CN111139216A - High-throughput, automated cell continuous culture method and uses thereof - Google Patents

Abstract

The invention discloses a high-throughput and automatic continuous cell culture method and application thereof. The method comprises the following steps: (1) providing a cell culture, inoculating and culturing; (2) settling the cells, and removing the supernatant in the reactor; (3) adding a fresh culture medium; (4) calculating a discharge rate (B) based on the cell density in the supernatant, maintaining a target cell density; (5) repeating the steps (2) to (4) until the continuous culture of the cells is completed. Compared with the traditional perfusion reactor, the cell continuous culture model provided by the invention has the advantages of large flux, high automation degree, small culture volume and low cost; compared with the culture by using a shake tube, the reactor has strong characteristics, high representativeness of the culture result, difficult occurrence of human errors and high reliability. In addition, the invention does not need to use an additional cell interception device in the perfusion reactor, thereby solving the problem that the cell interception device is easy to block.

Description

High-throughput, automated cell continuous culture method and uses thereof

PRIORITY AND RELATED APPLICATION

The present application claims priority from chinese patent application 201811295263.4 entitled high throughput, automated cell continuous culture process and uses thereof, filed 2018, month 11, day 1, which is incorporated herein by reference in its entirety, including the appendix.

Technical Field

The invention relates to the technical field of biological pharmacy and biology, in particular to the technical field of animal cell culture, and particularly relates to a high-throughput and automatic continuous cell culture method and application thereof in the field.

Background

Over the course of decades, cell culture techniques have become established for the production of many important proteins, particularly those that are glycosylated and complex macromolecular. These proteins are often used in the prevention and treatment of several serious human diseases, such as cancer, viral infections, genetic defect diseases and other chronic diseases.

At present, in vitro cell culture models mainly comprise three types of batch culture, fed-batch culture and continuous culture. Compared with batch culture and fed-batch culture, continuous culture (or open culture and perfusion culture) is increasingly gaining attention and popularizing because of its unique advantages in terms of production continuity, higher cell density obtained by culture, and better consistency of final products.

At present, the continuous culture of cells is mainly carried out in the industry by using a reactor (called a perfusion reactor) with a cell interception device, and the reactor has the advantages of high efficiency interception of cells and robustness of continuous culture. But also has its disadvantages, mainly expressed as: 1) the perfusion reactor with cell shut-off and pump/scale/piping for maintaining continuous culture and complex control components is highly prone to human error and requires a significant amount of labor and time for routine operation and monitoring. 2) Although the cell interception device has good interception effect, under the condition of long-time operation, the interception membrane hole is easy to block, so that the target protein in the reactor is intercepted, the protein yield is low, and the quality is reduced. 3) The scale of the perfusion reactor is usually large (3L to 200L are different), the culture time is long (30 days to 120 days are different), a culture medium with the culture volume 1-2 times of that of the reactor needs to be fed every day for continuous culture, the consumption of the culture medium is huge for long-term culture, and the cost is obviously increased.

Due to the above drawbacks, the perfusion reactor is not suitable for high throughput media screening and process optimization. In order to save cost and improve efficiency, the most used industry currently is to replace the perfusion reactor with a swing pipe for high throughput screening and optimization experiments. The shaking tube has small volume (20ml) and large flux, and can simulate continuous culture operation by centrifugal liquid exchange, thereby replacing a reactor to a certain extent to carry out primary screening and optimization of culture conditions. However, the disadvantages are still evident, mainly: 1) the reactor is not characterized by the absence of pH, dissolved oxygen, agitation and aeration control, so the culture result is poor in representativeness and the reactor cannot be effectively enlarged; 2) high-throughput experiments usually require a large number of experiments (20-100), and a large amount of labor is required for a large amount of pipe shaking operations, which wastes labor cost; 3) the manual operation error is easily caused by a large amount of liquid and frequent liquid replacement, and the authenticity and the representativeness of the experimental result are reduced.

Based on the shortcomings of the two continuous culture models in the industry (i.e., the perfusion reactor and the shake tube), a high-throughput and automatic continuous cell culture model is needed to overcome or balance the shortcomings, so that how to develop such an instructive culture model is of great significance to the industry.

Ambr15^TMThe system is a high-throughput automatic micro bioreactor culture system produced by Saedolis, and the design is primarily used for high-throughput screening and process optimization of fed-batch experiments, and the skilled person does not consider that the system has the function of continuous culture, and the prior art does not describe that the system has the function of continuous culture. The invention applies the continuous cell culture model to Ambr15 by technical innovation^TMThe system successfully realizes the high flux and automation of the continuous culture of the cells.

