Method for performing perfusion cell culture

Abstract

The present invention relates to a method and medium for performing perfusion cell culture, wherein a concentrated medium auxiliary substance containing phosphotyrosine is added to the basal medium in addition to the perfusion cell culture. This makes it possible to maintain or even increase the efficiency of cell culture while reducing the amount of culture medium consumed.

Description

[Technical Field]

[0001] The present invention relates to a method and medium for performing perfusion cell culture, wherein a concentrated medium auxiliary substance is added to the basal medium in addition to the perfusion cell culture. This makes it possible to maintain or even increase the efficiency of cell culture while reducing the amount of culture medium consumed. [Background technology]

[0002] The most common culture modes used in biomanufacturing are batch cell culture, fed batch, and perfusion cell culture. The reason for choosing one of these techniques lies in various factors related to the protein and / or host. Cells are cultured attached to a carrier or in suspension. The easiest mode to operate is probably the batch bioreactor. After inoculation, cells proliferate and are produced until a limit is reached due to medium consumption, and then the cell density begins to decrease. The second very common process is the fed batch, where nutrient limitation is prevented by adding high concentrations of feed at various points in the culture. Thus, the culture period is longer than in the batch mode, and volumetric productivity and space-time yield are increased.

[0003] The perfusion cell culture process allows for continuous operation over long periods, up to several months, by continuously perfusing the bioreactor with fresh medium throughout the culture, simultaneously providing fresh nutrients to the cells, and retaining a large number of viable cells while removing consumed medium and optionally dead cells and target products. Key advantages of perfusion technology include higher yield per bioreactor volume, increased flexibility, and more consistent product quality. However, achieving this requires very careful system and process design. Unlike fed-batch systems, perfusion systems do not accumulate waste. Expressed proteins can be rapidly removed and made available for purification. This is a significant advantage for easily destabilized proteins.

[0004] Removing consumed culture medium while retaining cells during culture can be done using various filtration techniques, such as alternating tangential flow (ATF) and standard tangential flow filtration (TFF). Other methods include the use of sedimentation devices, centrifuges, or acoustic devices. Another option is to retain cells by binding them to a substrate in a bioreactor (such as capillary fibers, membranes, or microcarriers in a fixed bed).

[0005] A review of perfusion cell culture providing details on preferred settings can be found in "Perfusion mammalian cell culture for recombinant protein manufacturing—A critical review" by Jean-Marc Bielser et al., Biotechnology Advances 36(2018)1328-1340. A filtration-based perfusion system that allows for the removal of only dead cells from the system through bleeding is described in "Potential of Cell Retention Techniques for Large-Scale High-Density Perfusion Culture of Suspended Mammalian Cells" by D. Voisard, F. Meuwly, P.-A. Ruffieux, G. Baer, ​​and A. Kadouri, Cytotechnology 28:163-175, 1998.

[0006] In some perfusion processes, ultrafiltration membranes are used to retain the product within the bioreactor. This process is also known as "concentrated fed-batch" or CFB. Concentrated fed-batch cell culture increases production capacity without additional volumetric capacity. Information on this particular perfusion process can be found in William C. Yanga,*, Daniel F. Minklera, Rashmi Kshirsagarb, Thomas Ryllb, Yao-Ming Huanga, Journal of Biotechnology 217(2016)1-11.

[0007] Figure 1 shows a schematic diagram of a state-of-the-art perfusion cell culture bioreactor. The bioreactor (1), containing a cell culture (2) with liquid cell culture medium and cells, is optionally agitated by a stirrer 3. New fresh medium can be added via Q-in, also called P. A harvest stream containing the liquid medium and target product exits the bioreactor (1) via the Q-harvest line. Q harvest is often referred to as H. A cell holding device (4) holds the cells in the manner described above, for example, so that cell-free harvests or cell-down harvests can be collected. Typically, in perfusion cell culture, medium is fed continuously or semi-continuously via Q-in, and harvests are taken continuously or semi-continuously via Q-harvest.

[0008] Once the cell density reaches the desired setpoint, excess cells are removed during the steady-state perfusion process to maintain the steady cell concentration and achieve steady-state operation. This is done via a bleed stream, also known as Q-bleed. This stream is a suspension containing liquid and solid portions. The solid portion contains viable and non-viable cells, as well as cell debris, while the liquid portion contains liquid cell culture medium, as well as waste components and target products such as cellular metabolites present in the liquid. To maintain a constant volume within the bioreactor, typically, P=H+B, also known as Q-in=Q-harvest+Q-bleed, meaning that the volume of cell culture medium newly added to the bioreactor via Q-in must be equivalent to the volume removed via Q-harvest and Q-bleed.

[0009] Dynamic perfusion cell culture is also possible. In this case, the cells are not removed, which means there is no bleeding. Such a process is typically performed in a shorter time compared to steady-state perfusion as described earlier.

[0010] Process performance and yield depend on various flow rates. Increasing the perfusion rate generally allows for the production of more biomass, and therefore more target products. The faster the cell growth, the greater the bleed rate and the lower the yield. Thus, stable operation is often defined as a state where the cell density is large enough to achieve economically viable productivity, but cell growth is controlled by either nutrient restriction or other environmental factors to minimize the bleed rate.

[0011] As a result, during the perfusion process, cells are held inside the bioreactor, and there is a constant exchange of medium, typically, with fresh medium supplied to the cells at the same rate as consumed medium (cellular waste and medium depleted of nutrients by cellular metabolism) is removed. This exchange is described as the perfusion rate and expressed as the vessel volume (VVD) of medium exchanged per day. Lowering the perfusion rate can also reduce the burden on the cell holding device by directly decreasing the amount of fresh medium handled and consumed by the system. However, higher perfusion rates can accommodate higher sustained viable cell density (VCD), productivity, and rapid product extraction, which can further increase product quality. The most common medium exchange rates are 1–3 vVD for cell concentrations of 30–100 million viable cells / ml.

[0012] The rate of medium exchange can also be called the perfusion rate. For each cell type and each perfusion process, there is a specific perfusion rate required to sustain the cells during that process. Lower perfusion rates typically result in reduced growth rates. Therefore, the high perfusion rates typically required in perfused cell culture are a disadvantage of this process, for example, compared to batch or fed-batch cell culture. Medium consumption in the perfusion process is much higher than in other cell culture processes.

[0013] Therefore, it would be advantageous to find a way to reduce medium consumption in the perfusion cell culture process while maintaining equivalent cell numbers and perfusion cell culture performance compared to a perfusion cell culture process using only basal medium.

[0014] It has been found that by using and adding two different medium compositions, rather than using only one perfusion medium, it is possible to lower the perfusion rate of a given perfusion process. In addition to the standard basal perfusion medium also used to initiate the perfusion process, a second, more concentrated medium composition, also referred to as a concentrated medium supplement, is added stepwise or gradually while increasing the cell concentration / viable cell density, while the perfusion rate of the standard basal perfusion medium typically decreases. Summary of the Invention

[0015] The present invention is a process for perfusion cell culture, comprising culturing cells in a bioreactor system comprising a bioreactor having a medium inlet and a harvest outlet, whereby, i. during the cell culture process, fresh basal cell culture medium is inserted into the bioreactor through the medium inlet continuously, or once or several times, ii. during the cell culture process, harvest is taken out of the bioreactor through the harvest outlet continuously, or once or several times, iii. during the cell culture process, a concentrated medium supplement containing at least phosphotyrosine is inserted into the bioreactor one or several times through the medium inlet or an additional inlet, The process is targeted.

