CN1778903A - A high-density continuous perfusion culture method for animal cells - Google Patents

Abstract

The invention opened a high density filling culture method of the animal cells. It study the metabolic rule of the cell growth, the nutrient consumption and the metabolic byproduct accumulation by studying theirs dynamics to determine and control the optimal nutritive condition. It feed the nutrient quality of cell growth by the stoichiometric relationship and the filling culture. The process can control the nutrient in a proper low concentration, so we can get the high density of the cells and the product concentration.

Description

A high-density continuous perfusion culture method for animal cells

technical field

The invention relates to a large-scale, high-density, serum-free continuous perfusion culture method for animal cells.

technical background

The cultivation process of animal cells in the bioreactor can have different operation modes, such as batch culture (Batch), continuous culture (Continuous), perfusion culture (Perfusion) and fed-batch culture (Fed-batch), etc., different operation modes has its different characteristics.

Continuous perfusion culture is to add fresh nutrient solution at a certain rate during the culture process, and discharge the supernatant at the same rate. The fragments are separated, and the living cells are returned to the reactor to continue culturing. Continuous perfusion culture not only maintains a relatively constant culture state, avoids the accumulation of harmful metabolic by-products, but also enables the cells in the reactor to reach a relatively high density.

In recent years, animal cell culture has made great progress in the study of cell physiology, especially in the understanding of unfavorable factors that affect cell growth and vitality. Through molecular genetic control, cells are more easily cultured in animal protein-free media. Obtaining high cell density and high cell viability has gradually become a common topic in engineering research. With regard to control schemes for feeding in fed and perfusion cultures, more and more efforts are being devoted to process control and monitoring, which is beneficial to ensure product quality and process consistency. Due to the metabolism of a large number of cells in large-scale animal cell culture, the cell culture environment changes rapidly during the culture process, and online process monitoring is very important. Large-scale animal cell culture can be successfully carried out by online measurement of a large amount of data such as culture conditions, metabolites and target products in the reactor, analysis and processing of the measurement results, and timely feedback control of the culture system.

For any given animal cell, the target product concentration is directly proportional to the integral of cell density and culture time, that is, P=q∫X _v dt, so there are two ways to increase the product concentration and its yield, the first is to increase the concentration of living cells The density, followed by maintaining the culture time as long as possible, which is closely related to the composition of the medium and the cell culture environment. As we all know, animal cells need more than 30 kinds of nutrients to maintain growth, survival and product synthesis, including glucose, essential amino acids, vitamins, some serum components and inorganic salts, etc. Among them, glucose and glutamine are the main components of cell growth, metabolism and product synthesis. It is the most important carbon source and energy substance in the synthesis. At the same time, animal cells also need a suitable culture environment, including pH, osmotic pressure, dissolved oxygen and temperature. As long as one nutrient is depleted during the cell culture process, cell growth, metabolism and product synthesis will be limited, so the supply of various nutrients during the cell culture process often determines the yield of the bioreactor. However, in addition to consuming nutrients, animal cell growth also generates many metabolic by-products, such as ammonia, lactic acid, non-essential amino acids, and _CO2 . Accumulation of these by-products to a certain extent often produces toxic effects on cells or changes the pH and osmotic pressure of the culture environment, thereby inhibiting cell growth and product synthesis. A large number of existing studies have proved that the contradiction between nutrient depletion and metabolic by-product accumulation during animal cell culture is the main factor limiting cell density, product concentration and yield of the culture process. In order to eliminate the nutrient limitation, some people have tried to concentrate or add nutrients to the medium within the permitted range of osmotic pressure. However, the final result is that although the nutrients are still abundant, the effect is minimal. The reason is that the medium in the medium High concentrations of nutrients such as glucose and glutamine promote the accumulation of metabolic by-products such as lactic acid, ammonia and alanine. In order to reduce the formation and accumulation of lactic acid and ammonia, researchers have also made various attempts in recent years. For example, some scholars use fructose, maltose and galactose as carbon sources instead of glucose. Although the production of lactic acid is significantly reduced, the energy metabolism of cells is more shifted to glutamine, thereby generating more ammonia. On the other hand, some researchers used dipeptides (alanyl-glutamine and glycyl-glutamine) that can generate glutamine by hydrolysis to replace free glutamine in the medium, although in the medium High-temperature disinfection and long-term storage have certain advantages, but failed to achieve the expected effect of reducing ammonia. On the contrary, it led to the accumulation of a large amount of alanine and glycine, which significantly increased the osmotic pressure of the medium to a level that was harmful to cell growth. Other attempts to reduce ammonia include: making cells completely do not need glutamine by inducing cells, cloning glutamine synthetase into cells, and using permeable membranes and electrolysis to selectively remove ammonia or ammonium ions, etc., but all These efforts have so far not yielded any desired results.

