HK1237365B - Method for increasing the specific production rate of eukaryotic cells - Google Patents

Description

Method for increasing the specific productivity of eukaryotic cells

The present invention belongs to the field of polypeptide production and cell culture media. Reported herein are the use of meta-tyrosine in a cell culture medium for increasing specific productivity (qP), methods for producing polypeptides in a cell culture medium containing meta-tyrosine, and cell culture media containing meta-tyrosine.

Background of the Invention

Cell cultures are used in fermentation processes to produce a variety of substances, particularly proteins. A distinction is made between processes in which the cell cultures are genetically unmodified and form their own metabolites, and processes in which organisms are genetically modified so that they produce larger quantities of their own substances, such as proteins, or produce foreign (heterologous) substances. Organisms producing a variety of substances are supplied with a nutrient medium that ensures their survival and enables the production of the desired target compound. Numerous culture media are known that allow for optimal cultivation of specific hosts for these purposes.

Protein biotherapeutics (e.g., monoclonal antibodies) are considered well-established drugs for the treatment of serious diseases and conditions such as cancer, multiple sclerosis, and rheumatoid arthritis (see review by Leader et al. (Leader et al., 2008)). Key molecular mass attributes of these highly active compounds must be closely monitored to ensure patient safety and functional efficacy. Unintended chemical modifications of target proteins can occur throughout the manufacturing process, starting with synthesis in microorganisms and cell cultures, and during protein purification, formulation, and storage. The most commonly observed chemical degradation pathways for protein drugs are asparagine deamidation, aspartate isomerization (Wakankar and Borchardt, 2006; Diepold et al., 2012; Dengl et al., 2013), and oxidation (Li et al., 1995; Ji et al., 2009; Hensel et al., 2011). Recently, several studies have reported an additional, related, enzymatic byproduct, the so-called protein SV, of recombinantly synthesized biotherapeutics (Khetan et al., 2010; Wen et al., 2009; Feeney et al., 2013). SVs are unintended amino acid substitutions at genetically predicted positions, resulting from either intrinsic nucleotide mutations (Bridges, 2001) or misincorporation of alternative amino acids during translation (Zeck et al., 2012). Translational misincorporation is thought to result from the promiscuity of structurally related amino acids by aminoacyl-tRNA synthetases (aaRSs) during endogenous substrate limitation (Feeney et al., 2013; Jakubowski, 2001). Gurer-Urhan et al. (2006) reported misincorporation of free meta-tyrosine into cellular proteins as a potential cytotoxic mechanism of oxidized amino acids.

SUMMARY OF THE INVENTION

It has been found that supplementing the culture medium with meta-tyrosine provides increased specific productivity (qP) for eukaryotic (host) cells producing (non-endogenous) polypeptides. It has been found that the addition of meta-tyrosine to eukaryotic cell cultures does not result in a significant decrease in cell viability or final product titer. Cell growth in such cultures is reduced/affected in an unfavorable manner (represented by a reduced viable cell density (VCD) and overall biomass production (as indicated by CTI)). In the methods of the present invention, no temperature shift, osmolality shift, or pH shift is required to increase the specific productivity of the cultured cells. Nor is it necessary to adjust the specific productivity by adding drugs such as valproic acid or sodium butyrate. The occurrence of amino acid sequence variants (SVs) caused by the misincorporation of alternative amino acids in the presence of meta-tyrosine during mRNA translation is controlled by the additional input of non-limiting concentrations of phenylalanine.

One aspect as reported herein is the use of meta-tyrosine for increasing the specific productivity of eukaryotic host cells producing/expressing/secreting a (non-endogenous/exogenous) polypeptide.

In one embodiment of this aspect, the eukaryotic host cell is a mammalian cell. In one embodiment of this aspect, the eukaryotic host cell is a Chinese hamster ovary (CHO) cell. In one embodiment of this aspect, the CHO cell is a CHO cell/cell line grown in suspension (=CHO suspension cells/cell line). In one embodiment of this aspect, the CHO cell is a CHO-K1 cell.

In one embodiment of this aspect, m-tyrosine is added to give a (final) concentration of 0.2 mM to 0.7 mM. In one embodiment of this aspect, m-tyrosine is added to give a (final) concentration of 0.25 mM to 0.6 mM. In one embodiment of this aspect, m-tyrosine is added to give a (final) concentration of 0.3 mM to 0.5 mM. In one embodiment of this aspect, m-tyrosine is added to give a (final) concentration of 0.3 mM to 0.4 mM.

In one embodiment of this aspect, the specific productivity is increased by at least 5% compared to the same production process without the supplementation of meta-tyrosine. In one embodiment of this aspect, the specific productivity is increased by at least 10% compared to the same production process without the supplementation of meta-tyrosine. In one embodiment of this aspect, the specific productivity is increased by at least 20% compared to the same production process without the supplementation of meta-tyrosine. In one embodiment of this aspect, the specific productivity is increased by at least 25% compared to the same production process without the supplementation of meta-tyrosine.

In one embodiment of this aspect, the use is in a protein-free culture medium. In one embodiment of this aspect, the use is in a chemically defined culture medium. In one embodiment of this aspect, the use is in a protein-free chemically defined culture medium.

In one embodiment of this aspect, use is carried out in a culture medium that additionally comprises phenylalanine at a non-limiting concentration. In one embodiment of this aspect, use is carried out in a culture medium that additionally comprises phenylalanine at a non-limiting concentration, wherein phenylalanine is added by continuous infusion or by one or more independent bolus shots of Phe stock solution at the beginning or during the fermentation process.

