Application of small peptides supplemented cell culture media for improved cell performance

Abstract

The present invention concerns a method of culturing a cell, wherein the basal cell culture medium and the feed cell culture medium, both, comprise at least one small peptide that comprises at least two alpha-bonded amino acids, wherein at least one of the amino acids is a tyrosine, a cysteine or a cystine. The remaining amino acids of the small peptide are selected from all natural alpha-amino acids or a salt thereof.

Description

[0001] Application of small peptides supplemented cell culture media for improved cell performance

[0002] The present invention concerns the right type of application of soluble amino acid derivatives such as small tyrosine, cysteine or cystine containing peptides that help to enrich cell culture media and feeds by delivering higher concentrations of these essential amino acids.

[0003] The optimization of cell culture basal media, cell culture feed media, and cell culture perfusion media is a key objective in recombinant protein production to achieve maximum productivity. Even small improvements in productivity can have significant economic benefits. CHO cell lines are commonly utilized for recombinant protein production due to their ability to grow well in both adherent and suspension cultures, and their efficient production of numerous proteins. Additionally, regulatory agencies have approved the use of CHO cells and recombinant proteins expressed in them, as they have been extensively characterized. Various methods can be employed to improve CHO cell culture performance, such as using enriched medium, monitoring, and adjusting osmolarity during production, reducing temperatures during specific phases of a cell culture, and / or introducing e.g., sodium butyrate to stimulate expression during the production phase (see, for example, US5705364 to Etcheverry et al.). Additionally, periodic feeding of essential nutrients during fed-batch cultures (see, for example, US5672502 to Birch et al.) and perfusion cultures can also increase production.

[0004] However, there is still a need in the field to continually improve the cell performance of recombinant protein producing cell cultures. The enrichment of cell culture media and feeds is a very promising option but limited by the very low solubility limit of several media and feed components such as amino acids like tyrosine and cystine.

[0005] To solve this solubility problem Barrett and Jacobia (WO2011133902 A2) propose a feed cell culture medium comprising at least one small peptide comprising at least two amino acids, wherein at least one of the amino acids is a cysteine or a tyrosine.

[0006] However, cell performances are not sufficient when cultured within a cell culture basal medium to which a cell culture feed medium comprising one tyrosine peptide and / or one cysteine / cystine peptide is continuously added, since a growth delay, called lag-phase in the early exponential growth phase, was observed.

[0007] The problem is solved by a method of culturing a cell, comprising contacting the cell with a basal cell culture medium and supplementing the basal cell culture medium with a feed cell culture medium or perfusion cell culture medium, wherein the basal cell culture medium and the feed cell culture medium or perfusion cell culture medium, both, comprise at least one small peptide, said small peptide comprising at least two alpha-bonded amino acids, wherein at least one of the amino acids is a tyrosine, a cysteine or a cystine and wherein the remaining amino acids of the small peptide are selected from all natural alpha-amino acids or a salt thereof.

[0008] This means, that in the method according to the present invention the basal cell culture medium comprising the cell already comprises the least one small peptide, said small peptide comprising at least two alpha-bonded amino acids, wherein at least one of the amino acids is a tyrosine, a cysteine or a cystine and wherein the remaining amino acids of the small peptide are selected from all natural alpha-amino acids or a salt thereof before the basal cell culture medium is supplemented with the feed cell culture medium or perfusion cell culture medium also comprising the small peptides mentioned before.

[0009] Advantageously, the basal cell culture medium comprises the least one small peptide that comprises at least two alpha-bonded amino acids, at least one of which being a tyrosine, a cysteine or a cystine in concentrations of 0.25 - 15 mM, preferably 0.5 - 10 mM or 0.75 - 5 mM, most preferable in a concentration of 1 - 2.5 mM.

[0010] A “culture medium”, according to the invention, shall be understood as being a liquid or solid medium containing nutrients, the medium being suitable for nourishing and supporting proliferation and / or productivity of cells in the culture, excluding media for parenteral nutrition. The cultured cells, according to the invention, may be bacterial cells, yeast cells, fungal cells, animal cells, such as mammalian cells or insect cells, and / or plant cells, e.g., algae. Typically, a culture medium provides essential and non-essential amino acids, vitamins, at least one energy source, lipids, and trace elements, all required by the cell for sustaining life, growth and / or product formation. The culture medium may also contain components that enhance growth and / or survival above the minimal rate, including hormones and growth factors. The culture medium has preferably a pH and a salt concentration which supports life, growth and / or product formation of the cells. A culture medium, according to the invention, preferably comprises all nutrients necessary to sustain life and proliferation of the cell culture. Preferred culture media are chemically defined media.

