the present invention include but are not limited to anti-CD2, anti-CD3, anti-CD20, anti-CD22, anti-CD30, anti-CD33, anti-CD37, anti-CD40, anti- CD44, anti-CD44v6, anti-CD49d, anti-CD52, anti-EGFR1 (HER1), anti-EGFR2 (HER2), anti- GD3, anti-IGF, anti-VEGF, anti-TNFalpha, anti-IL2, anti-IL-5R or anti-lgE antibodies, and are preferably selected from the group consisting of anti-CD20, anti-CD33, anti-CD37, anti-CD40, anti-CD44, anti-CD52, anti-HER2 / neu (erbB2), anti-EGFR, anti-IGF, anti-VEGF, anti- TNFalpha, anti-l L2 and anti-lgE antibodies.

[0070] The term “antibody”, "antibodies", or "immunoglobulin(s)" as used herein relates to proteins selected from among the globulins, which are naturally formed as a reaction of the host organism to a foreign substance (=antigen) from differentiated B-lymphocytes (plasma cells). There are various classes of immunoglobulins: IgA, IgD, IgE, IgG, IgM, IgY, IgW. Preferably the antibody is an IgG antibody, more preferably an IgG 1 or an lgG4 antibody. The terms immunoglobulin and antibody are used interchangeably herein. Antibodies include monoclonal, monospecific and multi-specific (such as bispecific or trispecific) antibodies, including other multi-specific antigen-binding proteins, such as antibody-like formats, a single chain antibody, an antigen-binding fragment of an antibody (e.g., a Fab or F(ab')2 fragment), a disulfide-linked Fv, etc. Antibodies can be of any species and include chimeric and humanized antibodies. “Chimeric” antibodies are molecules in which antibody domains or regions are derived from different species. For example, the variable region of heavy and light chain can be derived from rat or mouse antibody and the constant regions from a human antibody. In “humanized” antibodies only minimal sequences are derived from a non-human species. Often only the CDR amino acid residues of a human antibody are replaced with the CDR amino acid residues of a non-human species such as mouse, rat, rabbit or llama. Sometimes a few key framework amino acid residues with impact on antigen binding specificity and affinity are also replaced by non-human amino acid residues. Antibodies may be produced through chemical synthesis, via recombinant or transgenic means, via cell (e.g., hybridoma) culture, or by other means.

[0071] Typically, antibodies are tetrameric polypeptides composed of two pairs of a heterodimer each formed by a heavy and light chain. Stabilization of both the heterodimers as well as the tetrameric polypeptide structure occurs via interchain disulfide bridges. Each chain is composed of structural domains called “immunoglobulin domains” or “immunoglobulinregions” whereby the terms “domain” or “region” are used interchangeably. Each domain contains about 70 - 110 amino acids and forms a compact three-dimensional structure. Both heavy and light chain contain at their N-terminal end a “variable domain” or “variable region” with less conserved sequences which is responsible for antigen recognition and binding. The variable region of the light chain is also referred to as “VL” and the variable region of the heavy chain as “VH”.

[0072] Antigen-binding fragments include without being limited thereto e.g. “Fab fragments” (Fragment antigen-binding = Fab). Fab fragments consist of the variable regions of both chains, which are held together by the adjacent constant region. These may be formed by protease digestion, e.g. with papain, from conventional antibodies, but similarly Fab fragments may also be produced by genetic engineering. Further antibody fragments include F(ab‘)2 fragments, which may be prepared by proteolytic cleavage with pepsin.

[0073] Using genetic engineering methods, it is possible to produce shortened antibody fragments which consist only of the variable regions of the heavy (VH) and of the light chain (VL). These are referred to as Fv fragments (Fragment variable = fragment of the variable part). Since these Fv-fragments lack the covalent bonding of the two chains by the cysteines of the constant chains, the Fv fragments are often stabilized. It is advantageous to link the variable regions of the heavy and of the light chain by a short peptide fragment, e.g. of 10 to 30 amino acids, preferably 15 amino acids. In this way a single peptide strand is obtained consisting of VH and VL, linked by a peptide linker. An antibody protein of this kind is known as a single-chain-Fv (scFv). Examples of scFv-antibody proteins are known to the person skilled in the art. Thus, antibody fragments and antigen-binding fragments further include Fv- fragments and particularly scFv.