In one embodiment of this aspect, the molar ratio of meta-tyrosine to phenylalanine is less than or equal to 1.25. In one embodiment of this aspect, the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is greater than or equal to 95.0%. In one embodiment of this aspect, the molar ratio of meta-tyrosine to phenylalanine is less than or equal to 1.25 and the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is greater than or equal to 95.0%. This means that 5% or less of the meta-tyrosine residues are misincorporated into the protein sequence relative to the correctly incorporated phenylalanine residues. In one embodiment of this aspect, the molar ratio of meta-tyrosine to phenylalanine is less than or equal to 0.25. In one embodiment of this aspect, the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is greater than or equal to 99.0%. In one embodiment of this aspect, the molar ratio of meta-tyrosine to phenylalanine is less than or equal to 0.25 and the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is greater than or equal to 99.0%. In one embodiment of this aspect, the molar ratio of meta-tyrosine/phenylalanine is less than or equal to 0.125. In one embodiment of this aspect, the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is greater than or equal to 99.5%. In one embodiment of this aspect, the molar ratio of meta-tyrosine/phenylalanine is less than or equal to 0.125 and the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is greater than or equal to 99.5%. In one embodiment of this aspect, the molar ratio of meta-tyrosine/phenylalanine is less than or equal to 0.025. In one embodiment of this aspect, the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is greater than or equal to 99.9%. In one embodiment of this aspect, the molar ratio of meta-tyrosine/phenylalanine is less than or equal to 0.025 and the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is greater than or equal to 99.9%.

In one embodiment of this aspect, use is performed at a constant temperature.

In one embodiment of this aspect, use is performed at a reduced temperature during use.

In one embodiment of this aspect, use is performed at a constant pH.

In one embodiment of this aspect, the polypeptide is an immunoglobulin or a variant thereof or a fragment thereof or a fusion thereof. In one embodiment of this aspect, the polypeptide is a human immunoglobulin or a humanized immunoglobulin or a variant thereof or a fragment thereof or a fusion thereof. In one embodiment of this aspect, the polypeptide is a humanized antibody. In one embodiment of this aspect, the polypeptide is a humanized monoclonal antibody.

One aspect as reported herein is a method for producing a polypeptide in a eukaryotic host cell expressing a nucleic acid encoding the polypeptide, the method comprising culturing the eukaryotic host cell in a culture medium comprising meta-tyrosine.

In one embodiment of this aspect, m-tyrosine is added to give a (final) concentration of 0.2 mM to 0.7 mM. In one embodiment of this aspect, m-tyrosine is added to give a (final) concentration of 0.25 mM to 0.6 mM. In one embodiment of this aspect, m-tyrosine is added to give a (final) concentration of 0.3 mM to 0.5 mM. In one embodiment of this aspect, m-tyrosine is added to give a (final) concentration of 0.3 mM to 0.4 mM.

In one embodiment of this aspect, the specific productivity is increased by at least 5% compared to the same production process without the supplementation of meta-tyrosine. In one embodiment of this aspect, the specific productivity is increased by at least 10% compared to the same production process without the supplementation of meta-tyrosine. In one embodiment of this aspect, the specific productivity is increased by at least 20% compared to the same production process without the supplementation of meta-tyrosine. In one embodiment of this aspect, the specific productivity is increased by at least 25% compared to the same production process without the supplementation of meta-tyrosine.

In one embodiment of this aspect, the eukaryotic host cell is a mammalian cell. In one embodiment of this aspect, the eukaryotic host cell is a Chinese hamster ovary (CHO) cell. In one embodiment of this aspect, the CHO cell is a CHO suspension cell/a CHO cell grown in suspension. In one embodiment of this aspect, the CHO cell is a CHO-K1 cell.

In one embodiment of this aspect, the method is performed in a protein-free culture medium. In one embodiment of this aspect, the method is performed in a chemically defined culture medium. In one embodiment of this aspect, the method is performed in a protein-free chemically defined culture medium.

In one embodiment of this aspect, the method is performed in a culture medium that additionally comprises phenylalanine at a non-limiting concentration. In one embodiment of this aspect, the method is performed in a culture medium that additionally comprises phenylalanine at a non-limiting concentration, wherein phenylalanine is added by continuous infusion or by one or more independent boluses of a Phe stock solution at the beginning or during the fermentation process.

In one embodiment of this aspect, the molar ratio of meta-Tyrosine/phenylalanine is less than or equal to 1.25. In one embodiment of this aspect, the final accuracy of the protein sequence relative to Phe→m-Tyr misincorporation is greater than or equal to 95.0%. In one embodiment of this aspect, the molar ratio of meta-Tyrosine/phenylalanine is less than or equal to 1.25 and the final accuracy of the protein sequence relative to Phe→m-Tyr misincorporation is greater than or equal to 95.0%. This means that 5% or less of the meta-Tyrosine residues are misincorporated into the protein sequence relative to the correctly incorporated phenylalanine residues.

In one embodiment of this aspect, the molar ratio of m-Tyrosine/Phenylalanine is less than or equal to 0.25. In one embodiment of this aspect, the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is greater than or equal to 99.0%. In one embodiment of this aspect, the molar ratio of m-Tyrosine/Phenylalanine is less than or equal to 0.25 and the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is greater than or equal to 99.0%.

In one embodiment of this aspect, the molar ratio of m-Tyrosine/Phenylalanine is less than or equal to 0.125. In one embodiment of this aspect, the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is greater than or equal to 99.5%. In one embodiment of this aspect, the molar ratio of m-Tyrosine/Phenylalanine is less than or equal to 0.125 and the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is greater than or equal to 99.5%.

In one embodiment of this aspect, the molar ratio of m-Tyrosine/phenylalanine is less than or equal to 0.025. In one embodiment of this aspect, the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is greater than or equal to 99.9%. In one embodiment of this aspect, the molar ratio of m-Tyrosine/phenylalanine is less than or equal to 0.025 and the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is greater than or equal to 99.9%.

In one embodiment of this aspect, the method is performed at a constant temperature. In one embodiment of this aspect, the method is performed at a reduced temperature during use.

In one embodiment of this aspect, the method is performed at a constant pH.

In one embodiment of this aspect, the polypeptide is an immunoglobulin or a variant thereof or a fragment thereof or a fusion thereof. In one embodiment of this aspect, the polypeptide is a human immunoglobulin or a humanized immunoglobulin or a variant thereof or a fragment thereof or a fusion thereof. In one embodiment of this aspect, the polypeptide is a humanized antibody. In one embodiment of this aspect, the polypeptide is a humanized monoclonal antibody.

One aspect as reported herein is a culture medium comprising m-tyrosine and phenylalanine in a molar ratio lower than or equal to 1.25. One aspect as reported herein is a culture medium comprising m-tyrosine and phenylalanine in a molar ratio lower than or equal to 1.25 and a final accuracy of the protein sequence with respect to Phe→m-Tyr misincorporation higher than or equal to 95.0%. This means that 5% or less of the m-tyrosine residues were misincorporated into the protein sequence relative to the correctly incorporated phenylalanine residues.