[0016] In a preferred embodiment, the concentrated medium supplement comprises at least five different components.

[0017] In another preferred embodiment, the concentrated medium supplement containing at least phosphotyrosine is inserted into the bioreactor for at least 50% of the time of the cell culture, preferably for at least 75% of the time.

[0018] In a highly preferred embodiment, the concentrated medium adjunct is inserted without increasing the overall perfusion rate. This means that if the cell culture concentrated medium adjunct is inserted into the bioreactor at a specific perfusion rate, the perfusion rate of the basal medium is preferably decreased by at least the perfusion rate of the concentrated medium adjunct.

[0019] In one embodiment, the overall perfusion rate is decreased at least once during the course of the process.

[0020] In another embodiment, the concentrated medium adjunct and the basal perfusion medium are blended before being added to the bioreactor.

[0021] In a preferred embodiment, the concentrated medium adjunct comprises sulfocysteine and / or 2-oxoglutaric acid and salts thereof.

[0022] In a preferred embodiment, the concentration of the components of the concentrated medium adjunct is at least 3-fold, preferably 6-fold, the concentration of the equivalent components in the basal medium.

[0023] In a preferred embodiment, the overall perfusion rate (mostly expressed in vvd) calculated over the duration of the process is at least 15–50% lower than the lowest possible perfusion rate in the same process without auxiliary substances. The perfusion rate can also be expressed as cell-specific medium flow rate by dividing the perfusion rate (vvd) by the viable cell density (VCD), which results in the cell-specific perfusion rate (CSPR), describing the supply of medium per cell per day. The lowest CSPR that can still maintain a steady state is described as the critical CSPR and can be determined as described in Konstantinov K, Goudar C, Ng M, Meneses R, Thrift J, Chuppa S, Matanguihan C, Michaels J, Naveh D. The "push-to-low" approach for optimization of high-density perfusion cultures of animal cells. Adv Biochem Eng Biotechnol. 2006;101:75–98. It is important to note that critical CSPR depends on the combination of cell line and culture medium, and therefore needs to be determined for each combination of cell line and medium.

[0024] In a preferred embodiment, the process is initiated by inoculating a bioreactor with cells and basal cell culture medium, and performing batch cell culture until perfusion is initiated 2–5 days later, while the cells are still in the exponential growth phase. A suitable VCD for initiating perfusion is approximately 4–6 mio cells / ml.

[0025] In a preferred embodiment, the concentrated medium supplement is added to the bioreactor continuously, once, or several times after the start of perfusion.

[0026] In another embodiment, the CSPR during perfusion in either a steady-state or dynamic perfusion process is reduced by adding an auxiliary substance during the process compared to a process using only basal medium, and the performance of the two processes is comparable. For steady-state perfusion processes, the CSPR is preferably critical CSPR.

[0027] In another embodiment, in either a steady-state or dynamic perfusion process, the CSPR before entering the perfusion steady state is reduced by adding an auxiliary substance during the process, compared to a process using only basal medium, and the performance of the two processes is comparable. For steady-state perfusion processes, the CSPR is preferably critical CSPR.

[0028] In a preferred embodiment, the cell-specific perfusion rate of the process according to the present invention is lower than that of a perfusion process having the same or comparable VCD and / or productivity, but without the addition of concentrated medium auxiliary substances.

[0029] Furthermore, the present invention relates to a perfusion cell culture medium kit comprising a basal cell culture medium and a concentrated medium auxiliary substance containing at least phosphotyrosine.

[0030] In a preferred embodiment, the concentrated medium auxiliary material comprises sulfocysteine ​​and / or 2-oxoglutaric acid or a salt thereof.

[0031] In a preferred embodiment, the basal cell culture medium and the concentrated medium auxiliary are either liquid or in a dry state for rehydration with a defined amount of liquid, most preferably in a dry granulated state for rehydration with a defined amount of liquid before use. [Brief explanation of the drawing]

[0032] [Figure 1] Figure 1 shows a schematic diagram of a state-of-the-art perfusion bioreactor. [Figure 2]Figure 2 shows the results of a perfusion culture experiment comparing perfusion culture using only basal medium with perfusion culture in which the basal medium is blended with a concentrated medium supplement according to the present invention. [Figure 3] Figure 3 shows the results of a perfusion culture experiment comparing perfusion culture using only basal medium with perfusion culture in which the basal medium is blended with a concentrated medium supplement according to the present invention. [Figure 4] Figure 4 shows the results of a perfusion culture experiment comparing perfusion culture using only basal medium with perfusion culture in which the basal medium is blended with a concentrated medium supplement according to the present invention. [Figure 5] Figure 5 shows the results of a perfusion culture experiment comparing perfusion culture using only basal medium with perfusion culture in which the basal medium is blended with a concentrated medium supplement according to the present invention. Further details can be found in Example 1. [Figure 6] Figure 6 shows a steady-state perfusion cell culture comparing a state-of-the-art perfusion culture using only basal medium with a perfusion culture in which the basal medium is blended with a concentrated medium supplement according to the present invention. In this case, the perfusion rate when blending the basal medium with the concentrated medium supplement is maintained at a lower level from the outset compared to the state-of-the-art perfusion culture. Further details can be found in Example 2. [Figure 7] Figure 7 shows a dynamic perfusion cell culture comparing a state-of-the-art perfusion culture using only basal medium with a perfusion culture in which the basal medium is blended with a concentrated medium supplement according to the present invention. Blending begins immediately upon initiation of perfusion, and the perfusion rate when blending the basal medium with the concentrated medium supplement is maintained at a lower level compared to the final perfusion rate in the state-of-the-art perfusion culture. Further details can be found in Example 3. [Figure 8] Figure 8 shows that using perfusion aids can reduce media requirements by 30% without compromising process performance. [Figure 9] Figure 9 shows that using perfusion aids can reduce media requirements by 30% without compromising process performance. [Figure 10]Figure 10 shows that using perfusion aids can reduce media requirements by 30% without compromising process performance. [Figure 11] Figure 11 shows that the use of perfusion aids can reduce the culture medium requirement by 30% without compromising process performance. Further details can be found in Example 2. [Modes for carrying out the invention]

[0033] Cell culture is any setting in which cells are cultured, that is, maintained or proliferated.

[0034] Cell culture is typically performed in a bioreactor.

[0035] A bioreactor is any container suitable for cell culture, such as bottles, tubes, vessels, bags, flasks, and / or tanks. Typically, the container is sterilized before use. Cell culture is typically carried out by incubating cells in aqueous cell culture medium under conditions suitable for cell growth and / or maintenance, such as appropriate temperature, pH, osmolality, aeration, agitation, etc., which limit contamination by exogenous microorganisms from the environment. Those skilled in the art recognize suitable incubation conditions for culturing cells. The bioreactor used according to the present invention is preferably a bioreactor suitable for perfusion cell culture.