In fact, the metabolic pathways of animal cells to nutrients are strictly regulated by the culture environment and nutrient concentration during the culture process. Different nutrient metabolic pathways have significant differences in energy utilization and by-product generation. and glutamine metabolism. In the general batch culture process, due to the relatively high glucose concentration, most (more than 70%) glucose is metabolized through the glycolysis pathway, resulting in a large accumulation of lactic acid, and only 2mmolATP/mmol glucose can be generated; glutamine metabolism is also In this way, the high concentration of glutamine will accelerate the deamination degradation, so that glutamic acid and ammonia will accumulate in large quantities, and at the same time, the high concentration of glutamic acid will generate α-ketoglutarate into tricarboxylic acid through the glutamic acid-pyruvate transaminase pathway. Cycle, resulting in a large accumulation of alanine, the medium osmotic pressure increased, and only produce 9mmolATP/mmol glutamine. Studies on the kinetics of biochemical reactions in the process of cell metabolism have shown that the consumption of glucose by cells is mainly controlled by facilitated diffusion, and the concentration gradient of glucose on the cell membrane is its only driving force. When the glucose concentration is low, cells can also take up glucose through a high-affinity (Menten constant K _m lower than 0.2 mM) symport process driven by the Na ⁺ ion gradient. In addition, the mitochondrial hexokinase activity of transformed cells is high and not subject to feedback regulation by glucose-6-P, so high concentrations of glucose-6-P tend to enhance the glycolytic pathway. Similarly, the high concentration of glutamine in the medium also leads to the high-speed uptake of glutamine by cells, and the first step of glutamine deamination degradation is relatively high with the Michaelis constant K _m of glutaminase isoenzyme (2.2 to 4.5 mM), and this step is quite favorable thermodynamically (equilibrium constant K=320), but it is also very sensitive to reducing intracellular glutamine concentration. Both the glycolytic and glutamine deamination degradation pathways terminate in pyruvate, leading to its accumulation on both sides of the mitochondria. When excess pyruvate accumulation exceeds the oxidative capacity of mitochondria, the cells are forced to secrete excess carbon sources in the form of lactate and alanine. The above-mentioned metabolic characteristics of animal cells are the fundamental reasons why it is impossible to resolve the contradiction between nutrient depletion and by-product accumulation in batch culture.

The fed-batch culture or perfusion culture limited by glucose and glutamine is expected to eliminate the defect that the initial concentration is too high and then depleted in batch culture, and the concentration of glucose and glutamine in the medium can be controlled at a relatively low level by feeding. In recent years, the experimental results of many foreign researchers indicate that this culture method can help regulate cells entering high-energy metabolic pathways and reduce the accumulation of byproducts. For example, people such as Glacken (1998, Biotechnol. Bioeng. 32:491-506) make its concentration maintain low level in the microcarrier culture of MDCK cell by feeding glutamine, the generation of result ammonia reduces 60%, same, Ljunggren and Haggestrom (1992, Cytotechnology 8:45-56) used glutamine-limited fed-batch culture in myeloma cell culture, which reduced ammonia production by 50%. Furthermore, they studied the simultaneous limitation of glucose and glutamine fed-batch culture in hybridoma cell culture, and found that the production of lactic acid, ammonia and alanine was significantly reduced, while the energy utilization rate, cell density and product concentration were significantly improved ( 1994, Biotechnol. Bioeng. 44:808-818). However, there are still some deficiencies in the research work of these scholars. The main reason is that they often only consider glucose and/or glutamine. The limiting factor, in the end, often causes a large number of cells to die, the number of dead cells is greater than the number of living cells, and the increase of living cell density and product concentration is still limited. Although the medium continuous perfusion culture has more advantages than fed-batch culture, in addition to continuously supplementing nutrients, it can also remove metabolic byproducts in time, so the obtained cell density can be increased by one to two orders of magnitude compared with batch culture, but the current The perfusion culture in research and application, because its culture medium is non-balanced, often at the expense of high-speed culture medium perfusion, and the product concentration is greatly diluted, which is not worth the candle economically.

Contents of the invention

The technical problem to be solved in the present invention is to disclose a high-density continuous perfusion culture method for animal cells, so as to overcome the defects in the prior art and meet the needs of related parties.

Method of the present invention comprises the steps:

(1) Batch culture of the target cell line in the initial culture medium;

(2) Calculating kinetic parameters such as cell growth, consumption of various nutrients in the medium, generation of metabolic by-products, and generation of target products during the culture process;

(3) Redesign the initial medium and the nutrient composition of the perfusion medium in the perfusion culture process according to the calculation results, and carry out perfusion culture;

