WO2025124527A1 - Method and system for steady-state perfusion culture of cells - Google Patents

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

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application number CN202311732489.7 and filing date December 15, 2023, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention provides a method and system for steady-state perfusion culture of cells, belonging to the field of cell culture.

Background Art

Since the first commercialization of recombinant proteins in the 1980s, the biopharmaceutical industry has developed rapidly in the past 40 years. With the increasing demand for biopharmaceutical production capacity, continuous perfusion culture, a culture technology that has been used since the 1990s, has regained attention. The process principle of perfusion culture is to continuously add fresh culture medium on one side and continuously harvest the culture supernatant (protein) through a cell retention device on the other side, while continuously removing metabolic byproducts, so that the cells in the reactor are always in a good growth environment. Compared with the traditional fed-batch culture (Fed-batch) in which the culture supernatant is harvested once, perfusion culture can not only obtain protein products with more stable quality, but also make the cell density reach a very high level, thereby achieving a higher protein yield level. According to the presence or absence of cell bleeding, perfusion culture is divided into two culture methods: steady state perfusion and dynamic state perfusion. Since dynamic perfusion does not perform cell bleeding, due to nutrient limitation, the cell density and viability will decrease in the later stage of culture, and the culture cycle is generally 20-30 days. Steady-state perfusion continuously discharges cells while replenishing fresh culture medium, so that cells are always in a state of growth. The cell viability can be maintained at a high level. The culture cycle is generally 2-6 months, or even longer. For some easily degradable protein products, such as enzymes and factor products, steady-state perfusion is the preferred culture method. The operating variables involved in steady-state perfusion include cell specific perfusion rate (CSPR), perfusion rate (P, vessel volume per day, VVD) and viable cell density (VCD). CSPR = P/VCD, which represents the volume of culture medium required for each cell per day, in pL/cell/day. In the development of steady-state perfusion process, the most important step is to determine CSPRmin. For a given culture medium, there is a critical value for CSPR. Below this critical value, perfusion culture cannot be maintained; above this critical value, there is an excess of nutrients, resulting in waste of culture medium. It can be concluded that the best performance of the perfusion bioreactor is obtained at the minimum CSPR value at which cell growth is stable, which is called CSPRmin. How to determine CSPRmin? According to literature reports, it can be achieved by two methods: 1. Maintaining VCD unchanged, gradually reducing P (push to low), when the cell growth cannot be maintained stably, the previous CSPR is CSPRmin. 2. Maintaining P unchanged, gradually increasing VCD (push to high), when the cell growth cannot be maintained stably, the previous CSPR is CSPRmin. In the process of gradually reducing P or gradually increasing VCD, multiple CSPR inspection points need to be set. Each inspection cycle should be maintained for at least 7-10 days. The entire inspection cycle takes at least 1.5-2 months, resulting in a long cycle for the entire development process itself. Although each CSPR cycle is inspected for 7-10 days, the real steady-state maintenance time is much longer than this, usually at least 1-2 months, so the CSPRmin found by this method may not be able to maintain a real steady-state culture. Once the cells cannot maintain a 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

In order to solve the problem that the methods for finding CSPRmin in the prior art are time-consuming and labor-intensive, the present disclosure provides a novel cell steady-state perfusion culture method, in which a steady-state culture is attempted with any cell density as the starting cell density, and when the steady-state culture cannot be maintained, the final maintainable steady-state cell density is quickly found through continuous manual intervention, thereby determining CSPRmin, so that long-term steady-state culture can be carried out until the cell culture is terminated and the product is harvested.

Therefore, in one aspect, the present disclosure provides 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 at the initial perfusion rate P ₀ 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 was turned on, with the current cell density ρ _as the starting target steady-state density, and the perfusion rate P ₁ for steady-state culture, and the cell culture status was continuously monitored.

The said starting 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;

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 the cell culture is continued at a perfusion rate of P _1. 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 it is monitored that the cell density is 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 _ρFinally , the corresponding CSPR is CSPRmin;

Step 5. Maintainable Steady-State Perfusion Culture

The steady-state automatic feedback control system was turned on again, with ρfinal as the target _final steady-state density and the perfusion rate _P1 continuing the steady-state perfusion culture of cells until harvest.

In some embodiments, 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.

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

In some embodiments, 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.

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

In some embodiments, in step 2, the initial perfusion rate P ₀ 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.

In some embodiments, in step 3, the perfusion rate _P1 is in the range of about 2.0-5.0 VVD.

In some embodiments, in step 4, the cell density is stably maintained within a range of at most ±10.0×10 ⁶ cells/mL, such as at most ±5.0×10 ⁶ cells/mL, for at least 5 consecutive days.

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 ρ _final .

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 _ρfinal .

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.