Disclosure of Invention

Problems to be solved by the invention

One of the technical problems to be solved by the present invention is to provide a high-throughput, automated method for continuous cell culture, thereby overcoming the defects of the current perfusion reactor and shaking tube. For example, compared with the traditional perfusion reactor, the cell continuous culture method provided by the invention has higher flux and higher automation degree; compared with the method of culturing by using a shake tube, the method of continuously culturing the cells has the advantages of stronger reactor characteristics, high culture result representativeness, difficult occurrence of human errors and higher reliability.

The second technical problem to be solved by the present invention is to provide a method of utilizing gravity sedimentation to replace the cell retention device in the traditional reactor, thereby realizing high density cell culture. I.e., using gravity sedimentation to produce a cell-free or relatively low cell content supernatant, which is then removed and fresh medium is added. The gravity sedimentation method provided by the invention does not need an additional cell interception device, so that the problem of membrane interception blockage does not exist.

The third technical problem to be solved by the present invention is that Ambr15^TMIn the environment of continuous culture, a sequential gradient supernatant removal mode is provided, so that the problems that partial cell strains are not obvious in sedimentation effect in a short time and liquid replacement is difficult to perform in a gravity sedimentation mode are solved.

The fourth technical problem to be solved by the present inventionIn Ambr15^TMProvides an effective calculation method of the cell releasing rate under the environment of continuous culture, thereby solving the problem of maintaining constant cell density.

Means for solving the problems

In order to solve the above technical problems, the present invention provides a high-throughput, automated method for continuous cell culture, comprising the steps of:

a method for high throughput, automated continuous cell culture, the method comprising the steps of:

(1) providing a cell culture, inoculating it to sarbs 15^TMCulturing in a reactor of the system;

(2) carrying out cell sedimentation to form supernatant, removing the supernatant in the reactor once or for multiple times, wherein after removing the supernatant each time, the depth of a liquid taking gun head entering the liquid level of the supernatant is not more than 3 mm;

(3) adding a fresh culture medium;

(4) calculating a discharge rate (B) based on the cell density in the supernatant, maintaining a target cell density;

(5) repeating the steps (2) to (4) until the continuous culture of the cells is completed.

Preferably, Ambr15 is used prior to seeding the cell culture into the reactor^TMClean in place and sterilize step of System on Ambr15^TMThe system is cleaned.

More preferably, after said step (1), the aeration, agitation, pH and dissolved oxygen control are turned off while keeping the temperature control on to allow the cells to settle; preferably, the cell sedimentation is completed by allowing the cell culture solution to stand for 10-25 min.

According to the method for high-throughput and automatic continuous cell culture, when the continuous cell culture is carried out in a single reactor, the specific operation of removing the supernatant in the reactor in the step (2) is as follows:

①, calculating the total liquid taking quantity which is the culture volume multiplied by the liquid changing speed according to the preset liquid changing speed, ② calculating the liquid taking times which is the total liquid taking quantity/the liquid taking quantity of the liquid taking gun head according to the total liquid taking quantity and the liquid taking quantity of the liquid taking gun head each time, ③ adjusting the fixed height of the liquid taking gun head each time by program setting according to the corresponding relation between the culture liquid volume in the reactor and the fixed height of the liquid taking gun head until the removal of the supernatant liquid is completed in a single reactor, wherein the liquid changing speed is 0.5-2.0 VVD, and preferably the liquid changing speed is 0.9 VVD.

According to the method for high-throughput and automatic continuous cell culture, when the continuous cell culture is carried out in N reactors, wherein N is more than or equal to 2, the specific operation of removing the supernatant in the reactor in the step (2) is as follows:

①, calculating the total liquid taking quantity which is the culture volume multiplied by the liquid changing speed according to the preset liquid changing speed, ②, calculating the liquid taking times which is the total liquid taking quantity/the liquid taking quantity of the liquid taking gun head according to the total liquid taking quantity and the liquid taking quantity of the liquid taking gun head each time, ③, adjusting the fixed height of the liquid taking gun head each time according to the program setting according to the corresponding relation between the culture liquid volume in each reactor and the fixed height of the liquid taking gun head when taking liquid in each reactor, ④, determining the liquid taking sequence, sequentially removing the supernatant according to the sequence from the first reactor to the Nth reactor, sequentially removing the supernatant according to the sequence from the Nth reactor to the 1 st reactor, and removing the supernatant in a plurality of reactors, wherein the liquid changing speed is 0.5-2.0D, and preferably, the liquid changing speed is 0.9 VVD.