Repeat steps (3) to (4) to iteratively optimize the calculation results, and set the rate of continuous perfusion of the medium according to the cell density and production capacity required to achieve the culture process, so as to obtain high cell density and target product concentration. Kinetic Equation and Calculation of Batch Culture Parameters Calculation of Kinetic Equation Cells: $\frac{{\mathrm{wxya}}_V}{\mathrm{dt}}=\mathrm{\μ}{x}_V$ or $\mathrm{\μ}=\frac{1}{t_2-t_1}\ln \frac{x_{V2}}{x_{V1}}$ (Calculate specific growth rate of cells) Nutrients: $\frac{\mathrm{wxya}}{\mathrm{dt}}=-q_S{x}_V$ or $q_S=-\frac{1}{x_V}\frac{\mathrm{wxya}}{\mathrm{dt}}=-\frac{\mathrm{wxya}}{{\mathrm{wxya}}_V}$ (calculation of specific consumption rate of nutrients) product: $\frac{\mathrm{dP}}{\mathrm{dt}}=q_P^{\·}{x}_V$ or $q_P=\frac{1}{x_V}\frac{\mathrm{dP}}{\mathrm{dt}}=\mathrm{\μ}\frac{\mathrm{dP}}{{\mathrm{wxya}}_V}$ (Calculate the specific production rate of metabolic by-products produced by cells) Product concentration: P=q _P ∫X _v dt

Calculation of Kinetic Equations for Perfusion Culture Parameters

The inflow and outflow rates of materials are equal, most of the cells are trapped in the reactor, the total dilution rate D = F/V, the supernatant outflow rate D _s = F _s /V, and the cell outflow rate D _b = F _b /V.

cell: $\frac{d{x}_v}{\mathrm{dt}}=(\mathrm{\μ}-k_d-{\mathrm{D.}}_b)x_v;\frac{{\mathrm{wxya}}_d}{\mathrm{dt}}{k}_d{x}_v-{\mathrm{D.}}_b{x}_d$

Nutrients: $\frac{\mathrm{wxya}}{\mathrm{dt}}=\mathrm{D.}(S_f-S)-q_{\mathrm{the\; s}}{x}_v$

product: $\frac{\mathrm{dP}}{\mathrm{dt}}=q_P{x}_v-\mathrm{DP}$

In steady state, μ=k _d +D _b , $q_{\mathrm{the\; s}}=\frac{\mathrm{D.}(S_f-S)}{x_v},q_P=\frac{\mathrm{DP}}{x_v}.$ where the target product concentration can be expressed as

$\mathrm{<span\; class="notranslate">\; P</span>}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; q</span>}}_{\mathrm{<span\; class="notranslate">\; P</span>}}{\mathrm{<span\; class="notranslate">\; x</span>}}_{\mathrm{<span\; class="notranslate">\; v</span>}}}{\mathrm{<span\; class="notranslate">\; D.</span>}}<span\; class="notranslate">\; .</span>$

Symbol Description:

Xv viable cell density (10 ⁶ cells/mL)

Xt total cell density (10 ⁶ cells/mL)

Viability Cell Viability (%)

μ Cell specific growth rate (day ^-1 )

D perfusion rate or dilution rate (day ^-1 )

F Flow rate (mL/day)

Gluc, Gln Substrate Concentration (mM)

Lac, Amm By-product concentration (mM)

MAb Monoclonal antibody concentration (mg/L)

Specific consumption rate of q _Gluc q _Gln or q _s substrate (mmol/10 ⁹ cells/day)

p _Lac p _Amm p _Ala by-product specific formation rate (mmol/10 ⁹ cells/day)

Specific production rate of p _MAb monoclonal antibody (mg/10 ⁹ cells/day)

Cell yield of Y _x/Glue Y _x/Gln unit substrate consumption (10 ⁹ cells/mmol)

By-product yield of Y _lac/Gluc Y _Amm/Gln unit nutrient consumption (mmol/mmol)

V Culture volume (mL)

More specifically, the following steps are included:

(1) After the cells to be cultured are subcultured in a serum-free medium by a conventional method, they are inoculated in a bioreactor with an inoculation density of 1.5-3.0×10 ⁵ cells/ml;

(2) The reactor is set at a temperature of 37°C, a pH of 7.2, a dissolved oxygen of 50%, and a stirring speed of 40-80 rpm. In the serum-free initial medium, after culturing for 50-60 hours, the serum-free perfusion medium enters the bioreactor at a rate of perfusion rate D=1.0v/vday ^-1 for perfusion culture, and the culture period is 30-60 days. Collect liquid from the 10th day, the cell density can reach 1.0～2.40×10 ⁷ cells/ml, and the product concentration can reach more than 10 times of batch culture;

Said cells include hybridoma cells, recombinant CHO cells, 293 cells, insect cells, NSO and other engineered cells