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 the culture parameters measured by the cell culture monitoring device, including cell density.

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

Advantageous Effects of the Present Disclosure

Compared with the prior art, the method of steady-state perfusion culture of cells provided in the present invention can determine CSPRmin more quickly, usually only taking about 20 days, and under this CSPRmin, steady-state culture of cells can be maintained for at least 2 months, saving time and material costs, and the final recombinant protein yield is much higher than the original fed-batch culture (Fed-batch) process, and the quality is equivalent to or even better.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

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

Figure 2 shows the cell growth curve during the manual release phase.

FIG3 shows the cell growth curve during the steady-state perfusion phase.

Figure 4 shows the SEC purity analysis results.

FIG5 shows the results of icIEF charge heterogeneity analysis.

FIG6 shows the results of N-glycosylation analysis.

DETAILED DESCRIPTION

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.

Those skilled in the art will appreciate 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 any or all combinations of these.

Perfusion culture is alternating tangential flow perfusion culture or tangential flow perfusion culture. Alternating tangential flow perfusion culture refers to perfusion culture in which alternating tangential flow (ATF) filtration technology is used to intercept cells in cell culture; tangential flow perfusion culture refers to perfusion culture in which tangential flow (TFF) filtration technology is used to intercept cells during cell culture. In the perfusion culture in which tangential flow (TFF) filtration technology is used to intercept cells, the cell fluid forms a continuous annular flow direction through the peristaltic pump. After entering the fiber membrane, the waste liquid will be discharged from the system through the membrane, 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 intercept 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 interception device, and the metabolic waste will be discharged through the membrane with the culture medium and the cells will be trapped in the reactor. When using ATF, the alternating motion produces a flushing effect in the filter membrane, which helps to prevent the fiber membrane from clogging. In the present disclosure, perfusion culture in which alternating tangential flow (ATF) filtration technology is used to intercept cells is preferred.

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 constant cell growth and productivity, and cell bleeding is required to maintain a constant viable cell density or maintain the cells in a stable state. The typical viable cell density under the stable state is 10 to 100 × 10 ⁶ cells/mL.

As used herein, the term "cell bleeding" refers to the removal of cells and medium from a bioreactor to maintain a constant, sustainable viable cell density within the bioreactor. This cell bleeding can be accomplished using a dip tube and a peristaltic pump at a specified flow rate. The tubing should be of appropriate size; if it is too narrow, cells will tend to aggregate and clog, while if it is too large, the cells may settle. Cell bleeding can be determined based on growth rate so that the viable cell density can be limited to a desired volume in a continuous manner. Alternatively, cells can be removed at a certain frequency, such as once a day, and replaced with medium to maintain the cell density within a predictable range.

As used herein, the term "perfusion rate" refers to the amount of culture medium added and removed from a bioreactor in a given time, usually expressed as a portion or multiple of the working volume, usually measured every day. "Working volume" refers to the volume used for cell culture in a bioreactor. Perfusion usually begins on the 1st to 3rd day after inoculation, when the cells are still in the exponential growth phase, so the perfusion rate may increase during the culture process. The increase in perfusion rate can be incremental or continuous, i.e. based on cell density or nutrient consumption. Usually starting from 0.5 or 1.0 working volumes (VVD) per day, up to about 5.0 VVD may be achieved. Preferably, the perfusion rate is between 0.5 and 2.0 VVD. The increase may be 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.

As used in this article, the term "Cell Specific Perfusion Rate (CSPR)" refers to the volume of culture medium required per cell per day, expressed in units of "pL/cell/day". An ideal CSPR should result in optimal growth rate and productivity.

As used herein, the term "CSPRmin" refers to the critical CSPR value for maintaining stable proliferation and growth of cells. If the CSPR value is lower than the critical value, perfusion culture cannot be maintained; if the CSPR value is higher than the critical value, there will be excess nutrition, resulting in waste of culture medium.

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 the unit of "cells/mL", and is sometimes also referred to as "cell density (VCD)". A person skilled in the art can measure the viable cell density by any method known to those skilled in the art. Preferably, an online in situ live cell monitoring instrument, such as an ABER in situ live cell online detection instrument, is used to measure the viable cell density in real time.

When used herein, the terms "bioreactor" and "reactor" are used interchangeably and refer to any container that can be used for cell culture growth. The bioreactor can be of any size as long as it can meet the needs of cell culture. Typically, the volume of the bioreactor is at least 0.5L, 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, 12,000L or larger, or any volume between the above numerical ranges. In general, 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 lower than the volume of the bioreactor. The working volume for culturing cells in the bioreactor can be determined by those skilled in the art according to actual needs. The internal conditions of the bioreactor can be controlled during the culture period, including but not limited to pH and temperature. 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 cultures used in the methods of the present disclosure can be grown in any bioreactor suitable for perfusion culture.

