CN111560399A - Large-scale transient transfection method for cells - Google Patents

Large-scale transient transfection method for cells Download PDF

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CN111560399A
CN111560399A CN202010441846.4A CN202010441846A CN111560399A CN 111560399 A CN111560399 A CN 111560399A CN 202010441846 A CN202010441846 A CN 202010441846A CN 111560399 A CN111560399 A CN 111560399A
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cells
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CN111560399B (en
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何志娟
潘志卫
杨灵芝
周进伟
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Shanghai Junshi Bioengineering Co ltd
Suzhou Junmeng Biosciences Co Ltd
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Abstract

The present invention relates to a method for recombinant protein production by transient transfection of mammalian cells, comprising: shake flask culture of mammalian cell suspension; the bioreactor expands the mammalian cell suspension; the reaction tank expands the mammalian cell suspension in the bioreactor; adding plasmids and transfection reagents into the reaction tank mammal cell suspension for transient transfection; and culturing the transfected mammalian cell. The invention has the most outstanding advantages that the process can be amplified, the instantaneous conversion scale reaches 200L of single reaction, and in addition, the invention also has the advantages of short production period, high protein expression quantity, high yield, low production cost and the like.

Description

Large-scale transient transfection method for cells
Technical Field
The present invention relates to a method of transient transfection of cells on a large scale for the large scale production of recombinant proteins.
Background
Recombinant proteins, particularly therapeutic proteins represented by monoclonal antibody proteins, are produced mainly by expression in mammalian cells. The most widely used is the CHO cell (Chinese hamster ovary cell), which is widely used in research, development and industrial production of biotechnology products such as antibodies, gene recombinant protein drugs, and virus vaccines.
There are two main ways of performing transfection in mammalian cells: transient transfection and stable transfection.
Stable transfection is used to establish clonal cell lines in which the transfected gene of interest is integrated into chromosomal DNA and directs the synthesis of an appropriate amount of the protein of interest. Stable transfection allows efficient and stable expression for a long period of time, and yields are high. However, stable expression of protein requires construction of a stably expressed engineering cell strain, which takes 6-12 months and consumes a huge amount of cost; toxic proteins cannot be expressed and produced by stable cell lines; in addition, stable expression of protein genes in cell lines is a great challenge, and unstable protein expression is often caused due to gene silencing or copy number loss.
The transiently transfected plasmid DNA does not need to be stably integrated into the genome of the host cell after entering the cell, and expresses the foreign gene in an episomal state. The period of transient transfection is short, the target protein can be quickly obtained, and the method can be applied to exploration in the initial stage of research and development and large-scale high-throughput protein screening. However, the transfection efficiency of the conventional transient transfection method for cells is low, and the expression yield of the target gene in the transfected cells is low, which cannot meet the practical requirement.
Disclosure of Invention
The present invention relates to a large-scale production method of recombinant protein by using mammalian cell (in particular CHO cell), in particular, it relates to a large-scale transient transfection production method of recombinant protein by using mammalian cell (in particular CHO cell). The process of the invention can be amplified, the instantaneous conversion scale reaches 200L of single reaction, and in addition, the invention also has the advantages of short production period, high protein expression quantity, high yield, low production cost and the like. The invention provides a method for producing recombinant protein by transient transfection of mammalian cells, which comprises the following steps:
shake flask culture of mammalian cell suspension;
the bioreactor expands the mammalian cell suspension;
the reaction tank expands the mammalian cell suspension in the bioreactor;
adding plasmids and transfection reagents into the reaction tank mammal cell suspension for transient transfection;
culturing the transfected mammalian cell.
In some embodiments, the bioreactor is a WAVE bioreactor.
In some embodiments, the mammalian cell is a CHO cell.
In some embodiments, the CHO cell described above is a CHO 18 cell.
In some embodiments, the culture medium used for the CHO cell culture described above is CD CHO or Dynamis medium.
In some embodiments, the culture medium used for the CHO cell culture described above is CD CHO medium.
In some embodiments, the cryopreserved cells are thawed prior to shake flask culture.
In some embodiments, the shake flask culture is at about 3% to about 10% CO2Concentration, about 35-38 ℃ and about 100 and 140rpm for culture; preferably, it is between about 6% and 8% CO2Concentration, about 36-37 ℃ and about 110-.
In some embodiments, the shake flask culture described above comprises culturing using a shake flask having a volume of 25mL to 1L, preferably 50mL to 500mL, more preferably 125mL or 250 mL.