The components and contents of serum-free initial medium are as follows: Element Chinese chemical name Inorganic salt (mg/L) _CaCl2 calcium chloride 116.60 CuSO ₄ 5H ₂ O copper sulfate 0.0013 Fe(NO ₃ ) ₃ 9H ₂ O Ferric nitrate 0.05 FeSO ₄ 7H ₂ O ferrous sulfate 0.417 KCl potassium chloride 311.80 MgCl ₂ magnesium chloride 28.64 _MgSO4 magnesium sulfate 48.84 NaCl Sodium chloride 6995.50 NaHCO ₃ sodium bicarbonate 2440 NaH ₂ PO ₄ H ₂ O Sodium dihydrogen phosphate 62.50 Na ₂ HPO ₄ Disodium phosphate 71.02 ZnSO ₄ 7H ₂ O Zinc sulfate 0.432 L-amino acid (mg/L) Alanine Alanine 4.45 Arginine HCl arginine 147.5-210.5 Asparagine H ₂ O Asparagine 7.5-67.5 Aspartic acid aspartic acid 6.65-26.6 Cysteine H ₂ O cysteine 17.56-35.12 Cystein·2HCl cystine 31.29-62.58 Glutamic acid glutamic acid 7.35-29.4 Glutamine Glutamine 292-584 Glycine Glycine 18.75-37.5 Histidine·HCl·H ₂ O Histidine 31.48-104.93 Isoleucine Isoleucine 52.4-131.0 Leucine Leucine 58.95-157.2 Lysine HCl Lysine 91.25-182.5 Methionine Methionine 16.39-59.6 Phenylalanine Phenylalanine 33.0-82.5 Proline proline 17.25-34.5 Serine serine 26.25-94.5 Threonine threonine 53.55-95.2 Tryptophan Tryptophan 8.16-51.0 Tyrosine·2Na·2H ₂ O Tyrosine 55.79-139.5 Valine Valine 52.65-117.0 Vitamins/cofactors (mg/L) Biotin biotin 0.0035 Pantothenate·Ca thbrthdrexvbdr 2.24 Choline·Cl choline 8.98 Folic acid folic acid 2.65 Inositol Inositol 12.60 Niacinamide Nicotinamide 2.02 Pyridoxine·HCl Vitamin B6 2.031 Riboflavin riboflavin 0.219 Thiamine·HCl Thiamine 2.17 Thymidine Thymidine 0.365 Vitamin B ₁₂ Vitamin B12 0.68 Trace elements (mg/L) Na ₂ SeO ₃ Sodium Selenite 0.0008-0.0035 MnSO ₄ 4H ₂ O Manganese sulfate 0.00008-0.0008 Na2SiO ₃ 5H ₂ O Sodium silicate 0.002-0.01 (NH ₄ ) ₆ Mo ₇ O ₂₄ 4H ₂ O Ammonium molybdate 0.0017-0.017 NH ₄ VO ₃ Ammonium vanadate 0.00006-0.0006 NiCl ₂ 6H ₂ O nickel chloride 0.00002-0.00024 SnCl ₂ 2H ₂ O tin chloride 0.00002-0.00023 Other ingredients (mg/L) Glucose glucose 1000-1800 Na Hypoxanthine Hypoxanthine 2.39 Linoleic acid Linoleic acid 0.042 Lipoic acid lipoic acid 0.105 Phenol red Phenol red 8.10 Sodium Putrescine 2HCl Putrescine 0.081 Pyruvate Na sodium pyruvate 220.00 insulin 5-10 Transferrin 5-10 albumin 0-100 Hormone (mg/L) Hydrocortisone 0.036-0.362 Dexamethasone 0.039-0.392 progesterone 0.006-0.031 Estradiol 0.0027-0.027 B-Mercaptoethanol 0.61-1.83 ethanolamine 1.22-12.2