[0011] A “chemically defined medium”, is a medium that contains no cell extracts, cell hydrolysates, or protein hydrolysates. Chemically defined media comprise no components of unknown composition. As is commonly understood by the person skilled in the art, chemically defined media are usually free of animal-derived components. All components of a chemically defined medium have a known chemical structure. Culture media other than defined culture media may be referred to as “complex” culture media.

[0012] A “cell culture medium” shall be understood as being a culture medium suitable for sustaining life, proliferation and / or product formation of animal cells and / or plant cells. A “basal cell culture medium" shall be understood as being a solution or substance containing nutrients in which a culture of cells is initiated.

[0013] A “feed cell culture medium” shall be understood as being a solution or substance with which the cells are fed after the start of the cultivation process. In certain embodiments, a feed cell culture medium contains one or more components not present in a basal cell culture medium. The feed cell culture medium can also lack one or more components present in a basal cell culture medium. Preferably, the concentration of nutrients in the feed cell culture medium exceeds the concentration in the basal cell culture medium to avoid a loss of productivity by dilution.

[0014] A “perfusion medium” shall be understood as being a solution or substance containing nutrients that is continuously added after the beginning of a cell culture, in which harvest is continuously removed.

[0015] A “peptide” shall be understood as being a molecule comprising at least two amino acids covalently coupled to each other by alpha-peptide bonds (R1-CO-NH-R2).

[0016] A “small peptide” (also called oligopeptide) shall be understood as being a molecule comprising less than twenty, preferably two to ten amino acids covalently coupled to each other solely by alpha-peptide-bonds (R1-CO-NH-R2).

[0017] An "amino acid", in the context of the present invention, shall be understood as being a molecule comprising an amino functional group (-NH2) and a carboxylic acid functional group (-COOH), along with a side-chain specific to the respective amino acid. In the context of the present invention, both alpha- and beta-amino acids are included. Preferred amino acids of the invention are alpha-amino acids, in particular the 20 “natural amino” acids including cystine which are all alpha-amino acids as follows:

[0018] Alanine (Ala / A) Arginine (Arg I R) Asparagine (Asn I N) Aspartic acid (Asp I D) Cysteine (Cys I C) Cystine (Cyss / C2) Glutamic acid (Glu I E) Glutamine (Gln / Q) Glycine (Gly / G) Histidine (His I H) Isoleucine (lie / I) Leucine (Leu / L) Lysine (Lys / K)

[0019] Methionine (Met I M)

[0020] Phenylalanine (Phe I F)

[0021] Proline (Pro I P)

[0022] Serine (Ser I S)

[0023] Threonine (Thr / T)

[0024] Tryptophan (Trp I W)

[0025] Tyrosine (Tyr I Y)

[0026] Valine (Val / V)

[0027] Preferably, in the method according to the present invention at least one of the small peptides is a dipeptide, selected from the group consisting of X-tyrosine, tyrosine-X, X-cysteine, cysteine-X, and X-cystine-X, and wherein X is the remaining amino acid of the small peptide and selected from all naturally occurring alpha-amino acids.

[0028] A “dipeptide” shall be understood as being a molecule comprising two amino acids covalently coupled to each other by an alpha-peptide-bond (R1-CO-NH-R2).

[0029] In an embodiment the method of culturing a cell, comprises contacting the cell with a basal cell culture medium and supplementing the basal cell culture medium with a feed cell culture medium or perfusion cell culture medium, wherein the basal cell culture medium and the feed cell culture medium or perfusion cell culture medium, both, comprise at least one small peptide, said small peptide comprising at least two alpha-bonded amino acids, wherein at least one of the amino acids is a tyrosine, a cysteine or a cystine and wherein the remaining amino acids of the small peptide are selected from all natural alpha-amino acids or a salt thereof, wherein the basal cell culture medium comprising the cell already comprises the at least one small peptide before the basal cell culture medium is supplemented with the feed cell culture medium or perfusion cell culture medium also comprising the at least one small peptide.