[0074] In recent years, various strategies have been developed for preparing scFv as a multimeric derivative. This is intended to lead, in particular, to recombinant antibodies with improved pharmacokinetic and biodistribution properties as well as with increased binding avidity. In order to achieve multimerisation of the scFv, scFv were prepared as fusion proteins with multimerisation domains. The multimerisation domains may be, e.g. the CH3 region of an IgG or coiled coil structure (helix structures) such as Leucine-zipper domains. However, there are also strategies in which the interaction between the VH / VL regions of the scFv is used for the multimerisation (e.g. dia-, tri- and pentabodies). By diabody the skilled person means a bivalent homodimeric scFv derivative. The shortening of the linker in a scFv molecule to 5 - 10 amino acids leads to the formation of homodimers in which an inter-chain VH / VL- superimposition takes place. Diabodies may additionally be stabilized by the incorporation of disulphide bridges. Examples of diabody-antibody proteins are known from the prior art.

[0075] By minibody the skilled person means a bivalent, homodimeric scFv derivative. It consists of a fusion protein which contains the CH3 region of an immunoglobulin, preferablyIgG, most preferably lgG1 as the dimerisation region which is connected to the scFv via a Hinge region (e.g. also from I gG 1 ) and a linker region. Examples of minibody-antibody proteins are known from the prior art.

[0076] By triabody the skilled person means a: trivalent homotrimeric scFv derivative. ScFv derivatives wherein VH-VL is fused directly without a linker sequence lead to the formation of trimers.

[0077] The skilled person will also be familiar with so-called miniantibodies which have a bi-, tri- or tetravalent structure and are derived from scFv. The multimerisation is carried out by di- , tri- or tetrameric coiled coil structures. In a preferred embodiment of the present invention, the gene of interest is encoded for any of those desired polypeptides mentioned above, preferably for a monoclonal antibody, a derivative or fragment thereof.

[0078] The immunoglobulin fragments composed of the CH2 and CH3 domains of the antibody heavy chain are called “Fc fragments”, “Fc region” or “Fc” because of their crystallization propensity (Fc = fragment crystallizable). These may be formed by protease digestion, e.g. with papain or pepsin from conventional antibodies but may also be produced by genetic engineering. The N-terminal part of the Fc fragment might vary depending on how many amino acids of the hinge region are still present.

[0079] Antibodies comprising an antigen-binding fragment and an Fc region may also be referred to as full-length antibody. Full-length antibody may be mono-specific and multispecific antibodies, such as bispecific or trispecific antibodies.

[0080] Preferred therapeutic antibodies according to the invention are multispecific antibodies, particularly bispecific or trispecific antibodies. Bispecific antibodies typically combine antigen-binding specificities for target cells (e.g., malignant B cells) and effector cells (e.g., T cells, NK cells or macrophages) in one molecule. Exemplary bispecific antibodies, without being limited thereto are diabodies, BiTE (Bi-specific T-cell Engager) formats and DART (Dual-Affinity Re-Targeting) formats. The diabody format separates cognate variable domains of heavy and light chains of the two antigen binding specificities on two separate polypeptide chains, with the two polypeptide chains being associated non-covalently. The DART format is based on the diabody format, but it provides additional stabilization through a C-terminal disulfide bridge. Trispecific antibodies are monoclonal antibodies which combine three antigen-binding specificities. They may be built on bispecific-antibody technology that reconfigures the antigen-recognition domain of two different antibodies into one bispecific molecule. For example, trispecific antibodies have been generated that target CD38 on cancer cells and CD3 and CD28 on T cells. Multispecific antibodies are particularly difficult to product with high product quality.

[0081] Another preferred therapeutic protein is a fusion protein, such as an Fc-fusion protein. Thus, the invention can be advantageously used for production of fusion proteins, such as Fc-fusion proteins. Furthermore, the method of increasing protein producing according to the invention can be advantageously used for production of fusion proteins, such as Fc-fusion proteins.