As used herein, the terms "culture medium" and "cell culture medium" are used interchangeably and 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 to 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 usually 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 is most suitable for cell survival and proliferation. In certain embodiments, the culture medium of the present disclosure is a perfusion culture medium, including a basal culture medium, a feed culture medium, and a mixed culture medium of a basal culture medium and a feed culture 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 defined 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 day 2, and perfusion of the feed medium can start from day 3. Alternatively, perfusion of the basal medium can be started from day 1. For example, the basal medium can be perfused 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 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 in the present invention, 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 destroy the implementation of each step in the method disclosed in the present invention. 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.

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 defoamer, and for example, one or more selection agents that can be bound to resistance markers and viability markers in the host cell line, these selection agents include but are not limited to geneticin (G4118), neomycin, hygromycin B, puromycin, zeocin, methionine sulfenyl imide or methotrexate.

Steady-state cell perfusion culture method

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

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 at the initial perfusion rate P ₀ 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 was turned on, with the current cell density ρ _as the starting target steady-state density, and the perfusion rate P ₁ for steady-state culture, and the cell culture status was continuously monitored.

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 harvesting 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 10-15% of the cell culture fluid is manually discharged. This is repeated daily, and cell culture is continued at a perfusion rate of _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 it is monitored that the cell density is 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 _ρFinally , the corresponding CSPR is CSPRmin;

Step 5. Maintainable Steady-State Perfusion Culture

The steady-state automatic feedback control system was turned on again, with ρfinal as the _final target steady-state density and the perfusion rate _P1 continuing the steady-state perfusion culture of cells until harvest.

In the present disclosure, the cells to be cultivated are not specifically limited. The stable perfusion culture method of the present disclosure can be applicable to the cultivation of various cells, particularly for the cultivation of host cells for harvesting recombinant proteins. In certain embodiments, the host cell can be a mammalian host cell, such as selected from but not limited to HeLa, Cos, 3T3, myeloma cell lines (such as NS0, SP2/0) and Chinese hamster ovary (CHO) cells. In the exemplary embodiments of the present disclosure, the cells to be cultivated are CHO-K1 cells for expressing recombinant proteins, and what is harvested 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 stable perfusion culture method of the present disclosure can be used to cultivate other types of mammalian host cells, and the corresponding recombinant protein products can be harvested.

In the present disclosure, a cell culture is established by inoculating cells expressing a biological substance of interest, such as mammalian host cells expressing a recombinant protein, in a bioreactor at an inoculation density, for example, of at least 0.5×10 ⁶ cells/mL, such as about 0.5-4.0×10 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 method comprises the step of: using, for example, at least 0.5×10 ⁶ cells/mL, such as about 0.5-4.0×10 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, about 1.0-1.5×10 6 cells/mL, about 1.0-1.2×10 ⁶ cells/mL, or about 1.2-1.5×10 ⁶ cells/mL, such as about 1.0±0.2×10 ⁶ cells/mL, about 1.0±0.1×10 6 cells/mL, or about 1.2-1.5×10 ⁶ cells/mL, such as about 1.0±0.2×10 ⁶ cells/mL, about 1.0±0.1×10 ⁶ cells/mL, or about 1.0±0.1×10 ⁶ cells/mL. Cell cultures are established by inoculating cells/mL with cells expressing the biological substance of interest.

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, such as 1L, 2L, 3L, 4L, 5L. In the exemplary embodiments 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, without particular limitation. 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 in the present disclosure.

In the present disclosure, there is no specific limitation on the working volume for culturing cells in a bioreactor. It is to be understood 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 1L to 5L, such as 1L to 4L, 1L to 3L, 1L to 2L, such as 1.1L, 1.2L, 1.3L, 1.4L, 1.5L, 1.6L, 1.7L, 1.8L, 1.9L, 2.0L. In the non-limiting exemplary embodiments of the present disclosure, a working volume of 1.3L to 1.5L, such as 1.4L, is used.

In the present disclosure, there is no specific limitation on the culture medium, such as perfusion culture medium, for cell culture. 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 thereof. 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.

In the present disclosure, after cell inoculation, reactor control conditions are set, and the reactor control condition parameters for cell culture are not specifically limited. It is understood that those skilled in the art can select suitable reactor control conditions according to actual needs. In some embodiments, the temperature is, for example, controlled at 36.5°C to 37.5°C, such as 37.0°C. In some embodiments, 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 is constantly passed, 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, temperature 37.0°C, pH 7.00±0.25, DO 60%, initial stirring speed 300rpm, and bottom air flow 20mL/min. Those skilled in the art can adjust the reactor control condition parameters according to the actual needs of the host cell type actually cultured, the type of biological material expected to be harvested, such as the type of recombinant protein, etc.

