Method and system for steady-state perfusion culture of cells

Abstract

The present application provides a method and system for steady-state perfusion culture of cells. Compared with the prior art, the method of the present application can determine CSPRmin more quickly, thereby implementing stable perfusion cell culture, saving time and economic costs.

Description

Method and system for steady-state perfusion culture of cells

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application number CN202311732489.7, filed December 15, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure provides a method and system for steady-state perfusion culture of cells, belonging to the field of cell culture. Background Art

[0004] Since the first commercialization of recombinant proteins in the 1980s, the biopharmaceutical industry has experienced rapid growth over the past 40 years. With the growing demand for biopharmaceutical production capacity, continuous perfusion culture, a culture technique first used in the 1990s, has regained attention. The perfusion process involves continuously adding fresh culture medium while harvesting the culture supernatant (protein) through a cell retention device, while also continuously removing metabolic byproducts. This ensures that the cells within the reactor maintain an optimal growth environment. Compared to traditional fed-batch culture, where the culture supernatant is harvested once, perfusion culture not only produces more consistent protein products but also enables very high cell densities, thereby achieving higher protein yields. Depending on whether or not cells are bleeding, perfusion culture can be categorized into steady-state perfusion and dynamic state perfusion. Because dynamic perfusion does not involve cell bleeding, cell density and viability decrease in the later stages of culture due to nutrient limitation. The culture cycle typically lasts 20-30 days. Steady-state perfusion, on the other hand, continuously drains cells while constantly replenishing fresh culture medium, keeping cells in a constant state of growth. This allows for consistently high cell viability, and culture cycles typically range from 2 to 6 months, or even longer. For sensitive protein products, such as enzymes and factors, steady-state perfusion is the preferred culture method. The operational variables involved in steady-state perfusion include the cell-specific perfusion rate (CSPR), perfusion rate (P, measured in vessel volume per day (VVD), and viable cell density (VCD). CSPR = P / VCD, representing the volume of culture medium required per cell per day, measured in pL / cell / day. Determining the minimum CSPR is a crucial step in steady-state perfusion process development. For a given culture medium, the CSPR has a critical value. Below this critical value, perfusion culture cannot be sustained; above this critical value, nutrient overload results in medium waste. Therefore, optimal performance of a perfusion bioreactor is achieved at the minimum CSPR value at which cell growth is stable, termed the minimum CSPR. How is the minimum CSPR determined? According to literature, this can be achieved through two methods: 1. Maintaining VCD constant, gradually reducing P (push to low), and the CSPR before the cell growth cannot be maintained stably is defined as CSPRmin. 2. Maintaining P constant, gradually increasing VCD (push to high), and the CSPR before the cell growth cannot be maintained stably is defined as CSPRmin.In the process of gradually lowering P or gradually increasing VCD, multiple CSPR inspection points need to be set up. Each inspection cycle should last at least 7-10 days, and the entire inspection cycle takes at least 1.5-2 months, which makes the entire development process itself longer. Although each CSPR cycle is inspected for 7-10 days, the true steady-state maintenance time is much longer than this, usually requiring at least 1-2 months. Therefore, the CSPRmin found by this method may not be able to maintain true steady-state culture. Once the cells cannot maintain steady state during the culture process, the optimization work needs to start from scratch, and the time and material costs are very high. Summary of the Invention

[0005] To address the time-consuming and labor-intensive problem of finding CSPRmin in existing technologies, the present disclosure provides a novel steady-state perfusion culture method for cells. In this method, steady-state culture is attempted starting with any cell density. If the steady-state culture cannot be maintained, continuous manual intervention is used to quickly find the final maintainable steady-state cell density, thereby determining CSPRmin. This allows long-term steady-state culture to be carried out until the cell culture is completed and the product is harvested.

[0006] Therefore, in one aspect, the present disclosure provides a method for steady-state perfusion culture of cells, the method comprising:

[0007] Step 1. Reactor inoculation

[0008] Inoculating cells into a reactor containing culture medium, starting cell culture and monitoring cell culture status, wherein cell culture status parameters include cell density and viability;

[0009] Step 2. Start perfusion

[0010] On the second day, perfusion culture was started at the initial perfusion rate P0 and continued for several days until the Nth day, N = 4 to 6, during which the perfusion rate was gradually increased;

[0011] Step 3. Initial Steady-State Perfusion Culture

[0012] On day N+1, the steady-state automatic feedback control system is turned on, with the current cell density ρ 起始 As the initial target steady-state density, steady-state culture was performed at a perfusion rate of P1, and the cell culture status was continuously monitored.

[0013] The said starting of the steady-state automatic feedback control system includes starting the cell culture monitoring device and starting the coupling of the discharge pump and starting the coupling of the balance and the harvesting end pump;

[0014] Step 4. Manual platelet removal and determination of maintainable steady-state cell density

[0015] When it is monitored that the cell density begins to decrease or both the cell density and viability begin to decrease, the steady-state automatic feedback control system is turned off and about 10-15% of the cell culture medium is manually discharged. This is repeated daily and the cell culture is continued at the perfusion rate P1. The cell culture status is continuously monitored. The cell culture status parameters include cell density and viability.

[0016] The shutting down of the steady-state automatic feedback control system includes shutting down the coupling between the cell culture monitoring device and the discharge pump, but keeping the coupling between the balance and the harvesting end pump open.

[0017] When the cell density is monitored to be stably maintained within a certain range for at least 5 consecutive days, any cell density value within the range where the cell density is stably maintained for at least 5 consecutive days is selected as the maintainable steady-state cell density ρ 最终 , the corresponding CSPR is CSPRmin;

[0018] Step 5. Maintainable Steady-State Perfusion Culture

[0019] Start the steady-state automatic feedback control system again, with ρ 最终 To achieve the final steady-state density, cells were cultured at a perfusion rate of P1 until harvest.

[0020] In some embodiments, the cell is a mammalian cell, preferably selected from, but not limited to, HeLa, Cos, 3T3, myeloma cell lines (eg, NSO, SP2 / 0), and Chinese hamster ovary (CHO) cells.

[0021] In some embodiments, in step 1, the seeding density is at least 0.5×10 6 cells / mL.

[0022] In some embodiments, step 1 further includes setting reactor control conditions, wherein the reactor control conditions include any one or more of the following: temperature is controlled at 36.5°C to 37.5°C, preferably 37.0°C; pH is controlled at 7.00±1.00, 7.00±0.50 or 7.00±0.25; DO is controlled at 50-70%, 55-65% or 60%; initial stirring speed is controlled at 200-400rpm, 250-350rpm or 300rpm; and / or bottom air flow is controlled at 10-30mL / min, 15-25mL / min or 20mL / min.

[0023] In some embodiments, the cell culture status parameters further include glucose concentration, lactate concentration, amino acid concentration in the culture medium, and other common cell culture medium nutrient concentrations, cell metabolite concentrations, etc.

[0024] In some embodiments, in step 2, the initial perfusion rate P0 is in the range of about 0.1 to 1.0 VVD, and the perfusion rate is increased to ultimately not exceed 5.0 VVD.

[0025] In some embodiments, in step 3, the perfusion rate P1 is in the range of about 2.0 to 5.0 VVD.

[0026] In some embodiments, in step 4, the cell density is stably maintained at a maximum of ±10.0×10 6 cells / mL, for example, up to ±5.0×10 6 cells / mL.

[0027] In some embodiments, in step 4, any value or median value of the cell density for at least 5 consecutive days is selected as the maintainable steady-state cell density p 最终 .

[0028] In some embodiments, in step 4, the average of the highest and lowest cell densities for at least 5 consecutive days is selected as the maintainable steady-state cell density p. 最终 .

[0029] On the other hand, the present disclosure provides a system for steady-state perfusion culture of cells, which includes a reactor, a cell culture monitoring device, a balance, a liquid inlet pump, a discharge pump, a harvest end pump and a sampling port, wherein the balance is used to measure the weight of the reactor, the cell culture monitoring device is coupled to the discharge pump, the balance is coupled to the harvest end pump, and the sampling port is used for artificial discharge.

[0030] In some embodiments, the cell culture monitoring device is coupled to a discharge pump, and the discharge pump is turned on and off according to culture parameters measured by the cell culture monitoring device, including cell density.

[0031] In some embodiments, the balance is coupled to a harvest pump, and the opening and closing of the harvest pump is controlled according to the weight of the reactor measured by the balance.