[0082] The effector part of the fusion protein can be the complete sequence or any part of the sequence of a natural or modified heterologous protein. The immunoglobulin constant domain sequences may be obtained from any immunoglobulin subtypes, such as lgG1 , lgG2, lgG3, lgG4, lgA1 or lgA2 subtypes or classes such as IgG, IgA, IgE, IgD or IgM. Preferentially they are derived from human immunoglobulin, more preferred from human IgG and even more preferred from human IgG 1 and lgG2. Non-limiting examples of Fc-fusion proteins are MCP1- Fc, ICAM-Fc, EPO-Fc and scFv fragments or the like coupled to the CH2 domain of the heavy chain immunoglobulin constant region comprising the N-linked glycosylation site. Fc-fusion proteins can be constructed by genetic engineering approaches by introducing the CH2 domain of the heavy chain immunoglobulin constant region comprising the N-linked glycosylation site into another expression construct comprising for example other immunoglobulin domains, enzymatically active protein portions, or effector domains. Thus, an Fc-fusion protein according to the present invention comprises also a single chain Fv fragment linked to the CH2 domain of the heavy chain immunoglobulin constant region comprising e.g. the N-linked glycosylation site.

[0083] In a further aspect a method of producing a product of interest is provided using the methods of the invention and further comprising a step of isolating and / or purifying the product or interest and optionally formulating the product of interest into a pharmaceutically acceptable formulation. In one embodiment the product of interest is a recombinant protein. Specifically, a method of producing a recombinant protein is provided using the methods of the invention and further comprising a step of isolating and / or purifying the recombinant protein and optionally formulating the recombinant protein into a pharmaceutically acceptable formulation.

[0084] The recombinant protein may be a therapeutic protein, especially the antibody, antibody fragment, antibody derivative or Fc-fusion protein is preferably recovered / isolated from the culture medium as a secreted polypeptide. It is necessary to purify the therapeutic protein from other recombinant proteins and host cell proteins to obtain substantially homogenous preparations of the recombinant protein. As a first step, cells and / or particulate cell debris are removed from the culture medium. Further, the heterologous protein is purified from contaminant soluble proteins, polypeptides and nucleic acids, for example, by fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, Sephadex chromatography, and chromatography on silica or on a cation exchange resin such as DEAE. Methods for purifying a heterologous protein expressed by mammalian cells are known in the art.

[0085] Preferably the recombinant protein is an antibody or an antigen-binding fragment thereof, a multispecific antibody, such as a bispecific antibody or trispecific, or a multispecific antigen-binding fragment thereof or a fusion protein. The antibody or the multispecific antibody (e.g. bispecific or trispecific antibody) may be an lgG1 , lgG2a, lgG2b, lgG3 or lgG4 antibody, preferably an lgG1 or lgG4 antibody.EXAMPLESExample 1 : Detection of blue coloration and regeneration of capture resin

[0086] In an experimental fed-batch process using conditions as described in the literature, it was observed that loading HCCF after a prolonged HCCF hold time provided blue coloration on column pre-filters (Figure 1A) and at the top of capture column resins (Figure 1 B), such as MabSelect™ and MabSelect SuRe™. This coloration was intensified upon hold time of the HCCF at RT (about 18°C) prior to filtration or capture chromatography.

[0087] As may be taken from Figure 2 hold times of HCCF up to 7 h did not lead to a visible blue coloration of the filters. After 21 h HCCF hold time at RT the Sartopore 2 filter appeared blue. If the HCCF was filtrated 3 h after harvest and the filtrated HCCF was stored at RT for 23 h, the filter turned blue after a second filtration over a Sartopore 2.

[0088] A substance binding to a reused material in the purification process poses the threat of accumulation and subsequent break-through of the substance. To prevent this, carry-over from one cycle or batch to the next needs to be prevented by regenerating the column. This is commonly performed by stripping the column with 1M acetic acid and sanitizing the column with 0.2 M NaOH. While this has been shown to regenerate the column properly for many projects, this particular staining demanded the development of an additional regeneration step to prevent accumulation and carry-over. The solution that was found to remove the compound in question was 6 M urea in WFI (Figure 3). This is a commonly used regeneration solution in general. It was used as standard sanitization solution for the previous generation of the capture resin (MabSelect) and was replaced by 0.2 M NaOH for the current generation of capture resin (MabSelect SuRe). However, in order to remove the blue staining 6 M urea were required.