The components and contents of the serum-free perfusion medium are as follows: Element Chinese chemical name Inorganic salt (mg/L) _CaCl2 calcium chloride 116.60 CuSO ₄ 5H ₂ O copper sulfate 0.0013 Fe(NO ₃ ) ₃ 9H ₂ O Ferric nitrate 0.05 FeSO ₄ 7H ₂ O ferrous sulfate 0.417 KCl potassium chloride 311.80 MgCl ₂ magnesium chloride 28.64 _MgSO4 magnesium sulfate 48.84 NaCl Sodium chloride 6995.50 NaHCO ₃ sodium bicarbonate 2440 NaH ₂ PO ₄ H ₂ O Sodium dihydrogen phosphate 62.50 Na ₂ HPO ₄ Disodium phosphate 71.02 ZnSO ₄ 7H ₂ O Zinc sulfate 0.432 L-amino acid (mg/L) Alanine Alanine 4.45 Arginine HCl arginine 147.5-210.5 Asparagine H ₂ O Asparagine 7.5-67.5 Aspartic acid aspartic acid 6.65-26.6 Cysteine H ₂ O cysteine 17.56-35.12 Cystein·2HCl cystine 31.29-62.58 Glutamic acid glutamic acid 7.35-29.4 Glutamine Glutamine 584-1168 Glycine Glycine 18.75-37.5 Histidine·HCl·H ₂ O Histidine 31.48-104.93 Isoleucine Isoleucine 52.4-131.0 Leucine Leucine 58.95-157.2 Lysine HCl Lysine 91.25-182.5 Methionine Methionine 16.39-59.6 Phenylalanine Phenylalanine 33.0-82.5 Proline proline 17.25-34.5 Serine serine 26.25-94.5 Threonine threonine 53.55-95.2 Tryptophan Tryptophan 8.16-51.0 Tyrosine·2Na·2H ₂ O Tyrosine 55.79-139.5 Valine Valine 52.65-117.0 Vitamins/cofactors (mg/L) Biotin Biotin 0.0035 Pantothenate·Ca thbrthdrexvbdr 2.24 Choline·Cl choline 8.98 Folic acid folic acid 2.65 Inositol Inositol 12.60 Niacinamide Nicotinamide 2.02 Pyridoxine·HCl Vitamin B6 2.031 Riboflavin riboflavin 0.219 Thiamine·HCl Thiamine 2.17 Thymidine Thymidine 0.365 Vitamin B ₁₂ Vitamin B12 0.68 Trace elements (mg/L) Na ₂ SeO ₃ Sodium Selenite 0.0008-0.0035 MnSO ₄ 4H ₂ O Manganese sulfate 0.00008-0.0008 Na ₂ SiO ₃ 5H ₂ O Sodium silicate 0.002-0.01 (NH ₄ ) ₆ Mo ₇ O ₂₄ 4H ₂ O Ammonium molybdate 0.0017-0.017 NH4VO ₃ Ammonium vanadate 0.00006-0.0006 NiCl ₂ 6H ₂ O nickel chloride 0.00002-0.00024 SnCl ₂ 2H ₂ O tin chloride 0.00002-0.00023 Other ingredients (mg/L) Glucose glucose 1000-1800 Na Hypoxanthine Hypoxanthine 2.39 Linoleic acid Linoleic acid 0.042 Lipoic acid lipoic acid 0.105 Phenol red Phenol red 8.10 Sodium Putrescine 2HCl Putrescine 0.081 Pyruvate Na sodium pyruvate 220.00 insulin 5-10 Transferrin 5-10 albumin 0-100 Hormone (mg/L) Hydrocortisone 0.036-0.362 Dexamethasone 0.039-0.392 progesterone 0.006-0.031 Estradiol 0.0027-0.027 B-Mercaptoethanol 0.61-1.83 ethanolamine 1.22-12.2

Method of the present invention has following characteristics:

1. The medium used in the present invention is a serum-free medium;

2. Glucose, glutamine, essential amino acids and other nutrients required for cell growth were added to the serum-free medium;

3. The substances added in the serum-free medium are based on the characteristics of cell growth and metabolism during the culture process. After analyzing, calculating and optimizing the kinetic parameters, a nutritionally balanced serum-free medium is formed, which avoids nutrient deficiency. Create restrictions and avoid excessive accumulation of metabolic by-products caused by excess nutrients;

4. Using the optimized medium and optimized perfusion strategy of the present invention, higher cell density can be obtained at a lower perfusion rate, thereby significantly increasing the product concentration and avoiding the dilution of the target product at a high perfusion rate.

The serum-free medium for animal cell culture is composed of more than 30 nutrients such as glucose, glutamine, various amino acids, various growth factors, regulatory and transport proteins in addition to inorganic salts. The lack of any nutrient will cause Cell growth is limited or even leads to cell death; and excessive nutrient abundance can cause a large accumulation of metabolic by-products such as ammonia and lactic acid, which can also cause cells to stop growing or die. Studies have shown that to effectively reduce the production of ammonia and lactic acid, it is necessary to reduce the concentration of glucose and glutamine. The present invention studies the metabolic law between cell growth, nutrient consumption and metabolic by-product accumulation by studying the kinetic characteristics of cell growth, nutrient consumption and metabolic by-product accumulation, and determines and controls the optimal nutritional environment for cell growth. The contradiction between nutrient depletion and by-product accumulation is resolved by meeting the amount of nutrients required for cell growth through stoichiometric relationships and perfusion culture strategies. The process formed by the present invention can control various nutrients at a lower suitable concentration, which can ensure the nutrient supply of cells without limiting the growth of cells, and can reduce the generation of toxic waste, thereby obtaining high Cell density and product concentration.

Description of drawings

Fig. 1 is hybridoma cell continuous perfusion culture cell growth and product expression;

Figure 2 is CHO cell continuous perfusion culture cell growth and product expression;

Detailed ways

Example 1

Are HB58 hybridoma cells (obtained from ATCC) employed in serum-free medium? ? ? After subculture adaptation according to the method disclosed in the literature, inoculate in a 50-liter bioreactor with an inoculation density of 2.0×10 ⁵ cells/ml.

(2) After culturing for 60 hours in serum-free initial medium at 37°C and pH 7.2, perfuse the serum-free perfusion medium into the bioreactor at a perfusion rate of 1.0v/v day ^-1 for perfusion Culture, the culture time is 60 days, and the liquid is collected from the 10th day, the cell density can reach 2.40×10 ⁷ cells/ml, and the product concentration can reach 624mg/ml; the cell changes during the culture process are shown in Figure 1, in the figure, the curve 1 is the living cell density, curve 2 is the monoclonal antibody concentration.