[0036] A bioreactor system suitable for use in the present invention comprises a bioreactor and the following: - Stirring device - Devices for supplying and discharging components to and from the bioreactor, e.g., tubing, pumps, valves, storage tanks - Cell retention device (see above) - A system for monitoring bioreactor capacity, for example, bioreactor balance, level sensors, and other factors. - Apparatus for controlling and maintaining temperature, osmolality, aeration, stirring, and other parameters. - Computer systems for automated or partially automated operation of cell culture bioreactors The bioreactor is equipped with additional equipment essential for performing perfusion cell culture, as described in one or more of the above.

[0037] A cell culture medium (synonymously used: culture medium) according to the present invention is any mixture of components that maintain and / or support the in vitro growth of cells and / or support or maintain a particular physiological state.

[0038] Cell culture media may contain undefined components such as plasma, serum, embryo extracts, or other undefined biological extracts or peptones. Alternatively, they may preferably be chemically defined media. Cell culture media may contain all components essential for maintaining and / or supporting in vitro cell growth, or they may be used for the addition of selected components, either in combination with or without additional components (media supplements) added separately. The components of cell culture media are also called cell culture medium components.

[0039] The basal cell culture medium according to the present invention comprises all components for maintaining and / or supporting in vitro cell proliferation and / or supporting or maintaining a specific physiological state in perfusion cell culture.

[0040] The concentrated medium supplement according to the present invention contains at least phosphotyrosine. Typically, the concentrated medium supplement according to the present invention contains three or more components, and the concentration of the components is at least three times higher than the concentration of the equivalent component in the basal cell culture medium. Typically, the concentrated medium supplement contains components at concentrations 3 to 10 times higher than the concentration of the equivalent component in the basal cell culture medium. The equivalent component may be the same chemical component, a component that can be used as a substitute for the component, or a mixture of both. For example, phosphotyrosine is equivalent to tyrosine, and sulfocysteine ​​is equivalent to cysteine ​​or cystine. In some cases, the concentrated medium supplement cannot contain only the same components as the basal medium, because the component is not sufficiently soluble in the 3 to 10-fold concentrate. In this case, it is advantageous to completely or partially replace the component with an equivalent component that has higher solubility but performs the same function in cell culture and is suitable for replacing the component of the basal medium.

[0041] The concentrated medium auxiliary may include, for example, one or more amino acids and / or amino acid equivalents, one or more sugars and / or sugar equivalents. In any case, the overall concentration of the equivalent in the concentrated medium auxiliary, i.e., the same component and / or suitable substitute, is preferably at least three times, for example, three, four, five, six, seven, eight, nine, ten, twenty, or fifty times higher than in the basal cell culture medium. Typically, the concentration is three to fifty times, preferably five to twenty times higher than in the basal cell culture medium.

[0042] All concentrations refer to the concentrations in the liquid medium added to the cell culture. Typically, the overall concentration of the components in the concentrated medium auxiliary is greater than 100 g / L, preferably 100-400 g / L, and most preferably 150-250 g / L.

[0043] The cell culture media and processes according to the present invention are designed to be suitable for the growth or maintenance / support of growth of prokaryotic cells such as bacterial cells, as well as eukaryotic cells such as yeast, fungi, algae, plants, insects, and / or mammalian cells, and optionally archaea. Preferred cells are mammalian cells, particularly CHO cells.

[0044] A chemically defined cell culture medium is a cell culture medium composed of chemically well-characterized, "defined" raw materials. This means that the chemical composition of all chemicals used in the medium is known. A chemically defined medium does not consist of chemically undefined substances such as yeast, animal, or plant tissue; a chemically defined medium does not contain peptones, feeder cells, serum, undefined extracts or digests, or other components that may contribute chemically undefined proteins and / or peptides and / or hydrolysates to the medium. In some cases, a chemically defined medium may contain chemically defined proteins or peptides. One example is insulin (see others below).

[0045] Liquid cell culture media are typically produced by dissolving powdered cell culture media in a suitable liquid.

[0046] Powdered cell culture media, or dry powdered or dehydrated cell culture media, are typically cell culture media resulting from a grinding or freeze-drying process. This means that powdered cell culture media are typically fine granular particulate media rather than liquid media. The term “dry powder” may be used interchangeably with the term “powder”; however, “dry powder” as used herein simply refers to the overall appearance of the granulated material and is not intended to mean that the material is completely free of solvents that have complexed or aggregated, unless otherwise specified. Powdered cell culture media can also be granulated cell culture media, e.g., dry granulated by roller compression or wet granulated by fluidized bed spray granulation. Such media can also be prepared by spray drying. Dry powdered media resulting from grinding or freeze-drying processes typically have particle sizes less than 0.5 mm, e.g., 0.05–0.5 mm.

[0047] For example, dry powdered culture media resulting from dry or wet granulation processes such as spray drying, wet granulation, or dry compression typically have particle sizes greater than 0.5 mm, for example, 0.5–5 mm.

[0048] The culture medium, which is either dry powdered or preferably in a dry granulated state, is dissolved in an appropriate amount of liquid before use. The concentrations of the culture medium components provided herein always refer to the concentrations in the respective liquid culture mediums, and those skilled in the art will recognize that a dry powdered culture medium dissolves in a specific amount of aqueous liquid to give the respective liquid culture medium with components at specific concentrations.

[0049] The solvent, also called the liquid, used to prepare liquid cell culture media is typically water (most specifically, distilled water and / or deionized water or purified water or water for injection or water purified by reverse osmosis (Milli-Q®)) or aqueous buffer. The solvent may also contain physiological saline, soluble acids or base ions that provide a suitable pH range (typically within the range of pH 1 to pH 10), stabilizers, surfactants, preservatives, and alcohols or other polar organic solvents.

[0050] The pH of the lysis medium before adding cells is typically pH 2–12, more preferably pH 4–10, even more preferably pH 6–8, most preferably pH 6.5–7.5, and ideally pH 6.8–7.3.

[0051] A cell culture medium containing all the components essential for maintaining and / or supporting in vitro cell growth, such as a basal cell culture medium used in the present invention, typically includes at least one sugar component, one or more amino acids, one or more vitamins or vitamin precursors, one or more salts, one or more buffer components, one or more cofactors, and one or more nucleic acid components (nitrogen bases), or derivatives thereof. It may also contain recombinant proteins, such as r-insulin, r-BSA, r-transferrin, r-cytokines, and other chemically defined biochemical substances.

[0052] The culture medium may also contain sodium pyruvate, highly purified and chemically well-defined extracts, fatty acids and / or fatty acid derivatives and / or poloxamer product components (block copolymers based on ethylene oxide and propylene oxide), in particular Poloxamer 188, sometimes called Pluronic F 68, Kolliphor P 188, or Lutrol F 68, and / or chemically prepared nonionic surfactants and other surface active components. An example of a suitable nonionic surfactant is a primary hydroxyl-terminated bifunctional block copolymer surfactant, also called poloxamer, available, for example, from BASF, Germany under the trade name Pluronic®. Such poloxamer product components will be simply referred to as poloxamer or Pluronic below. Chelating agents, hormones, and / or growth factors may be added.