[0032] Advantageous Effects of the Present Disclosure

[0033] Compared with the existing technology, the method of steady-state cell perfusion culture provided by the present disclosure can determine CSPRmin more quickly, usually only taking about 20 days. Under this CSPRmin, steady-state cell culture can be maintained for at least 2 months, saving time and material costs. The final recombinant protein yield is much higher than the original fed-batch culture (Fed-batch) process, and the quality is comparable to or even better. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Those skilled in the art should understand that the drawings described in the present disclosure are for illustration purposes only and are not intended to limit the scope of the present invention in any way.

[0035] FIG1 shows a schematic diagram of automatic feedback control of steady-state culture in a reactor.

[0036] Figure 2 shows the cell growth curve during the manual release stage.

[0037] Figure 3 shows the cell growth curve during the steady-state perfusion phase.

[0038] Figure 4 shows the SEC purity analysis results.

[0039] Figure 5 shows the results of icIEF charge heterogeneity analysis.

[0040] FIG6 shows the results of N-glycosylation analysis. DETAILED DESCRIPTION

[0041] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0042] Those skilled in the art will understand that a "perfusion" culture process is one in which the cell culture receives additions of fresh medium and spent medium is removed from the bioreactor. Perfusion can be continuous, stepwise, intermittent, or a combination of any or all of these.

[0043] Perfusion culture is alternating tangential flow perfusion culture or tangential flow perfusion culture. Alternating tangential flow perfusion culture refers to the perfusion culture in which alternating tangential flow (ATF) filtration technology is used to retain cells in cell culture; tangential flow perfusion culture refers to the perfusion culture in which tangential flow (TFF) filtration technology is used to retain cells during cell culture. In the perfusion culture in which tangential flow (TFF) filtration technology is used to retain cells, the cell fluid is formed into a continuous annular flow direction by the action of a peristaltic pump. After entering the fiber membrane, the waste liquid will pass through the membrane and be discharged outside the system, and the cells will return to the culture system along the loop. In the perfusion culture in which alternating tangential flow (ATF) filtration technology is used to retain cells, the reciprocating blowing and suction action of the diaphragm pump is used to realize the reciprocating flow of the culture medium in the tank in the retention device, while metabolic waste will be discharged through the membrane along with the culture medium and the cells will be retained in the reactor. When using ATF, the alternating motion produces a flushing effect in the filter membrane, which helps to prevent clogging of the fiber membrane. In the present disclosure, perfusion culture in which alternating tangential flow (ATF) filtration technology is used to retain cells is preferred.

[0044] As used herein, the term "steady state" refers to a condition in which the cell density and bioreactor environment remain relatively constant. This can be achieved by cell bleeding, nutrient restriction, and / or temperature reduction. In most perfusion cultures, nutrient supply and waste removal will allow for constant cell growth and productivity, and cell bleeding is required to maintain a constant viable cell density or to maintain the cells in a steady state. Typical viable cell densities in the steady state are 10 to 100 × 10 6 cells / mL.

[0045] When used herein, term " cell bleeding " refers to removing cells and culture medium from bioreactor, to maintain constant, sustainable viable cell density in bioreactor. This cell bleeding can be completed with the flow velocity of regulation using a dip tube and a peristaltic pump. The pipeline should have a suitable size, and cell aggregation and obstruction may occur easily in too narrow pipelines, and if too large, cells may settle. Cell bleeding can be determined according to growth rate, and therefore viable cell density can be limited to required volume in a continuous manner. Alternatively, cells can be removed with a certain frequency, for example, once a day, and replaced with culture medium so that cell density is maintained within a predictable range.

[0046] As used herein, the term "perfusion rate" refers to the amount of culture medium added and removed from a bioreactor within a given time period, typically expressed as a portion or multiple of the working volume, typically measured daily. "Working volume" refers to the volume used for cell culture in a bioreactor. Perfusion typically begins on day 1-3 after inoculation, when cells are still in the exponential growth phase, and therefore the perfusion rate may increase during the culture process. The increase in perfusion rate may be incremental or continuous, i.e., based on cell density or nutrient consumption. Typically, starting from 0.5 or 1.0 working volumes per day (VVD), the rate may reach approximately 5.0 VVD at most. Preferably, the perfusion rate is between 0.5 and 2.0 VVD. The increase may be in the range of 0.1 to 1.0 VVD, or greater, such as 0.1 VVD, 0.2 VVD, 0.3 VVD, 0.4 VVD, 0.5 VVD, 0.6 VVD, 0.7 VVD, 0.8 VVD, 0.9 VVD, 1.0 VVD, etc.

[0047] As used herein, the term "cell specific perfusion rate (CSPR)" refers to the volume of culture medium required per cell per day, expressed in pL / cell / day. An ideal CSPR should result in optimal growth rate and productivity.

[0048] As used herein, the term "CSPRmin" refers to the critical CSPR value for maintaining stable cell proliferation and growth. Below this critical value, perfusion culture cannot be maintained; above this critical value, there is excess nutrition, resulting in waste of culture medium.

[0049] As used herein, the term "viable cell density (VCD)" refers to the number of cells present in a given volume of culture medium, expressed in units of "cells / mL," and is sometimes also referred to as "cell density (VCD)." Those skilled in the art can measure viable cell density by any method known to those skilled in the art. Preferably, viable cell density is measured in real time using an online in situ viable cell monitoring instrument, such as the ABER in situ viable cell online detection instrument.

[0050] As used herein, the terms "bioreactor" and "reactor" are used interchangeably to refer to any vessel that can be used to grow cell cultures. A bioreactor can be of any size, as long as it meets the requirements of cell culture. Typically, the volume of a bioreactor is at least 0.5 L, and can be 1, 2, 3, 4, 5, 10, 50, 100, 200, 250, 500, 1,000, 1,500, 2,000, 2,500, 5,000, 8,000, 10,000, 12,000 L or larger, or any volume within the aforementioned ranges. Generally, the working volume is less than the volume of the bioreactor, for example, 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, or even less of the volume of the bioreactor. The working volume used to culture cells in a bioreactor can be determined by one skilled in the art based on actual needs. The internal conditions of the bioreactor, including but not limited to pH and temperature, can be controlled during the culture period. Those skilled in the art will be able to select a suitable bioreactor for practicing the methods of the present disclosure based on relevant requirements.The cell culture used in the methods of the present disclosure can be grown in any bioreactor suitable for perfusion culture.

[0051] As used herein, the terms "culture medium" and "cell culture medium" are used interchangeably to refer to a nutrient solution that nourishes cells, particularly mammalian cells. Cell culture medium formulations are well known in the art. Typically, cell culture media provide the essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required for minimal cell growth and / or survival, as well as buffers and salts. The culture medium may also contain supplementary ingredients that enhance growth and / or survival above the minimum rate, including but not limited to hormones and / or other growth factors, specific ions (e.g., sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds are typically present at very low final concentrations), amino acids, lipids, and / or glucose or other energy sources. In certain embodiments, the culture medium is advantageously formulated to a pH and salt concentration that are optimal for cell survival and proliferation. In certain embodiments, the culture medium of the present disclosure is a perfusion medium, including a basal medium, a feed medium, and a mixed medium of a basal medium and a feed medium. The term "basal medium" refers to a cell culture medium used to start cell culture and can support cell growth, including but not limited to Dynamis medium, CD OptiCHO AGT (Invitrogen), CD CHO AGT (Invitrogen), etc. The term "feed medium" refers to the continued addition of nutrients with limited components to support cell growth during the vigorous cell growth period, including but not limited to 3×FeedB+ medium, Cell boost7a / Cell boost7b (Invitrogen) (i.e., CB7a and CB7b), etc. Cell perfusion culture is maintained by replenishing basal medium and feed medium. Prior to the feed medium, cells can be cultured in the basal medium for 1 day, 2 days, 3 days or more. For example, perfusion of the basal medium can start from the 2nd day, and perfusion of the feed medium starts from the 3rd day. Alternatively, perfusion of the basal medium can be started from the 1st day. Another example can be given, that the basal medium can be perfused starting from the 1st day, the 2nd day, the 3rd day, the 4th day, the 5th day or the 6th day, and the feed medium can be perfused starting from the 2nd day, the 3rd day, the 4th day, the 5th day, the 6th day or the 7th day. The basal medium and the feed medium can be mixed in a suitable ratio in advance and then added to the bioreactor. In the perfusion culture method disclosed herein, those skilled in the art can adjust the composition of the perfusion medium according to actual needs and experience to improve the cell culture conditions. It should be understood that the adjustment of the composition of the perfusion medium may affect the cell proliferation, change the cell density and / or the change trend, but will not undermine the implementation of each step in the method disclosed herein. For example, in step 4, when those skilled in the art optionally adjust the composition of the perfusion medium, the number of days for the implementation of this step may change, but this does not affect the determination of the steady-state density that can be maintained in this step.

