Method for increasing the specific production rate of eukaryotic cells

Abstract

The current invention reports the use of meta-tyrosine for increasing the specific productivity of a eukaryotic cell that produces/expresses a polypeptide. In the current method it is not necessary to perform a temperature-, osmolality- or pH shift or to add drugs like valproic acid or sodium butyrate to modulate the specific productivity of the cultivated cells. The method does not affect cell viability or product titer.

Claims

1. A method for increasing specific productivity (qP) of a Chinese Hamster Ovary (CHO) cell, the method comprising culturing the cell in culture medium, wherein the culture medium contains meta-tyrosine.

2-9. (canceled)

10. The method of claim 1, wherein the culture medium further comprises phenylalanine in a non-limiting concentration.

11-15. (canceled)

16. A method for producing an exogenous polypeptide in a Chinese Hamster Ovary (CHO) cell, wherein the CHO cell expresses a nucleic acid encoding the exogenous polypeptide, the method comprising culturing the CHO cell in a culture medium comprising meta-tyrosine.

17. The method of claim 16, wherein the meta-tyrosine in the culture medium is at a concentration of from 0.2 mM to 0.7 mM.

18. The method of claim 16, wherein the meta-tyrosine in the culture medium is at a concentration of from 0.25 mM to 0.6 mM.

19. The method of claim 16, wherein the meta-tyrosine in the culture medium is at a concentration of from 0.3 mM to 0.5 mM.

20. The method of claim 16, wherein the meta-tyrosine in the culture medium is at a concentration of from 0.3 mM to 0.4 mM.

21-22. (canceled)

23. The method of claim 16, wherein the polypeptide is an immunoglobulin or a variant thereof or a fragment thereof or a fusion thereof.

24. The method of claim 16, wherein the culture medium additionally comprises phenylalanine in a non-limiting concentration.

25. The method of claim 24, wherein the molar ratio of meta-tyrosine/phenylalanine is lower than or equal to 1.25.

26. The method of claim 24, wherein the molar ratio of meta-tyrosine/phenylalanine is lower than or equal to 0.25.

27. The method of claim 24, wherein the molar ratio of meta-tyrosine/phenylalanine is lower than or equal to 0.125.

28. The method of claim 24, wherein the molar ratio of meta-tyrosine/phenylalanine is lower than or equal to 0.025.

29. The method of claim 16, wherein the culture medium temperature is kept constant during the process.

30. (canceled)

31. (canceled)

Description

DESCRIPTION OF THE FIGURES

[0082] FIGS. 1A, 1B, 1C, and 1D

[0083] Meta-Tyr modulates CHO biomass generation under Phe limitation conditions. (FIG. 1A) For the fed-batch process two serial continuous feeds, feed 1 and feed 2, were used. CHO fed-batch cultivations were supplemented with either non (control), 0.1 mM, 0.3 mM, or 0.4 mM para-Tyr, ortho-Tyr, or meta-Tyr in the beginning of the process. The cell time integral (CTI), as measure of CHO cell biomass generation, is shown for para-Tyr (FIG. 1B), ortho-Tyr (FIG. 1C) and meta-Tyr (FIG. 1D) supplementation.

[0084] FIGS. 2A, 2B, and 2C

[0085] Different roles of meta-Tyr and ortho-Tyr in CHO cell growth regulation under Phe limitation conditions. The viable cell density is shown for para-Tyr (FIG. 2A), ortho-Tyr (FIG. 2B) and meta-Tyr (FIG. 2C) supplementation. Fed-batch cultivation with no para-Tyr, ortho-Tyr or meta-Tyr supplementation is shown as control.

[0086] FIGS. 3A, 3B, and 3C

[0087] Supplementation of meta-Tyr and ortho-Tyr in CHO fed-batch cultivations does not alter product yield under Phe limitation conditions. The product concentration is shown for para-Tyr (FIG. 3A), ortho-Tyr (FIG. 3B) and meta-Tyr (FIG. 3C) supplementation. Fed-batch cultivation with no para-Tyr, ortho-Tyr or meta-Tyr supplementation is shown as control.

[0088] FIGS. 4A, 4B, and 4C

[0089] Meta-Tyr supplementation increases cell-specific product formation rate qP under Phe limitation conditions. The cell-specific product formation rate qP is shown for para-Tyr (FIG. 4A), ortho-Tyr (FIG. 4B) and meta-Tyr (FIG. 4C) supplementation. Fed-batch cultivation with no para-Tyr, ortho-Tyr or meta-Tyr supplementation is shown as control.