One aspect as reported herein is a culture medium comprising m-tyrosine and phenylalanine in a molar ratio lower than or equal to 0.25. One aspect as reported herein is a culture medium comprising m-tyrosine and phenylalanine in a molar ratio lower than or equal to 0.25 and a final accuracy of the protein sequence with respect to Phe→m-Tyr misincorporation higher than or equal to 99.0%.

One aspect as reported herein is a culture medium comprising m-tyrosine and phenylalanine in a molar ratio lower than or equal to 0.125. One aspect as reported herein is a culture medium comprising m-tyrosine and phenylalanine in a molar ratio lower than or equal to 0.125 and a final accuracy of the protein sequence with respect to Phe→m-Tyr misincorporation higher than or equal to 99.5%.

One aspect as reported herein is a culture medium comprising m-tyrosine and phenylalanine in a molar ratio lower than or equal to 0.025. One aspect as reported herein is a culture medium comprising m-tyrosine and phenylalanine in a molar ratio lower than or equal to 0.025 and a final accuracy of the protein sequence with respect to Phe→m-Tyr misincorporation higher than or equal to 99.9%.

In one embodiment of this aspect, the culture medium is a protein-free culture medium. In one embodiment of this aspect, the culture medium is a chemically defined culture medium. In one embodiment of this aspect, the culture medium is a protein-free chemically defined culture medium.

Detailed Description of the Invention

Modulating the specific productivity (qP) of eukaryotic cells is considered a powerful tool to address changes in product quality attributes such as protein N-glycosylation microheterogeneity, charge variants, protein aggregates, and fragment characteristics. For example, a moderate qP is assumed to favor post-translational N-glycosylation because the target protein can have a longer residence time in the glycan-forming compartments, namely the endoplasmic reticulum (ER) and the Golgi apparatus (Hossler et al., 2009).

Currently, qP regulation is achieved in biotechnological practice through drugs such as the histone deacetylase (HDAC) inhibitors valproic acid and sodium butyrate (Lee et al., 2009; Murray-Beaulieu et al., 2009) or through chemo-physical parameters such as temperature shifts (Hendrick et al., 2001), pH shifts (Yoon et al., 2005), and increased osmolality (Han et al., 2009). These approaches have significant drawbacks. On the one hand, HDAC inhibitors severely reduce cell viability and induce apoptosis, which can lead to secondary problems such as target protein fragmentation and poor clearance of host cell proteins and DNA during protein purification. On the other hand, chemo-physical parameters are difficult to control in GMP facilities and often vary in terms of transfer kinetics between different bioreactors due to vessel limitations and different mass transfer. Here, we report that the specific supplementation of biotechnological processes with m-tyrosine is a promising alternative to existing processes for increasing qP in eukaryotic cells, particularly CHO cells, without affecting cell viability and without the need for chemo-physical regulation.

However, Gurer-Orhan et al., (2006) have reported that certain levels of meta-tyrosine concentration induce cytotoxic effects in adherently grown CHO cells.

The invention as reported herein is based at least in part on the observation that supplementation of the culture medium with meta-tyrosine leads to an increase in the specific productivity (qP) of eukaryotic suspension cells, in particular CHO suspension cells, producing exogenous polypeptides.

In contrast to the observations of Gurer-Orhan et al., addition of meta-tyrosine did not result in a cytotoxic effect (i.e., cell viability and final product titer were not significantly affected), although cell growth was adversely affected, as can be seen from the reduced viable cell density (VCD) and total biomass production (as indicated by CTI) (see Figures 1 to 5).

Thus, one aspect as reported herein is the use of meta-tyrosine to increase the specific productivity of a polypeptide producing eukaryotic host cell.

One aspect as reported herein is a method for producing a polypeptide in a eukaryotic host cell expressing a nucleic acid encoding the polypeptide, the method comprising culturing the eukaryotic host cell in a culture medium comprising meta-tyrosine.

In one embodiment of all aspects, the eukaryotic host cell is a mammalian cell. In one embodiment, the mammalian cell is selected from the group consisting of CHO cells (e.g., CHO-K1 or CHO DG44), BHK cells, NSO cells, SP2/0 cells, HEK 293 cells, HEK 293EBNA cells, PER.C6 cells, and COS cells. In one embodiment of all aspects, the mammalian cell is a Chinese hamster ovary (CHO) cell. In one embodiment of all aspects, the CHO cell is a CHO suspension cell. In one embodiment of all aspects, the CHO cell is a CHO-K1 cell.

It has been found that an increase in specific productivity can be achieved within a certain concentration range of meta-tyrosine added to the culture medium.

In one embodiment of all aspects, m-tyrosine is added to produce a concentration of 0.2 mM to 0.7 mM. In one embodiment of all aspects, m-tyrosine is added to produce a concentration of 0.25 mM to 0.6 mM. In one embodiment of all aspects, m-tyrosine is added to produce a concentration of 0.3 mM to 0.5 mM. In one embodiment of all aspects, m-tyrosine is added to produce a concentration of 0.3 mM to 0.4 mM.

In one embodiment of all aspects, the specific productivity is increased by at least 5% compared to the same production process without meta-tyrosine supplementation. In one embodiment of all aspects, the specific productivity is increased by at least 10% compared to the same production process without meta-tyrosine supplementation. In one embodiment of all aspects, the specific productivity is increased by at least 20% compared to the same production process without meta-tyrosine supplementation. In one embodiment of all aspects, the specific productivity is increased by at least 25% compared to the same production process without meta-tyrosine supplementation.

It has been found that in order to achieve increased specific productivity, no temperature shift, osmolality shift or pH shift is required, and no addition of drugs such as valproic acid or sodium butyrate is required to adjust the specific productivity, as reported in the art. However, it will be appreciated by those skilled in the art that these adjustments to the culture process may also be additionally performed/included in the method as reported herein.

In one embodiment of all aspects, the use or method is performed at a constant temperature.In one embodiment of all aspects, the use or method is performed at a reduced temperature during use.

In one embodiment of all aspects the use or method is performed at a constant pH.

Potential amino acid sequence variants (SVs) due to misincorporation of alternative amino acids that may occur when meta-tyrosine is added to the culture medium during translation were controlled by the additional input of non-limiting concentrations of phenylalanine.