According to the method for continuously culturing the high-flux and automatic cells, the cell culture is continuously cultured after the step (2), so that the cell culture solution is in a control state again. The specific operation of the step (3) is as follows: adding an equal amount of fresh culture medium to the reactor as the supernatant removed in step (2) to complete the liquid change; preferably, the fresh medium is selected from any one of an Advanced CHO production medium, a tannin S5 production medium, a tannin S5 production medium containing 5% Feed210, a tannin S5 production medium containing 5% FM016, a tannin S5 production medium containing 3% CB7a, a GE prototype production medium, and a GE prototype production medium containing 3% CB7 a; more preferably, the medium is a tannin S5 production medium or GE prototype production medium containing 5% feed 210.

According to the method for high-throughput and automatic continuous cell culture, when the cell density in the supernatant is less than 2% of the original cell liquid density, the bleeding rate (B) in the step (4) is calculated by the following method:

firstly, the cell density N3 after cell discharge is calculated according to the following formula

N₃＝(N₁×N)/N₂Wherein said N is₁Cell density of the previous day, N is target cell density, N₂Is the current cell density;

then N is added₃Substituting into the following formula to calculate the release rate (B)

B＝(N₂-N₃)/N₂。

When the cell density in the supernatant is 2% or more of the original cell fluid density, the bleeding rate (B) in the step (4) is calculated as follows:

will N₂、N₁N, V and B₁Substituting the numerical value of (a) into the formula:

wherein said N is₁Cell density of the previous day, N is target cell density, N₂At the current cell density, B₁The current releasing rate B to the next day can be obtained by substituting the data into the releasing rate V from the previous day to the current day and the culture volume V₂The release rate B₂I.e. the release rate (B).

In a specific embodiment of the present invention, the cell continuous culture method comprises the steps of:

(1) ambr15 using Sadoris^TMSystem for controlling a power supply

As shown in FIG. 1, Ambr15^TMThe system comprises: the reactor comprises four hardware elements, namely a reactor workstation 1, an orifice plate placing area 2, a gun head placing area 3 and a mechanical arm 4. One reactor station 1 can accommodate 12 reactors, Ambr15^TMThe system comprises 4 reactor workstations 1, and 48 reactors; the well plate placing area 2 is used for placing a sampling plate (for containing a culture solution sample taken out of the reactor) and a deep well plate (for containing process solutions such as culture medium, supplementary materials, sugar, alkali, antifoaming agents, and the like); a lance head placing area 3 for placing a lance head, Ambr15^TMThe system has 1ml and 4ml gun heads for sampling and sample adding; the mechanical arm 4 is Ambr15^TMThe automatic core part of the system, all sampling and sample adding operations are completed by a mechanical arm 4.

When the sampling operation needs to be completed, the mechanical arm 4 firstly absorbs 1ml of gun head from the gun head placing area 3, then moves to the position above the reactor to be taken, removes the reactor cap, inserts the gun head into the reactor to absorb a sample with a preset volume, then covers the reactor cap, moves the mechanical arm 4 to the sampling plate, and puts the sample into the plate hole to finish sampling. Correspondingly, when the sample adding operation needs to be completed, the mechanical arm 4 firstly sucks 1ml or 4ml of gun head from the gun head placing area 3, then moves to the position above the deep hole plate filled with the process solution, sucks a certain amount of process solution by using the gun head, then moves to the position above the reactor, and adds the process solution, so that the sample adding is completed.

(2) Stop Ambr15^TMProcess control of reactors in a system

Set up the program, close Ambr15^TMAnd (3) ventilating and stirring the bottom of the reactor in the system, controlling the pH value and dissolved oxygen, and standing the cell culture solution for 10-25 min. Temperature control is maintained during the process. After 10-25 min, a section of clarified liquid is formed on the top layer of the culture solution, and the clarified liquid contains no cells or less cells. The effect of sedimentation of cells after closing the bottom aeration of the reactor and agitation, pH and dissolved oxygen control is shown in figure 2.

(3) Removal of Ambr15^TMSupernatant in reactor in system

To achieve the most efficient and reasonable removal of the supernatant, the following steps and methods need to be followed:

a) implementation of fixed height sampling

Due to Ambr15^TMThe default sampling mode of the reactor in the system is to insert a 1ml tip into the bottom of the tank for sampling, but in the gravity settling mode, cells are aggregated at the bottom of the tank, and supernatant is generated at the upper layer, so the method cannot remove the supernatant generated after the culture solution is settled. Reconstitution of Ambr15 according to the invention^TMThe system is sampled in a way that can realize fixed-height sampling, namely Ambr15 programmed by an engineer^TMThe sampling method carries out the operation of sampling at a fixed height, and the tail end of a 1ml sampling gun head is fixed at the position of supernatant, wherein the depth is not more than 3mm, so that the sampling at the fixed height can be realized. Fig. 3 illustrates the difference between conventional sampling and fixed height sampling.