[0090] FIGS. 5A, 5B, 5C, 5D, 5E, and 5F

[0091] Different roles of meta-Tyr and ortho-Tyr for CHO cell viability under Phe limitation conditions. The cell viability and supernatant LDH activity are shown for para-Tyr (FIG. 5A, FIG. 5D), ortho-Tyr (FIG. 5B, FIG. 5E) and meta-Tyr (FIG. 5C, FIG. 5F) supplementation. Fed-batch cultivation with no para-Tyr, ortho-Tyr or meta-Tyr supplementation is shown as control.

[0092] FIGS. 6A, 6B, 6C, and 6D

[0093] Meta-Tyr modulates CHO biomass generation under Phe non-limitation conditions. (FIG. 6A) For the fed-batch process two serial continuous feeds, feed 1 and feed 2 with high Phe concentration, were used. CHO fed-batch cultivations were supplemented with either non (control), 0.1 mM, 0.3 mM, or 0.4 mM para-Tyr, ortho-Tyr, or meta-Tyr in the beginning of the process. The cell time integral (CTI), as measure of CHO cell biomass generation, is shown for para-Tyr (FIG. 6B), ortho-Tyr (FIG. 6C) and meta-Tyr (FIG. 6D) supplementation.

[0094] FIGS. 7A, 7B, and 7C

[0095] Supplementation of meta-Tyr and ortho-Tyr in CHO fed-batch cultivations does not alter product yield under Phe non-limitation conditions. The product concentration is shown for para-Tyr (FIG. 7A), ortho-Tyr (FIG. 7B) and meta-Tyr (FIG. 7C) supplementation. Fed-batch cultivation with no para-Tyr, ortho-Tyr or meta-Tyr supplementation is shown as control.

[0096] FIGS. 8A, 8B, and 8C

[0097] Meta-Tyr supplementation increases cell-specific product formation rate qP under Phe non-limitation conditions. The cell-specific product formation rate qP is shown for para-Tyr (FIG. 8A), ortho-Tyr (FIG. 8B) and meta-Tyr (FIG. 8C) supplementation. Fed-batch cultivation with no para-Tyr, ortho-Tyr or meta-Tyr supplementation is shown as control.

EXAMPLES

[0098] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Reagents and Material

[0099] DL-ortho-tyrosine (2-hydroxy-DL-phenylalanine), DL-meta-tyrosine (3-hydroxy-DL-phenylalanine), L-para-tyrosine, L-phenylalanine, and guanidinium hydrochloride were obtained from Sigma-Aldrich (Munich, Germany). L-ortho-tyrosine (2-hydroxy-L-phenylalanine) and L-meta-tyrosine (3-hydroxy-L-phenylalanine) were purchased from RSP Amino Acids, LLC (Shirley, Mass., USA). All other reagents were purchased from Merck (Darmstadt, Germany) and Sigma-Aldrich (Munich, Germany).

Cells and Cell Cultivation

[0100] For all cell culture experiments a recombinant CHO-K1 cell line, called clone 1, expressing a humanized monoclonal antibody was used. The recombinant CHO-K1 cell line was generated using an L-methionine sulfoximine sensitive CHO-K1 host cell line (Lonza, Cologne, Germany). The cells were cultivated in protein free, chemically defined CD-CHO medium (Life Technologies, Darmstadt, Germany) supplemented with 50 μM L-methionine sulfoximine (Sigma Aldrich, Munich, Germany) during seed train cultivation. Seed train cultivation was performed in shake flasks using a humidified incubator, 7% CO.sub.2, and 37° C. setting. The cells were splitted every three to four days for subcultivation and culture expansion. For all experiments, cells with identical age in culture (appr. 21 generations) until start of the experiment were used.

Meta-Tyr and Ortho-Tyr Supplementation Experiments

[0101] All supplementation experiments were performed using shake flask cultivation systems, chemically defined CD-CHO medium without the selection pressure L-methionine sulfoximine and two appropriate serial continuous applied feeds (feed 1 and feed 2). Recombinant CHO-K1 cells producing a humanized monoclonal antibody were inoculated with 3×10.sup.5 viable cells/mL and cultured for 14 days. CD-CHO base medium was sterile supplemented before inoculation with 0.1 mM, 0.3 mM or 0.4 mM of either ortho-Tyr, meta-Tyr or para-Tyr. Control cultivation with no supplementation of ortho-Tyr, meta-Tyr or para-Tyr was used as reference. Phenylalanine (Phe) non-limitation conditions were realized by increasing concentration of Phe in feed 2.