[0053] Other components that may be included are lactic acid, thioglycolic acid, thiosulfate, tetrathionate, diaminobutane, myo-inositol, phosphatidylcholine (lecithin), sphingomyelin, iron-containing compounds (such as compounds with iron-sulfur clusters), uric acid, carbamoyl phosphate, succinic acid, thioredoxin (single or multiple), orotic acid, phosphatidic acid, polyamines (such as putrescine, spermidine, spermine, and / or cadaverine), triglycerides, and steroids (such as cholesterol, but not limited to these). These include metallothione (not specified), oxygen, glycerol, urea, alpha-ketoglutarate, ammonia, glycerophosphate, starch, glycogen, glyoxylate, isoprenoids, methanol, ethanol, propanol, butanol, acetone, lipids (including, but not limited to, those in micelles), triptyline, butyline, cholic acid, deoxycholic acid, polyphosphate, acetate, tartrate, malatate, and / or oxalates.

[0054] All sugar components are monosaccharides or disaccharides such as glucose, galactose, ribose, or fructose (examples of monosaccharides), or sucrose, lactose, or maltose (examples of disaccharides), or derivatives thereof such as sugar alcohols. Alternatively, the sugar components may be oligosaccharides or polysaccharides.

[0055] Examples of amino acids according to the present invention include proteinogenic amino acids, particularly essential amino acids such as leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine, as well as non-proteinogenic amino acids such as D-amino acids. Where an amino acid is mentioned without defining whether it is a D- or L-variant, both are covered, and L-amino acids are preferred.

[0056] Therefore, tyrosine means L- or D-tyrosine, preferably L-tyrosine.

[0057] Cysteine ​​refers to L- or D-cysteine, preferably L-cysteine.

[0058] Amino acids can exist as free acids or their metal salts.

[0059] This also includes amino acid precursors and equivalents.

[0060] A suitable equivalent of tyrosine is phosphotyrosine. Phosphotyrosine refers to (S)-2-amino-3-(4-phosphonooxyphenyl)-propionic acid, as well as its salts such as monosodium, disodium, monopotassium, dipotassium, calcium, and magnesium salts. Phosphotyrosine, also called O-phospho-L-tyrosine, has CAS number 21820-51-9.

[0061] Suitable derivatives of cysteine ​​are those sulfonated at the SH group of cysteine, such as (S)-2-amino-3-sulfosulfanylpropionic acid or its salts, also known as sulfocysteine. The CAS number for L-cysteine ​​S-sodium sulfate sesquihydrate is 150465-29-5.

[0062] Other suitable equivalents of a particular amino acid are alpha-keto acids among the group of 4-methyl-2-oxopentanoic acid (ketoLeu), 3-methyl-2-oxopentanoic acid (ketoIle), alpha-ketoisovaleric acid (ketoVal), phenylpyruvic acid (ketoPhe), and alpha-keto-gammamethylthiobutyrate (ketoMet), and / or their derivatives. Suitable derivatives are metal salt derivatives, peptide derivatives such as dipeptides or tripeptides containing alpha-keto acids, ester derivatives, and other derivatives, most preferably metal salt derivatives such as sodium, potassium, calcium, or magnesium salts, preferably sodium salts.

[0063] Another suitable equivalent of amino acids is N-lactoyl amino acids and their Na + , K + Mg 2+ Ca 2+ Li + A salt such as, preferably its Na + There is a salt. Those skilled in the art can also use free amino acids and H so that a salt is produced. + Na + We are aware that substitution can also be performed with metal-to-metal pairions such as those mentioned above.

[0064] In a preferred embodiment, the N-lactoyl amino acid is selected from N-lactoylleucine, N-lactoylisoleucine, N-lactoylvaline, N-lactoylphenylalanine, N-lactoyltyrosine, and / or N-lactoylmethionine, most preferably N-lactoylleucine and / or N-lactoylisoleucine.

[0065] In a preferred embodiment, the N-lactyl amino acid is a component represented by Formula I: [Chemical formula] and is one or more components, where R1 + is H + or Na + K + Mg 2+ Ca 2+ Li + preferably a metal ion such as Na + . R2 is a characteristic residue of an amino acid. When the amino acid is leucine, the component of Formula I is [Chemical formula] . When the amino acid is isoleucine, the component of Formula I is [Chemical formula] .

[0066] In a preferred embodiment, the equivalent is the sodium salt of the N-lactyl amino acid. That is, preferably, R1 + is Na + .

[0067] Examples of vitamins include vitamin A (retinol, retinal, various retinoids, and four carotenoids), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin, niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxamine, pyridoxal), vitamin B7 (biotin), vitamin B9 (folic acid, folinic acid), vitamin B 12These include vitamins (cyanocobalamin, hydroxycobalamin, methylcobalamin), vitamin C (ascorbic acid) (ascorbic acid phosphate is an example), vitamin D (ergocalciferol, cholecalciferol), vitamin E (tocopherol, tocotrienol), and vitamin K (phylloquinone, menaquinone). Vitamin precursors and analogues are also included.

[0068] Examples of salts include inorganic ions such as bicarbonate, calcium, chloride, magnesium, phosphoric acid, potassium, and sodium, or components containing trace elements such as Co, Cu, F, Fe, Mn, Mo, Ni, Se, Si, Ni, Bi, V, and Zn. Examples include copper(II) sulfate pentahydrate (CuSO4·5H2O), sodium chloride (NaCl), calcium chloride (CaCl2·2H2O), potassium chloride (KCl), iron(II) sulfate, monobasic sodium phosphate anhydrous (NaH2PO4), anhydrous magnesium sulfate (MgSO4), dibasic sodium phosphate anhydrous (Na2HPO4), magnesium chloride hexahydrate (MgCl2·6H2O), and zinc sulfate heptahydrate (ZnSO4·7H2O).

[0069] Examples of buffers include carbonic acid, citrate, phosphoric acid, HEPES, PIPES, ACES, BES, TES, MOPS, and TRIS.

[0070] Examples of cofactors include thiamine, biotin, vitamin C, calciferol, choline, NAD / NADP (reduced and / or oxidized), cobalamin, vitamin B12, flavin mononucleotides and derivatives, flavin adenine dinucleotides and derivatives, glutathione (reduced and / or oxidized, and / or as dimers), heme, hemin, hemoglobin, ferritin, nucleotide phosphates and / or derivatives (e.g., adenosine phosphate), coenzyme F420, s-adenosylmethionine, coenzyme B, coenzyme M, coenzyme Q, acetyl-Co-A, molybdopterin, pyrroloquinoline quinone, and compounds, salts, complexes, and / or derivatives of tetrahydrobiopterin.

[0071] Nucleic acid components include nucleic acid bases such as cytosine, guanine, adenine, thymine, uracil, xanthine, and / or hypoxanthine; nucleosides such as cytidine, uridine, adenosine, xanthosine, inosine, guanosine, and thymidine; and nucleotides such as adenosine monophosphate, adenosine diphosphate, or adenosine triphosphate, as well as deoxyphosphates and / or phosphate derivatives, and / or dimers, trimers, and / or polymers thereof, such as RNA and / or DNA.