[0052] In certain embodiments, the perfusion medium of the present disclosure is composed of a basal medium and a feed medium, and the basal medium and the feed medium are described below. In certain embodiments, the perfusion medium of the present disclosure includes other components in addition to the basal medium and the feed medium. For example, a defoaming agent, and for example, one or more selective agents capable of binding to resistance markers and viability markers in the host cell line, such selective agents include but are not limited to geneticin (G4118), neomycin, hygromycin B, puromycin, zeocin, methionine sulfenyl imide or methotrexate.

[0053] Steady-state cell perfusion culture method

[0054] The method for steady-state perfusion culture of cells disclosed herein comprises the following steps:

[0055] Step 1. Reactor inoculation

[0056] Inoculating cells into a reactor containing culture medium, starting cell culture and monitoring cell culture status, wherein cell culture status parameters include cell density and viability;

[0057] Step 2. Start perfusion

[0058] On the second day, perfusion culture was started at the initial perfusion rate P0 and continued for several days until the Nth day, N = 4 to 6, during which the perfusion rate was gradually increased;

[0059] Step 3. Initial Steady-State Perfusion Culture

[0060] On day N+1, the steady-state automatic feedback control system is turned on, with the current cell density ρ 起始 As the initial target steady-state density, steady-state culture was performed at a perfusion rate of P1, and the cell culture status was continuously monitored.

[0061] Said starting of the steady-state automatic feedback control system includes starting the coupling between the cell culture monitoring device and the discharge pump and starting the coupling between the balance and the harvesting end pump;

[0062] Step 4. Manual platelet removal and determination of maintainable steady-state cell density

[0063] When it is monitored that the cell density begins to decrease or both the cell density and viability begin to decrease, the steady-state automatic feedback control system is turned off and 10-15% of the cell culture medium is manually discharged. This is repeated daily and the cell culture is continued at the perfusion rate P1. The cell culture status is continuously monitored. The cell culture status parameters include cell density and viability.

[0064] The shutting down of the steady-state automatic feedback control system includes shutting down the coupling between the cell culture monitoring device and the discharge pump, but keeping the coupling between the balance and the harvesting end pump open.

[0065] When the cell density is monitored to be stably maintained within a certain range for at least 5 consecutive days, any cell density value within the range where the cell density is stably maintained for at least 5 consecutive days is selected as the maintainable steady-state cell density ρ 最终 , the corresponding CSPR is CSPRmin;

[0066] Step 5. Maintainable Steady-State Perfusion Culture

[0067] Start the steady-state automatic feedback control system again, with ρ 最终 To achieve the final target steady-state density, cells were cultured at a steady-state perfusion rate of P1 until harvest.

[0068] In the present disclosure, the cells for cultivating are not specifically limited. Steady-state perfusion culture method of the present disclosure can be applicable to the cultivation of various cells, particularly for the cultivation of host cells for gathering recombinant protein. In certain embodiments, the host cell can be a mammalian host cell, for example, selected from but not limited to HeLa, Cos, 3T3, myeloma cell line (such as NS0, SP2 / 0) and Chinese hamster ovary (CHO) cell. In the exemplary embodiments of the present disclosure, the cells for cultivating are CHO-K1 cells for expressing recombinant protein, and what are gathered in the crops is the recombinant protein produced by CHO-K1 cells. It should be understood that the above exemplary embodiments are not intended to limit the scope of the present disclosure, and in fact the steady-state perfusion culture method of the present disclosure can be adopted to cultivate other types of mammalian host cells, and gather in the crops corresponding recombinant protein products.

[0069] In the present disclosure, a cell culture is established by inoculating a bioreactor with cells expressing a biological substance of interest, such as mammalian host cells expressing a recombinant protein, at a density of, for example, at least 0.5×10 6 cells / mL, for example, about 0.5 to 4.0 × 10 6 cells / mL, about 0.5~3.5×10 6 cells / mL, about 0.5~3.0×10 6 cells / mL, about 0.5~2.5×10 6 cells / mL, 0.5~2.0×10 6 cells / mL, about 0.5~1.5×10 6 cells / mL, about 1.0~3.0×10 6 cells / mL, about 1.0-2.0×10 6 cells / mL, or any cell density within the above range. In at least one embodiment, the cells are grown in a bioreactor using, for example, at least 0.5×10 6cells / mL, for example, about 0.5 to 4.0 × 10 6 cells / mL, approximately 0.5 to 3.5 × 10 6 cells / mL, about 0.5~3.0×10 6 cells / mL, about 0.5~2.5×10 6 cells / mL, 0.5~2.0×10 6 cells / mL, about 0.5~1.5×10 6 cells / mL, about 1.0~3.0×10 6 cells / mL, about 1.0-2.0×10 6 cells / mL, about 1.0~1.5×10 6 cells / mL, about 1.0~1.2×10 6 cells / mL, or about 1.2 to 1.5 × 10 6 cells / mL, for example, about 1.0±0.2×10 6 cells / mL, approximately 1.0±0.1×10 6 Cell cultures are established by inoculating cells expressing the biological substance of interest at 50 cells / mL.

[0070] In the present disclosure, there is no specific limitation on the size of the bioreactor. It is understood that those skilled in the art can select a bioreactor with a suitable volume according to actual needs. In some embodiments, the volume of the bioreactor is 1L to 5L, for example 1L, 2L, 3L, 4L, 5L. In the exemplary embodiment of the present disclosure, a bioreactor with a volume of 3L is used. It should be understood by those skilled in the art that various bioreactors suitable for cell culture in the art can be used in the present disclosure, and there is no particular limitation on this. Those skilled in the art can set the conditions for cell culture and the operating conditions of the bioreactor according to actual needs, and these adjustments do not affect the core step procedures of the cell steady-state perfusion culture method disclosed herein.

[0071] In the present disclosure, there is no specific limitation on the working volume for culturing cells in a bioreactor. It will be appreciated that those skilled in the art can select a suitable working volume for culturing cells according to actual needs. In some embodiments, the working volume for culturing cells is 1 L to 5 L, for example, 1 L to 4 L, 1 L to 3 L, 1 L to 2 L, for example, 1.1 L, 1.2 L, 1.3 L, 1.4 L, 1.5 L, 1.6 L, 1.7 L, 1.8 L, 1.9 L, 2.0 L. In the non-limiting exemplary embodiments of the present disclosure, a working volume of 1.3 L to 1.5 L, for example, 1.4 L, is used.

[0072] In the present disclosure, there is no specific limitation on the culture medium used for cell culture, such as perfusion culture medium. It is understood that those skilled in the art can select a suitable culture medium according to actual needs. In some embodiments, the perfusion culture medium includes a basal culture medium, a feed culture medium or a culture medium mixed in an appropriate proportion. In an exemplary embodiment of the present disclosure, the basal culture medium is Dynamis culture medium and the feed culture medium is 3×FeedB+ culture medium. In an optional embodiment, the perfusion culture medium is a mixture of a basal culture medium and a feed culture medium in an appropriate proportion, for example, the volume ratio of the basal culture medium to the feed culture medium is 90:10, 88:12, 85:15, 83:17 or 80:20. In some embodiments, the volume ratio of the basal culture medium to the feed culture medium is (80-90):(10-20), or (85-90):(10-15). Optionally, during the perfusion culture process, those skilled in the art can adjust the volume ratio of the basal culture medium to the feed culture medium according to actual conditions.

[0073] In the present disclosure, after cell inoculation, the reactor control conditions are set, and the reactor control condition parameters for cell culture are not specifically limited. It will be understood that those skilled in the art can select appropriate reactor control conditions according to actual needs. In some embodiments, the temperature is, for example, controlled at 36.5°C to 37.5°C, for example, 37.0°C. In some embodiments, the pH is, for example, controlled at 7.00±1.00, 7.00±0.50, 7.00±0.25. In some embodiments, DO is, for example, controlled at 50-70%, 55-65%, 60%. In some embodiments, the initial stirring speed is, for example, controlled at 200-400rpm, 250-350rpm, 300rpm. In some embodiments, the bottom air flow is, for example, controlled at 10-30mL / min, 15-25mL / min, 20mL / min. In the exemplary embodiment of the present disclosure, the reaction control conditions are, for example, a temperature of 37.0°C, a pH of 7.00±0.25, a DO of 60%, an initial stirring speed of 300 rpm, and a constant bottom air flow of 20 mL / min. Those skilled in the art will be able to adjust the reactor control condition parameters based on the actual host cell type being cultured, the type of biological material to be harvested, such as the type of recombinant protein, and other actual requirements.