Through the optimization of the cultivation process in the present invention, the utilization rate of cells for nutrients such as glucose, glutamine and other various amino acids has been greatly improved, and the generation rate of metabolic by-products such as lactic acid, ammonia and alanine has been greatly reduced , cell density and product concentration were significantly increased. Compared with the results of batch culture, the cell density in the reactor was 11.32 times that of batch culture, the product concentration was 9.18 times, and the process yield was 38.24 times (see Table 1). Table 1 Comparison of characteristic parameters between continuous perfusion culture and batch culture in 50L bioreactor Batch perfusion culture Perfusion Culture/Batch Working volume (liter) Culture cycle (day) Product production cycle (day) Perfusion rate (d ^-1 ) Viable cell density (10 ⁵ cells/ml) 355521.221 3560501.024026 11.321.24 Monoclonal antibody production rate (μg/10 ⁶ cell/day) Monoclonal antibody concentration (mg/L) Yield (mg/day) Yield (mg/L _medium /day) 6847613.6 62418200520 9.1838.2438.24

Example 2

Recombinant CHO cells (rCHO SS3 A2, expressing human anticoagulant factor III) were seeded in a 2-liter bioreactor of B.BRAUN at a seeding density of 2.75×10 ⁵ cells/ml.

(2) After culturing for 40 hours in serum-free initial medium at 37°C and pH 7.2, the serum-free perfusion medium was perfused into the bioreactor at a perfusion rate of 0.58v/v day ^-1 for perfusion Culture, the culture time is 28 days, and the liquid is collected from the 8th day, the highest cell density can reach 1.1×10 ⁷ cells/ml, and the product concentration can reach 390U/ml; the cell changes during the culture process are shown in Figure 2, in the figure, Curve 3 is the living cell density, curve 4 is the total cell density, and curve 5 is the expression level of the product (ATIII).

Through the optimization of the culture medium and the culture process of the present invention, various nutrients in the culture medium are controlled at lower appropriate levels, so that the generation rate of metabolic by-products is greatly reduced, so the cell density and product concentration are significantly increased. Compared with the results of ordinary medium batch culture, the cell density and product concentration were increased by 6 times and 5 times, respectively.

Claims (5)

1. a high-density continous pouring culture of animal cell is characterized in that, comprises the steps:

(1) cultivation is criticized in the target cell strain in initial medium;

(2) various nutrition consumption and kinetic parameters such as metabolic by-prods generation and target product generation in the cell growth in the calculating culturing process, the substratum;

(3) form according to the initial medium in the calculation result redesign perfusion culture process and the nutrition of perfusion culture base, and carry out perfusion culture;

Repeat (3)～(4) step calculation result is carried out iteration optimization, and set the speed of substratum continous pouring, to obtain high cell density and target product concentration according to the required cell density that reaches of culturing process and throughput.

Kinetic equation and calculating

Criticize culture parameters computational dynamics equation

Cell: $\frac{{\mathrm{dX}}_V}{\mathrm{dt}}=\mathrm{\&mu;}{X}_V$ Or $\mathrm{\&mu;}=\frac{1}{t_2-t_1}\ln \frac{X_{V2}}{X_{V1}}$ (calculating the specific growth rate of cell)

Nutrition: $\frac{\mathrm{dS}}{\mathrm{dt}}=-q_S{X}_V$ Or $q_S=-\frac{1}{X_V}\frac{\mathrm{dS}}{\mathrm{dt}}=-\mathrm{\&mu;}\frac{\mathrm{dS}}{d{X}_V}$ (calculating nutraceutical specific consumption rate)

Product: $\frac{\mathrm{dP}}{\mathrm{dt}}=q_P{X}_V$ Or $q_P=\frac{1}{X_V}\frac{\mathrm{dP}}{\mathrm{dt}}=\mathrm{\&mu;}\frac{\mathrm{dP}}{d{X}_V}$ (calculating the specific production rate that cell produces metabolic by-prods)

Production concentration: P=q _P∫ X _vDt

Perfusion culture calculation of parameter kinetic equation

Material turnover speed equates, most of cell retention in reactor, total thinning ratio D=F/V, supernatant rate of outflow D _s=F _s/ V, cell rate of outflow D _b=F _b/ V.