The fixed height sampling needs to represent the relation between the fixed height of the tail end of the gun head and the culture volume, and the invention accurately measures the corresponding relation between the volume and the height of a series of culture solutions. The corresponding fixed height of the tip end when the culture volume is 5-10 ml is listed in table 1:

TABLE 1 corresponding relationship between culture fluid volume and fixed height

Volume of culture solution	Fixed height of tail end of gun head
		5ml	13.1mm
6ml	15.9mm
		7ml	17.8mm
8ml	20.5mm
		9ml	23.0mm
10ml	25.3mm

When only a single reactor is required, selecting the operation of b) removing the supernatant in the reactor; when a plurality of reactors are required, the operation described in c) is selected to remove the supernatant from the reactor.

b) Implementation of gradient sampling

Assuming that the culture volume is 10ml and the liquid change rate is 0.3VVD (1 VVD represents one culture volume), that is, the liquid change amount in one day is 0.3X 10ml to 3ml, 3ml of supernatant liquid needs to be sucked up in total, and the suction is finished in 3 times because the 1ml tip sucks up 1ml at a time. The 1 st 1ml fixes the gun head at the position with the liquid loading capacity of 9ml for suction, the 2 nd 1ml fixes the gun head at the position with the liquid loading capacity of 8ml for suction, and the 3 rd 1ml fixes the gun head at the position with the liquid loading capacity of 7ml for suction. The above gradient sampling process is shown in fig. 4.

c) Implementation of gradient sequential sampling

As shown in the upper graph of FIG. 5, assuming that there are 6 reactors, the culture volume is 10ml, the liquid change rate is 0.3VVD (i.e., the liquid change amount is 3ml), and samples are taken in 3 portions after 5 to 25min of sedimentation. The setting program fixes the gun head at the position with the liquid loading amount of 9ml to suck the 1 st 1ml supernatant of the 1 st reactor, then continues to fix the gun head at the position with the liquid loading amount of 9ml to suck the 1 st 1ml supernatant of the 2 nd reactor, and so on until the 1 st 1ml supernatant of the 6 th reactor is taken out. After 1ml in the 6 th reactor is taken, the time is 6-7 minutes, and then the supernatant of 1ml in the 2 nd reactor in the 6 th reactor is clearer. This minimizes the settling time before sampling.

At this point, we changed the 2 nd 1ml sampling sequence from the 6 th reactor to the 1 st reactor, so that due to the time interval, the supernatant was also clearer when it was taken to the 1 st reactor. The difference in the cell residual rate in the supernatant of each reactor can be minimized by such a sequence. The above sampling process is shown in fig. 5.

(4) Opening Ambr15^TMProcess control of reactors in a system

The procedure was set up, and when the supernatant was completely removed, the bottom of the reactor was vented and agitation, pH and dissolved oxygen control were started.

(5) Adding fresh culture medium

Assuming a culture volume of 10ml and a single exchange rate of 0.3VVD, i.e.3 ml of supernatant had been removed, the program was set up after the process control was turned on, and 3ml of fresh medium (i.e.an amount of fresh medium equal to the amount of supernatant removed) was added to each reactor and the exchange was complete.

(6) Adjustment of liquid changing rate and liquid changing times

The liquid changing rate and the liquid changing times can be adjusted, for example, the liquid changing rate can be set to be 0.5-2.0 VVD, and the liquid changing frequency can be set to be 2-8 times per day. Preferably, the liquid change rate can be set to 0.9VVD for maintaining high density culture, and the liquid change can be performed 3 times a day, with a single liquid change rate of 0.3VVD and 8 hours intervals. Like this, flexible transformation.

In order to solve the second technical problem, refer to step (1) and step (2) in the first step of solving the technical problem.

To solve the third technical problem, refer to steps (1) to (3) in one of the steps for solving the technical problem.

To solve the fourth technical problem, the following release rate calculation method is detailed:

first, the specific growth rate (μ) calculation formula is introduced:

wherein, t₁And t₂Respectively different cell culture time points (t)₂>t₁)，N₂Is t₂Cell live cell density of (2) time, N₁Is t₁Viable cell density.

Under the condition of sufficient nutrition, the cells can continuously grow and reproduce, and the cell density can continuously rise. However, in continuous culture it is desirable to maintain a constant cell density in order to achieve steady state culture, and if this constant cell density is to be maintained, a certain amount of cell liquid must be discharged over a period of time, which is referred to as the discharge rate (B).