In Table 1 , the number/frequency/score of Phe→xTyr (x means meta-Tyr and/or ortho-Tyr) sequence variant formation of the tracer peptides is shown (maximum levels of Phe→xTyr misincorporation at day 14 when supplemented with 0.1 mM, 0.3 mM and 0.4 mM ortho-Tyr and meta-Tyr).

From the data of supernatant meta-Tyr, o-Tyr and L-Phe and the corresponding Phe→xTyr misincorporation from the supplementation experiments, threshold ratios of meta-Tyr/Phe and o-Tyr/Phe were calculated that resulted in a final accuracy of the generated sequences of 99.9%, 99.5%, 99.0% and 95.0% (referring to o-Tyr and meta-Tyr misincorporation).

Table 1

It has been found that adjusting the maximum threshold ratio of Tyr/Phe in the culture medium to 1.25, 0.25, 0.125 or 0.025 can control/avoid unwanted SV in the produced polypeptides (with 95.0%, 99.0%, 99.5% or 99.9% sequence accuracy, respectively) while increasing specific productivity. The specific productivity was increased regardless of whether additional Phe supplementation was performed (see Figure 8).

In one embodiment of all aspects the culture medium additionally comprises phenylalanine at non-limiting concentrations.

In one embodiment of all aspects, the molar ratio of m-Tyrosine/Phenylalanine is lower than or equal to 1.25. In one embodiment of all aspects, the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is higher than or equal to 95.0%. In one embodiment of this aspect, the molar ratio of m-Tyrosine/Phenylalanine is lower than or equal to 1.25 and the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is higher than or equal to 95.0%.

In one embodiment of all aspects, the molar ratio of m-Tyrosine/Phenylalanine is lower than or equal to 0.25. In one embodiment of all aspects, the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is higher than or equal to 99.0%. In one embodiment of this aspect, the molar ratio of m-Tyrosine/Phenylalanine is lower than or equal to 1.25 and the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is higher than or equal to 99.0%.

In one embodiment of all aspects, the molar ratio of m-Tyrosine/Phenylalanine is lower than or equal to 0.125. In one embodiment of this aspect, the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is higher than or equal to 99.5%. In one embodiment of this aspect, the molar ratio of m-Tyrosine/Phenylalanine is lower than or equal to 1.25 and the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is higher than or equal to 99.5%.

In one embodiment of all aspects, the molar ratio of m-Tyrosine/Phenylalanine is less than or equal to 0.025. In one embodiment of this aspect, the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is greater than or equal to 99.9%. In one embodiment of this aspect, the molar ratio of m-Tyrosine/Phenylalanine is less than or equal to 1.25 and the final accuracy relative to the protein sequence of Phe→m-Tyr misincorporation is greater than or equal to 99.9%.

definition

As used herein, "biomass" refers to the amount or weight of cells cultured in a culture medium. Biomass can be measured directly or indirectly by determining viable cell density, total cell density, cell time integral (relative to viable cell density and total cell density), cell volume time integral (relative to viable cell density and total cell density), packed cell volume, dry weight, or wet weight.

As used herein, "bioreactor" refers to any container for growing mammalian cell cultures. Typically, a bioreactor will be at least 1 liter and can be 10, 100, 250, 500, 1000, 2500, 5000, 8000, 10,000, 12,000 liters or larger or any volume therebetween. The internal conditions of the bioreactor include, but are not limited to, pH, dissolved oxygen, and temperature, which are generally controlled during the incubation time. The bioreactor can comprise any material suitable for accommodating mammalian cell cultures suspended in the culture medium under the culture conditions of the present invention, including glass, plastic, or metal.

As used herein, "cell density" refers to the number of cells present in a given volume of culture medium.

"Cell viability" refers to the fraction of cells that are alive at a particular time relative to the total number of live and dead cells in culture at that time.

The term "cell culture" refers to cells grown in suspension or adherently in roller bottles, flasks, glass or stainless steel culture vessels, and the like. The term "cell culture" also encompasses large-scale protocols, such as bioreactors. The present invention encompasses cell culture methods for large-scale and small-scale production of polypeptides. Various methods can be used and alternatively run in batch, split-batch, fed-batch, or perfusion modes, including but not limited to fluidized bed bioreactors, shake flask cultures, or stirred tank bioreactor systems.

As used interchangeably within the present invention, the terms "cell culture medium," "culture medium," or "culture medium" refer to a nutrient solution for growing mammalian cells. Such nutrient solutions typically include a variety of factors necessary for growth and maintaining a cellular environment. For example, common nutrient solutions can include basal medium formulations, a variety of supplements depending on the type of culture, and occasionally a selection agent. Generally, such solutions provide the essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required for minimum cell growth and/or survival. Such solutions may also contain supplementary components that enhance growth and/or survival above the minimum rate, including but not limited to hormones and/or other growth factors, specific ions such as sodium, chloride, calcium, magnesium, and phosphate, buffering components, vitamins, nucleosides or nucleotides, trace elements, amino acids, lipids, and/or glucose or other energy sources. The culture medium is advantageously formulated to an optimal pH and salt concentration for cell survival and proliferation. The culture medium may be a protein-free medium, meaning that the culture medium will not contain full-length proteins but will contain undefined peptides, such as peptides from plant hydrolysates. The culture medium may comprise human serum albumin and human transferrins, but potentially comprise animal-derived insulin and lipids, or contain xeno-free medium containing human serum albumin, human transferrins, human insulin and chemically defined lipids. Alternatively, the culture medium may be a chemically known culture medium, i.e., a culture medium in which all substances are determined and present at a determined concentration. These culture media may contain only recombinant proteins and/or hormones or protein-free chemically known culture media, i.e., if desired, only contain low molecular weight components and synthetic peptides/hormones. Chemically known culture media may also be completely free of any protein.