In some embodiments, the above shake flask culture further comprises culturing using a shake flask having a volume of 1-10L, preferably about 2-8L, preferably about 4-6L, preferably about 1L, 3L, or 5L.
In some embodiments, the shake flask culture is performed 2 or more times, and the cell density is about (2.0-6.0) × 106When the cell/mL and the cell viability are more than or equal to 90.0 percent, the shake flask is replaced to carry out shake flask amplification.
In some embodiments, the shake flask culture is expanded to 5L-10L shake flasks, preferably 5L shake flasks, by 2 or more than 2 expansion steps.
In some embodiments, the cell seeding density per shake flask culture described above is 0.2 × 106-0.6×106Individual cells/mL.
In some embodiments, the shake flask culture is performed at 35-38 deg.C, 100-2Under the condition of concentration, preferably at 36-37 deg.C, 110-130rpm and 6-8% CO2Under the condition of concentration.
In some embodiments, a cell density of about 2.0 × 10 is used6～6.0×106Carrying out bioreactor amplification on the shake flask culture with each cell/mL and the cell viability rate of more than or equal to 90.0%.
In some embodiments, the bioreactor is expanded with a seeding density of 0.2 × 106-0.6×106Individual cells/mL. In some embodiments, the parameters of the bioreactor are: temperature of about 35-37 deg.C, CO2Concentration of about 0-5%, aeration flow rate of 0.2-0.6lpm, angle of about 6-8 °; preferably, the temperature is about 35.5-36.5 ℃.
In some embodiments, the bioreactor has a volume of about 10-100L, preferably about 20-80L, preferably about 40-60L, preferably about 50L.
In some embodiments, a cell density of about 2.0 × 10 is used6～6.0×106The culture with cell per mL and cell activity rate more than or equal to 90.0 percent is subjected to the amplification in the reaction tank.
In some embodiments, the parameters of the reaction tank are: the temperature is about 35.5-36.5 ℃, the pH is about 6.7-7.3, the rotating speed is about 50-100 rpm, and the Dissolved Oxygen (DO) is about 30-50%; preferably, the temperature is about 36.0 ℃, the pH is about 7.0, the rotating speed is about 60rpm, and the dissolved oxygen is about 40%; more preferably, the depth air is about 1.0lpm and the surface air is about 1.0 lpm.
In some embodiments, the bioreactor-derived cells are identified as (0.7-1.1) × 106The reaction tank was seeded with a seeding density of individual cells/mL.
In some embodiments, the reaction tank has a volume of about 100L-500L, preferably about 200L-400L, and preferably about 200L.
In some embodiments, the cell density in the reaction tank is about 3.0 × 106～5.0×106At individual cells/mL, transient transfection was performed.
In some embodiments, the plasmid concentration is not less than 0.5 μ g/. mu.L.
In some embodiments, the transfection reagent is Polyethyleneimine (PEI) at a concentration of about 1-3 mg/mL.
In some embodiments, before the transient transfection, the plasmid is added to the culture medium of the cell and mixed uniformly to obtain a plasmid mixture, then the transfection reagent is added to the plasmid mixture for about 0.5-2.0 minutes, and the obtained transfection mixture is injected into a reaction tank after the mixing is continued for about 1.0-2.0 minutes; preferably, the transfection mixture is added for a period of time not exceeding 15 minutes.
In some embodiments, feeding is performed on days 2, 4, 6, and 8 post-transfection.
In some embodiments, after transfection, if the sugar content of the culture medium in the reaction tank is less than 2g/L, glucose is fed to the culture medium at a concentration of about 2-3g/L so that the sugar content in the culture medium is 2g/L or more.
In some embodiments, the transient expression process is terminated 6-10 days after transfection.
In some embodiments, the recombinant protein is a monoclonal antibody, preferably a fully human anti-2019-nCOV monoclonal antibody.
Drawings
FIG. 1: 200L transient production process flow chart.
FIG. 2: schematic diagram of transient process.
FIG. 3: the 200L transient produced expression profiles.
FIG. 4: A200L transient production Viable Cell Density (VCD) plot.
FIG. 5: A200L transient production cell Viability (VIA) map was generated.
FIG. 6: the 200L transient produced residual sugar (Gluc) maps.
FIG. 7: A200L transient generated cell Diameter (DIA) map.
FIG. 8: 200L transient production lactic acid (Lac) map.
FIG. 9: dynamics and CD CHO culture medium contrast expression (Titer) chart.
FIG. 10: dynamics and CD CHO media versus specific Viable Cell Density (VCD) plots.