[0089] A regeneration with 6 M urea in downflow mode demonstrated that the compound is actually moved along the column and out of the column outlet rather than being bleached while remaining on the column (Figure 3). This experiment also showed that the movement of the color band was too slow (approximately 1 cm per CV of regeneration solution) to be applicable in large scale. Thus, the direction of the flow had to be switched from downflow to upflow during this regeneration step. In this mode of operation, 3 CVs of 6 M urea solution wereshown to be sufficient for column regeneration. Thus, this setup was proposed and accepted for process transfer.

[0090] Isotope labeling experiments surprisingly identified two important amino acids (Cysteine CYS and Tryptophane TRP) from the cell culture media, as most probable root cause for the formation of the blue colored compounds.Example 2: Partial identification of blue substances and analysis of upstream intermediates using LC-MS / MS

[0091] Further investigation of the blue substances identified the substances as low molecular weight compounds that are formed in the supernatant of CHO cultures (Figure 4B). After a structural identification study (1 D and 2D-NMR data) the compounds “Blue 1” (2 indole groups) and “Blue 2” (3 indole groups) could be partially identified as shown in Figure 4A (top). Both compounds can be extracted after basic separation with 0.1 % ammonium bicarbonate (Figure 4C).

[0092] As a following step an analytical method was developed to quantify the blue compounds from process till final drug substance using liquid-chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) (as illustrated in Figure 5A) and the method for ion generation was electro-spray ionization (ESI) (as illustrated in Figure 5B).

[0093] A tandem mass spectrometer with triple quadrupole analyzer was used in this study. A quadrupole consists of four parallel rods with varying voltages, which allow only a narrow band of m / z values to be moved along the axis of the rods. By varying the voltages, a wide range of m / z values are scanned. Hence, ions of a particular m / z ratio in the first quadrupole (Q1) are selected as precursor ions. These ions are split into smaller fragment ions e.g. by collision with an inert gas - collision induced dissociation (CID) in a collision cell (Q2). Fragmented ions are introduced into a second quadrupole mass analyzer (Q3), where they are separated by their m / z ratio for a second time and where the final product ion is selected. Product ions were identified by the detector and results were displayed as mass spectra as a function of the m / z ratio. Identification follows by correlating known masses to the identified masses. The use of two mass analyzers coupled with fragmentation enables the identification and separation of ions with similar m / z ratios and leads to higher resolution and thus higher specificity.

[0094] The developed LC-MS / MS method allows relative quantification of both identified blue compounds even in complex biological matrices, such as cell culture supernatants. High sensitivity is ensured by three dimensions of targeted analysis (retention time, Q1 mass to Q3 massed of two fragmentation reactions per analyte). Polysorbate 20 introduced a noticeablebackground signal (ghost peaks). The method shows linear response in spiking studies and is thus suitable for relative quantification with acceptable reproducibility.

[0095] This method was used to investigate formation of the blue substances in upstream intermediates and drug substance and drug product clearance. Formation kinetics in upstream intermediates where therefore analyzed using this LC-MS / MS method. It was shown that the formation already starts during the cell culture upstream process slowly at about day 3 and more prominently at about day 7 (Figure 4B). All samples were incubated for 24 hours at room temperature prior to analysis, which is about the time when color formation peaks.

[0096] Color formation was further analysed in an experiment investigating the formation of the blue substances in HCCF samples during storage, which typically last from between 6 to 48 hours, preferably from about 24 to 30 hours. HCCF samples of 3 L runs for two different recombinant proteins were stored for up to 48 h at room temperature (RT), frozen and analyzed together after all samples were collected by LC-MS / MS measurements targeted against blueOl and blue02. Color formation further increased after about three hours and reached a plateau after about 24 hours (Figure 4D). The recombinant proteins produced (labelled as protein 1 and protein 2) were all antigen-binding proteins, of which protein 2 is a monoclonal antibodies and protein 1 is a multi-specific antigen binding protein. Due to the high sensitivity of the LC-MS / MS method, an increase in color intensity could be detected as early as after three hours, while upon visual inspection of the filter color formation was only detected after 21 hours (compare Figure 4D and Figure 2). Further a temperature dependency was observed with faster color formation and higher color intensity at 37°C and slower color formation and less color intensity at lower temperatures (2-8°C) compared to room temperature (data not shown). No blued or blue 02 was detected in the product pool after protein A affinity chromatography (data not shown).