The term "cell" or "host cell" refers to a cell into which a nucleic acid (e.g., a nucleic acid encoding a heterologous polypeptide) can be introduced/transfected. Host cells include prokaryotic cells for propagating vectors/plasmids and eukaryotic cells for expressing nucleic acids. In one embodiment, the eukaryotic cell is a mammalian cell. In another embodiment, the mammalian host cell is selected from the following mammalian cells, including CHO cells (e.g., CHO-K1 or CHO DG44), BHK cells, NSO cells, SP2/0 cells, HEK 293 cells, HEK 293EBNA cells, PER.C6 cells, and COS cells. For host cell fermentation and therefore for expressing the target polypeptide, culture medium is used. Typically, CHO cells are widely used to express pharmaceutical polypeptides in the laboratory or on a large scale during production. Due to its widespread distribution and use, the characteristic properties and genetic background of CHO cells are well known. Therefore, CHO cells are approved by regulatory authorities for the production of therapeutic proteins for humans. In one embodiment, the mammalian cell is a CHO cell. In one embodiment, the mammalian cell is a CHO suspension cell line/a CHO cell line grown in suspension.

The method of the present invention is suitable for producing secreted heterologous polypeptides on a large scale, ie, in an industrial manner.

The cultivation of cells used to produce the desired polypeptide on a large scale generally consists of a series of independent cultivations, wherein all cultivations except the final cultivation (i.e., large-scale cultivation, i.e., the last cultivation in this series) are carried out until a certain cell density is reached in the culture vessel. If the predetermined cell density is reached, the entire culture or its fraction is used to inoculate the next culture vessel, which has a larger volume, which is at most 100 times that of the previous culture. The entire cultivation that serves as the basis for at least one further cultivation with a larger volume is called a "seed fermentation" or "seed culture". Only in large-scale cultivation, i.e., in a cultivation that is not intended to serve as the basis for further cultivation with a larger volume (also known as a "main fermentation"), is there a cultivation endpoint determined by the concentration of secretory heterologous immunoglobulin produced in the culture medium or during the cultivation time. As used in this application, the term "large-scale" refers to the final cultivation of an industrial production process. In one embodiment, large-scale cultivation is carried out in a volume of at least 100 liters, in another embodiment in a volume of at least 500 liters, and in another embodiment in a volume of at least 1000 liters up to 25,000 liters. In one embodiment, the final (i.e., large-scale) culture medium does not contain a eukaryotic selection agent.

As used herein, "splitting" is also known as passaging or subculturing cells. This involves transferring a small number of cells into fresh culture medium, thereby inoculating the new culture with the split cells. In suspension cultures, a small amount of culture containing some cells is diluted into a larger volume of fresh culture medium.

As used herein, "titer" refers to the total amount of recombinantly expressed polypeptide produced by mammalian cell culture in a given volume of culture medium. Titer is generally expressed in units of: mg polypeptide/ml culture medium.

"Gene" refers to a nucleic acid that is a segment that can express a peptide, polypeptide or protein, such as on a chromosome or on a plasmid. Beside the coding region (i.e., structural gene), the gene contains other functional elements, such as signal sequences, promoters, introns and/or terminators.

"Structural gene" refers to the region of a gene without a signal sequence, ie, the coding region.

As used herein, the term "expression" refers to transcription and/or translation occurring inside a cell. The transcription level of the target product in a host cell can be determined based on the amount of the corresponding mRNA present in the cell. For example, mRNA transcribed from a selected nucleic acid can be quantified by PCR or by Northern blot hybridization (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). The protein encoded by the selected nucleic acid can be quantified by a variety of methods, such as by ELISA, by measuring the biological activity of the protein, or by using antibodies that recognize and bind to the protein, using an assay method (such as Western blotting or radioimmunoassay) that is unrelated to this type of activity and is quantified (see Sambrook et al., 1989, above).

The term "polypeptide" is a polymer composed of amino acid residues joined by peptide bonds, whether naturally or synthetically produced. A polypeptide having fewer than about 20 amino acid residues may be referred to as a "peptide." A polypeptide comprising two or more amino acid chains or comprising amino acid chains of 100 amino acids or more in length may be referred to as a "protein." A polypeptide or protein may also contain non-peptide components, such as sugar groups or metal ions. Sugars and other non-peptide substituents may be added to the protein by the cell producing the protein and may vary with the type of cell. Proteins and polypeptides are defined herein according to their amino acid backbone structure; appendages such as sugar groups are not generally specified, but may be present. In one embodiment, the polypeptide is an immunoglobulin or immunoglobulin fragment or immunoglobulin conjugate. In one embodiment, the polypeptide is an immunoglobulin heavy chain or immunoglobulin light chain or fragment, a fusion or conjugate thereof. An "exogenous" or "non-endogenous" polypeptide is a polypeptide that does not originate from within the host cell used.

As used herein, the term "nucleic acid" is a polymer composed of independent nucleotides, i.e., a polynucleotide. It refers to naturally occurring nucleic acids or partially or completely non-naturally occurring nucleic acids, which, for example, encode polypeptides that can be recombinantly produced. Nucleic acids can be composed of isolated or chemically synthesized DNA fragments. Nucleic acids can be integrated into another nucleic acid, for example, into an expression plasmid or genome/chromosome of a host cell. Plasmids include shuttle vectors and expression vectors. Generally, plasmids will also include a prokaryotic propagation unit comprising a replication origin (e.g., ColE1 replication origin) and a selectable marker (e.g., ampicillin or tetracycline resistance gene) for replication and selection of vectors in bacteria, respectively.

The term "immunoglobulin" refers to a molecule comprising at least two so-called light chain polypeptides (light chain) and two so-called heavy chain polypeptides (heavy chain). Each heavy chain polypeptide and light chain polypeptide comprises a variable domain (variable region) (usually the amino-terminal portion of the polypeptide chain), which comprises a binding region capable of interacting with an antigen. Each heavy chain polypeptide and light chain polypeptide also comprises a constant region (usually the carboxyl-terminal portion). The constant region of the heavy chain mediates the binding of the immunoglobulin i) to cells (such as phagocytes) carrying Fc gamma receptors (FcγRs) or ii) to cells carrying the neonatal Fc receptor (FcRn) (also known as the Brambell receptor). It also mediates binding to certain factors, including factors of the classical complement system such as component C1q.

The term "immunoglobulin" is used herein in the broadest sense and encompasses a variety of immunoglobulin structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and immunoglobulin fragments, so long as they exhibit the desired antigen-binding activity.