Viable Cell Densities, Viability and Cell Time Integral

[0102] For analysis of viable and total cell densities an automated Cedex HiRes system (Roche Diagnostics, Mannheim, Germany) was used. Discrimination of viable and total cell densities were evaluated using the trypan blue exclusion staining method and analyzing more than 10 pictures per sample and day according to the manufacturer's specifications. Viable cell density (VCD) and cell viability were calculated as described in equation 1 (Equ 1) and equation 2 (Equ 2), respectively.

Viable cell density=N.sub.Trypan blue negative×(10.sup.5 viable cells/ml)  (Equ 1)

Cell viability=N.sub.Trypan blue negative/(N.sub.Trypan blue negative+N.sub.Trypan blue positive cells)×100%  (Equ 2)

[0103] As indicator for overall biomass generation in the process a cumulative cell time integral (CTI) was calculated as followed (Equ 3).

Cell time integral=Σ(0.5×(VCD.sub.n−1+VCD.sub.n)×(t.sub.n−t.sub.n−1))×(10.sup.5 viable cells×d/ml)  (Equ 3)

[0104] Lactate dehydrogenase (LDH) activity in the cell-free supernatant was analyzed using a Cobas Integra 400 plus system (Roche Diagnostics, Mannheim, Germany).

Quantification of IgG Titer and Calculation of qP

[0105] Product titer war either quantified by a Cobas Integra 400 plus system (Roche, Mannheim, Germany) according to the manufacture's protocol or by PorosA HPLC method as described previously (Zeck et al. 2012). The overall specific productivity qP was calculated for the analysis of cell production capacity according to equation 4.

qP=(Titer.sub.n−Titer.sub.n−1)/(CTI.sub.n−CTI.sub.n−1)×(pg/(viable cell×d))  (Equ 4)

Determination of Peptide Sequence Variants and Identification of Meta- and Ortho-Tyr Sequence Variants with Synthetic Peptides

[0106] Quantification of peptide sequence variants were performed as described previously (Zeck et al. 2012). Briefly, antibody samples (250 μg) were denatured by addition of denaturing buffer (0.4 M Tris, 8 M guanidinium hydrochloride, pH 8) to a final volume of 240 μL. Reduction was achieved by addition of 20 μL of 0.24 M DTT freshly prepared in denaturing buffer and incubation at 37° C. for 60 min. Subsequently, the sample was alkylated by addition of 20 μL of 0.6 M iodoacetic acid in water for 15 min at room temperature in the dark. The excess of alkylation reagent was inactivated by addition of 30 μL of DTT solution. The sample was than buffer exchanged to approximately 480 μL of 50 mM Tris/HCl, pH 7.5 using NAP 5 Sephadex G-25 DNA grade columns (GE Healthcare, Munich, Germany). Digestion was performed with trypsin for 5 h at 37° C. (ratio 1:37). The peptide mixture obtained was injected and separated without pretreatment using reversed phase HPLC (Agilent 1100 Cap LC, Agilent Technologies, Böblingen, Germany). A Polaris 3 C18-ether column (1×250 mm; 3 μm particle diameter, 180 Å pore size) from Varian (Darmstadt, Germany) was used for separation. The solvents were 0.1% formic acid in water (A) and in acetonitrile (B) (Sigma Aldrich, Munich, Germany). A linear gradient from 2 to 38% B was run over 80 min at 37° C. The HPLC eluate was split using Triversa NanoMate (Advion, Ithaca, N.Y.) and 380 nL/min were infused into a LTQ Orbitrap classic tandem mass spectrometer (Thermo Fisher Scientific, Dreieich, Germany) operating in positive ion mode. For confirmation of ortho- and meta-Tyr peaks in extracted ion chromatograms we used synthetic peptides of mAb HC66-72 DQFTISR (unmodified), DQpYTISR, DQmYTISR, and DQoYTISR. Synthetic peptides were purchased from Biosyntan GmbH (Berlin, Germany).