[0097] The LC-MS / MS method is further illustrated in Figure 5. It was further found that the blue compounds are not present in the drug substance or drug product and therefore do not pose a risk for the produced recombinant product.

[0098] Additional investigations of the full structure identified two important amino acids (cysteine (CYS) and tryptophane (TRP)), which were verified via isotopic labeling cell culture experiments, as most probable root cause for the formation of the blue substances.

[0099] If the full molecular structure of both blue compounds is resolved in the future, the method could be further improved to allow absolute quantification.

[0100] These results were used as a basis to set up a final cell culture DoE study in an Ambr250 with the amino acids CYS and TRP to identify if the formation of the Blue 1 and 2 could be influenced. The aim was to reduce the blue coloration with no impact on process performance.Example 3: Cell culture DoE study in Ambr 250®

[0101] To further analyze the impact of media adjustments on the formation of blue coloration, we performed regular fed- batch processes. The cell culture verification experiment was executed as a Design of Experiments (DoE) study in an Ambr250® bioreactor system. A DoE study is a data collection and analysis tool that allows varying multiple input factors and determines their combined and single effects on different output parameters. Thus, this study can identify interactions of multiple factors in a process by altering the levels of multiple inputs simultaneously in the process. The seed train cultures were processed in shake flasks and the seeding densities were set at 0.7x10E06 cells / ml. 24 vessels were tested with varying CYS and TRP levels (3 Levels = -1 / 0 / +1) using CHO-K1 cells deficient in glutamine synthetase (GS - / -) (CHO-K1 GS) producing an IgG 1 monoclonal antibody (mAb) and cell culture media in a 14 day fed-batch process. The DoE study was based on an l-optimal design and included the factors cysteine (combining cysteine and cystine concentrations) and tryptophane as well as exogenously added lactate. Since lactate revealed no effect on blue coloration, this factor was excluded from DoE evaluation.

[0102] Cells were fed with two feeds, Feed 1 and Feed 2. Feed 1 (comprising TRP and cysteine) was added continuously from day 2 at 30 mL / L / day (of the culture starting volume) until the end of the process on day 14 (12 days of feeding). Feed 2 (comprising cystine) was also added continuously from day 2 at 10 mL / L / day (of the culture starting volume) until the end of the process on day 14 (12 days of feeding). Glucose was added to the process on demand. Cell counting and cell viability determination was performed using a ViCell Blu analyzer (Beckman Coulter, Germany). Glucose, lactate and product titer (IgG) were determined using a Konelab Prime60i (Termo Scientific, USA).

[0103] Feed 1 was added at 30 ml / L / day comprising 6.26 mM cysteine and varying tryptophane concentrations of 9.67 mM, 7.25 mM and 4.83 mM, referred to as conditions (+1), (0) and (-1) herein, respectively. Feed 2 is a cystine feed and was added at 10 ml / L / day comprising varying cystine concentrations of 8.33 mM, 4.17 mM and 0 mM referred to as conditions (+1), (0) and (-1) herein, respectively. The standard medium comprises tryptophane in Feed 1 and cysteine in Feed 2 at the highest tested concentration. The different conditions, including the varying concentrations, the cystine concentrations as cysteine equivalents (Cys equ.), the cumulative amounts and the daily additions are summarized in Table 2 (at the bottom cumulative amounts and daily additions are calculated with the values for the highest CYS concentration and the two lower TRP concentrations underlined).Table 2: Overview on experimental setting and concentrations:

[0104] The blue compound concentrations (Blue 1 and Blue 2) were measured in the final HCCF after storage for 24 hours at room temperature.

[0105] The DoE study showed a direct impact of CYS and TRP on the formation of Blue 1 (Figures 10 and 11) and Blue 2 (Figures 12 and 13). Blue 1 and Blue 2 show the same effects. Surprisingly, formation of Blue 1 and Blue 2 was reduced with lower TRP concentration (Figure 10A and 12A, respectively), but increased with lower CYS concentration (Figure 10B and 12A, respectively). Thus, lowest formation of Blue 1 and Blue 2 was achieved with low TRP concentrations and high CYS concentrations (Figures 11 and 13, respectively).