[0074] The setting method of the reactor control condition parameters depends on the bioreactor used and can be operated according to the instructions provided by the manufacturer.

[0075] In the present disclosure, cell perfusion culture can be carried out in a manner familiar to those skilled in the art, for example, by using methods well known to those skilled in the art such as ATF or TFF. In some embodiments, the present disclosure adopts an ATF cell perfusion culture method. In the present disclosure, when describing pumps for inputting and discharging liquids such as culture medium and cell culture fluid, the term peristaltic pump is used in some cases, but it should be understood that this is not intended to limit the scope of the present disclosure, and other types of pumps, as long as they are applicable to the field of cell culture, can be used in the methods and systems of the present disclosure.

[0076] In step 1, the cell culture parameters include cell density and viability. In some embodiments, the cell culture parameters may also include biochemical parameters, such as, but not limited to, glucose concentration, lactate concentration, amino acid concentration, and other common cell culture medium nutrient concentrations and cell metabolite concentrations in the culture medium.

[0077] In step 1, the culture medium may be a basal culture medium. In an exemplary embodiment of the present disclosure, the basal culture medium is Dynamis culture medium. The working volume after cell inoculation is V0 and the weight is W0. The balance will continuously monitor the weight. It should be understood that a balance usually measures the total weight of the reactor and the cell culture fluid contained therein. In the method of the present disclosure, this total weight can be used as a benchmark. In addition, since the dead weight of the reactor may be known, another way is to obtain the weight of the cell culture fluid contained in the reactor in real time by calculation, with the weight of the cell culture fluid as a benchmark. In the present disclosure, unless otherwise specified, the description is based on the weight of the reactor, that is, W0 is the total weight of the reactor and the cell culture fluid contained therein.

[0078] In step 1, perfusion culture is not initiated, but those skilled in the art can determine whether to activate a cell retention device, such as tangential flow filtration (TFF) or alternating tangential flow filtration (ATF), based on actual needs. It should be understood that since perfusion culture is not initiated, even if the cell retention device is activated, only the filtration circulation flow can be activated without discharging the cell-free harvest fluid.

[0079] In step 2, perfusion culture is started, and the culture medium added by perfusion can be a basal culture medium. In an exemplary embodiment of the present disclosure, the basal culture medium is Dynamis culture medium.

[0080] After perfusion is initiated, the cells in the reactor begin to proliferate rapidly, resulting in a continuous increase in the cell density within the reactor. The initial perfusion rate P0 can be determined by those skilled in the art based on practical needs, typically within the range of 0.1 to 1.0 VVD, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 VVD, and any of the aforementioned values ​​as endpoints, as well as any values ​​within such ranges. In some embodiments, the initial perfusion rate P0 is 0.5 VVD. The duration of the perfusion culture in step 2 can be determined by those skilled in the art based on practical needs, such as 3, 4, 5, or more days. In some embodiments, the perfusion culture is performed for 3 days. The "gradually increasing the perfusion rate during this period" can be performed by those skilled in the art in various ways based on practical needs, such as increasing the perfusion rate by a certain amount at a fixed time each day. It should be understood that there are no specific limitations on how many times the perfusion rate is adjusted daily and how much the perfusion rate is increased each time, and these can be determined by those skilled in the art. In some embodiments, at a fixed time each day, for example, at the same or similar time as the start of step 1, the perfusion rate is increased once, for example, by 0.5 VVD, or by other values ​​determined by those skilled in the art. In some embodiments, the perfusion culture is carried out for 3 days, and the perfusion rate is increased once a day by 0.5 VVD, i.e., on the 2nd day (the first day of reactor inoculation), the perfusion rate is 0.5 VVD; on the 3rd day, the perfusion rate is 1.0 VVD; and on the 4th day, the perfusion rate is 1.5 VVD. The perfusion rate at the end of step 2 can be determined by those skilled in the art, and is generally not more than 5.0 VVD, 4.5 VVD, 4.0 VVD, 3.5 VVD, 3.0 VVD, 2.5 VVD, 2.0 VVD or 1.5 VVD.

[0081] In the present disclosure, the adjustment of the perfusion rate should also be understood to be within a certain range of fluctuation, for example, the fluctuation range can be 0.05VVD, 0.04VVD, 0.03VVD, 0.02VVD, 0.01VVD. For example, in step 2, the initial perfusion rate P0 for starting perfusion culture can be 0.5±0.05VVD, and the perfusion rate can be adjusted to 1.0±0.05VVD on day 3, and other situations are similar.

[0082] In the present disclosure, the description of each time should not be understood as a fixed time point, but can be a time period. For example, the description "day 2" in step 2 should be understood as 24 ± 6 hours from the start of culture after the cells are inoculated in step 1, such as 24 ± 5 ​​hours, 24 ± 4 hours, 24 ± 3 hours, 24 ± 2 hours, 24 ± 1 hour, 24 ± 0.5 hours, 24 ± 0.3 hours, 24 ± 0.2, 24 ± 0.1 hours. Other descriptions of time in the present disclosure are also similar, for example, "day 3" should be understood as 48 ± 6 hours from the start of culture after the cells are inoculated in step 1, such as 48 ± 5 hours, 48 ​​± 4 hours, 48 ​​± 3 hours, 48 ​​± 2 hours, 48 ​​± 1 hour, 48 ± 0.5 hours, 48 ​​± 0.3 hours, 48 ​​± 0.2, 48 ± 0.1 hours, hours, and so on. In some embodiments, operations are performed at the same time of each day or within a time period before or after, for example, ±2 hours, ±1 hour, ±0.5 hours, ±0.3 hours, ±0.2, ±0.1 hours, including but not limited to reading cell density readings, turning on or off the steady-state automatic feedback control system, manual release, etc.

[0083] It should be understood that after perfusion is initiated, the peristaltic pump for inlet will remain on to continuously infuse liquid. A balance is coupled to the peristaltic pump at the harvest end, which is turned on or off based on the changes in the cell culture fluid weight / reactor weight continuously measured by the balance. During the perfusion culture process in step 2, the continuous inlet and the harvest discharge are in dynamic equilibrium, and the weight of the cell culture fluid in the reactor remains constant, and accordingly, the reactor weight remains constant. It should be understood that, in this disclosure, "cell culture fluid weight / reactor weight remains constant" should not be interpreted as meaning that the cell culture fluid weight / reactor weight always remains constant. Rather, it can fluctuate within a certain range above or below this value. As long as the fluctuation does not exceed the upper or lower limits of this range, the cell culture fluid weight is considered to remain constant. These upper and lower limits can be determined by those skilled in the art based on actual needs. In some embodiments, the upper and lower limits range, for example, are ±2%, ±1%, ±0.5%, ±0.1%, etc. In some embodiments, the upper and lower limits can be set based on the sensitivity of the balance. For example, if the balance measures a significant figure of 0.1 kg or other values, the weight within W ± 0.1 kg can be considered to remain constant.

[0084] In the present disclosure, steady-state culture refers to culture performed while maintaining a constant cell density and a constant cell culture fluid weight / reactor weight, which can be achieved in the following ways.

[0085] Establish a steady-state automatic feedback control system, including coupling the cell culture monitoring device with the discharge peristaltic pump and coupling the balance with the harvesting end peristaltic pump. Under the condition of maintaining the perfusion rate of continuous liquid inflow of culture medium, the target steady-state density needs to be maintained at ρ目标 , when the cell density detected in the bioreactor is at the target steady-state density ρ 目标 The target steady-state density is considered to be maintained when it is within ±10%, for example, within 8%, within 5%, within 2%, or within 1%. Another way is to define it in the form of a specific numerical value. When the cell density detected in the bioreactor is within the target steady-state density ρ 目标 ±10.0×10 6 cells / mL is considered to maintain the target steady-state density, for example, 8.0×10 6 cells / mL, less than 5.0×10 6 cells / mL, less than 4.0×10 6 cells / mL, less than 3.0×10 6 cells / mL, less than 2.0×10 6 cells / mL, less than 1.0×10 6 cells / mL, less than 0.8×10 6 cells / mL, less than 0.5×10 6 cells / mL, less than 0.1×10 6 cells / mL. In actual work, those skilled in the art can determine the applicable cell density variation range based on the detection sensitivity of the instrument used to monitor cell density. Those skilled in the art can also determine the applicable cell density variation range based on actual needs. In some embodiments, when the cell density detected in the bioreactor is within the target steady-state density p 目标 The target steady-state density is considered to be maintained within ±5.0%. When a change in cell density is detected, such as an increase or decrease in cell density reading, ρ 目标 × 5.0%, it can be considered that the cell density will continue to increase or decrease to destroy the steady state, at which point the steady-state automatic feedback control system will begin to operate to adjust the cell density. In some embodiments, when the cell density detected in the bioreactor is within the target steady-state density ρ 目标 ±5.0×10 6 cells / mL, the target steady-state density is maintained. When the detected cell density changes, such as the cell density reading increases or decreases by 5.0×10 6 cells / mL, it can be assumed that the cell density will continue to increase or decrease to destroy the steady state, at which point the steady-state automatic feedback control system will begin to operate to regulate the cell density.