[0030] The skilled person in the field of cell culture media knows at what intervals the feed medium should be supplemented to the basal cell culture medium. For example, the basal cell culture medium is supplemented with the feed cell culture medium if the glucose concentration of the basal cell culture medium falls below 2 g / L.

[0031] In an embodiment the basal cell culture medium is supplemented with the feed cell culture medium 0.5 to 10% on day 1 to 3, preferably 1 .5% on day 3, 0.5 to 10% on day 4, preferably 2% on day 4, 0.5 to 10% on day 5, preferably 2.5% on day 5, and 0.5 to 10% daily from day 6 to the end of the cultivation, preferably 3% daily from day 6 to the end of the cultivation.

[0032] In a particular embodiment of the method according to the present invention at least one of the one of the remaining amino acids of the small peptide are selected from the group consisting of lysine, glycine, alanine, serine, valine, proline, aspartic acid, and glutamic acid; preferably, at least one of the remaining amino acids of the small peptide is lysine, glycine, or alanine.

[0033] In a preferred embodiment of the method according to the present invention at least one of the small peptides is glycine-tyrosine or tyrosine-glycine or alanine-tyrosine or tyrosine-alanine, i.e. Gly-Tyr, Tyr-Gly, Ala-Tyr and Tyr-Ala, respectively.

[0034] In a further preferred embodiment of the method of the present invention at least one of the small peptides is lysine-cysteine, cysteine-lysine or their oxidized dimers or alanine-cysteine, cysteinealanine, or their oxidized dimers.

[0035] In an embodiment at least one of the small peptides of the basal cell culture medium is selected from lysine-cysteine or alanine-cysteine and at least one of the small peptides of the feed cell culture medium is selected from lysine-cysteine, glycine-tyrosine, alanine-tyrosine or alanine- cysteine.

[0036] Under oxidative conditions (e.g. atmospheric oxygen), peptides comprising cysteine are forming disulfide bonds via oxidized -S-H residues and, therefore, peptide dimers, such as lysine-cysteine, cysteine-lysine, alanine-cysteine, or cysteine-alanine dimers in the present case, i.e. (Lys-Cys)2, Cys-Lys)2, (Ala-Cys)2 and (Cys-Ala)2, respectively. These peptides may also be present as salts or in hydrate form.

[0037] The method according to the present invention is suitable for culturing animal cells or plant cells, most preferred mammalian cells. In specific embodiments the cells to be cultured are CHO cells, COS cells, VERO cells, BHK cells, HEK cells, HELA cells, AE-1 cells, insect cells, fibroblast cells, muscle cells, nerve cells, stem cells, skin cells, endothelial cells and hybridoma cells. Preferred cells of the invention are CHO cells and hybridoma cells. Most preferred cells of the invention are CHO cells.

[0038] Description of the drawing

[0039] Figure 1 shows viable cell density (*106cells / mL) (depicted by solid curves), and IgG titer (g / L) (depicted by dashed curves) of IgG-producing CHO cells grown in (i) a basal cell culture medium (HyClone™ ActiPro™) supplemented with a cell culture feed (Cell Boost™ 7a) containing a cysteine as well as tyrosine dipeptide (DP) or (ii) HyClone™ ActiPro™ containing a cysteine DP supplemented with Cell Boost™ 7a containing a cysteine as well as tyrosine DP. (Lys-Cys)2 and Gly-Tyr are used as the cysteine and tyrosine DP, respectively. See general experimental part as well as experiment 1 . Figure 2 shows viable cell density (*106cells / mL) (depicted by solid curves), and IgG titer (g / L) (depicted by dashed curves) of IgG-producing CHO cells grown in (i) a basal cell culture medium (HyClone™ ActiPro™) supplemented with a cell culture feed (Cell Boost™ 7a) containing a cysteine as well as tyrosine dipeptide (DP) or (ii) HyClone™ ActiPro™ containing a cysteine DP supplemented with Cell Boost™ 7a containing a cysteine as well as tyrosine DP. (Lys-Cys)2 and Ala-Tyr are used as the cysteine and tyrosine DP, respectively. See general experimental part as well as experiment 2.