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.

In the present disclosure, cell perfusion culture can be carried out in a manner familiar to those skilled in the art, such as by ATF or TFF or other methods well known to those skilled in the art. In some embodiments, the present disclosure adopts ATF cell perfusion culture. 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 suitable for the field of cell culture, can be used in the methods and systems of the present disclosure.

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

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 V ₀ and the weight is W _0. The balance will continuously monitor the weight. It should be understood that the balance usually measures the total weight of the reactor and the cell culture solution contained therein. In the method of the present disclosure, the total weight can be used as a reference. In addition, since the dead weight of the reactor may be known, another way is to obtain the weight of the cell culture solution contained in the reactor in real time by calculation, with the weight of the cell culture solution as a reference. In the present disclosure, unless otherwise specified, the description is based on the weight of the reactor, that is, W ₀ is the total weight of the reactor and the cell culture solution contained therein.

In step 1, perfusion culture is not started, but those skilled in the art can determine whether to start the cell retention device according to actual needs, such as starting tangential flow filtration (TFF) or alternating tangential flow filtration (ATF). It should be understood that since perfusion culture is not started, even if the cell retention device is turned on, only the filtration circulation flow can be turned on without discharging the cell-free harvest liquid.

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.

After the perfusion is turned on, the cells in the reactor begin to proliferate in large quantities, so the cell density in the reactor tends to continue to increase. The initial perfusion rate _P0 can be determined by those skilled in the art according to actual needs, usually in 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 above values as endpoints and any values therein. In some embodiments, the initial perfusion rate _P0 is 0.5 VVD. The time of perfusion culture in step 2 can be determined by those skilled in the art according to actual needs, for example, it can be 3 days, 4 days, 5 days or more days. In some embodiments, perfusion culture is performed for 3 days. The "gradually increasing the perfusion rate during the period" can be performed by those skilled in the art in various ways according to actual needs, for example, the perfusion rate can be increased by a certain value at a fixed time every day. It should be understood that there is no particular restriction on how many times the perfusion rate is adjusted each day and how much the perfusion rate is increased each time, and it can be determined by those skilled in the art. In some embodiments, at a fixed time every day, for example, the same or similar time as the start time of step 1, the perfusion rate is increased once, for example, by 0.5 VVD, or other values of VVD 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 every day by 0.5 VVD, that is, on the second day (the first day of reactor inoculation), the perfusion rate is 0.5 VVD; on the third day, the perfusion rate is 1.0 VVD; on the fourth 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, usually not exceeding 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.

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 floating 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 is adjusted to 1.0±0.05VVD on the third day, and other situations are similar.

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. 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, such as ±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.

It should be understood that after the perfusion is turned on, the inlet peristaltic pump will remain in the open state to continuously inlet the liquid. The balance is coupled to the peristaltic pump at the harvest end, and the peristaltic pump at the harvest end is turned on or off according to the change in the weight of the cell culture fluid/reactor weight continuously measured by the balance. During the perfusion culture process of step 2, the continuous inlet and the harvest discharge are in dynamic equilibrium, the weight of the cell culture fluid in the reactor remains constant, and the weight of the reactor remains constant accordingly. It should be understood that in the present disclosure, the cell culture fluid weight/reactor weight remains constant should not be understood as the cell culture fluid weight/reactor weight always remaining at a value without changing, but may fluctuate within a certain range above and below a value, as long as the fluctuation amplitude does not exceed the upper and lower limits of the range, it can be considered that the cell culture fluid weight remains constant. The upper and lower limits can be determined by those skilled in the art according to actual needs. In some embodiments, the upper and lower limits are, for example, ±2%, ±1%, ±0.5%, ±0.1%, etc. In some embodiments, the upper and lower limits can be set according to the sensitivity of the balance, for example, if the significant figures measured by the balance are 0.1kg or other values, the weight can be considered to remain constant at W±0.1kg.