[0106] The effects of cysteine and / or tryptophane on product titer and viability are displayed in Figure 6 and Figure 8, respectively. Viability was increased with low concentrations of tryptophane (-1) and cystine feeding (-1). By contrast, product titer was strongly increased with high cystine feeding (+1), while higher TRP only had a small impact on product titer. Thus, particularly reducing CYS negatively impacted titer. At the same time, it is known from previous observations that even higher CYS concentrations may negatively affect product quality and particularly increase charge variants.

[0107] The effects of combinational tryptophane and cystine feeding in terms of viability and product titer are shown in Figure 7 and Figure 9. These results confirm that titer was strongly impacted with an optimum for high cystine feeding level and only a minor effect for high tryptophane feeding levels. The effects on viability were generally very small with slightly higher viability with lower tryptophane and cystine feed levels.

[0108] Cell cultivation data for optimized conditions are displayed in Figure 14 including viable cell density (VCD) [106cells / ml], total cell density (TCD) [106cells / ml], viability [%], product concentration [normalized], glucose concentration [g / L], lactate [g / L], osmolarity [mOsmol / kg] and pH low tryptophane (-1) and high cysteine (+1) (grey circles) compared to standard medium (black circles). The process parameters are highly comparable between the two setups. High cysteine in combination with low tryptophane levels do not seems to have a negative impact on the process, while simultaneously reducing the formation of Blue 1 and Blue 2. For product titer the only minor difference was detected with 8 % lower titer for the adjusted medium, which is still acceptable.

[0109] Overall, the CYS and TRP level need to be carefully chosen to reduce the blue coloration without negatively impacting process performance (e.g., viability and product concentration) or product quality (e.g. acidic peak groups (APGs)). Overall, increasing or maintaining high CYS feed levels together with reduced TRP levels prevent or reduce Blue 1 and Blue 2 formation without negatively impacting process performance, including viability and titer. Thus, a certain ratio between CYS and TRP of the cumulative amounts of CYS and TRP as well as the daily feed seems to be important as may be taken from Tables 3 and 4, respectively.Table 3: Ratios of cumulative amounts (based on Cys equivalents)Table 4: Ratios of daily addition (based on Cys equivalents)

[0110] These results can therefore directly be used for media optimization to reduce the formation of the compounds Blue 1 and Blue 2 in future processes. High cystine feeding (hence high cysteine concentrations) with daily additions of at least 250 pM or even 300 pM cysteine equivalence (or cumulative amounts of 5.5 mM or even 6 mM over the course of the process) in combination with lower tryptophane concentrations in feed with daily additions of less than 230 pM or even less than 200 pM (or cumulative amounts of less than 3.8 mM or even less than 3.5 mM) and hence keeping the ratio of CYS to TRP between 1 .6 and 3.0 can reduce the formation of blue coloration while still maintaining an efficient fed-batch process with only minor impact on overall process performance in terms of e.g. growth, titer production and metabolism (see Figure 14).

Claims

CLAIMS1. A method for reducing formation of one or more blue colored compounds in a fed- batch culture of CHO cells, wherein the method comprises:(a) inoculating CHO cells encoding a recombinant protein in a basal cell culture medium to form a cell culture;(b) cultivating the CHO cells in the cell culture under conditions that allow expression of the recombinant protein and secretion into the cell culture fluid (CCF), wherein the cell culture is regularly fed with nutrients by adding a feed medium and / or one or more feed supplement(s);(c) harvesting the CCF to obtain the harvested cell culture fluid (HCCF) comprising the recombinant protein and storing the HCCF until purification; and(d) optionally purifying the recombinant protein from the HCCF, wherein(i) the cell culture comprises until harvesting the CCF in step (c) a cumulative amount of• cysteine of about 5.5-7.5 mM, preferably about 6.0-7.0 mM;• tryptophane of about 2.5-3.8 mM, preferably of about 2.5-3.5 mM; and• a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about1.7 to 3.0, more preferably about 2.0 to 2.8; and / or(ii) the feed medium and / or one or more feed supplement(s) together provide for at least 10 days of the culture an average daily addition of• cysteine of about 250-400 pM, preferably about 300-380 pM;• tryptophane of about 120-230 pM, preferably of about 130-200 pM; and• a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about1.7 to 3.0, more preferably about 2.0 to 2.8; wherein the molar amount of cysteine refers to the molar cysteine equivalent provided by cysteine and / or cystine or a salt or hydrate thereof.