Depending on the amino acid sequence of the heavy chain constant region, immunoglobulins are divided into different classes: IgA, IgD, IgE, IgG, and IgM. Some of these classes are further divided into subclasses (isotypes), namely IgG1, IgG2, IgG3, and IgG4 for IgG, or IgA1 and IgA2 for IgA. Depending on the class to which the immunoglobulin belongs, the heavy chain constant region is referred to as α (IgA), δ (IgD), ε (IgE), γ (IgG), and μ (IgM), respectively. In one embodiment, the immunoglobulin is an IgG class immunoglobulin. In another embodiment, the immunoglobulin has a human constant region or a constant region derived from a human source. In yet another embodiment, the immunoglobulin belongs to the IgG4 subclass or the IgG1, IgG2, or IgG3 subclass, and the antibody is modified in such a way that Fcγ receptor (e.g., FcγRIIIa) binding and/or C1q binding cannot be detected. In one embodiment, the immunoglobulin is a human IgG4 subclass or a mutated human IgG1 subclass. In one embodiment, the immunoglobulin is of the human IgG1 subclass with mutations L234A and L235A. In another embodiment, for Fcγ receptor binding, the immunoglobulin is of the IgG4 subclass, or of the IgG1 or IgG2 subclass, with mutations in L234, L235, and/or D265, and/or contains the PVA236 mutation. In yet another embodiment, the immunoglobulin has a mutation selected from S228P, L234A, L235A, L235E, SPLE (S228P and L235E), and/or PVA236 (PVA236 means replacement of the amino acid sequence ELLG (given in the single-letter amino acid code) from amino acid positions 233 to 236 of IgG1 or EFLG of IgG4 with PVA). In one embodiment, the immunoglobulin is of the IgG4 subclass and has the IgG4 mutation S228P, or the immunoglobulin is of the IgG1 subclass and has the mutations L234A and L235A.

The variable domain of an immunoglobulin light or heavy chain in turn comprises different segments, namely four framework regions (FR) and three hypervariable regions (CDR).

An "immunoglobulin fragment" refers to a polypeptide comprising at least one domain from a group of domains, including the variable domain, _CH1 domain, hinge region, _CH2 domain, _CH3 domain, _CH4 domain, or the variable domain or _CL domain of an immunoglobulin heavy chain. This also includes derivatives and variants thereof. Additionally, variable domains may contain one or more amino acids or amino acid regions deleted.

"Immunoglobulin conjugate" refers to a polypeptide comprising at least one domain of an immunoglobulin heavy chain or light chain conjugated to another polypeptide via a peptide bond. The other polypeptide is a non-immunoglobulin peptide, such as a hormone, growth receptor, antifusogenic peptide, etc.

As used herein, "Phe non-limiting" or "Phe non-limiting" concentration means that phenylalanine is fed in excess, i.e., the culture is supplemented with, for example, 0.6 mM Phe daily starting on day 6 until day 14. Without additional feeds, Phe will typically be limiting ("Phe limited") by day 10 or 11. Phenylalanine supplementation can be added by continuous feed or, alternatively, by one or more separate bolus injections of Phe stock solution at the beginning or during the fermentation process.

The term "increased specific productivity" means that, under the conditions described herein, the specific productivity of the corresponding host cells is higher relative to the same production process without the supplementation of meta-tyrosine. Specific productivity (qP) is calculated as reflected in the Examples as a measure of the daily production capacity of the cells (amount of polypeptide/protein produced, e.g., in picograms).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Meta-Tyr regulates CHO biomass production under Phe-limited conditions. (A) Fed-batch process using two series of continuous feeds: Feed 1 and Feed 2. At the beginning of the process, CHO fed-batch cultures were not supplemented (control), supplemented with 0.1 mM, 0.3 mM, or 0.4 mM p-Tyr, o-Tyr, or m-Tyr. As a measure of CHO cell biomass production, the cell time integral (CTI) for p-Tyr (B), o-Tyr (C), and m-Tyr (D) supplementation is shown.

Figure 2 shows the different roles of m-Tyr and o-Tyr in regulating CHO cell growth under Phe-limited conditions. Viable cell density is shown following supplementation with p-Tyr (A), o-Tyr (B), and m-Tyr (C). A fed-batch culture without supplementation with p-Tyr, o-Tyr, or m-Tyr is shown as a control.

FIG3 : Supplementation of m-Tyr and o-Tyr in CHO fed-batch cultures does not alter product yields under Phe-limited conditions. Product concentrations are shown for p-Tyr (A), o-Tyr (B), and m-Tyr (C) supplementation. Fed-batch cultures without p-Tyr, o-Tyr, or m-Tyr supplementation are shown as controls.

Figure 4 shows that m-Tyr supplementation increases the cell-specific product formation rate qP under Phe-limiting conditions. The cell-specific product formation rate qP is shown for p-Tyr (A), o-Tyr (B), and m-Tyr (C) supplementation. A fed-batch culture without p-Tyr, o-Tyr, or m-Tyr supplementation is shown as a control.

Figure 5 Differential effects of m-Tyr and o-Tyr on CHO cell viability under Phe-limited conditions. Cell viability and supernatant LDH activity are shown for cells supplemented with p-Tyr (A, D), o-Tyr (B, E), and m-Tyr (C, F). Fed-batch cultures without p-Tyr, o-Tyr, or m-Tyr supplementation are shown as controls.

Figure 6: Meta-Tyr regulates CHO biomass production under Phe-non-limiting conditions. (A) A fed-batch process uses two continuous feeds: Feed 1 and Feed 2 with a high Phe concentration. At the beginning of the process, CHO fed-batch cultures were not supplemented (control), supplemented with 0.1 mM, 0.3 mM, or 0.4 mM p-Tyr, o-Tyr, or m-Tyr. As a measure of CHO cell biomass production, the cell time integral (CTI) for p-Tyr (B), o-Tyr (C), and m-Tyr (D) supplementation is shown.

FIG7 : Supplementation of m-Tyr and o-Tyr in CHO fed-batch cultures does not alter product yields under Phe-non-limiting conditions. Product concentrations are shown for p-Tyr (A), o-Tyr (B), and m-Tyr (C) supplementation. Fed-batch cultures without p-Tyr, o-Tyr, or m-Tyr supplementation are shown as controls.

Figure 8 shows that m-Tyr supplementation increases the cell-specific product formation rate qP under Phe-non-limiting conditions. The cell-specific product formation rate qP of p-Tyr (A), o-Tyr (B), and m-Tyr (C) is shown. A fed-batch culture without supplementation of p-Tyr, o-Tyr, or m-Tyr is shown as a control.