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

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 harvest end peristaltic pump. Under the condition of maintaining the perfusion rate of the culture medium continuously, it is necessary to maintain the target steady-state density as _ρtarget . When the cell density detected in the bioreactor is within ±10% of the _target steady-state density ρtarget, it is considered to maintain the target steady-state density, for example, within 8%, within 5%, within 2%, within 1%. Another way is to limit it in the form of specific numerical values. When the cell density detected in the bioreactor is within ±10.0×10 ⁶ cells/mL of the _target steady-state density ρtarget, it is considered to maintain the target steady-state density. For example, within 8.0×10 ⁶ cells/mL, within 5.0×10 ⁶ cells/mL, within 4.0×10 ⁶ cells/mL, within 3.0×10 ⁶ cells/mL, within 2.0×10 ⁶ cells/mL, within 1.0×10 ⁶ cells/mL, within 0.8×10 ⁶ cells/mL, within 0.5×10 ⁶ cells/mL, and within 0.1×10 ⁶ cells/mL. In actual work, according to the detection sensitivity of the instrument used to monitor the cell density, those skilled in the art can determine the applicable cell density variation range. Those skilled in the art can also determine the applicable cell density variation range according to actual needs. In some embodiments, when the cell density detected in the bioreactor is within ±5.0% of the _target steady-state density ρtarget, it is considered to maintain the target steady-state density. When the detected cell density changes, such as the cell density reading increases or decreases _{by ρtarget} ×5.0%, it can be considered that the cell density will continue to increase or decrease to disrupt the steady state, and 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 ±5.0×10 ⁶ cells/mL of the _target steady-state density ρtarget, it is considered that 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 considered that the cell density will continue to increase or decrease to disrupt the steady state, and the steady-state automatic feedback control system will begin to operate to adjust the cell density.

When it is detected that the cell density change reaches the upper limit (for example, an increase of 5.0% of the target steady-state density; for example, ρ _target + 5.0×10 ⁶ 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 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 in the reactor/a constant weight of the reactor, when the weight of the cell culture fluid/the weight of the reactor reaches the set lower limit of change (for example, in some embodiments, W-0.1kg), the balance sends a signal to the peristaltic pump at the harvest end, and the peristaltic pump at the harvest end is turned off. Since the inlet peristaltic pump and the discharge peristaltic pump can match or not match, when the discharge peristaltic pump discharge rate is less than the inlet peristaltic pump, after the peristaltic pump at the harvest end is turned off, the weight of the cell culture solution in the reactor will rise again, and when the cell culture solution weight/reactor weight reaches the set upper limit of change (for example, W+0.1kg in some embodiments), the peristaltic pump at the harvest end will be turned on. Thus, relying on the coupling of the weight measuring scale and the peristaltic pump at the harvest end, the cell culture solution weight/reactor weight in the reactor can be kept constant.

As the cell culture fluid containing cells is continuously discharged, the cell density in the reactor decreases. When the cell density reaches the set lower limit of change (for example, in some embodiments, _ρtarget ×95%, or in some embodiments, _ρtarget -5.0×10 ⁶ cells/mL), the cell culture monitoring device sends a signal to the discharge peristaltic pump, and the discharge peristaltic pump is turned off to stop discharging the cell culture fluid. The coupling of the weight measuring balance and the peristaltic pump at the harvesting end keeps working, so that the weight of the cell culture fluid in the reactor can be kept constant. After stopping the discharge of the cell culture fluid, as the cells proliferate, the cell density in the reactor rises again. When the cell density change reaches the upper limit again, 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, and another round of adjusting the cell density begins.

Through the steady-state automatic feedback control system, that is, the coupling of the cell culture monitoring device with the discharge peristaltic pump and the coupling of the balance with the peristaltic pump at the harvest end, a constant cell density and a constant cell culture fluid weight/reactor weight are maintained, that is, a steady-state culture is achieved. In the steady-state culture, the cell viability can also be kept constant, usually above 90%, above 95%, above 96%, above 97%, above 98%, above 99%, etc. In general, a cell viability of more than 95% is considered to be a better proliferation and growth state of the cells.

However, in step 3, since the target steady-state density maintained at this time, i.e., the _starting cell density ρstart, is the cell density in the reactor at the end of step 2, this cell density is usually not a steady-state density that can be stably maintained 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 replenish and 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.

During 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, the cell density and cell viability are checked at intervals of 24 hours, i.e., at the same time every day. When it is observed that the cell density can still be maintained at the target steady-state density and the cell viability is maintained, it is considered to be still 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 begin to decline, the cell density is lower than the target steady-state density, and the cell viability cannot be maintained, for example, when the cell viability is lower than 95%, it is considered that the steady-state culture cannot be maintained, and adjustments need to be made at this time, 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, for example, when the cell viability is maintained above 95%, it is also considered that the steady-state culture cannot be maintained, and adjustments need to be made at this time, i.e., step 3 ends, step 4 is started, and manual discharge is performed. In other words, when the cell density is observed to decrease for two consecutive times (two consecutive days in some embodiments) 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, adjustments need to be made, that is, step 3 ends and step 4 begins, manual discharge.

In some embodiments, when it is monitored that the cell density begins to decrease or both the cell density and the 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, but begins to decrease, and the cell viability can no longer be maintained at at least above 95%.

In step 3, the perfusion rate _P1 is in the range of 2.0 to 5.0 VVD, for example, 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.