[0086] When the cell density change reaches the upper limit (e.g., an increase of 5.0% of the target steady-state density; for example, 目标 +5.0×106 cells / mL), the cell culture monitoring device sends a signal to the discharge peristaltic pump, and the discharge peristaltic pump is turned on to discharge the cell culture fluid. The rate at which the discharge peristaltic pump discharges the cell culture fluid can be set by the reactor manufacturer, or can be set by those skilled in the art. Usually, the discharge rate of the discharge peristaltic pump is not greater than the inlet rate of the inlet peristaltic pump. After the discharge peristaltic pump is turned on, as the cell culture fluid is discharged, the weight of the cell culture fluid in the reactor / the weight of the reactor decreases. In order to maintain a constant weight of the cell culture fluid / a constant weight of the reactor in the reactor, when the weight of the cell culture fluid / the weight of the reactor reaches a set lower limit of change (for example, in some embodiments, W-0.1kg), the balance sends a signal to the harvesting end peristaltic pump, and the harvesting end peristaltic pump is turned off. Because the inlet peristaltic pump's inflow rate and the outlet peristaltic pump's discharge rate can match or mismatch, when the outlet peristaltic pump's discharge rate is lower than the inlet peristaltic pump's inflow rate, the weight of the cell culture fluid in the reactor will recover after the peristaltic pump at the harvest end is shut down. When the cell culture fluid weight / reactor weight reaches a set upper limit (e.g., W+0.1 kg in some embodiments), the peristaltic pump at the harvest end will be turned on. Thus, by coupling the weight-measuring scale with the peristaltic pump at the harvest end, the weight of the cell culture fluid in the reactor / reactor weight can be maintained constant.

[0087] As the cell culture solution containing cells is continuously discharged, the cell density in the reactor decreases. When the cell density reaches the set lower limit of variation (for example, in some embodiments, p 目标 × 95%, or in some embodiments, ρ 目标 -5.0×10 6 When the cell culture medium reaches a certain level (cells / mL), the cell culture monitoring device sends a signal to the discharge peristaltic pump, shutting it down and ceasing discharge of the cell culture fluid. The weight-measuring balance coupled to the harvesting peristaltic pump remains operational, maintaining a constant weight of the cell culture fluid within the reactor. After cessation of cell culture fluid discharge, the cell density in the reactor rebounds as cells proliferate. When the cell density reaches its upper limit again, the cell culture monitoring device sends a signal to the discharge peristaltic pump, restarting it to discharge the cell culture fluid and begin another round of cell density adjustment.

[0088] Through a steady-state automatic feedback control system, namely the coupling of the cell culture monitoring device with the discharge peristaltic pump and the coupling of the balance with the harvesting peristaltic pump, a constant cell density and a constant cell culture fluid weight / reactor weight are maintained, thus achieving steady-state culture. During steady-state culture, cell viability is also maintained constant, typically above 90%, above 95%, above 96%, above 97%, above 98%, above 99%, etc. In general, a cell viability above 95% is considered to indicate an optimal state of cell proliferation and growth.

[0089] However, in step 3, since the target steady-state density maintained at this time is the starting cell density ρ 起始 The cell density in the reactor at the end of step 2 is usually not a steady-state density that can be maintained stably for a long time. As the cell culture time increases, the cell culture conditions in the reactor deteriorate, cell proliferation slows down, or even stops, or cell proliferation is insufficient to replace dead cells. At this time, the cell density in the reactor will continue to decrease, and the cell viability will also begin to decrease, making it impossible to maintain steady-state culture.

[0090] In the duration of step 3, monitoring the cell culture state can be to check the cell density and cell viability in the reactor at fixed time intervals, such as every 24 hours. In some embodiments, with 24 hours as intervals, i.e., check the cell density and cell viability at the same time every day. When it is observed that the cell density can also be maintained at the target steady-state density and the cell viability is maintained, it is considered to be in steady-state culture. When it is observed twice in a row (in some embodiments for two consecutive days) that the cell density and viability all begin to decline, the cell density is lower than the target steady-state density, and the cell viability can not be maintained, such as when the cell viability is lower than 95%, it is considered that steady-state culture cannot be maintained, and now it is necessary to make adjustments, i.e., step 3 ends, step 4 is started, and manual discharge is performed. In some cases, when it is observed twice in a row (in some embodiments for two consecutive days) that the cell density begins to decline, the cell density is lower than the target steady-state density, but the cell viability can still be maintained, such as when the cell viability is maintained above 95%, it is also considered that steady-state culture cannot be maintained, and now it is necessary to make adjustments, i.e., step 3 ends, step 4 is started, and manual discharge is performed. In other words, when the cell density is observed to begin to decrease for two consecutive times (in some embodiments, for two consecutive days) and the cell density is lower than the target steady-state density, even if the cell viability can still be maintained, it is considered that the steady-state culture cannot be maintained. At this time, adjustment is required, that is, step 3 ends and step 4 begins, manual release.

[0091] In some embodiments, when the cell density is monitored to begin to decrease or both the cell density and viability begin to decrease, it means that when the steady-state automatic feedback control system is turned on, the monitored cell density can no longer be maintained at the target steady-state density and begins to decrease, and the cell viability can no longer be maintained at at least above 95%.

[0092] In step 3, the perfusion rate P1 is in the range of 2.0 to 5.0 VVD, such as 2.0 VVD, 2.5 VVD, 3.0 VVD, 3.5 VVD, 4.0 VVD, 4.5 VVD, 5.0 VVD, or a range with any of the above values ​​as endpoints, or any value within these ranges.

[0093] In step 4, manual discharge refers to the discharge of a predetermined volume of cell culture fluid from the reactor through the sampling port within a relatively short period of time, such as 1 hour, 0.5 hour, 0.2 hour, 10 minutes, 5 minutes, 2 minutes, or 1 minute. Those skilled in the art can determine the predetermined volume. In some embodiments, the predetermined volume is (10-15) ± 0.5% of the total volume of the cell culture fluid in the reactor, such as 10%, 11%, 12%, 13%, 14%, or 15%. In some embodiments, the predetermined volume is 10 ± 0.5% of the total volume of the cell culture fluid in the reactor. Manual discharge is repeated at fixed time intervals, such as every 24 hours. In some embodiments, manual discharge is performed at 24-hour intervals, i.e., at the same time each day. It should be understood that because the inlet peristaltic pump continuously inputs fresh culture medium, the total volume of the cell culture fluid in the reactor has been restored before each manual discharge after the first manual discharge and is maintained constant by the coupling of the balance to the peristaltic pump at the harvesting end.

[0094] Before manual discharge, disable the automatic cell density feedback. This means that the coupling between the cell culture monitoring device and the discharge peristaltic pump is stopped, and the discharge peristaltic pump is no longer turned on or off based on changes in cell density. In step 4, the discharge peristaltic pump remains off. The coupling between the balance and the peristaltic pump at the harvesting end remains on to maintain a constant cell culture medium weight / reactor weight.

[0095] After each manual discharge, the weight of the cell culture fluid / reactor weight decreases, the peristaltic pump at the harvest end is turned off, and the liquid inlet peristaltic pump continues to infuse liquid until the weight of the cell culture fluid in the reactor / reactor weight reaches the same. Then, with the help of the coupling between the balance and the peristaltic pump at the harvest end, the peristaltic pump at the harvest end is turned on or off to maintain the weight of the cell culture fluid / reactor weight constant.