FIG. 11: graphs comparing activity rates (VIA) for dynamics and CD CHO media.
FIG. 12: transient transfection plasmid backbone map; the upper panel is a plasmid for the heavy chain and the lower panel is a plasmid for the light chain.
200L 20200224 JM, 200L 20200306-1 JM, and 20200306-2 JM in FIGS. 3 to 8 represent production lot numbers at different times, respectively.
Detailed Description
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.
In order that the present invention may be more readily understood, certain technical and scientific terms are specifically defined as follows. Unless otherwise defined herein, all 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. As used herein, including the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
The term "about," when used in conjunction with a numerical value, is intended to encompass a numerical value within a range having a lower limit that is 5% less than the stated numerical value and an upper limit that is 5% greater than the stated numerical value.
The term "transfection" is used herein to refer to the delivery of a nucleic acid, protein or other macromolecule to a target cell such that the nucleic acid, protein or other macromolecule is expressed or biologically functional in the cell.
The term "transient transfection" or "transient expression" is one of the ways of introducing DNA into eukaryotic cells. In transient transfection, recombinant DNA is introduced into a highly infectious cell line to obtain transient but high level expression of the gene of interest. Transfected DNA need not be integrated into the host chromosome, transfected cells can be harvested in a shorter time than stable transfection, and the lysates tested for expression of the gene of interest.
The term "stable transfection" is used to establish a cloned cell line in which the transfected gene of interest is integrated into chromosomal DNA and directs the synthesis of an appropriate amount of the protein of interest.
The term "cell" is meant to include all types of eukaryotic and prokaryotic cells. In a preferred embodiment, the term refers to a eukaryotic cell, in particular a mammalian cell. Preferred cells are cells that can be grown in suspension, especially mammalian cells that can be grown in suspension. In certain exemplary but non-limiting embodiments, the term "cell" means a CHO cell or variant thereof, such as a suspension-grown CHO 18 cell.
The term "cell culture" or "culturing" means maintaining cells in an artificial in vitro environment.
The term "feeding, feeding or supplementing medium" means adding a volume of fresh cell culture medium to the medium already present in the culture and/or supplementing the medium already present in the culture with new medium.
The term "recombinant protein" refers to a protein encoded by a nucleic acid introduced into a host cell. The host cell expresses the nucleic acid. The term "expressing a nucleic acid" is synonymous with "expressing a protein from an RNA encoded by a nucleic acid". "protein" as used herein broadly refers to polymeric amino acids, such as peptides, polypeptides, proteins, lipoproteins, glycoproteins, and the like.
The term "protein yield" refers to the amount of protein expressed by the cultured cells and can be measured, for example, in terms of grams of protein produced per liter of culture medium. If the protein is not secreted by the cell, the protein can be isolated from the interior of the cell by methods known to those skilled in the art. If the protein is secreted by the cell, the protein can be isolated from the culture medium by methods known to those skilled in the art. The amount of protein expressed by the cell can be readily determined by one of skill in the art. The protein may be a recombinant protein.
The term "cell suspension" means a cell culture in which most or all of the cells in the culture vessel are present in suspension, and few or no cells in the culture vessel are attached to the surface of the vessel or another surface within the vessel. Preferably, "suspension culture has 75% or more of the cells in the culture vessel in suspension form and does not adhere to surfaces on or in the culture vessel. More preferably, the "cell suspension" has 85% or more of the cells in suspension in the culture vessel and does not adhere to surfaces on or in the culture vessel. Even more preferably, the "cell suspension" has 95% or more of the cells in suspension in the culture vessel and does not adhere to surfaces on or in the culture vessel.
The terms "cell culture medium" and "culture medium" refer to a nutritive solution used to cultivate cells or tissues. These phrases may be used interchangeably.
The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which other DNA segments can be ligated. One type of vector is a phage vector. Another type of vector is a viral vector, in which additional DNA segments can be joined into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors for use in recombinant DNA techniques are typically in the form of plasmids. In the present specification, "plasmid" and "expression vector" may be used interchangeably, since plasmids are the most commonly used form of vector. Non-limiting examples of the types of vector modifications that may be suitable for the practice of the present invention include, but are not limited to, modifications such as the addition of one or more enhancers, one or more promoters, one or more ribosome binding sites, one or more origins of replication, and the like. In certain preferred but non-limiting embodiments, and in the practice of the present invention, the expression vector used may include one or more enhancer elements selected to improve the expression of the protein of interest in the transient expression system of the invention. The enhancer element selected may be located 5 'or 3' to the expressible nucleic acid sequence for expression of the protein of interest.