In step 4, manual discharge refers to the discharge of a predetermined volume of cell culture fluid from the reactor through the sampling port in 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 by themselves. 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%, etc. 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, etc. In some embodiments, manual discharge is performed at intervals of 24 hours, i.e., at the same time every day. It should be understood that, since 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 kept constant by coupling the balance with the peristaltic pump at the harvesting end.

Before manual discharge, turn off the automatic feedback of cell density, that is, stop the coupling between the cell culture monitoring device and the discharge peristaltic pump, and no longer turn on or off the discharge peristaltic pump according to the change of 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 fluid weight/reactor weight.

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 inlet peristaltic pump continues to infuse fluid until the weight of the cell culture fluid in the reactor/reactor weight reaches a certain value. 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.

During the duration of step 4, monitoring the cell culture status may be to check the cell density and cell viability in the reactor at fixed time intervals, such as every 24 hours, etc. In some embodiments, the cell density and cell viability are checked at intervals of 24 hours, i.e., at the same time every day.

After manual discharge, due to the discharge of a large number of cells, as the inlet peristaltic pump continues to feed liquid, the cell density in the reactor will drop temporarily, but due to the improvement of the cell culture environment, the cell proliferation situation recovers, and the cell density will gradually increase again. With the manual discharge every day, the 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 in a certain interval within a certain time period. Those skilled in the art determine the length of the above time period and the range of the cell density interval by themselves. In some embodiments, the time period is at least 5 days, such as 5 days, 6 days, 7 days, 8 days or longer. In some embodiments, the cell density range is at most ±10.0×10 ⁶ cells/mL, that is, any day is taken as D1, the recorded cell density is ρ ₁ , and in the subsequent period of time, the cell density is recorded every day, ρ ₂ , ρ ₃ ...ρ _m , if the difference between the highest value and the lowest value in ρ ₁ to ρ _m is ≤20.0×10 ⁶ cells/mL, then the cell density is considered to be stably maintained within the range of at most ±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 cells/mL ^. cells/mL. In some embodiments, the cell density range is at most ±5.0×10 ⁶ cells/mL. When the cell density is observed to be stably maintained in a certain range within a certain period of time, for example, in some embodiments, the cell density is observed to be stably maintained in a range of at most ±10.0×10 ⁶ cells/mL for at least 5 days, for example, at most ±5.0×10 ⁶ cells/mL, any cell density value in the range is determined as _the maintainable steady-state cell density ρfinal, that is, the VCD that can be maintained at the perfusion rate P ₁ , and the corresponding CSPR is CSPRmin. For example, in some embodiments, the cell density is observed to be stably maintained in a range of at most ±10.0×10 ⁶ cells/mL for at least 5 days, for example, at most ±5.0×10 ⁶ cells/mL, and the median value from ρ ₁ to ρ _m is selected to be determined as the maintainable steady-state cell density _ρfinal , that is, the VCD that can be maintained at the perfusion rate P ₁ , and the corresponding CSPR is CSPRmin. For example, in some embodiments, the cell density is observed to be stably maintained in a range of up to ±10.0×10 ⁶ cells/mL, for example, up to ±5.0×10 ⁶ cells/mL for at least 5 days, and the average of the highest and lowest values from ρ ₁ to ρ _m is selected as the maintainable steady-state cell density ρ _final , that is, the VCD that can be maintained at the perfusion rate P ₁ , and the corresponding CSPR is CSPRmin.

Step 4 ends.

Those skilled in the art will appreciate that during the cell proliferation in step 4, the cell density can remain stable and/or continue to grow, and the cell viability can generally remain at a high level, such as above 95%.

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 peristaltic pump at the harvesting end are turned on to start steady-state culture until the end of the culture.

Those skilled in the art can decide the time to end the culture according to actual needs, such as obtaining the desired amount of protein.

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

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

Step 1. Reactor inoculation

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

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 Chinese hamster ovary (CHO) cells,

Optionally, the seeding density is at least 0.5×10 ⁶ cells/mL, such as about 0.5-4.0×10 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, about 1.0-1.5×10 ⁶ cells/mL, about 1.0-1.2×10 ⁶ cells/mL, or about 1.2-1.5×10 ⁶ cells/mL, such as about 1.0±0.2×10 ⁶ cells/mL, about 1.0±0.1× ^{10 6 cells} /mL,

Optionally, the culture medium is a basal medium,

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 is constantly controlled at 10-30mL/min, 15-25mL/min or 20mL/min,

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.;

Step 2. Start perfusion

On the second day, perfusion culture was started at the initial perfusion rate P ₀ and continued for several days until the Nth day, N = 4-6, during which the perfusion rate was gradually increased.

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.

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

Step 3. Initial Steady-State Perfusion Culture

On day N+1, the steady-state automatic feedback control system was turned on, with the current cell density ρ _as the starting target steady-state density, and the perfusion rate P ₁ for steady-state culture, and the cell culture status was continuously monitored.