[0096] During the duration of step 4, monitoring the cell culture status can be checking the cell density and cell viability in the reactor at fixed time intervals, for example, every 24 hours. In some embodiments, the cell density and cell viability are checked at intervals of 24 hours, i.e., at the same time every day.

[0097] After manual discharge, due to the discharge of a large number of cells, as the peristaltic pump continues to feed liquid, the cell density in the reactor will drop briefly, but due to the improvement of the cell culture environment, cell proliferation will recover and the cell density will gradually increase again. With daily manual discharge, cell density and viability begin to rise. With each manual discharge, the cell density in the reactor continues to rise, but the amplitude of change gradually slows down until it is stably maintained within a certain range within a certain period of time. Those skilled in the art will determine the length of the above time period and the range of the cell density interval. In some embodiments, the time period is at least 5 days, for example 5 days, 6 days, 7 days, 8 days or longer. In some embodiments, the cell density interval is at most ±10.0×10 6 cells / mL, that is, any day is D1, the recorded cell density is ρ1, and in the following time period, the cell density is recorded every day, ρ2, ρ3…ρ m , if ρ1 to ρ m The difference between the highest and lowest values ​​in the 6 cells / mL, the cell density is considered to be stably maintained at a maximum of ±10.0×10 6 cells / mL, for example, the cell density range is ±9.0×10 6 cells / mL, ±8.0×10 6 cells / mL, ±7.0×10 6 cells / mL, ±6.0×10 6 cells / mL, ±5.0×10 6 cells / mL, ±4.0×10 6 cells / mL, ±3.0×10 6 cells / mL, ±2.0×10 6 cells / mL, or ±1.0×10 6 In some embodiments, the cell density range is up to ±5.0×10 6 When the cell density is observed to be stably maintained within a certain range for a certain period of time, for example, in some embodiments, the cell density is observed to be stably maintained at a maximum of ±10.0×10 cells / mL for at least 5 days. 6 cells / mL, for example, up to ±5.0×10 6 cells / mL, any cell density value within this interval is determined as the maintainable steady-state cell density ρ 最终 , that is, the VCD that can be maintained at the perfusion rate P1, and its corresponding CSPR is CSPRmin. For example, in some embodiments, it is observed that the cell density is stably maintained at a maximum of ±10.0×106 cells / mL, for example, up to ±5.0×10 6 cells / mL, select ρ1 to ρ m The median value in was determined as the maintainable steady-state cell density ρ 最终 , that is, the VCD that can be maintained at the perfusion rate P1, and its corresponding CSPR is CSPRmin. For example, in some embodiments, it is observed that the cell density is stably maintained at a maximum of ±10.0×10 6 cells / mL, for example, up to ±5.0×10 6 cells / mL, select ρ1 to ρ m The average of the highest and lowest values ​​in the α-cell density was determined as the maintainable steady-state cell density ρ. 最终 , that is, the VCD that can be maintained at the perfusion rate P1, and its corresponding CSPR is CSPRmin.

[0098] Step 4 ends.

[0099] Those skilled in the art will appreciate that during the cell proliferation period in step 4, the cell density can remain stable and / or continue to increase, and the cell viability can generally be maintained at a high level, for example, above 95%.

[0100] In step 5, the steady-state automatic feedback control system is turned on again, that is, the coupling between the cell culture monitoring device and the discharge peristaltic pump and the coupling between the balance and the harvesting end peristaltic pump are turned on to start steady-state culture until the end of the culture.

[0101] Those skilled in the art can determine the time to end the culture according to actual needs, such as obtaining the desired protein amount.

[0102] According to the method disclosed herein, CSPRmin can be found quickly, for example, usually within about 20 days, and its value can usually be below 30 pL / cell / day, thereby maintaining steady-state culture for up to 2 months, or even longer.

[0103] In one embodiment, the method of steady-state perfusion culture of cells disclosed herein comprises the following steps:

[0104] Step 1. Reactor inoculation

[0105] The cells are inoculated into a reactor containing culture medium, cell culture is started and the cell culture status is monitored. The cell culture status parameters include cell density and viability.

[0106] Optionally, the cell is a mammalian cell, for example selected from, but not limited to, HeLa, Cos, 3T3, myeloma cell lines (e.g., NSO, SP2 / 0), and Chinese hamster ovary (CHO) cells,

[0107] Optionally, the seeding density is at least 0.5×10 6 cells / mL, for example, about 0.5 to 4.0 × 10 6 cells / mL, approximately 0.5 to 3.5 × 10 6 cells / mL, about 0.5~3.0×10 6 cells / mL, about 0.5~2.5×10 6 cells / mL, 0.5~2.0×10 6 cells / mL, about 0.5~1.5×10 6 cells / mL, about 1.0~3.0×10 6 cells / mL, about 1.0-2.0×10 6 cells / mL, about 1.0~1.5×10 6 cells / mL, about 1.0~1.2×10 6 cells / mL, or about 1.2 to 1.5 × 10 6 cells / mL, for example, about 1.0±0.2×10 6 cells / mL, approximately 1.0±0.1×10 6 cells / mL,

[0108] Optionally, the culture medium is a basal medium,

[0109] Optionally, the reactor control conditions are set, and the reactor control conditions include any one or more of the following: temperature is controlled at 36.5°C to 37.5°C, preferably 37.0°C; pH is controlled at 7.00±1.00, 7.00±0.50 or 7.00±0.25; DO is controlled at 50-70%, 55-65% or 60%; initial stirring speed is controlled at 200-400rpm, 250-350rpm or 300rpm; and / or bottom air flow is controlled at 10-30mL / min, 15-25mL / min or 20mL / min.

[0110] Optionally, the cell culture status parameters further include glucose concentration, lactate concentration, amino acid concentration, and other common cell culture medium nutrient concentrations, cell metabolite concentrations, etc.;

[0111] Step 2. Start perfusion

[0112] On the second day, perfusion culture was started at the initial perfusion rate P0 and continued for several days until the Nth day, N = 4 to 6, during which the perfusion rate was gradually increased.

[0113] Optionally, the initial perfusion rate P0 is within the range of 0.1 to 1.0 VVD, and the perfusion rate is increased to a final value not exceeding 5.0 VVD. Optionally, the perfusion rate is increased every day to a final value not exceeding 5.0 VVD.

[0114] Optionally, the perfusion medium is a basal medium or a mixed medium of a basal medium and a feed medium;

[0115] Step 3. Initial Steady-State Perfusion Culture

[0116] On day N+1, the steady-state automatic feedback control system is turned on, with the current cell density ρ 起始 As the initial target steady-state density, steady-state culture was performed at a perfusion rate of P1, and the cell culture status was continuously monitored.

[0117] The said starting of the steady-state automatic feedback control system includes starting the coupling between the cell culture monitoring device and the discharge pump and starting the coupling between the balance and the harvesting end pump.

[0118] Optionally, the perfusion rate P1 is in the range of about 2.0 to 5.0 VVD,

[0119] Optionally, the perfusion medium is a basal medium or a mixed medium of a basal medium and a feed medium.

[0120] Optionally, the discharge pump is a discharge peristaltic pump, and the harvesting end pump is a harvesting end peristaltic pump;

[0121] Step 4. Manual platelet removal and determination of maintainable steady-state cell density

[0122] When it is monitored that the cell density begins to decrease or both the cell density and viability begin to decrease, the steady-state automatic feedback control system is turned off and about 10-15% of the cell culture medium is manually discharged. This is repeated daily and the cell culture is continued at the perfusion rate P1. The cell culture status is continuously monitored. The cell culture status parameters include cell density and viability.

[0123] The shutting down of the steady-state automatic feedback control system includes shutting down the coupling between the cell culture monitoring device and the discharge pump, but keeping the coupling between the balance and the harvesting end pump open.

[0124] When the cell density is monitored to be stable within a certain range for at least 5 consecutive days, any cell density value within the range is selected as the maintainable steady-state cell density ρ 最终 , the corresponding CSPR is CSPRmin,

[0125] Optionally, the cell culture state parameters also include glucose concentration, lactate concentration, amino acid concentration, and other common cell culture medium nutrient concentrations, cell metabolite concentrations, etc.