In the present invention, the protein of interest or the protein produced by transient transfection of the CHO cell of the present invention may be a protein having a biological function of interest widely used in the fields of agriculture and industry, medical health, and the like, and includes, but is not limited to, various enzymes, antibodies, growth hormones, and the like. The enzyme of interest can be a variety of enzymes known in the art, including but not limited to synthetases, lyases, isomerases, phospholipases, translocases, oxidoreductases, and the like. The antibody of interest can be a variety of antibodies known in the art, including but not limited to therapeutic antibodies and antibodies for detection. The antibody may be a monoclonal antibody. In some embodiments, the antibody is a single chain antibody.
A variety of techniques and reagents are available for introducing macromolecules into target cells in a process known as "transfection". Commonly used agents include, for example, calcium phosphate, DEAE dextran, and liposomes. Examples of detailed Protocols for the use of these types of reagents are described in Current Protocols in molecular Biology, Chapter 9, Osebe et al, John Wiley father, John and Sons, 1998 a. Other methods of transfecting cells are known in the art and may include electroporation (gene electrotransfer), sonoporation, optical transfection, protoplast fusion, ballistic infection (immunoperfection), magnetic transfection, or viral transduction.
"agent for introducing a macromolecule into a cell" or "transfection agent" refers to any substance, formulation, or composition known to facilitate entry of a macromolecule into a cell. In some embodiments, the agent may be a "transfection agent" and may be any compound and/or composition that increases the uptake of one or more nucleic acids into one or more target cells. Suitable transfection reagents may include, but are not limited to, one or more compounds and/or compositions comprising cationic polymers (e.g., Polyethyleneimine (PEI)), polymers of positively charged amino acids (e.g., polylysine and polyarginine), positively charged dendrimers and fragmented dendrimers, polymers containing cationic white cyclodextrins (CD polymers), DEAE-dextran, and the like.
The invention provides a large-scale transient cell transfection method, which sequentially comprises the steps of shake flask culture, bioreactor amplification, reaction tank amplification and transient transfection of cells in a reaction tank. Optionally, a cell recovery step is further included before the shake flask culture.
In the present invention, the cells suitable for use in the present invention may be any of the various cells known in the art for transient transfection, in particular eukaryotic and prokaryotic cells, preferably mammalian cells. Preferably, the cells for use in the present invention are suspension-growable cells, such as suspension-growable mammalian cells. In a particularly preferred embodiment, CHO cells are cultured and transiently transfected using the methods of the present invention. The CHO cell may be a variety of CHO cells commonly used in the art, including but not limited to CHO 18 cells. In the invention, "amplification" refers to culturing cells to make the cells proliferate and increase in number; "Shake flask expansion" refers to culturing lower density cells in a shake flask to obtain higher density cells to achieve an increase in cell number.
In the present invention, the medium used for the cell culture and expansion at each stage is a medium known in the art to be suitable for the maintenance, culture and expansion of the cultured cells. For example, when the cells are CHO cells, suitable media include, but are not limited to, dynamics media and CD CHO media, preferably CD CHO media. It is to be understood that the cell culture media at each stage may be the same or different, and may be determined according to the actual culture conditions.
Before the shake flask culture, the frozen cells can be revived. Can be recovered by conventional method. For example, cryopreserved cells are thawed at about 37 ℃ for no more than 2 minutes, then centrifuged to obtain a pellet, and the cells are resuspended in a pre-warmed medium to resuscitate the cells.
Subculture is required before bioreactor expansion. The subculture is usually carried out in shake flasks, this stage also being referred to as shake flask culture. The number of shake flask culture times can be determined according to actual conditions. When the number of cells is expanded by shake flask culture to a number sufficient for bioreactor expansion, the shake flask culture can be stopped and bioreactor expansion performed using a suitable shake flask culture.
Generally, the revived cells are inoculated into a shake flask with a volume of 25mL-5L, preferably 50mL-500mL, more preferably 125mL or 250mL for the first shake flask culture/expansion when the VCD of the cells reaches (2.0-6.0) × 106The volume of the flask for subsequent shake flask expansion may be 1L to 10L, preferably 2-8L, preferably 4-6L, preferably about 5L the inoculation density may be determined by one skilled in the art from different cells, different flask volumes, according to conventional cultivation methods.an exemplary inoculation density may be about 0.2 × 106～0.6×106Individual cells/mL medium. The culture/amplification conditions for each flask can be set at 35-38 deg.C, 100-2Concentration; preferably 36-37 ℃, 110-130rpm and 6-8% CO2And (4) concentration.