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 to the procedures described without departing from the spirit of the invention.

Reagents and materials

DL-o-tyrosine (2-hydroxy-DL-phenylalanine), DL-m-tyrosine (3-hydroxy-DL-phenylalanine), L-p-tyrosine, L-phenylalanine, and guanidine hydrochloride were obtained from Sigma-Aldrich (Munich, Germany). L-o-tyrosine (2-hydroxy-L-phenylalanine) and L-m-tyrosine (3-hydroxy-L-phenylalanine) were purchased from RSP Amino Acids, LLC (Shirley, MA, USA). All other reagents were purchased from Merck (Darmstadt, Germany) and Sigma-Aldrich (Munich, Germany).

Cells and cell culture

For all cell culture experiments, a recombinant CHO-K1 cell line expressing a humanized monoclonal antibody was used, referred to as clone 1. Recombinant CHO-K1 cell line was generated using a CHO-K1 host cell line (Lonza, Cologne, Germany) sensitive to L-methionine sulfoximine. During seed series cultivation, cells were cultivated in a protein-free, chemically defined CD-CHO culture medium (Life Technologies, Darmstadt, Germany) supplemented with 50 μM L-methionine sulfoximine (Sigma Aldrich, Munich, Germany). Seed series cultivation was performed in a humidified incubator, 7% CO ₂ , and 37°C environment in a shake flask. Every 3 to 4 days, cells were sub-bottled to culture and expand the culture. For all experiments, cells of the same age (approximately 21 generations) were used until the start of the experiment.

m-Tyr and o-Tyr supplementation experiments

All supplementation experiments were performed using a shake flask culture system, a chemically defined CD-CHO medium containing no selective pressure L-methionine sulfoximine, and two suitable series of continuously applied feeds (feed 1 and feed 2). Recombinant CHO-K1 cells producing humanized monoclonal antibodies were inoculated with 3×10 ⁵ viable cells/mL and cultured for 14 days. Prior to inoculation, CD-CHO basal medium was aseptically supplemented with 0.1 mM, 0.3 mM, or 0.4 mM o-Tyr, m-Tyr, or p-Tyr. A control culture not supplemented with o-Tyr, m-Tyr, or p-Tyr was used as a reference. Phenylalanine (Phe) non-limiting conditions were achieved by increasing the Phe concentration in feed 2.

Live cell density, viability and cell time integral

To analyze viable cell density and total cell density, an automated Cedex HiRes system (Roche Diagnostics, Mannheim, Germany) was used. Each sample and more than 10 images per day were analyzed using trypan blue exclusion according to the manufacturer's instructions to evaluate the difference between viable cell density and total cell density. Viable cell density (VCD) and cell viability were calculated as described in Equation 1 (Equ1) and Equation 2 (Equ2), respectively.

(Equ1) Viable cell density = N _{trypan blue negative} × (10 ⁵ viable cells/ml)

(Equ2) Cell viability = N _{trypan blue negative} / (N _{trypan blue negative} + N _{trypan blue positive} cells) × 100%

As an indicator of the total biomass produced during the process, the cumulative cell time integral (CTI) was calculated as follows (Equ 3).

(Equ3) Cellular time integral = Σ(0.5 × (VCD _n-1 + VCD _n ) × (t _n – _{t n-1} )) × (10 ⁵ viable cells × d/ml)

The cell-free supernatant was analyzed for lactate dehydrogenase (LDH) activity using the Cobas Integra 400plus system (Roche Diagnostics, Mannheim, Germany).

IgG titer quantification and qP calculation

Product titer was quantified by Cobas Integra 400plus system (Roche, Mannheim, Germany) according to the manufacturer's protocol or by PorosA HPLC method as described previously (Zeck et al., 2012). The overall specific productivity qP was calculated according to Equation 4 to analyze cell production capacity.

(Equ 4) qP = (titer _n - titer _n-1 )/( _{CTIn - CTIn-1} ) × (pg/(viable cells × d))

Determination of peptide sequence variants and identification of m-Tyr and o-Tyr sequence variants using synthetic peptides

Quantification of peptide sequence variants was performed as previously described (Zeck et al., 2012). In short, antibody samples (250 μg) were denatured by adding denaturing buffer (0.4M Tris, 8M guanidine hydrochloride, pH 8) to a final volume of 240 μL. Reduction was achieved by adding 20 μL 0.24M DTT freshly prepared in denaturing buffer and incubating at 37°C for 60 minutes. Subsequently, the sample was alkylated for 15 minutes at room temperature in the dark by adding 20 μL 0.6M iodoacetic acid in water. Excess alkylating agent was inactivated by adding 30 μL DTT solution. Subsequently, NAP 5 Sephadex G-25 DNA grade columns (GE Healthcare, Munich, Germany) were used to exchange the sample's buffer into approximately 480 μL 50mM Tris/HCl, pH 7.5. Digestion was performed with trypsin at 37°C for 5 hours (ratio 1:37). The peptide mixture obtained was injected and separated using reverse phase HPLC (Agilent 1100 Cap LC, Agilent Technologies, Germany) without pretreatment. A Polaris 3 C18-ether column (1x 250mm; 3 μm particle size, pore size) from Varian (Darmstadt, Germany) was used for separation. The solvent was 0.1% formic acid (SigmaAldrich, Munich, Germany) in water (A) and acetonitrile (B). A linear gradient of 2% B to 38% B was run at 37°C over 80 minutes. The HPLC eluate was split using a Triversa NanoMate (Advion, Ithaca, NY) and 380 nL/min and input into a LTQ Orbitrap classic tandem mass spectrometer (ThermoFisherScientific, Dreieich, Germany) running in positive ion mode. To confirm the o-Tyr and m-Tyr peaks in the extracted ion chromatograms, we used the following synthetic peptides: mAb HC66-72DQFTISR (unmodified), DQpYTISR, DQmYTISR, and DQoYTISR. The synthetic peptides were purchased from Biosyntan GmbH (Berlin, Germany).