[0126] Optionally, the cell density is stably maintained at a maximum of ±5.0 × 10 6 cells / mL,

[0127] Optionally, any value or median of the cell density for at least 5 consecutive days is selected as the maintainable steady-state cell density p 最终 ,

[0128] Optionally, the average of the highest and lowest cell densities for at least 5 consecutive days is selected as the maintainable steady-state cell density p 最终 ,

[0129] Optionally, the perfusion medium composition is adjusted during the process;

[0130] Step 5. Maintainable Steady-State Perfusion Culture

[0131] Start the steady-state automatic feedback control system again, with ρ 最终 For the final target steady-state density, the cells are continuously cultured in a steady-state perfusion culture at a perfusion rate P1, for example, for at least 1 month, 2 months, or 3 months, or for at least 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, or 90 days, until harvested.

[0132] In one embodiment, the method of steady-state perfusion culture of cells disclosed herein comprises the following steps:

[0133] Step 1. Reactor inoculation

[0134] The cells are inoculated into a reactor containing culture medium, cell culture is started and the cell culture status is monitored. The cell culture status parameters include cell density and viability.

[0135] Optionally, the cell is a mammalian cell, for example selected from, but not limited to, HeLa, Cos, 3T3, a myeloma cell line (e.g., NS0, SP2 / 0), and a Chinese hamster ovary (CHO) cell, such as a CHO-K1 cell,

[0136] Optionally, the seeding density is about 1.0 to 1.5 × 10 6 cells / mL, for example, about 1.0±0.1×10 6 cells / mL,1.0±0.2×10 6 cells / mL,

[0137] Optionally, the culture medium is a basal medium, such as Dynamis basal medium,

[0138] Optionally, the reactor control conditions are set, wherein the reactor control conditions include any one or more of the following: temperature 37.0°C, pH 7.00±0.25, DO 60%, initial stirring speed 300 rpm, and / or bottom air flow constant 20 mL / min,

[0139] Optionally, the cell culture status parameters further include glucose concentration, lactate concentration, amino acid concentration, and other common cell culture medium nutrient concentrations, cell metabolite concentrations, etc.;

[0140] Step 2. Start perfusion

[0141] On the second day, perfusion culture was started with an initial perfusion rate P0 of about 0.5 VVD and continued for 3 days, during which the perfusion rate was increased by about 0.5 VVD every day, that is, the perfusion rate on the third day was about 1.0 VVD, and the perfusion rate on the fourth day was about 1.5 VVD.

[0142] Optionally, the perfusion medium is a basal medium or a mixed medium of a basal medium and a feed medium, such as Dynamis basal medium;

[0143] Step 3. Initial Steady-State Perfusion Culture

[0144] On the 5th day, the steady-state automatic feedback control system was turned on, and the current cell density ρ 起始 As the initial target steady-state density, steady-state culture was performed at a perfusion rate of P1, and the cell culture status was continuously monitored.

[0145] The said starting of the steady-state automatic feedback control system includes starting the coupling between the cell culture monitoring device and the discharge peristaltic pump and starting the coupling between the balance and the harvesting end peristaltic pump.

[0146] Optionally, the perfusion rate P1 is about 2.0 VVD,

[0147] Optionally, the perfusion medium is a basal medium or a mixed medium of a basal medium and a feed medium, for example, a mixed medium consisting of Dynamis and 3×FeedB+ in a volume ratio of 85:15;

[0148] Step 4. Manual platelet removal and determination of maintainable steady-state cell density

[0149] When it is monitored that the cell density begins to decrease or both the cell density and viability begin to decrease, the steady-state automatic feedback control system is turned off, and about 10%-15% of the cell culture medium is manually discharged, for example, about 10% of the cell culture medium, and this is repeated daily. Cell culture is continued at the perfusion rate P1, and the cell culture status is continuously monitored. The cell culture status parameters include cell density and viability.

[0150] The shutting down of the steady-state automatic feedback control system includes shutting down the coupling between the cell culture monitoring device and the discharge peristaltic pump, but keeping the coupling between the balance and the harvesting end peristaltic pump open.

[0151] When the cell density is monitored to be stable at ±5.0×10 6 cells / mL, any cell density value within this range is selected as the maintainable steady-state cell density ρ 最终 , the corresponding CSPR is CSPRmin,

[0152] Optionally, the cell culture state parameters also include glucose concentration, lactate concentration, amino acid concentration, and other common cell culture medium nutrient concentrations, cell metabolite concentrations, etc.

[0153] Optionally, any value or median of the cell density for at least 5 consecutive days is selected as the maintainable steady-state cell density p 最终 ,

[0154] Optionally, the average of the highest and lowest cell densities for at least 5 consecutive days is selected as the maintainable steady-state cell density p 最终 ,

[0155] Optionally, the composition of the perfusion medium is adjusted during the period, for example, a mixed medium consisting of Dynamis and 3×FeedB+ in a volume ratio of 90:10;

[0156] Step 5. Maintainable Steady-State Perfusion Culture

[0157] Start the steady-state automatic feedback control system again, with ρ 最终 For the final target steady-state density, the cells are continuously cultured in a steady-state perfusion culture at a perfusion rate P1, for example, for at least 1 month, 2 months, or 3 months, or for at least 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, or 90 days, until harvested.

[0158] Steady-state cell perfusion culture system

[0159] Figure 1 shows a schematic diagram of an embodiment of a cell steady-state perfusion culture system according to the present disclosure. The system includes a reactor, a cell culture monitoring device, a balance, an inlet pump, a discharge pump, a harvesting end pump and a sampling port. The cell culture monitoring device, such as an Aber in situ live cell online detector, is used to monitor the cell density and viability in the reactor. The balance is located at the bottom of the reactor and is used to measure the weight of the reactor. The inlet pump is used to input fresh culture medium into the reactor. The discharge pump is used to discharge cell culture fluid. The harvesting end pump is used to discharge cell-free harvest to facilitate subsequent separation and purification of desired biological substances, such as recombinant proteins. The cell culture monitoring device is coupled to the discharge peristaltic pump to control the opening and closing of the discharge pump according to the culture parameters measured by the cell culture monitoring device, including cell density. The balance is coupled to the harvesting end pump to control the opening and closing of the discharge peristaltic pump according to the reactor weight measured by the balance. The sampling port is used to discharge cell culture fluid from the reactor during artificial discharge.

[0160] Those skilled in the art should understand that Figure 1 only exemplifies the cell steady-state perfusion culture system disclosed herein, and does not limit the technical solution of the present invention. According to actual needs, those skilled in the art can make modifications to the cell steady-state perfusion culture system disclosed herein without destroying the implementation of the cell steady-state perfusion culture method disclosed herein.

[0161] The technical solutions of the present disclosure are further described in detail below through examples and in conjunction with the accompanying drawings. Unless otherwise stated, the methods and materials of the embodiments described below are conventional products that can be purchased on the market. Those skilled in the art will understand that the methods and materials described below are merely exemplary and should not be construed as limiting the scope of the present disclosure.

[0162] Example

[0163] Example 1 Determining CSPRmin and Steady-State Perfusion Culture of Cells

[0164] Preparation of seed solution

[0165] CHO-K1 cells were revived in Dynamis medium in an incubator at 37.0°C, 5.0% CO2, and 120 rpm. Cells were sampled and counted 72 ± 4 hours after recovery. The viable cell density before each cell passage should be greater than 1.0 × 10 6 cells / mL, cell viability should be greater than 90%. Cell passage should be performed every 72±4 hours.

[0166] The reactor structure is shown in Figure 1: Fresh culture medium is pumped into the reactor at a constant flow rate. A peristaltic pump connected to the rear end of the ATF is coupled to the reactor weight, setting the reactor weight to a constant value. When the weight exceeds the set value, the peristaltic pump turns on and cells are harvested; when it falls below the set value, the peristaltic pump turns off. Furthermore, the reading of an Aber online cell density meter is coupled to the discharge peristaltic pump, setting the Aber cell density to a constant value. When the cell density exceeds the set value, the peristaltic pump turns on and discharges the cells; when it falls below the set value, the peristaltic pump turns off. With these settings, steady-state automatic control is achieved.

[0167] Step 1. Reactor inoculation

[0168] Cells were inoculated into a 3 L Applikon perfusion bioreactor containing Dynamis medium and the ATF controller was turned on. The inoculation density was 1.0 × 10 6 cells / mL, the reactor temperature was controlled at 37.0°C, and the pH was controlled at 7.0±0.25.

[0169] Step 2. Start perfusion

[0170] On day 2 of culture, the peristaltic pump was turned on, the perfusion rate was set to 0.5 V / D, and perfusion was started using Dynamis basal medium. On day 3, the perfusion rate was adjusted to 1.0 V / D. On day 4, the perfusion rate was adjusted to 1.5 V / D.