[0072] Components that improve the physicochemical properties of the culture medium, such as increasing the clarity and / or solubility of the medium and / or one or more of its components, without significantly adversely affecting the cell proliferation characteristics at the concentrations used, may be added. Such components include, but are not limited to, chelating agents (e.g., EDTA), antioxidants, detergents, surfactants, emulsifiers (such as polysorbate 80), neutralizing agents (such as polysorbate 80), micelle-forming agents, micelle inhibitors, and / or polypropylene glycol, polyethylene alcohol, and / or carboxymethylcellulose.

[0073] As used herein, the terms “cell density,” “viable cell density,” and “cell concentration” are interchangeable in meaning the number of metabolically active cells per unit volume of cell culture.

[0074] The term “perfusion” or “perfusion process” refers to a cell culture process used to produce a target product, e.g., an antibody or recombinant protein, where a high concentration of cells in a bioreactor continuously or once or more receive fresh growth medium during cell culture, thereby harvesting the consumed medium, which may contain the target product, meaning that the cells are continuously or once or more removed from the bioreactor during cell culture. Preferably, fresh growth medium is continuously fed into the bioreactor, and the consumed medium, which may contain the target product, is continuously harvested.

[0075] The amount of fresh medium exchanged or fed into the bioreactor per day (perfusion rate) is expressed as the daily vessel volume (VVD) of medium exchanged. For example, 2 liters of medium perfused daily into a system with a working capacity of 2 liters is expressed as 1 VVD. Unless otherwise specified, the given perfusion rate is the overall perfusion rate for the total amount of medium fed. The perfusion rate may consist of the perfusion rates of the various media fed into the bioreactor. For example, a perfusion rate of 2 VVD may consist of a perfusion rate of 1 VVD for basal medium and a perfusion rate of 1 VVD for medium concentrates.

[0076] Cells cultured in the systems and processes of the present invention may be cells capable of expressing target products, such as therapeutic biomolecules, including immunoglobulins (e.g., monoclonal antibodies or antibody fragments), fusion proteins, coagulation factors, interferons, insulin, growth hormone, or other recombinant proteins. Such cells may include, for example, CHO cells, baby hamster kidney (BHK) cells, PER.C.6 cells, myeloma cells, HEK cells, and others.

[0077] A "steady state" is typically a stable state in which there is no change over time, or where change in one direction is constantly balanced by change in another direction. In perfusion, a steady state can be defined by a "constant viable cell density." A constant viable cell density, combined with a constant perfusion rate, results in a constant cell-specific perfusion rate (CSPR), which is generally considered a critical criterion for achieving a steady state. Furthermore, since a system of living cells cannot be kept absolutely constant, a "steady state" is reached when the VCD, and therefore the CSPR, at a constant perfusion rate is stable within a range of ±20%, preferably around ±10%. For example, if the VCD is approximately 50 × 10⁻⁶ 6 If set to cells / ml, the VCD in steady state will be 40 × 10 6 cells / ml~60×10 6 The cell / ml ratio may vary.

[0078] Typically, to perform perfusion cell culture, a small number of cells and liquid cell culture medium are introduced into a bioreactor, and the culture conditions are selected so that the cell density increases while the cells express the target product through cell division. The culture can be carried out according to methods known in the art, for example, with appropriate levels of agitation, addition of oxygen / air, CO2, and removal of other gaseous metabolites. During culture, various parameters such as pH, conductivity, metabolite concentration, cell density, etc., can be controlled to provide conditions suitable for a given cell type. The cell density can be appropriately increased to a level where the cell concentration in the bioreactor is at least 1 million cells / ml, preferably at least 10 million cells / ml, typically 10 million to 250 million cells / ml. The upper limit will be set mainly by the rheological properties of the cell suspension at very high cell densities, and as it approaches a paste-like consistency, agitation and gas exchange may be inhibited. Other limitations on the perfusion process may prevent the operator from achieving this physical limitation, e.g., maximum cell retention device flow rate, maximum bioreactor oxygen transfer rate, product stability limitations, and the minimum cell-specific perfusion rate (CSPR) tolerated by the culture medium. Cell viability may be, for example, at least 50% (at least 80% or at least 90%).

[0079] The concentration of the target product expressed by cells within the bioreactor can be at least 0.1 g / l or at least 2 g / l. Typically 0.1–5 g / l, but in some processes such as CFB where the product is retained in the bioreactor without being harvested, product concentrations of up to 10–30 g / l can be achieved.

[0080] An exemplary bioreactor suitable for perfusion cell culture includes a cell retention device for keeping cells within the bioreactor during harvest. This cell retention device can be acoustic, alternating tangential flow (ATF), sedimentation device, centrifuge, and the like. In some examples, disposable, reusable, or semi-disposable bioreactors may be used. Any combination of hardware designs may be used. In one example, a disposable cell retention device may be used. In some embodiments, disposable conduits, tubing systems, pumps, bag assemblies, and cell retention devices are used rather than rigid piping and reusable devices.

[0081] The bioreactor of the bioreactor system of the present invention may have any suitable capacity, but is not limited to this exemplary range, including but not limited to about 1 L to about 2000 L. Specific exemplary bioreactor capacities include, but are not limited to, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 500, 1000, 1500 L, any intermediate capacity, and similar.

[0082] An exemplary bioreactor may have any suitable minimum and maximum working volumes, depending, for example, the total vessel volume, the ratio of vessel height to diameter, the vessel configuration (e.g., whether the bioreactor is a bag bioreactor), the growth rate, and the like. For example, in a 5 L bioreactor, the exemplary minimum working volume may range from about 10 mL to about 1 L, and the exemplary maximum working volume may range from about 3.5 L to about 5 L. In a 20 L bioreactor, the exemplary minimum working volume may range from about 100 mL to about 5 L, and the exemplary maximum working volume may range from about 15 L to about 19 L. In a 200 L bioreactor, the exemplary minimum working volume may range from about 20 mL to about 50 L, and the exemplary maximum working volume may range from about 150 L to about 190 L. Those skilled in the art will recognize that the above figures and ranges are illustrative and not intended to limit the scope of the present invention.

[0083] A bioreactor may include one or more inlets, also called inlet ports, for introducing one or more feeds (e.g., cell culture media), chemicals (e.g., pH buffers), defoamers, and the like. It may also include one or more outlets, also called outlet ports, for removing cells and / or liquids from the bioreactor. Each inlet and / or outlet in the bioreactor may be provided with any suitable mechanism for initiating and guiding the fluid flow through the inlet and / or outlet, including but not limited to one or more peristaltic pumps, one or more pressurizing mechanisms, and the like. Each inlet and / or outlet may be provided with any suitable mechanism for monitoring and controlling the fluid flow through the inlet, including but not limited to one or more mass flowmeters, one or more flow control valves, and the like. For example, a bioreactor may include a flow control mechanism for controlling the flow rate of substances entering and leaving the bioreactor.

[0084] The bioreactor may also be equipped with means for controlling its capacity and / or level.

[0085] The bioreactor includes a medium inlet, which may operate discretely or continuously, to introduce new cell culture medium into the cell culture. The bioreactor includes one or more harvest outlets for releasing the consumed cell culture, cells, and / or target products. The harvest outlets may include flow control valves for controlling the harvest rate. In one embodiment, the harvested material may be stored in a harvest bottle or container.