The bleed rate required to maintain a constant cell density was calculated in two models as follows:

a) taking a rocking tube as an example, suppose that yesterday's viable cell density is N₁Today's viable cell density is N₂The cell density to be maintained is N, and the cell density after the cell discharge is N today₃Yesterday to today specific growth rate is μ₁The specific growth rate from today to tomorrow is μ₂. The cell culture time interval from yesterday to today and from today to tomorrow are both Δ t. When the continuous culture reached a steady state, μ was considered₁＝μ₂。

Therefore, there are:

since Δ t is 1, N₁，N₂N is known and substituting the correlation value yields N₃The value of (c).

Then, assuming that the culture volume of the shake tube is V and the volume required to be released is V₁The release rate is B, and the release rate is calculated according to the formula (1)

Corresponding release rate

b) Taking the reactor as an example, assuming that the culture volume of the reactor is V, the viable cell density in the reactor is x, the culture time is t, the discharge rate is B, and the reactorMedium cell growth rate of mu_realApparent specific growth rate of mu_app。

The cell number balance equation is listed as follows according to "the amount of change in cell number-the number of growing cells-the number of cells flowing out" in the reactor:

V×dx＝V×μ_real×x×dt-x×B×dt

both sides were simultaneously divided by V × x × dt to give:

according to the calculation formula of the apparent ratio growth rate

Further obtaining:

suppose that yesterday's reactor has a viable cell density of N₁The viable cell density in today's reactors is N₂The cell density in the reactor to be maintained is N, and the release rate from yesterday to today is B₁The calculated release rate from today to tomorrow is B₂When the continuous culture reached a steady state, μ was considered to be two days before and after_realAnd (3) substituting the equation (2) to obtain:

so that the discharge rate B required to maintain cell density homeostasis can be calculated₂。

Whether method a) or method b) is carried out in the presence of Ambr15^TMAnd (4) judging by taking the density value of the living cells in the supernatant generated by sedimentation of the system reactor as a standard. When the cell density in the supernatant is at the original cell density

Below 2%, the tube shaking model, i.e. the method described in method a), can be selected to calculate the bleed rate B required to maintain a constant cell density; when the cell density in the supernatant is more than 2% of the original cell density, the cell density can be

This is considered to be cell bleed, so the reactor model, i.e.the method described in method B), is selected to calculate the bleed rate B required to maintain a constant cell density₂。

ADVANTAGEOUS EFFECTS OF INVENTION

Compared with the prior art, the invention has the beneficial results that:

1. compared with the traditional perfusion reactor, the method has the advantages of large flux, high automation degree, small culture volume and low cost, and is more suitable for large-scale culture condition screening and process optimization; compared with the shaking tube continuous culture, the method has the advantages of strong reactor characteristics, high culture result representativeness, difficult occurrence of human errors and high reliability.

2. The invention adopts the gravity settling mode to intercept the cells, thereby avoiding an additional cell intercepting device of the perfusion reactor and avoiding the problem that the cell intercepting device is easy to block.

3. The invention provides a sequential gradient supernatant removal mode, which solves the problem that part of cell strains cannot be deposited in Ambr15 due to poor sedimentation effect^TMThe gravity settling and liquid changing are carried out in the system.

4. The invention provides a method for calculating the cell releasing rate, and the solution is Ambr15^TMThe maintenance of constant cell density during continuous culture of the system.

The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The embodiments are presented as examples of the invention and the invention is not limited to the embodiments described below. Any equivalent modifications and substitutions to the embodiments described below are within the scope of the present invention for those skilled in the art. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Drawings

FIG. 1 shows Ambr15^TMThe system is schematic.

FIG. 2 shows Ambr15 being closed^TMAnd (4) a cell sedimentation effect diagram after process control of a reactor in the system.

FIG. 3 shows Ambr15^TMThe difference between the conventional sampling (left) and the fixed height sampling (right) of the system is shown.

FIG. 4 shows Ambr15^TMThe effect graph of gradient sampling of a single reactor of the system.

FIG. 5 shows Ambr15^TMThe effect graph is sampled by a plurality of reactors in a gradient sequence.

FIG. 6 is a graph showing the change in the number of viable cells of a CHO-K1 cell cultured for various periods of time.

FIG. 7 is a graph showing the change in antibody titer of certain CHO-K1 cells cultured for various periods of time.

FIGS. 8 to 11 are graphs showing the change of viable cell density, cell viability, lactate concentration and product protein concentration of clone A after culturing for different periods of time, respectively.

FIGS. 12 to 15 are graphs showing the change of viable cell density, cell viability, lactate concentration and product protein concentration of clone B cultured for different periods of time, respectively.

Description of reference numerals: 1 a reactor workstation; 2, a hole plate placing area; 3, a gun head placing area; 4 mechanical arm.

Detailed Description

The invention utilizes Ambr15^TMThe automatic continuous culture model established by the system is mainly used for overcoming the defects of a perfusion reactor model and a shaking tube model, has partial advantages of a reactor and a shaking tube, and has the characteristics of strong reactor characteristics, high flux, high automation degree and the like.