[0171] Step 3. Establish a steady-state automatic feedback control system

[0172] On the fifth day of culture, the culture medium was replaced with perfusion production medium consisting of Dynamis and 3×FeedB+ in a volume ratio of 85:15, and the perfusion rate was adjusted to 2.0VVD. Aber automatic feedback control was turned on and the cell density was set at (45±5)×10 6 cells / mL.

[0173] The cell status and bleeding volume were observed daily. Table 1 records the cell density, viability, and bleeding volume from day 6 to day 12. As can be seen from the table, the cell density, viability, and bleeding volume gradually decreased. On day 12, the cell viability had dropped from 99.2% at the beginning to 95.9%. This indicates that at a perfusion rate of 2.0VVD, using Dynamis and 3×FeedB+ medium in a volume ratio of 85:15, it is impossible to maintain a cell density of (45±5)×10 6 cells / mL (CSPR 44.4pL / cell / day).

[0174] Table 1 Cell status and discharge volume

[0175] Step 4. Manual release

[0176] Turn off the Aber automatic feedback control and initiate manual draining at a rate of approximately 10%-15% of the reactor culture volume. Simultaneously, change the perfusion medium from 85:15 Dynamis:3×FeedB+ to 90:10 Dynamis:3×FeedB+. Repeat manual draining daily.

[0177] The results of cell culture under manual release are shown in Figure 2. With manual release, cell growth gradually recovered. From day 20 to day 25, the cell density was basically maintained at 75×10 6 cells / mL (CSPR 26.7 pL / cell / day), so this density was determined as the steady-state density to be maintained subsequently.

[0178] Step 5. Steady-state perfusion culture

[0179] Turn on Aber automatic feedback control and set the Aber control density to (75±5)×10 6 cells / mL. Samples were taken every day to test the protein production and quality outside the tank. The results are shown in Figure 3. From D19 to D78, a total of 60 days, the cell density was always maintained at 75×10 6 cells / mL, with cell viability generally maintained above 95%. Steady state lasted for up to 2 months, with CSPR as low as 26.7pL / cell / day, which is the CSPRmin under these culture conditions.

[0180] The protein yield results are shown in Table 2. As of day 78, the cumulative protein yield was about 120 g / L, which was about 31 times that of the original process. The original process used a Fed-batch culture method. The culture process was as follows: 0.5×10 6 The cells were inoculated into a 3 L reactor containing Dynamis medium at a target density of 10 cells / mL. 3×FeedB+ supplements equivalent to 3%, 4%, 5%, 5%, 5%, and 5% of the current volume were added on culture days 3, 5, 7, 9, 11, and 13, respectively. On culture day 5, the temperature was lowered to 33.0°C. On culture day 14, the supernatant was harvested and analyzed for protein yield and quality.

[0181] Table 2 Protein yield

[0182] The protein purity results are shown in Figure 4. The protein purity at different time points was tested. It can be seen that the protein purity was maintained above 98% throughout the entire culture cycle, which is comparable to the original Fed-batch process.

[0183] The results of protein charge heterogeneity are shown in Figure 5. The protein charge heterogeneity at different time points was detected. Throughout the entire culture cycle, the protein acid region was less than 25%, and the main peak was basically above 60%, which is better than the original Fed-batch process.

[0184] The results of protein glycoform detection are shown in Figure 6. Protein glycoforms were detected at different time points. Throughout the entire culture cycle, Man5 was less than 1%, which was lower than the original Fed-batch process.

[0185] The embodiments of the present invention are not limited to the above embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and details, and these are all considered to fall within the scope of protection of the present invention.

Claims

1. A method for steady-state perfusion culture of cells, the method comprising: Step 1. Reactor inoculation Inoculating cells into a reactor containing a culture medium, starting cell culture and monitoring the cell culture status, wherein the cell culture status parameters include cell density and viability; Step 2. Start perfusion On the second day, perfusion culture was started with an initial perfusion rate P0 and continued for several days until the Nth day, N = 4 to 6, during which the perfusion rate was gradually increased; Step 3. Initial Steady-State Perfusion Culture On day N+1, the steady-state automatic feedback control system is turned on, with the current cell density ρ 起始 The cell culture status was continuously monitored at a perfusion rate of P1 to achieve the initial target steady-state density. The said starting the steady-state automatic feedback control system includes starting the coupling between the cell culture monitoring device and the discharge pump and starting the coupling between the balance and the harvest end pump; Step 4. Manual platelet release and determination of maintainable steady-state cell density When it is monitored that the cell density begins to decrease or both the cell density and viability begin to decrease, the steady-state automatic feedback control system is turned off, and about 10-15% of the cell culture fluid is manually discharged. This is repeated daily, and cell culture is continued at the perfusion rate P1. The cell culture status is continuously monitored. The cell culture status parameters include cell density and viability. The shutting down of the steady-state automatic feedback control system includes shutting down the coupling between the cell culture monitoring device and the discharge pump, but keeping the coupling between the balance and the harvest end pump open. When the cell density is monitored to be stably maintained within a certain range for at least 5 consecutive days, any cell density value within the range where the cell density is stably maintained for at least 5 consecutive days is selected as the maintainable steady-state cell density ρ 最终 , the corresponding CSPR is CSPRmin; Step 5. Maintainable Steady-State Perfusion Culture Start the steady-state automatic feedback control system again, with ρ 最终 To achieve the final target steady-state density, cells were cultured at a steady-state perfusion rate of P1 until harvest.

2. The method for steady-state perfusion culture of cells according to claim 1, wherein: The cell is a mammalian cell, preferably selected from but not limited to HeLa, Cos, 3T3, myeloma cell lines (eg, NS0, SP2 / 0) and Chinese hamster ovary (CHO) cells.

3. The method for steady-state perfusion culture of cells according to claim 1 or 2, wherein: In step 1, the seeding density is at least 0.5×10 6 cells / mL.

4. The method for steady-state perfusion culture of cells according to any one of claims 1 to 3, wherein: Step 1 also includes setting reactor control conditions, which include any one or more of the following: temperature is controlled at 36.5°C to 37.5°C, preferably 37.0°C; pH is controlled at 7.00±1.00, 7.00±0.50 or 7.00±0.25; DO is controlled at 50-70%, 55-65% or 60%; initial stirring speed is controlled at 200-400rpm, 250-350rpm or 300rpm; and / or bottom air is constantly controlled at 10-30mL / min, 15-25mL / min or 20mL / min.

5. The method for steady-state perfusion culture of cells according to any one of claims 1 to 4, wherein: The cell culture state parameters also include glucose concentration, lactic acid concentration, amino acid concentration in the culture medium, and other common cell culture medium nutrient concentrations, cell metabolite concentrations, etc.

6. The method for steady-state perfusion culture of cells according to any one of claims 1 to 5, wherein: In step 2, the initial perfusion rate P0 is in the range of about 0.1 to 1.0 VVD, and the perfusion rate is increased to eventually not exceed 5.0 VVD.

7. The method for steady-state perfusion culture of cells according to any one of claims 1 to 6, wherein: In step 3, the perfusion rate P1 is in the range of about 2.0 to 5.0 VVD.

8. The method for steady-state perfusion culture of cells according to any one of claims 1 to 7, wherein: In step 4, the cell density was maintained stably at a maximum of ±10.0×10 6 cells / mL, for example, up to ±5.0×10 6 cells / mL range.

9. The method for steady-state perfusion culture of cells according to any one of claims 1 to 8, wherein: In step 4, any value or median value of the cell density for at least 5 consecutive days is selected as the maintainable steady-state cell density p 最终 .

10. The method for steady-state perfusion culture of cells according to any one of claims 1 to 8, wherein: In step 4, the average of the highest and lowest cell densities for at least 5 consecutive days is selected as the maintainable steady-state cell density ρ 最终 .

11. A system for steady-state perfusion culture of cells, comprising a reactor, a cell culture monitoring device, a balance, a liquid inlet pump, a discharge pump, a harvesting end pump and a sampling port, wherein: The balance is used to measure the reactor weight, the cell culture monitoring equipment is coupled to the discharge pump, and the balance is coupled to the harvest end pump.

12. The system for steady-state perfusion culture of cells according to claim 11, wherein: The cell culture monitoring device is coupled to the discharge pump, and the discharge pump is turned on and off according to the culture parameters measured by the cell culture monitoring device, including cell density.

13. The system for steady-state perfusion culture of cells according to claim 11 or 12, wherein: The balance is coupled to the harvest end pump, and the opening and closing of the harvest end pump is controlled according to the reactor weight measured by the balance.

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