Calculate Penalty Factors and Surrogate Makers for Phe→o-Tyr and Phe→meta-Tyr Sequence Variant Prediction

We hypothesized that the incorporation of m-Tyr and/or o-Tyr instead of Phe can be described by a simplified model that assumes a penalty for the use of m-Tyr and/or o-Tyr but not for the use of Phe. This penalty coefficient may arise from better transport of different sources such as L-Phe into the cell and/or from editing mechanisms that try to prevent the use of m-Tyr and/or o-Tyr during protein synthesis. This assumption leads to the equation

(Equ 5)p×r×[x]＝[y]，

Where p is the penalty coefficient, r is the ratio of the average meta-Tyr concentration or o-Tyr concentration to the Phe concentration (during a given time interval), [x] is the concentration of the produced protein (in the time interval), and [y] is the concentration of the produced protein with the sequence variant. Note that this model does not include any dependence on time, process stage, Phe and meta-Tyr concentration or o-Tyr concentration ratio that could lead to, for example, phase shifts. The calculation of the penalty coefficient is straightforward (Equation 7).

(Equ 6) p = [y] / (r × [x])

Similarly, knowing the penalty coefficient, it is also possible to calculate the ratio of the concentration of meta-Tyr or ortho-Tyr to the concentration of Phe for the desired percentage of product without sequence variants.

Example 1

Effect of m-tyrosine supplementation on the regulation of specific productivity (qP) under phenylalanine-limited conditions

Gurer-Orhan et al. have previously reported that supplementation of CHO cells with inter-Tyr exhibits dose-dependent cytotoxicity. In a concentration screen, a 50% reduction in the ability of MTX to reduce CHO cells was observed when 0.5 mM inter-Tyr was supplemented (Gurer-Orhan et al., (2006)). To date, no data or concentrations for o-Tyr supplementation in cell culture have been reported. A dose-dependent culture protocol was used to determine the relevance and tolerable concentrations of inter-Tyr and o-Tyr for CHO cell growth performance. To this end, the CHO culture model described in the materials and methods was supplemented with 0.1 mM, 0.3 mM or 0.4 mM p-Tyr, o-Tyr or inter-Tyr. Following the so-called "control" or "positive control", we used an unsupplemented standard culture method as a reference. Here, Phe was brought into restriction as of day 10/11.

In the first approach, the effects of inter-Tyr and o-Tyr on macroscopic cell growth markers, viable cell density (VCD), cell viability, and cell time integral (CTI) as total biomass production were analyzed. On day 9/10, all cultures were tested except one culture supplemented with inter-Tyr, which reached a maximum VCD of approximately 180 x 10 ⁵ cells/ml, while inter-Tyr treated CHO clone 1 showed a dose-dependent decrease in maximum VCD ( FIG2 ). A cumulative decrease in CTI was observed for inter-Tyr supplemented CHO cultures ( FIG1 ).

In contrast to data previously published by Gurer-Orhan et al., no significant effect of m-Tyr supplementation on cell viability was observed in our fed-batch CHO culture model. However, o-Tyr supplementation showed lower cell viability, less than 60% on day 14, and higher final lactate dehydrogenase activity in the supernatant compared to the control and m-Tyr and p-Tyr supplementation (Figure 5).

Total culture productivity, determined by product concentration analysis, revealed no differences between experimental conditions ( FIG3 ). All cultures showed plateauing titers from day 11/12 onward. Furthermore, single meta-Tyr exhibited an overall higher specific productivity, qP ( FIG4 ). Under Phe-limited conditions, supplementation with 0.1 mM, 0.3 mM, and 0.4 mM meta-Tyr increased qP by +5%, +26%, and +36%, respectively, compared to the control.

Example 2

Effect of m-tyrosine supplementation on the regulation of specific productivity (qP) under non-phenylalanine-limiting conditions

In the second approach, the effect of meta-Tyr and o-Tyr supplementation in CHO fed-batch cultures under Phe non-limiting conditions was analyzed. To this end, the amount of Phe in feed 2 was increased to prevent Phe limitation (Figure 6).

Again, all cultures were tested, with the exception of one culture supplemented with meta-Tyr, which achieved similar CTIs of approximately 35,000 to 40,000 x 10 ⁵ cells * h / ml, while meta-Tyr treated CHO clone 1 showed a dose-dependent reduction in CTI ( FIG. 6 ). No differences in product titer were observed for all conditions tested ( FIG. 7 ). However, providing sufficient Phe to the CHO cultures prevented stagnation in product titer compared to the Phe-limited conditions described above. Meta-Tyr supplementation showed an overall higher specific productivity qP ( FIG. 4 ), even under Phe-unlimited conditions ( FIG. 8 ). Supplementation with 0.1 mM, 0.3 mM, and 0.4 mM meta-Tyr under Phe-unlimited conditions increased qP by +3%, +26%, and +28%, respectively, compared to the control.

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Claims (10)

1. Use of meta-tyrosine to increase the specific productivity of Chinese hamster ovary (CHO) cells that produce exogenous peptides, wherein meta-tyrosine is added to obtain a concentration of 0.1 mM to 0.4 mM. 2. The use according to claim 1, wherein the CHO cells are CHO suspension cells. 3. The use according to claim 1 or 2, wherein the use is in a culture medium additionally containing a non-limiting concentration of phenylalanine, and the molar ratio of meta-tyrosine to phenylalanine is less than or equal to 1.25. 4. The use according to claim 1, wherein the polypeptide is an immunoglobulin or a variant thereof, a fragment thereof, or a fusion thereof. 5. A method for producing exogenous polypeptides in eukaryotic host cells expressing nucleic acids encoding polypeptides, the method comprising culturing eukaryotic host cells in a medium containing meta-tyrosine, wherein meta-tyrosine is added to produce a concentration of 0.1 mM to 0.4 mM. 6. The method according to claim 5, wherein the eukaryotic host cell is a Chinese hamster ovary (CHO) cell. 7. The method of claim 5, wherein the polypeptide is an immunoglobulin or a variant thereof, a fragment thereof, or a fusion thereof. 8. The method of claim 5, wherein the culture medium additionally contains a non-limiting concentration of phenylalanine, wherein the molar ratio of meta-tyrosine to phenylalanine is less than or equal to 1.25. 9. The method according to any one of claims 5 to 8, wherein the temperature remains constant during the method. 10. A method for increasing the specific productivity of Chinese hamster ovary (CHO) cells that produce exogenous peptides, wherein the culture medium contains 0.1 mM to 0.4 mM meta-tyrosine.