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 harvesting end pump.

Optionally, the perfusion rate _P1 is in the range of about 2.0 to 5.0 VVD,

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

Optionally, the discharge pump is a discharge peristaltic pump, and the harvest end pump is a harvest end peristaltic 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 the cell culture is continued at a perfusion rate of P _1. 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 is selected as the maintainable steady-state cell density ρ. _Finally , the corresponding CSPR is CSPRmin.

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.

Optionally, the cell density is stably maintained within a range of at most ±5.0×10 ⁶ cells/mL for at least 5 consecutive days,

Optionally, any value or median value of the cell density for at least 5 consecutive days is selected as the maintainable steady-state cell density _pFinally ,

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.

Optionally, adjusting the perfusion medium composition during the process;

Step 5. Maintainable Steady-State Perfusion Culture

The steady-state automatic feedback control system is started again, with ρfinal as the _final target steady-state density and the perfusion rate _P1 continuously performing steady-state perfusion culture of the cells, for example, continuing the steady-state perfusion culture for at least 1 month, 2 months, or 3 months, or for example, continuing the steady-state perfusion culture for at least 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, or 90 days, until harvest.

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

Step 1. Reactor inoculation

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

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,

Optionally, the seeding density is about 1.0 to 1.5×10 ⁶ cells/mL, such as about 1.0±0.1×10 ⁶ cells/mL, 1.0±0.2×10 ⁶ cells/mL,

Optionally, the culture medium is a basal medium, such as Dynamis basal medium,

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 300rpm, and/or bottom air constant flow 20mL/min,

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.;

Step 2. Start perfusion

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.

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;

Step 3. Initial Steady-State Perfusion Culture

On the fifth day, the steady-state automatic feedback control system was turned on, with the current cell density _ρstart as the starting target steady-state density, and the perfusion rate _P1 for steady-state culture, and the cell culture status was continuously monitored.

The said starting 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 harvest end peristaltic pump.

Optionally, the perfusion rate _P1 is about 2.0 VVD,

Optionally, the perfusion medium is a basal medium or a mixed medium of a basal medium and a feed medium, such as a mixed medium consisting of Dynamis and 3×FeedB+ in a volume ratio of 85:15;

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 the 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, for example, about 10% of the cell culture fluid, and this is repeated daily. Cell culture is continued at a perfusion rate of P ₁ , and 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 peristaltic pump, but keeping the coupling between the balance and the harvest end peristaltic pump open.

When the cell density is monitored to be stably maintained within the range of ±5.0×10 ⁶ cells/mL for at least 5 consecutive days, any cell density value within this range is selected as the maintainable steady-state cell density ρ. _Finally , the corresponding CSPR is CSPRmin.

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.

Optionally, any value or median value of the cell density for at least 5 consecutive days is selected as the maintainable steady-state cell density _pFinally ,

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.

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

Step 5. Maintainable Steady-State Perfusion Culture

The steady-state automatic feedback control system is started again, with ρfinal as the _final target steady-state density and the perfusion rate _P1 continuously performing steady-state perfusion culture of the cells, for example, continuing the steady-state perfusion culture for at least 1 month, 2 months, or 3 months, or for example, continuing the steady-state perfusion culture for at least 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, or 90 days, until harvest.

Steady-state cell perfusion culture system

FIG1 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 harvest 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 the cell culture fluid. The harvest end pump is used to discharge the cell-free harvest to facilitate subsequent separation and purification of the desired biological substance, such as recombinant protein. 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 harvest 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 the cell culture fluid from the reactor during artificial discharge.

Those skilled in the art should understand that FIG. 1 is only an exemplary display of the cell steady-state perfusion culture system of the present invention, 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 of the present invention without destroying the cell steady-state perfusion culture method of the present invention.

The technical scheme of the present disclosure is further described in detail below by way of examples and in conjunction with the accompanying drawings. Unless otherwise specified, the methods and materials of the embodiments described below are conventional products that can be purchased on the market. It will be understood by those skilled in the art that the present invention belongs to that the methods and materials described below are merely exemplary and should not be construed as limiting the scope of the present disclosure.

Example

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

Preparation of seed solution

CHO-K1 cells were revived in Dynamis medium in an incubator at 37.0°C, 5.0% CO ₂ and 120 rpm. Samples were taken and counted 72±4 hours after cell resuscitation. The viable cell density before each cell passaging should be greater than 1.0×10 ⁶ cells/mL and the cell viability should be greater than 90%. Cell passaging was performed every 72±4 hours.