[0086] The bioreactor system may additionally include a bleed recovery device. The bleed recovery device comprises a bleed inlet that leads the bleed out of the bioreactor into means for separating the cells of the bleed from the liquid portion of the bleed, and a bleed liquid portion outlet that leads the cells of the bleed from means for separating the liquid portion of the bleed to the bioreactor and / or harvest outlet. In this case, the harvest outlet means any part of the harvest outlet, e.g., a tube or harvest container. The bleed recovery device can be made of a metal or preferably a soft or hard material such as plastic that forms a defined closed sterile volume.

[0087] A flow control valve may be provided at the inlet to control the bleed rate and the duration of bleed extraction.

[0088] Typically, a bioreactor system also includes pumps and valves installed via piping. The pumps are used to transport liquids, suspensions, or cell slurries from the bioreactor to, for example, harvested products, or to transport liquids, suspensions, or cell slurries from, for example, harvested products or breech recovery equipment to the bioreactor or other locations. Suitable pumps include peristaltic pumps, magnetically coupled pumps, membrane pumps, and others.

[0089] Valves are positioned to obstruct, allow, or guide the flow of liquids, cell suspensions, or cell slurries, for example. Examples of suitable valves include solenoid valves or pinch valves.

[0090] The bioreactor system may include one or more sensors or probes for detecting one or more operating parameters in real time, including, but not limited to, the status of the inlet port, the status of the outlet port, the status of the multidirectional manifold, a capacitance probe, a cell culture volume sensor, a cell culture bioreactor weight sensor, a liquid level sensor, a thermometer, a pH probe, an oxygen probe, a lactate probe, an ammonia probe, a stir rate sensor, a metabolic flux sensor, a metabolic rate sensor, a perfusion rate sensor, a carbon monoxide sensor, a mass spectrometer, a gas chromatograph, a combination thereof, and the like. These sensors may detect one or more operating parameters, including, but not limited to, viable cell density (using a capacitance probe or any alternative method that provides online measurement of cell density), cell culture volume, cell culture weight, cell culture medium level, temperature, pH, dissolved oxygen, a stir rate, a metabolic flux, a metabolic rate, a perfusion rate of the perfusion device, an oxygen uptake rate, carbon dioxide production (using, for example, a gas chromatograph, a mass spectrometry), a lactate level, an ammonia level, a combination thereof, and the like. The bioreactor may also be equipped with soft sensors.

[0091] The bioreactor and its inlet ports, outlet ports, and similar components may be connected to one or more process control systems configured or programmed to perform multivariate analysis of sensor data and to automatically control the operation of the bioreactor in real time based on the analysis. The process control system may control the operation by, for example, opening and closing inlet or outlet ports, changing the state of a multidirectional manifold, changing the perfusion rate of the bioreactor system, changing the stirring rate of the cell culture, temperature, pH, dissolved oxygen level, combinations thereof, and similar components.

[0092] The present invention is a process for perfusion cell culture, comprising culturing cells in a bioreactor system having a culture medium inlet and a harvest outlet, thereby, i. During the cell culture process, new cell culture medium is continuously, or once or several times, inserted into the bioreactor through the medium inlet. ii. During the cell culture process, the harvested material is continuously, or once or several times, removed from the bioreactor through the harvest outlet. iii. During the cell culture process, a concentrated medium auxiliary containing at least phosphotyrosine is inserted into the bioreactor once or several times via the medium inlet or an additional inlet. The target is the process.

[0093] The inventors have found that the use of concentrated medium supplements, when phosphotyrosine is used as the tyrosine equivalent, can be advantageously applied to perfused cell culture without adversely affecting the steady state of the perfusion process. While phosphotyrosine cannot be effectively used in basal media for perfused cell culture, the combination of using concentrated medium supplements and using phosphotyrosine as the tyrosine equivalent in said supplements has proven particularly beneficial. By adding concentrated medium supplements containing components that are consumed more rapidly by cells than other components in the basal medium, it is not necessary to add the entire basal medium to supply those components. Rather, by adding the concentrate, only those components are supplied in larger quantities, while other components are then sufficiently supplied through the basal medium, which can then be supplied in reduced quantities compared to a perfusion process using only the basal medium.

[0094] The above describes a suitable system for carrying out the process according to the present invention. Perfusion is preferably achieved using a cell holding device. The cell culture perfusion process can be initiated as in known perfusion processes. This is typically done by inoculating a bioreactor with basal medium and cells. The inoculation cell density is typically 0.2 to 10 mio cells / ml, preferably 0.5 to 1.0 mio cells / ml. It is also possible to start with more inoculation material, and therefore with a higher cell density.

[0095] It is possible to perform the process in perfusion mode from the beginning, directly, i.e., immediately after cell inoculation. However, the process is preferably carried out first in batch mode for a period of time while the number of cells, VCD, is increasing. Typically, the process is carried out in batch mode for 2 to 8 days, preferably 3 to 5 days, before perfusion is initiated. In either case, it is preferable to turn on perfusion before proliferation ceases to be exponential. This can be evaluated, for example, in prior cell line characterization experiments.

[0096] Next, perfusion is turned on. The ultimate goal is to set a constant perfusion rate and a constant bleed rate to reach a certain VCD. In the process according to the present invention, perfusion can be initiated by feeding only basal cell culture medium, or by feeding a combination of basal medium and concentrated medium auxiliary substances. Preferably, the perfusion phase is initiated by feeding only basal cell culture medium. The perfusion rate is preferably about 0.5 to 3 vvd, preferably 1 to 1.5. After reaching a certain VCD, the composition and amount of the medium can then be changed by lowering the overall perfusion rate and increasing the proportion of concentrated medium auxiliary substances in the total volume of medium. The medium composition can be changed once or several times, for example, two, three, or four times, thereby typically increasing the proportion of concentrated medium auxiliary substances and lowering the overall perfusion rate. The overall perfusion rate is the perfusion rate of the basal medium and medium concentrate. The bleed rate is, of course, adjusted accordingly so that the outflow stream, which is the harvested material and bleed, is equivalent to the inflow stream, which is the basal medium and concentrated medium supplements. The medium fed to the bioreactor may vary between 100% basal medium and basal medium and concentrated medium supplements in a 50:50 (v / v) ratio, where the ratio of basal medium to concentrated medium supplements is preferably 99:1 to 60:40 (v / v), and preferably 98:2 to 80:20. By adding a certain proportion of concentrated medium supplements, the perfusion rate can be reduced by 10 to 80%, preferably 15 to 40%, compared to the perfusion rate when using only basal medium to maintain the same VCD.

[0097] The basal medium and the concentrated medium auxiliary are preferably added via the same medium inlet or two different medium inlets. If the concentrated medium auxiliary and the basal medium are added via the same medium inlet, they are preferably pre-blended. The supply can be continuous, semi-continuous (meaning continuous over a certain time frame), or partial.

[0098] Alternatively, it is possible to increase the overall perfusion rate during the entire perfusion process or during a certain period of the perfusion process by adding the concentrated medium supplement while maintaining a constant perfusion rate of the basal medium, or by adding the concentrated medium supplement while lowering the perfusion rate of the basal medium compared to the addition of the concentrated medium supplement.