The technical solution of the present invention is further explained by the following embodiments. It should be emphasized that the invention is not limited to the specific embodiments illustrated. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms (such as "includes" and "including") is not limiting. Moreover, the ranges provided in the specification and the appended claims include all values between the endpoints and breakpoints.

Example 1

The implementation process is detailed by taking the example of selecting recombinant CHO-K1 cell to produce a monoclonal antibody with the IgG1 type (the CHO-K1 is a cell strain proprietary to Shanghai Yao Ming biotechnology company).

(1) A CHO-K1 cell line required in this example was recovered: the culture was carried out in CD CHO medium (purchased from ThermoFisher) in 125 ml-1L shake flasks (purchased from Corning). The cell strain is inoculated into a culture container containing CDCHO culture medium and is cultured in a Kuhner or INFORS Multitron constant temperature shaker. The parameters of shaking culture are set at 36.5 deg.C, humidity 80%, and CO₂Concentration 60%, shaker rotation speed 125 rpm.

After recovery, every 2-4 days, the cell density is 1.0-3.0 multiplied by 10⁶The cells/ml range is passed to the next generation. When the generation is 4-6, the culture medium can be expanded for two generations by using Advanced CHO production medium (purchased from Thermo Fisher), and when the cell density reaches 2.0-4.0 multiplied by 10⁶cells/ml, inoculated to Ambr15^TMIn the reactor of the system (purchased from Sartorius), the target inoculation density was 0.4X 10⁶cells/ml。

(2) Using Ambr15^TMThe in situ cleaning and sterilization step of the System was carried out with Ambr15^TMCleaning of the system safety cabinet and the machine itself, then according to Ambr15^TMThe system user manual performs the reactor installation.

This example is in Ambr15 ^TM4 reactors, designated Ambr15-1, Ambr15-2, Ambr15-3 and Ambr1, are installed in the reactor station (1) of the system5-4, then using Ambr15^TMThe program setup of the system and the robotic arm (4) add 8-9 ml of Advanced CHO production medium to each reactor. Open Ambr15^TMThe temperature of the system is controlled to be 36.5 ℃, the stirring speed is set to be 700-900 rpm, the aeration quantity at the fixed bottom is 0.1ml/min, and the aeration is carried out overnight.

The next day samples were taken for blood gas analyzer (BGA, from Siemens) testing, pH, pCO were checked₂And pO₂Level when pO₂And correcting the dissolved oxygen display value to be 100% at 130-150 mmHg, and then correcting and displaying the pH value to be the BGA pH value. After completion of the operation, the inoculation operation was carried out so that the volume of the medium in the reactor after inoculation was 10 ml.

(3) Inoculating and culturing to the 3 rd day, and starting continuous liquid changing operation, wherein the liquid changing speed is set to be 0.855VVD, and the liquid changing speed is changed by 0.285VVD every 8 hours for three times. The appropriate bleed rate was calculated daily to maintain a target culture density of 30X 10⁶cells/ml, and the specific liquid changing and discharging operations refer to the technical scheme. The specific calculation process of the bleeding rate is described below by taking Ambr15-1 as an example:

the culture volume was 10ml, the rate of liquid change was 0.855VVD, and the liquid change was performed 3 times a day. The VCD (viable cell density) at day 6 of Ambr15-1 was 17.47X 10⁶cells/mL, cell density of supernatant after cell sedimentation (10 min sedimentation) 1.59X 10⁶cells/mL, cell loss was (1.59X 10)⁶cells/mL)×(0.855/3×10ml)/10ml/(17.47×10⁶cells/mL)×100％＝2.6％>2% the release rate on the next day will be calculated with reference to the cell loss rate on the previous day, so a reactor model (2.6%)>2%) was performed for the discharge rate calculation. VCD on day 7 was 25.93X 10⁶cells/mL, it is expected that the VCD at day 8 could be maintained at 30.00X 10⁶cells/mL。

Release rate at day 7, according to equation (3), B₂/V＝ln(N₂/N₁)+B₁/V–ln(N/N₂) In which N is₂＝25.93×10⁶cells/mL，N₁＝17.47×10⁶cells/mL，N＝30.00×10⁶cells/mL，B₁B is obtained by substituting 0 (no release operation on day 6) into the formula₂When the/V is 0.25VVD, the release volume is 0.25 × 10ml to 2.5 ml.