[0040] Figure 3 shows viable cell density (*106cells / mL) (depicted by solid curves), and IgG titer (g / L) (depicted by dashed curves) of IgG-producing CHO cells grown in (i) a basal cell culture medium (HyClone™ ActiPro™) supplemented with a cell culture feed (Cell Boost™ 7a) containing a cysteine as well as tyrosine dipeptide (DP) or (ii) HyClone™ ActiPro™ containing a cysteine DP supplemented with Cell Boost™ 7a containing a cysteine as well as tyrosine DP. (Ala-Cys)2 and Gly-Tyr are used as the cysteine and tyrosine DP, respectively. See general experimental part as well as experiment 3.

[0041] Figure 4 shows viable cell density (*106cells / mL) (depicted by solid curves), and IgG titer (g / L) (depicted by dashed curves) of IgG-producing CHO cells grown in (i) a basal cell culture medium (HyClone™ ActiPro™) supplemented with a cell culture feed (Cell Boost™ 7a) containing a cysteine as well as tyrosine dipeptide (DP) or (ii) HyClone™ ActiPro™ containing a cysteine DP supplemented with Cell Boost™ 7a containing a cysteine as well as tyrosine DP. (Ala-Cys)2 and Ala-Tyr are used as the cysteine and tyrosine DP, respectively. See general experimental part as well as experiment 4.

[0042] General experimental Part

[0043] Fed batch testing was performed using IgG-producing CHO cells grown either in a basal cell culture medium HyClone™ ActiPro™ (Cat. No. SH31037; Cytiva, Marlborough, Massachusetts, US) or in basal cell culture medium HyClone™ ActiPro™ additionally containing a cysteine- containing small DP (Evonik Operations GmbH, Essen, Germany), both supplemented with a cell culture feed Cell Boost™ 7a (Cat. No. SH31026; Cytiva, Marlborough, Massachusetts, US) additionally containing a cysteine- as well as tyrosine-containing small DP.

[0044] The small peptides present in the cell culture media or supplemental feeds of this invention, which contain cysteine and tyrosine, are meant to be utilized with any exemplary basal cell culture medium, with any exemplary feed cell culture medium, and with any exemplary perfusion cell culture medium. These media can comprise a balanced mixture of concentrated amino acids, glucose, vitamins, trace elements, or other appropriate components suitable for the growth of a desired cell type, as can be deemed by an expert in the field. Spinning tube bioreactors (Cat. No. 227245; Greiner Bio-One International GmbH, Frickenhausen, Germany) with 25 mL working volume, without a pH and a pO2 control were used to grow the cells. Glucose levels were kept manually above 2 g / L by adding a desired volume of a glucose stock solution (450 g / L in ddH2O) to each cell culture each day. According to the feeding schedule used, CHO cells were fed with the cell culture feed Cell Boost™ 7a containing a cysteine- as well as tyrosine-containing small DP 1 .5% on day 3, 2% on day 4, 2.5% on day 5, and 3% daily from day 6 to day 13.

[0045] Experiment 1

[0046] IgG-producing CHO cells grown in HyClone™ ActiPro™ medium, which additionally contained a cysteine-containing small DP achieved a more than 45% higher viable cell density

[0047] (21 .67*106cells / mL on day 9) and a ~9% better IgG productivity (5.07 g / L on day 14), as compared to HyClone™ ActiPro™ medium without a cysteine DP (14.87*106cells / mL on day 9; 4.66 g / L on day 14), while both cultures were supplemented with Cell Boost™ 7a feed medium, which additionally contained a cysteine as well as a tyrosine DP (Figure 1). In particular, in this example, the dipeptide L-lysyl-L-cysteine, which was added as it’s dimer (N, / '-di-L-lysy l-L-cystine) , was added as dry powder at a concentration of 1 .5 mM to basal cell culture medium and mixed until dissolved. In the same way, 15 mM N, A / '-di-L-lysyl-L-cystine and 25 mM glycyl-L-tyrosine were added to the feed cell culture medium.