Cell: $\frac{{\mathrm{dX}}_v}{\mathrm{dt}}=(\mathrm{\&mu;}-k_d-D_b)X_v;\frac{d{X}_d}{\mathrm{dt}}=k_d{X}_v-D_b{X}_d$

Nutrition: $\frac{\mathrm{dS}}{\mathrm{dt}}=D(S_f-S)-q_s{X}_v$

Product: $\frac{\mathrm{dP}}{\mathrm{dt}}=q_P{X}_v-\mathrm{DP}$

Under steady state, $\mathrm{\&mu;}=k_d+D_b,q_s=\frac{D(S_f-S)}{X_v},q_P=\frac{\mathrm{DP}}{X_v}.$ Wherein target product concentration can be expressed as

$P=\frac{q_P{X}_v}{D}$

Nomenclature:

Xv viable cell density (10 ⁶Cells/mL)

The total cell density (10 of Xt ⁶Cells/mL)

Viability cytoactive (%)

μ specific cell growth rate (day ^-1)

D irrigation rate or title thinning ratio (day ^-1)

F flows rate of acceleration (mL/day)

Gluc, Gln concentration of substrate (mM)

Lac, Amm by-product concentration (mM)

MAb monoclonal antibody concentration (mg/L)

q _Glucq _GlnOr q _sSubstrate specific consumption rate (mmol/10 ⁹Cells/day)

p _Lacp _Ammp _AlaBy product specific production rate (mmol/10 ⁹Cells/day)

p _MAbMonoclonal antibody specific production rate (mg/10 ⁹Cells/day)

Y _X/GlucY _X/GlnThe cell yield (10 of unit base consumption ⁹Cells/mmol)

Y _Lac/GlucY _Amm/GlnThe by-product yields (mmol/mmol) that the unit nutrition consumes

V volume of culture (mL).

2. method according to claim 1 is characterized in that, comprises the steps:

(1) will need cultured cells in serum free medium, to adopt conventional method to carry out adaptation of virus after, be inoculated in the bio-reactor, inoculum density is 1.0～3.0 * 10 ⁵Cell/ml;

(2) reactor is set to 37 ℃ of temperature, and pH is 7.0～7.2, dissolved oxygen 40～60%, 40～80 rev/mins of mixing speed.In the serum-free initial medium, cultivate after 50～60 hours, with serum-free perfusion culture base with irrigation rate D=1.0v/v day ^-1Speed enter bio-reactor and carry out perfusion culture, incubation time is 10～60 days.

3. method according to claim 2 is characterized in that, said cell comprises hybridoma, recombinaant CHO cell, 293 cells, insect cell or NSO cell.

4. method according to claim 2 is characterized in that, the component and the content of serum-free initial medium are as follows: Composition The Chinese chemical name Inorganic salt (mg/L) CaCl ₂ Calcium chloride 116.60 CuSO ₄·5H ₂O Copper sulfate 0.0013 Fe(NO ₃) ₃·9H ₂O Iron nitrate 0.05 FeSO4·7H ₂O Ferrous sulfate 0.417 KCl Repone K 311.80 MgCl ₂ Magnesium chloride 28.64 MgSO ₄ Sal epsom 48.84 NaCl Sodium-chlor 6995.50 NaHCO ₃ Sodium bicarbonate 2440 NaH ₂PO ₄H ₂O SODIUM PHOSPHATE, MONOBASIC 62.50 Na ₂HPO ₄ Sodium phosphate dibasic 71.02 ZnSO ₄·7H ₂O Zinc sulfate 0.432 L-amino acid (mg/L) Alanine L-Ala 4.45 Arginine·HCl Arginine 147.5-210.5 Asparagine·H ₂O Asparamide 7.5-67.5 Aspartic?acid Aspartic Acid 6.65-26.6 Cysteine·H ₂O Halfcystine 17.56-35.12 Cystein·2HCl Gelucystine 31.29-62.58 Glutamic?acid L-glutamic acid 7.35-29.4 Glutamine Glutamine 292-584 Glycine Glycine 18.75-37.5 Histidine·HCl·H ₂O Histidine 31.48-104.93 Isoleucine Isoleucine 52.4-131.0 Leucine Leucine 58.95-157.2 Lysine·HCl Methionin 91.25-182.5 Methionine Methionine(Met) 16.39-59.6 Phenylalanine Phenylalanine 33.0-82.5 Proline Proline(Pro) 17.25-34.5 Serine Serine 26.25-94.5 Threonine Threonine 53.55-95.2 Tryptophan Tryptophane 8.16-51.0 Tyrosine·2Na·2H ₂O Tyrosine 55.79-139.5 Valine Xie Ansuan 52.65-117.0 Vitamins/cofactors(mg/L) Biotin Vitamin H 0.0035 Pantothenate·Ca Calcium pantothenate 2.24 Choline·Cl Choline 8.98 Folic?acid Folic acid 2.65 Inositol Inositol 12.60 Niacinamide Niacinamide 2.02 Pyridoxine·HCl Vitamin B6 2.031 Riboflavin Riboflavin 0.219 Thiamine·HCl Thiamines 2.17 Thymidine Thymidine 0.365 Vitamin?B ₁₂ Vitamin B12 0.68 Trace element (mg/L) Na ₂SeO ₃ Sodium Selenite 0.0008-0.0035 MnSO ₄·4H ₂O Manganous sulfate 0.00008-0.0008 Na ₂SiO ₃·5H ₂O Water glass 0.002-0.01 (NH ₄) ₆Mo ₇O ₂₄·4H ₂O Molybdenum acid ammonia 0.0017-0.017 NH ₄VO ₃ Vanadic acid ammonia 0.00006-0.0006 NiCl ₂·6H ₂O Nickelous chloride 0.00002-0.00024 SnCl ₂·2H ₂O Tin chloride 0.00002-0.00023 Other composition (mg/L) Glucose Glucose 1000-1800 Na?Hypoxanthine Xanthoglobulin 2.39 Linoleic?acid Linolic acid 0.042 Lipoic?acid Thioctic Acid 0.105 Phenol?red Phenol red 8.10 Sodium?Putrescine·2HCl Putrescine 0.081 Pyruvate·Na Sodium.alpha.-ketopropionate 220.00 Regular Insulin 5-10 Transferrins,iron complexes 5-10 Albumin 0-100 Hormone (mg/L) Hydrocortisone 0.036-0.362 Dexamethasone 0.039-0.392 Progesterone 0.006-0.031 Estradiol 0.0027-0.027 The B-mercaptoethanol 0.61-1.83 Thanomin 1.22-12.2