The supernatant density after sedimentation (sedimentation for 20 minutes) on day 7 was 0.79X 10⁶cells/mL, cell loss 0.79X 10⁶cells/mL×(0.855/3×10ml)/10ml/(25.93×10⁶cells/mL)×100％＝0.87％<2% the release rate on day two will be calculated with reference to the cell loss rate on day one before, so a shake tube model (0.87%)<2%) was performed for the discharge rate calculation. VCD at day 8 was 31.12X 10⁶cells/mL, it is expected that the VCD at day 8 could be maintained at 30.00X 10⁶cells/mL。

According to the formula ln (N)₂/N₁)＝ln(N/N₃) To obtain N₃＝N×N₁/N₂Substituting formula B ═ N₂-N₃)/N₂The release rate B can be obtained. Wherein N is₁＝25.93×10⁶cells/mL,N₂＝31.12×10⁶cells/mL，N＝30.00×10⁶cells/mL, calculating to obtain N₃＝25.00×10⁶cells/mL, B0.20 VVD, bleed volume 0.20 × 10mL 2.0 mL.

(4) This culture was continued for 16 days, and live Cell Density (VCD) and antibody Titer (Titer) were measured during the culture using a Cell counter (Vi-CELL XR, available from BECKMAN), and Titer and biochemical measurements using an automated multifunctional biochemical analyzer (Cedex, available from Roche). The graphs of the change in VCD and titer are shown in FIGS. 6 and 7, respectively, and it can be seen that the number of living cells in the 4 reactors was maintained relatively stably at 30X 10 from day 7⁶cells/ml; the protein concentration was maintained at about 0.8-1.0g/L from day 8, and the culture was successful.

Example 2

The detailed implementation process is exemplified by the selection of perfusion medium containing two CHO-K1 clones encoding different foreign protein coding genes

(1) Two different CHO-K1 cell lines, clone A and clone B, required in this example were recovered, wherein clone A produced a protein of the Fc fusion protein type and clone B produced a protein of the IgG4 monoclonal antibody type: the clone A and gInoculating LongB into culture containers containing CD CHO culture medium (purchased from Thermo Fisher) respectively, wherein the culture containers are 125 ml-1L (purchased from Corning) shake flasks, and culturing in a constant temperature shaking table of Kuhner or INFORS Multitron, and the setting parameters of shaking table culture are 36.5 deg.C, humidity 80%, and CO₂Concentration 60%, shaker rotation speed 125 rpm.

After recovery, every 2-4 days, the cell density is 1.0-3.0 multiplied by 10⁶The cells/ml range is passed to the next generation. As generations 4-6, Duoning S5, Duoning S5 (containing 5% Feed210), Duoning S5 (containing 5% FM016), Duoning S5 (containing 3% CB7a), GE prototype and GE prototype (containing 3% CB7a) were used (Duoning S5 is a trial culture medium from Shanghai Duoning Biotech Co., Ltd., GE prototype is a trial culture medium from GE Co., Ltd.), Feed210 is from Merck, FM016 and CB7a are from HyClone^TM) Performing two generations of expansion until the cell density reaches 2.0-4.0 × 10⁶cells/ml, inoculated to Ambr15^TMIn the reactor of the system, the target inoculation density is 0.4X 10⁶cells/ml。

(2) The procedure was the same as in (2) in example 1, except that the culture temperature was set to 34.5 ℃ and the culture volume after inoculation was adjusted to 8 ml.

(3) Inoculating and culturing to the 3 rd day, starting continuous liquid changing operation, wherein the liquid changing rate is set to be 0.72VVD, liquid is changed three times a day, the interval is 8 hours, each time is 0.24VVD, and the proper discharging rate is calculated every day to maintain the target culture density, and the specific liquid changing and discharging operation refers to the technical scheme and the example 1.

(4) The main process conditions of this example are described in table 2.

Table 2 main process conditions in example 2

(5) The culture lasts for 17 days, and the change curve graphs of the number of living cells, the cell viability, the lactic acid content and the target protein content of the clone A after being cultured for different time are respectively shown in FIGS. 8 to 11; the graphs of the number of viable cells, the cell viability, the lactic acid content and the target protein content of clone B after being cultured for different periods are shown in FIGS. 12 to 15, respectively. As shown in FIGS. 8 to 15)

As can be seen from fig. 8 and 12: both clone A and clone B reached a steady state of viable cell number after 9 days of culture, and most reactors were able to reach the target culture density, i.e., viable cell number of 30X 10⁶cells/ml, the cell culture density can only be maintained at 15-25 × 10 under individual culture conditions⁶cells/ml.

As can be seen from fig. 9 and 13: the cell viability of the two clones is more than 95% when the two clones are harvested; from the viewpoint of the target protein concentration and lactic acid expression level, the experimental group using GE prototype and tannin S5 (5% Feed210) in clone A and clone B was not only high in protein concentration but also low in lactic acid expression level, and thus GE prototype and tannin S5 (5% Feed210) were suitable for the continuous culture mode of clone A and clone B and could be used as the final production medium.