[0099] In another embodiment of the process of the present invention, it is also possible to increase the VCD for a given perfusion rate by increasing the proportion of concentrated medium auxiliary substances. This means that the change in the overall medium composition by increasing the proportion of concentrated medium auxiliary substances results in an increase in VCD and a decrease in CSPR, respectively. For this application as well, for most perfusion processes, the process is preferably started by first running it in batch mode for a while while the number of cells, i.e., the VCD, is increasing. Typically, the process is run in batch mode for 2 to 8 days, preferably 3 to 5 days, before perfusion is started. In any case, it is preferable that perfusion is turned on before cell proliferation ceases to be exponential. This can be evaluated, for example, in a prior cell line characterization experiment. A preferred cell density for starting perfusion is 4 to 6 million cells / ml.

[0100] Next, perfusion is turned on. As described above, perfusion can be initiated by feeding only basal cell culture medium, or by feeding a combination of basal medium and medium concentrate. Preferably, the perfusion phase is initiated by feeding only basal cell culture medium. The perfusion rate is preferably about 0.5 to 3, preferably 1 to 1.5. After reaching a certain VCD, the medium composition is then changed by increasing the proportion of concentrated medium auxiliary substances. The perfusion rate is kept constant. This results in a higher and more stable cell density compared to a perfusion process with the same process parameters, otherwise only basal medium is applied.

[0101] It is also possible to combine the two process variants by, on the one hand, adding a certain proportion of concentrated medium supplements, and on the other hand, by reducing the perfusion rate to a range where the VCD is still high compared to the same setting with basal medium alone. In this case, the reduction in perfusion rate is typically not as high as when the VCD is maintained, but by adjusting the proportion of concentrated medium supplements, those skilled in the art can adjust the perfusion rate and VCD to optimal values, resulting in a lower perfusion rate and a higher VCD compared to a process in which only basal medium is fed to the cells.

[0102] Overall, the process is flexible. The perfusion rate can be kept constant during the perfusion state, and can be increased or decreased stepwise or continuously. This concept can be applied to both steady-state and dynamic perfusion approaches. Those skilled in the art can adapt the scheme to the specific needs of their respective cell cultures.

[0103] As can be seen from the process variants above, the addition of concentrated medium auxiliary substances can be used, on the one hand, to make the process as efficient as possible by achieving higher productivity compared to a similar process using only basal medium. On the other hand, it can be used to reduce volumetric medium consumption, which is a known drawback in perfused cell culture.

[0104] The composition of the concentrated medium auxiliary was found to be critical for achieving optimal results in optimizing process performance and / or reducing medium consumption. The concentrated medium auxiliary preferably comprises at least three components, and more preferably five to thirty components.

[0105] The concentrated culture medium auxiliary preferably contains three or more amino acids and / or their equivalents.

[0106] Preferably, it contains 3 to 10 amino acids and / or equivalents thereof selected from the group consisting of asparagine, arginine, cysteine, glutamic acid, histidine, leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine.

[0107] Preferably, it contains 2-oxoglutaric acid or an equivalent such as its disodium salt.

[0108] Preferably, it contains one or more vitamins.

[0109] It may also contain at least one type of sugar.

[0110] In a very preferred embodiment, the concentrated medium auxiliary material comprises phosphotyrosine, as well as 2-oxoglutaric acid and / or sulfocysteine, in particular (S)-2-amino-3-(4-phosphonooxyphenyl)-propionate sodium salt, (S)-2-amino-3-sulfosulfanyl-propionate sodium salt, and / or 2-oxoglutaric acid disodium salt.

[0111] The concentrated medium auxiliary substance may be a dry powder or liquid medium that is dissolved in a liquid before use. It is configured so that the pH of the liquid medium is 6.5 to 10, preferably 7 to 8.

[0112] A basal medium can be any cell culture medium suitable for maintaining perfused cell culture. Suitable media are known to those skilled in the art. Typically, such a basal medium comprises one or more sugar components, one or more amino acids, one or more vitamins or vitamin precursors, one or more salts, one or more buffer components, one or more cofactors, and one or more nucleic acid components (nitrogen bases) or derivatives thereof.

[0113] Furthermore, the present invention relates to a perfusion cell culture medium kit of parts comprising a basal cell culture medium and a concentrated medium auxiliary substance containing at least phosphotyrosine.

[0114] In a preferred embodiment, the concentration of the components of the concentrated medium auxiliary substance is at least three times, preferably six times, the concentration of the equivalent components in the basal medium.

[0115] In a preferred embodiment, the basal cell culture medium and the concentrated medium auxiliary are either liquid or in a dry state for pre-use rehydration with a defined amount of liquid, and are most preferably granulated.

[0116] The present invention is further illustrated by the following examples, without limitation. All references cited above or below are incorporated herein by reference.

[0117] example Concentrated medium supplements, also known as perfusion aids or "PS," can be used for both steady-state and dynamic perfusion.

[0118] Steady state Example 1 To test the use of concentrated medium supplements in steady-state perfusion, two stirred tank glass bioreactors with a capacity of 1 L and a working capacity of 0.89 L were used. Both were equipped with pH probes, DO probes, temperature probes, and biomass probes and controlled by an Applikon MyControl system (bioreactor settings: pH=7.0±0.05; DO=40%; temperature=37℃±0.1℃, stirring=300~500 rpm; stirring type: marine impeller). An ATF device was used as the cell retention device. Both bioreactors were filled with EX-CELL Advanced HD Perfusion® medium and 0.5 × 10⁶ 6 Cells were inoculated at a cell density of viable cells / ml. The cell line used was our company's proprietary CHO-K1 cell line. Cells were grown in batch mode until day 5. Subsequently, perfusion was started in both bioreactors using only EX-CELL Advanced HD Perfusion® medium at a perfusion rate of 0.9 vvd.

[0119] On day 17, the perfusion rate for one bioreactor (condition "Perfusion Supplement") was reduced to 0.8 vvd while blending 2% [v / v] perfusion supplement into the basal medium. The remaining condition (condition "Control") was maintained at 0.9 vvd. On day 26, the perfusion rate of the "Perfusion Supplement" bioreactor was further reduced to 0.6 vvd while increasing the concentration of the perfusion supplement in the basal medium to 6% [v / v].

[0120] Throughout the run, viable cell density, viability, and IgG concentration were monitored. Cells were 50 × 10⁶ 6 Cells proliferated exponentially until the viable cell density reached 0.77 vvd in the bioreactor (Figure 2). The viable cell density was then kept constant using a "bleed" function implemented by the bioreactor controller. Growth under both conditions was comparable. Survival rates under both conditions (Figure 3) were comparable up to day 21, and after a 30% reduction in perfusion rate, there was a slight decrease for the "Perfusion Supplement" condition, but remained constant during this operating state. IgG concentrations (Figure 4) were comparable for both conditions up to day 17. When the perfusion rate for the "Perfusion Supplement" condition was reduced to 0.77 vvd, IgG concentrations increased through a longer residence time in the bioreactor. Further reduction of the perfusion rate for the "Perfusion Supplement" condition to 0.63 vvd resulted in a further increase in IgG concentrations. Cell-specific productivity (qP, calculated according to Bausch M, Schultheiss C, Sieck JB. Recommendations for Comparison of Productivity Between Fed-Batch and Perfusion Processes. Biotechnol J. 2019 Feb;14(2):e1700721) could be maintained throughout the run and even increased after 20 days under the "Perfusion Supplement" condition (Figure 5).