5. method according to claim 2 is characterized in that, the component and the content of serum-free perfusion culture base are as follows: Composition The Chinese chemical name Inorganic salt (mg/L) CaCl ₂ Calcium chloride 116.60 CuSO ₄·5H ₂O Copper sulfate 0.0013 Fe(NO ₃) ₃·9H ₂O Iron nitrate 0.05 FeSO4·7H ₂O Ferrous sulfate 0.417 KCl Repone K 311.80 MgCl ₂ Magnesium chloride 28.64 MgSO ₄ Sal epsom 48.84 NaCl Sodium-chlor 6995.50 NaHCO ₃ Sodium bicarbonate 2440 NaH ₂PO ₄H ₂O SODIUM PHOSPHATE, MONOBASIC 62.50 Na ₂HPO ₄ Sodium phosphate dibasic 71.02 ZnSO ₄·7H ₂O Zinc sulfate 0.432 L-amino acid (mg/L) Alanine L-Ala 4.45 Arginine·HCl Arginine 147.5-210.5 Asparagine·H ₂O Asparamide 7.5-67.5 Aspartic?acid Aspartic Acid 6.65-26.6 Cysteine·H ₂O Halfcystine 17.56-35.12 Cystein·2HCl Gelucystine 31.29-62.58 Glutamic?acid L-glutamic acid 7.35-29.4 Glutamine Glutamine 584-1168 Glycine Glycine 18.75-37.5 Histidine·HCl·H ₂O Histidine 31.48-104.93 Isoleucine Isoleucine 52.4-131.0 Leucine Leucine 58.95-157.2 Lysine·HCl Methionin 91.25-182.5 Methionine Methionine(Met) 16.39-59.6 Phenylalanine Phenylalanine 33.0-82.5 Proline Proline(Pro) 17.25-34.5 Serine Serine 26.25-94.5 Threonine Threonine 53.55-95.2 Tryptophan Tryptophane 8.16-51.0 Tyrosine·2Na·2H ₂O Tyrosine 55.79-139.5 Valine Xie Ansuan 52.65-117.0 Vitamins/cofactors(mg/L) Biotin Vitamin H 0.0035 Pantothenate·Ca Calcium pantothenate 2.24 Choline·Cl Choline 8.98 Folic?acid Folic acid 2.65 Inositol Inositol 12.60 Niacinamide Niacinamide 2.02 Pyridoxine·HCl Vitamin B6 2.031 Riboflavin Riboflavin 0.219 Thiamine·HCl Thiamines 2.17 Thymidine Thymidine 0.365 Vitamin?B ₁₂ Vitamin B12 0.68 Trace element (mg/L) Na ₂SeO ₃ Sodium Selenite 0.0008-0.0035 MnSO ₄·4H ₂O Manganous sulfate 0.00008-0.0008 Na ₂SiO ₃·5H ₂O Water glass 0.002-0.01 (NH ₄) ₆Mo ₇O ₂₄·4H ₂O Molybdenum acid ammonia 0.0017-0.017 NH ₄VO ₃ Vanadic acid ammonia 0.00006-0.0006 NiCl ₂·6H ₂O Nickelous chloride 0.00002-0.00024 SnCl ₂·2H ₂O Tin chloride 0.00002-0.00023 Other composition (mg/L) Glucose Glucose 1000-1800 Na?Hypoxanthine Xanthoglobulin 2.39 Linoleic?acid Linolic acid 0.042 Lipoic?acid Thioctic Acid 0.105 Phenol?red Phenol red 8.10 Sodium?Putrescine·2HCl Putrescine 0.081 Pyruvate·Na Sodium.alpha.-ketopropionate 220.00 Regular Insulin 5-10 Transferrins,iron complexes 5-10 Albumin 0-100 Hormone (mg/L) Hydrocortisone 0.036-0.362 Dexamethasone 0.039-0.392 Progesterone 0.006-0.031 Estradiol 0.0027-0.027 The B-mercaptoethanol 0.61-1.83 Thanomin 1.22-12.2

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