2. The method of claim 1 , wherein the feed medium and / or one or more feed supplement(s) together provide cysteine and tryptophane per day (pM / L / day) and / or cumulative until harvest (pM / L) at a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about 1.7 to 3.0, more preferably about 2.0 to 2.8.

3. The method of claims 1 or 2 wherein the method results in reduced formation of one or more blue colored compounds in the CCF and / or HCCF compared to cysteine andtryptophane provided by a feed medium and / or one or more feed supplement(s) together per day (pM / L / day) and / or cumulative until harvest (pM / L) at a molar ratio of cysteine / tryptophane of less than 1.6.

4. The method of any one of the preceding claims, wherein in (i) more than 15% of the cysteine are provided as cystine, preferably more than 20%, even more preferably more than 30% are provided as cystine and / or in (ii) more than 30% of the cysteine are provided as cystine, preferably more than 40%, even more preferably more than 45% are provided as cystine.

5. The method of any one of the preceding claims, wherein the feeding with nutrients by adding a feed medium and / or one or more feed supplement(s) starts between day 0 and day 4, preferably at days 2-3.

6. The method of any one of the preceding claims, wherein cysteine and tryptophane are provided daily by the feed medium and / or one or more feed supplement(s) starting at least on days 2-4 at a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about 1.7 to 3.0, more preferably about 2.0 to 2.8.

7. The method of any one of the preceding claims, wherein the feed medium and / or one or more feed supplement(s) together provide for at least 12 days of the culture an average daily addition of• cysteine of about 250-400 pM, preferably about 300-380 pM; and• tryptophane of about 120-230 pM, preferably of about 130-200 pM and a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about 1.7 to 3.0, more preferably about 2.0 to 2.8.

8. The method of any one of the preceding claims, wherein the average daily addition is provided per day or at a respective higher concentration at larger intervals.

9. The method of any one of the preceding claims, wherein the basal medium comprises cysteine at about 2-2.5 mM and tryptophane at about 0.8-1.5 mM.

10. The method of any one of the preceding claims, wherein the one or more blue colored compounds are low molecular weight organic chemical compounds comprising 2-3 indole residues.

11. The method of any one of the preceding claims, wherein the one or more blue colored compounds comprise a compound of the following structure:

12. The method of claim 10 or 11, wherein the one or more blue colored compounds comprise a compound comprising three indole residues and the sum formula C34H20N5O5S2.

13. The method of any one of the preceding claims, wherein the one or more blue colored compounds are present in the HCCF or in the CCF and the HCCF.

14. The method of any one of the preceding claims, wherein the one or more blue colored compounds if present in the HCCF result in blue coloration of filters and / or of one or more chromatography columns in step (d).

15. The method of claim 13 or 14, wherein the one or more blue colored compounds if present in the HCCF result in blue coloration of an affinity chromatography column in step (d).

16. A method of manufacturing a recombinant protein of interest comprising the steps of a) inoculating CHO cells encoding a recombinant protein in a basal cell culture medium to form a cell culture; b) cultivating the CHO cells in the cell culture under conditions that allow expression of the recombinant protein and secretion into the cell culture fluid (CCF), wherein the cell culture is regularly fed with nutrients by adding a feed medium and / or one or more feed supplement(s); c) harvesting the CCF to obtain the harvested cell culture fluid (HCCF) comprising the recombinant protein and storing the HCCF until purification; and d) optionally purifying the recombinant protein from the HCCF, wherein(i) the cell culture comprises until harvesting the CCF in step (c) a cumulative amount of cysteine of about 5.5-7.5 mM, preferably about 6.0-7.0 mM;• tryptophane of about 2.5-3.8 mM, preferably of about 2.5-3.5 mM; and• a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about1.7 to 3.0, more preferably about 2.0 to 2.8; and / or(ii) the feed medium and / or one or more feed supplement(s) together provide for at least 10 days of the culture an average daily addition of• cysteine of about 250-400 pM, preferably about 300-380 pM;• tryptophane of about 120-230 pM, preferably of about 130-200 pM; and• a molar ratio of cysteine / tryptophane of about 1.6 to 3.0, preferably about 1.7 to 3.0, more preferably about 2.0 to 2.8; wherein the molar amount of cysteine refers to the molar cysteine equivalent provided by cysteine and / or cystine or a salt or hydrate thereof.

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