[0048] Experiment 2

[0049] IgG-producing CHO cells grown in HyClone™ ActiPro™ medium, which additionally contained a cysteine-containing small DP achieved a more than 38% higher viable cell density

[0050] (13.95*106cells / mL on day 9) and a -15% better IgG productivity (5.15 g / L on day 14), as compared to HyClone™ ActiPro™ medium without a cysteine DP (10.12*106cells / mL on day 9;

[0051] 4.49 g / L on day 14), while both cultures were supplemented with Cell Boost™ 7a feed medium, which additionally contained a cysteine as well as a tyrosine DP (Figure 1). In particular, in this example, the dipeptide L-lysyl-L-cysteine, which was added as it’s dimer (N, / '-di-L-lysy l-L-cystine) , was added as dry powder at a concentration of 1 .5 mM to basal cell culture medium and mixed until dissolved. In the same way, 15 mM N, A / '-di-L-lysyl-L-cystine and 25 mM L-alanyl-L-tyrosine were added to the feed cell culture medium.

[0052] Experiment 3

[0053] IgG-producing CHO cells grown in HyClone™ ActiPro™ medium, which additionally contained a cysteine-containing small DP achieved a more than 82% higher viable cell density

[0054] (15.73*106cells / mL on day 9) and a -27% better IgG productivity (5.27 g / L on day 14), as compared to HyClone™ ActiPro™ medium without a cysteine DP (8.62*106cells / mL on day 9;

[0055] 4.16 g / L on day 14), while both cultures were supplemented with Cell Boost™ 7a feed medium, which additionally contained a cysteine as well as a tyrosine DP (Figure 1). In particular, in this example, the dipeptide L-alanyl-L-cysteine, which was added as it’s dimer (N, N -di-L-alany l-L- cystine), was added as dry powder at a concentration of 1 .5 mM to basal cell culture medium and mixed until dissolved. In the same way, 15 mM A / ,A / '-di-L-alanyl-L-cystine and 25 mM glycyl-L- tyrosine were added to the feed cell culture medium.

[0056] Experiment 4

[0057] IgG-producing CHO cells grown in HyClone™ ActiPro™ medium, which additionally contained a cysteine-containing small DP achieved a more than 63% higher viable cell density (14.17*106cells / mL on day 9) and a -21% better IgG productivity (5.18 g / L on day 14), as compared to HyClone™ ActiPro™ medium without a cysteine DP (8.69*106cells / mL on day 9;

[0058] 4.29 g / L on day 14), while both cultures were supplemented with Cell Boost™ 7a feed medium, which additionally contained a cysteine as well as a tyrosine DP (Figure 1). In particular, in this example, the dipeptide L-alanyl-L-cysteine, which was added as it’s dimer (N, N -di-L-alany l-L- cystine), was added as dry powder at a concentration of 1 .5 mM to basal cell culture medium and mixed until dissolved. In the same way, 15 mM A / ,A / '-di-L-alanyl-L-cystine and 25 mM L-alanyl-L- tyrosine were added to the feed cell culture medium.

Claims

Claims1 . A method of culturing a cell, comprising contacting the cell with a basal cell culture medium and supplementing the basal cell culture medium with a feed cell culture medium or perfusion cell culture medium, wherein the basal cell culture medium and the feed cell culture medium or perfusion cell culture medium, both, comprise at least one small peptide, said small peptide comprising at least two alpha-bonded amino acids, wherein at least one of the amino acids is a tyrosine, a cysteine or a cystine and wherein the remaining amino acids of the small peptide are selected from all natural alpha-amino acids or a salt thereof.

2. The method of claim 1 , wherein at least one of the small peptides is selected from the group consisting of X-tyrosine, tyrosine-X, X-cysteine, cysteine-X, and X-cystine-X, and wherein X is the remaining amino acid of the small peptide and selected from all naturally occurring alphaamino acids.

3. The method of any of claim 1 or claim 2, wherein at least one of the remaining amino acids of the small peptide are selected from the group consisting of lysine, glycine, alanine, serine, valine, proline, aspartic acid, and glutamic acid.

4. The method of claim 3, wherein the at least one of the remaining amino acids of the small peptide is lysine, glycine, or alanine.

5. The method of any of the preceding claims, wherein at least one of the small peptides is glycine-tyrosine or tyrosine-glycine or alanine-tyrosine or tyrosine-alanine.

6. The method of any of the preceding claims, wherein at least one of the small peptides is lysine-cysteine, cysteine-lysine or their oxidized dimers or alanine-cysteine, cysteine-alanine, or their oxidized dimers.

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