Calculation of Penalty Factors and Surrogate Makers for Phe→Ortho-Tyr and Phe→Meta-Tyr Sequence Variant Prediction

[0107] We hypothesize that the incorporation of meta- and/or ortho-Tyr instead of Phe can be described by a simplified model which assumes that the use of meta- and/or ortho-Tyr instead of Phe is penalized. This penalty factor can result from different sources such as a better transport of L-Phe into the cells and/or an editing mechanism during protein synthesis which tries to prevent the use of meta- and/or ortho-Tyr. This assumption leads to the equation

p×r×[x]=[y]  (Equ 5)

where p is the penalty factor, r is the ratio of average meta- or ortho-Tyr vs. Phe concentrations (during a given time interval), [x] is the concentration of protein produced (in the time interval) and [y] is the concentration of protein produced which has the sequence variant. Note that this model does not include any dependencies on time, process stage, Phe and meta- or ortho-Tyr concentration ratios that lead to, e.g. phase shifts. The calculation of the penalty factor is straightforward (Equ 7).

p=[y]/(r×[x])  (Equ 6)

[0108] Similarly, knowing the penalty factor it is also possible to calculate ratios of meta- or ortho-Tyr to Phe concentrations for a desired percentage of product without sequence variants.

Example 1

Effects of Meta-Tyrosine Supplementation on Modulation of Specific Productivity (qP) Under Phenylalanine Limitation Conditions

[0109] Previously, Gurer-Orhan et al. reported that meta-Tyr supplementation of CHO cells showed dose-dependent cell cytotoxicity. In concentration screens, a 50% reduction of the MTX reduction capacity of CHO cells was observed when supplemented with 0.5 mM meta-Tyr (Gurer-Orhan et al., (2006)). No data or concentration have been reported for ortho-Tyr supplementation in cell cultures to date. Using a dose-dependent cultivation approach, it was aimed to determine the relevance and tolerable concentrations of meta-Tyr and ortho-Tyr on CHO cell growth performance. For this, a CHO cultivation model described in material and methods was supplemented with either 0.1 mM, 0.3 mM or 0.4 mM para-Tyr, ortho-Tyr or meta-Tyr. We used the standard cultivation process with no supplementation as reference, following so-called “control” or “positive control”. Here, Phe will go into limitation by day 10/11.

[0110] In a first approach, the role of meta- and ortho-Tyr on the macroscopic cell growth markers, viable cell density (VCD), cell viability and cell time integral (CTI) as marker for overall biomass production was analyzed. On day 9/10, all cultures tested, except the one with meta-Tyr supplementation, reached a maximum VCD of approximately 180×10.sup.5 cells/ml, while CHO clone 1 treated with meta-Tyr showed dose dependent reduced maximal VCD (FIG. 2). A decreased cumulative CTI for the meta-Tyr supplemented CHO cultures was observed (FIG. 1). In contrast to data published by Gurer-Orhan et al. previously, no significant impact of meta-Tyr supplementation on cell viability in our fed-batch CHO cultivation model was observed. However, supplementation of ortho-Tyr showed lower cell viability with less than 60% on day 14 and higher final LDH activity in the supernatant compared to control and meta-Tyr, as well para-Tyr supplementation (FIG. 5).

[0111] The overall productivity of the cultures, determined by product concentration analysis, revealed no differences between the test cases (FIG. 3). All cultures showed titer stagnation from day 11/12 on. Additionally, meta-Tyr alone showed an overall higher specific productivity qP (FIG. 4). Supplementation of 0.1 mM, 0.3 mM, and 0.4 mM meta-Tyr under Phe limitation conditions increased qP compared to control by +5%, +26%, and +36%, respectively.

Example 2

Effects of Meta-Tyrosine Supplementation on Modulation of Specific Productivity (qP) Under Phenylalanine Non-Limitation Conditions

[0112] In a second approach, the role of meta- and ortho-Tyr supplementation under Phe non-limitation conditions in CHO fed-batch cultivations was analyzed. For that, the amount of Phe in feed 2 was increased to prevent Phe limitation (FIG. 6).

[0113] Again, all cultures tested, except the one with meta-Tyr supplementation, reached similar CTIs of approximately 35,000 to 40,000×10.sup.5 cells*h/ml, while CHO clone 1 treated with meta-Tyr showed dose dependent reduction in CTI (FIG. 6). No differences were observed in product titer for all tested conditions (FIG. 7). However, compared to Phe limitation conditions described before, provision of CHO cultures with sufficient Phe prevent stagnation of product titer. Meta-Tyr supplementation showed an overall higher specific productivity qP (FIG. 4) even under Phe non-limitation conditions (FIG. 8). Supplementation of 0.1 mM, 0.3 mM, and 0.4 mM meta-Tyr under Phe non-limitation conditions increased qP compared to control by +3%, +26%, and +28%, respectively.

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