[0121] Example 2 Rather than gradually decreasing the perfusion rate, a perfusion aid can be added during exponential growth to avoid the high perfusion rate required in steady state when using only basal medium (compared to Example 1). This example shows two options for obtaining the same growth performance in a steady-state perfusion process. Add 0.5 × 10 to the basal medium in the perfusion setting bioreactor. 6 Inoculate with viable cells / ml. Grow cells in batch mode until day 3. On day 3, start perfusion at a perfusion rate of 0.2, for example, and increase during cell growth. 100 × 10 6 To obtain a stable steady state of viable cells / ml, it is essential to maintain the steady state by increasing the perfusion rate to 1 vvd in the prior art "basal medium only" option. To reduce this medium requirement, a perfusion aid is already added during exponential growth (option 2 according to the present invention), allowing a 30% reduction in the overall final perfusion rate essential for maintaining the steady state. Figure 6 shows an example with two options that result in the same VCD.

[0122] In addition, the perfusion process was performed in two stirred-tank glass bioreactors with a capacity of 1 L and a working capacity of 0.8 L (Figures 8-11). Both were equipped with pH probes, DO probes, temperature probes, and biomass probes and controlled by an Applikon MyControl system (bioreactor settings: pH=6.90±0.05; DO=40%; temperature=37.0℃±0.1℃, stirring=300~500 rpm; stirring type: marine impeller). An ATF device was used as the cell retention device. Both bioreactors were filled with EX-CELL Advanced HD Perfusion® medium and 2.2~2.4 × 10⁶ cells. 6Cells were inoculated at a cell density of viable cells / ml. The cell line used was our proprietary CHO-K1 cell line. Cells were grown in batch mode until day 1. Subsequently, perfusion was initiated in both bioreactors using only EX-CELL Advanced HD Perfusion® medium at a perfusion rate of 0.5 vvd for both conditions. On day 4, for the "basal medium + perfusion aid" condition, the basal medium was blended with 6% perfusion aid, and the perfusion rate (still in the growth phase) was increased to 1.26 vvd for both conditions. On day 6, while maintaining the "basal medium + perfusion aid" condition at 1.26 vvd, the perfusion rate (still in the growth phase) for the control was further increased to 1.8 vvd. Throughout the run, viable cell density, viability, and IgG concentration were monitored. Cells were 100 × 10⁶ 6 Cells proliferated exponentially until the viable cell density reached 1 / ml (Figure 8). A "bleed" function, implemented by a bioreactor controller, was then used to maintain a constant viable cell density. Growth under both conditions was comparable. Survival rates under both conditions (Figure 9) were comparable up to day 10, and slightly lower for the perfusion aid condition. However, over 90% was maintained until the end of the run. IgG concentration (Figure 10) was lower for the control from day 6 onwards. Through the calculation of cell-specific productivity (qP, Figure 11), the increased titer for the perfusion aid condition was attributed not only to the lower perfusion rate and longer residence time of the product, but also to the higher qP, indicating that media requirements can be reduced by 30% using perfusion aids without compromising process performance.

[0123] Dynamic perfusion (no bleeding) Example 3 Perfusion aids can also be used in dynamic perfusion. This example shows two options for achieving the same growth performance in dynamic perfusion. Add 0.5 × 10 to the basal medium in the perfusion setting bioreactor. 6Inoculate with viable cells / ml. Grow cells in batch mode until day 3. On day 3, start perfusion at a perfusion rate of 1 vvd (Option 1, latest technology). Cells initially grow exponentially until they reach maximum viable cell density. Afterward, the cell density decreases. To obtain the same growth performance with reduced medium requirements, in Option 2, blend a perfusion aid with the basal medium at a concentration of 2-10%. This reduces the perfusion rate to 0.7 vvd in this example without affecting the growth behavior. Figure 7 shows the two options that yield the same VCD.

Claims

1. A process for perfusion cell culture, comprising culturing cells in a bioreactor system comprising a bioreactor having a culture medium inlet and a harvest outlet, wherein i. Continuously during the cell culture process, or once or several times, new basal cells The culture medium is inserted into the bioreactor through the medium inlet. ii. During the cell culture process, the harvested material is continuously, or once or several times, removed from the bioreactor through the harvest outlet. iii. During the cell culture process, a concentrated medium auxiliary containing at least phosphotyrosine is inserted into the bioreactor once or several times via the medium inlet or an additional inlet. The aforementioned process.

2. The process according to claim 1, wherein the concentrated medium supplement is inserted for at least 50%, preferably at least 75%, of the cell culture time without increasing the overall perfusion rate.

3. The perfusion cell culture process according to claim 1 or 2, wherein the overall perfusion rate is reduced at least once during the course of the process.

4. The perfusion cell culture process according to any one of claims 1 to 3, wherein the concentrated culture medium auxiliary substance comprises at least five different components.

5. The perfusion cell culture process according to any one of claims 1 to 4, wherein the concentrated medium auxiliary substance comprises sulfocysteine ​​and / or 2-oxoglutaric acid and / or a salt thereof.

6. The perfusion cell culture process according to any one of claims 1 to 5, wherein the concentration of the components of the concentrated medium auxiliary substance is at least three times the concentration of the equivalent components in the basal medium.

7. A perfusion cell culture process according to any one of claims 1 to 6, wherein the overall perfusion rate (usually expressed in vvd) calculated over the duration of the process is at least 15–50% lower than the lowest possible perfusion rate in the same process without concentrated medium supplements.

8. The perfusion cell culture process according to any one of claims 1 to 7, wherein the process is initiated by inoculating a bioreactor with cells and basal cell culture medium and performing batch cell culture until perfusion is initiated 2 to 5 days later while the cells are still in the exponential growth phase.

9. The perfusion cell culture process according to any one of claims 1 to 8, wherein the concentrated medium auxiliary substance is added to the bioreactor continuously, once, or several times after the start of perfusion.

10. A perfusion cell culture process according to any one of claims 1 to 9, wherein the CSPR during perfusion is reduced stepwise or gradually by the addition of a concentrated medium supplement, compared to a process using only basal medium.

11. A perfusion cell culture process according to any one of claims 1 to 9, wherein the CSPR before entering the perfusion state is reduced by the addition of a concentrated medium supplement compared to a process using only basal medium.

12. The perfusion cell culture process according to any one of claims 1 to 11, wherein the critical cell-specific perfusion rate of this process is lower than that of the same perfusion process having the same VCD but without the addition of concentrated medium auxiliary substances.

13. A perfusion cell culture medium kit comprising a basal cell culture medium and a concentrated medium supplement containing at least phosphotyrosine.

14. The perfusion cell culture medium kit according to claim 13, wherein the concentration of the components of the concentrated medium auxiliary substance is at least three times the concentration of the equivalent components in the basal medium.

15. The perfusion cell culture medium kit according to claim 13 or 14, wherein the concentrated medium auxiliary substance comprises one or more components selected from sulfocysteine ​​and 2-oxoglutaric acid and salts thereof.

16. A perfusion cell culture medium kit according to any one of claims 13 to 15, wherein the basic cell culture medium and the concentrated medium auxiliary substance are granulated.

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