The structure of the reactor is shown in Figure 1: fresh culture medium is pumped into the reactor at a constant flow rate, and the peristaltic pump connected to the rear end of the ATF is coupled to the weight of the reactor, and the weight of the reactor is set to a constant value. When the weight exceeds the set value, the peristaltic pump is turned on to harvest cells; when it is lower than the set value, the peristaltic pump is turned off. In addition, the reading of the Aber online cell density meter is coupled to the discharge peristaltic pump, and the Aber cell density is set to a constant value. When the cell density exceeds the set value, the peristaltic pump is turned on to discharge; when it is lower than the set value, the peristaltic pump is turned off. Through the above settings, steady-state automatic control can be achieved.

Step 1. Reactor inoculation

The cells were inoculated into a 3L 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℃, and the pH was controlled at 7.0±0.25.

Step 2. Start perfusion

On the second day of culture, the culture medium peristaltic pump was turned on, the perfusion rate was set to 0.5 VVD, and perfusion was started. The perfusion medium was Dynamis basal medium. On the third day, the perfusion rate was adjusted to 1.0 VVD. On the fourth day, the perfusion rate was adjusted to 1.5 VVD.

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

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

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 shows that at a perfusion rate of 2.0VVD, using Dynamis and 3×FeedB+ medium with a volume ratio of 85:15, it is impossible to maintain a steady-state culture with a cell density of (45±5)×10 ⁶ cells/mL (CSPR 44.4pL/cell/day).

Table 1 Cell status and discharge volume

Step 4. Manually release

Turn off the Aber automatic feedback control and start manual discharge, with the discharge ratio being about 10%-15% of the reactor culture volume. At the same time, change the perfusion medium from 85:15 Dynamis and 3×FeedB+ to 90:10 Dynamis and 3×FeedB+. Repeat manual discharge once a day.

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 around 75×10 ⁶ cells/mL (CSPR 26.7pL/cell/day) for 5 consecutive days. Therefore, this density was determined as the steady-state density to be maintained subsequently.

Step 5. Steady-state perfusion culture

Turn on Aber automatic feedback control and set the Aber control density to (75±5)×10 ⁶ cells/mL. Samples were kept every day to detect the protein yield 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 around 75×10 ⁶ cells/mL, and the cell viability was basically maintained above 95%. The steady state lasted for up to 2 months, and the CSPR was as low as 26.7pL/cell/day, which is the CSPRmin under this culture condition.

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

Table 2 Protein yield

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

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

The results of protein glycoform detection are shown in Figure 6. The protein glycoforms at different time points were detected respectively. During the entire culture period, Man5 was lower than 1%, which was lower than the original Fed-batch process.

The implementation of the present invention is not limited to the above-mentioned 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 detail, and these are considered to fall within the protection scope of the present invention.

Claims (13)

A method for steady-state perfusion culture of cells, 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 at the initial perfusion rate P ₀ 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 was turned on, with the current cell density ρ _as the starting target steady-state density, and the perfusion rate P ₁ for steady-state culture, and the cell culture status was continuously monitored. 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 the cell culture is continued at a perfusion rate of P _1. 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 it is monitored that the cell density is 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 _ρFinally , the corresponding CSPR is CSPRmin; Step 5. Maintainable Steady-State Perfusion Culture The steady-state automatic feedback control system was turned on again, with ρfinal as the _final target steady-state density and the perfusion rate _P1 continuing the steady-state perfusion culture of cells until harvest. The method for steady-state perfusion culture of cells according to claim 1, wherein the cells are mammalian cells, preferably selected from but not limited to HeLa, Cos, 3T3, myeloma cell lines (e.g., NS0, SP2/0) and Chinese hamster ovary (CHO) cells. The method for steady-state perfusion culture of cells according to claim 1 or 2, wherein in step 1, the inoculation density is at least 0.5×10 ⁶ cells/mL. The method for steady-state perfusion culture of cells according to any one of claims 1 to 3, wherein step 1 further comprises setting reactor control conditions, wherein the reactor control conditions comprise 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. 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, lactate concentration, amino acid concentration in the culture medium, and other common cell culture medium nutrient concentrations, cell metabolite concentrations, etc. 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 a final rate not exceeding 5.0 VVD. 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. 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 is stably maintained within a range of at most ±10.0×10 ⁶ cells/mL, for example, within a range of at most ±5.0×10 ⁶ cells/mL for at least 5 consecutive days. 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 ρ _final . 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 _final . A system for steady-state perfusion culture of cells comprises 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 weight of the reactor, the cell culture monitoring device is coupled with the discharge pump, and the balance is coupled with the harvesting end pump. According to the system for steady-state perfusion culture of cells according to claim 11, the cell culture monitoring device is coupled to the discharge pump, and the opening and closing of the discharge pump are controlled according to the culture parameters measured by the cell culture monitoring device, including cell density. 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.