Claims (10)

1. A method for high throughput, automated continuous cell culture, the method comprising the steps of:

(1) providing a cell culture, inoculating it to sarbs 15^TMCulturing in a reactor of the system;

(2) carrying out cell sedimentation to form supernatant, removing the supernatant in the reactor once or for multiple times, wherein after removing the supernatant each time, the depth of a liquid taking gun head entering the liquid level of the supernatant is not more than 3 mm;

(3) adding a fresh culture medium;

(4) calculating a discharge rate (B) based on the cell density in the supernatant, maintaining a target cell density;

(5) repeating the steps (2) to (4) until the continuous culture of the cells is completed.

2. The method of claim 1, wherein: use of Ambr15 prior to seeding the cell culture into the reactor^TMClean in place and sterilize step of System on Ambr15^TMThe system is cleaned.

3. The method of claim 1, wherein: after the step (1), under the condition of keeping the temperature control on, closing aeration, stirring, pH and dissolved oxygen control to enable cells to settle; preferably, the cell sedimentation is completed by allowing the cell culture solution to stand for 10-25 min.

4. The method according to claim 1, wherein the continuous cell culture is performed in a single reactor, and the operations of removing the supernatant in the reactor in the step (2) are as follows:

①, calculating the total liquid taking quantity which is the culture volume and the liquid changing speed according to the preset liquid changing speed, ② calculating the liquid taking times which is the total liquid taking quantity/the liquid taking quantity of the liquid taking gun head according to the total liquid taking quantity and the liquid taking quantity of the liquid taking gun head each time, ③ adjusting the fixed height of the liquid taking gun head each time by program setting according to the corresponding relation between the culture liquid volume in the reactor and the fixed height of the liquid taking gun head until the supernatant is removed in a single reactor.

5. The method according to claim 1, wherein the continuous cell culture is performed in N reactors, wherein N ≧ 2, and the operations of removing the supernatant in the reactor in step (2) are:

①, calculating the total liquid taking amount which is the culture volume and the liquid changing rate according to the preset liquid changing rate, ② calculating the liquid taking times which is the total liquid taking amount/the liquid taking amount of the liquid taking gun head each time according to the total liquid taking amount and the liquid taking amount of the liquid taking gun head each time, ③ adjusting the fixed height of the liquid taking gun head each time through program setting according to the corresponding relation between the culture liquid volume and the fixed height of the liquid taking gun head in each reactor when liquid is taken in each reactor, ④ determining the liquid taking sequence, namely, sequentially finishing the first removal of supernatant liquid according to the sequence from the first reactor to the Nth reactor, then, sequentially finishing the second removal of the supernatant liquid according to the sequence from the Nth reactor to the 1 st reactor until the removal of the supernatant liquid is finished in a plurality of reactors.

6. The method of claim 4 or 5, wherein the liquid change rate is 0.5-2.0 VVD; preferably, the liquid change rate is 0.9 VVD.

7. The method of claim 1, wherein the culturing of the cell culture is continued after step (2) to bring the cell culture fluid back into control.

8. The method according to claim 1, characterized in that the specific operation of step (3) is: adding an equal amount of fresh culture medium to the reactor as the supernatant removed in step (2) to complete the liquid change; preferably, the fresh medium is selected from any one of an Advanced CHO production medium, a tannin S5 production medium, a tannin S5 production medium containing 5% Feed210, a tannin S5 production medium containing 5% FM016, a tannin S5 production medium containing 3% CB7a, a GE prototype production medium, and a GE prototype production medium containing 3% CB7 a; more preferably, the medium is a tannin S5 production medium or GE prototype production medium containing 5% feed 210.

9. The method according to claim 1, wherein the bleeding rate (B) in the step (4) is calculated by the following method when the cell density in the supernatant is 2% or less of the original cell fluid density:

firstly, the cell density N after cell discharge is calculated according to the following formula₃

N₃＝(N₁×N)/N₂Wherein said N is₁Cell density of the previous day, N is target cell density, N₂Is the current cell density;

then N is added₃Substituting into the following formula to calculate the release rate (B)

B＝(N₂-N₃)/N₂。

10. The method according to claim 1, wherein the bleeding rate (B) in the step (4) is calculated as follows when the cell density in the supernatant is 2% or more of the original cell fluid density:

will N₂、N₁N, V and B₁Substituting the numerical value of (a) into the formula:

wherein said N is₁Cell density of the previous day, N is target cell density, N₂At the current cell density, B₁The current releasing rate B to the next day can be obtained by substituting the data into the releasing rate V from the previous day to the current day and the culture volume V₂The release rate B₂I.e. the release rate (B).

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