For example, cells obtained from a 1L shake flask culture can be transferred to several 2L shake flasks for amplification or directly to a 3L shake flask for amplification, in a preferred embodiment, the cells are amplified several times to a 5L to 10L shake flask, and in a more preferred embodiment, the shake flask culture comprises 125mL shake flask culture, 1L shake flask culture, 3L shake flask culture, and 5L shake flask culture6When the cell/mL and the cell viability are more than or equal to 90.0 percent, the shake flask can be replaced for the next shake flask amplification.
When the shake flask is expanded to obtain a certain amount of subculture (for example, 0.2 × 10 can be achieved in a bioreactor)6～0.6×106Seeding Density of Individual cells/mL Medium), VCD of cells (2.0-6.0) × 106Cell/mBioreactor expansion is performed on shake flask cultures with a cell viability ≧ 90.0% in the present invention, the bioreactor is the device that enables the bioreaction to be achieved, including various cell culture devices for cell culture and fermentation, the volume of the bioreactor can typically be about 10-100L, preferably about 20-80L, preferably about 40-60L, preferably about 50L6～0.6×106Individual cells/mL medium.
The reaction conditions in the bioreactor may include: temperature of about 35-37 deg.C, CO2Concentration of about 0-5%, aeration flow rate of 0.2-0.6lpm, angle of about 6-8 °; preferably, the temperature is about 35.5-36.5 ℃. The rocking speed of the bioreactor may be 15-25rpm, preferably 18-22 rpm.
When the cell density in the bioreactor is about 2.0 × 106～6.0×106Individual cells/mL, preferably 4.0 × 106～6.0×106The exemplary reactor includes a reactor from Sartorius Stedim (Biostat STR), the reactor may have a volume of about 100L to 500L, preferably about 200L to 400L, and preferably about 200L, and the seeding density for the reactor expansion may be 0.7 × 106～1.1×106Individual cells/mL medium. The parameters of the reaction tank can be set as: the temperature is about 35.5-36.5 ℃, the pH is about 6.7-7.3, the rotating speed is about 50-100 rpm, and the Dissolved Oxygen (DO) is about 30-50%; preferably, the temperature is about 36.0 ℃, the pH is about 7.0, the rotating speed is about 60rpm, and the dissolved oxygen is about 40%; more preferably, the deep air flow rate is about 1.0lpm and the superficial air flow rate is about 1.0 lpm.
It is understood that the culture temperature, rotation speed, pH and other relevant process parameters of different cells in the shake flask culture, bioreactor and reaction tank are different, and the optimal process parameters can be selected by the skilled person according to the actual reaction conditions.
In the present invention, when the cell density in the reaction tank is about 3.0 × 106～5.0×106At individual cells/mL, transient transfection was performed. The vector used for transient transfection may be any of a variety of vectors well known in the art which contain the coding sequence for the protein of interest. The vector to be transfected can be constructed by selecting an appropriate backbone vector according to the cell selected and cloning the coding sequence for the protein of interest and associated regulatory elements such as promoter, enhancer, origin of replication and optionally one or more markers into the backbone vector using conventional methods. The protein of interest is preferably a monoclonal antibody, more preferably a fully human monoclonal antibody, as described herein. In some embodiments, the protein of interest is a fully human anti-2019-nCOV monoclonal antibody.
When transfection is performed, the cell culture medium and the carrier can be mixed first, and then mixed with the transfection reagent after being mixed uniformly. Usually, the carrier is mixed with the medium at a concentration of not less than 0.5. mu.g/. mu.L. The transfection reagent may be added to the mixture of the cell culture medium and the carrier for about 0.5 to 2.0 minutes, and then the mixture of the three is mixed for about 1.0 to 2.0 minutes, after which the resulting mixture is added to the reaction tank. Preferably, the mixture is added to the reaction tank for a period of time not exceeding 15 minutes. The concentration of the transfection reagent can be determined according to the type of the transfection reagent, the type of the cell, the type of the vector, etc., and the concentration of the transfection reagent prepared is usually 1 to 3 mg/mL. In a preferred embodiment, the transfection reagent is Polyethyleneimine (PEI).
In a preferred embodiment, the cells are CHO cells and the transfection reagent is Polyethyleneimine (PEI); before instantaneous transfection, a carrier is added into a culture medium and uniformly mixed, then a transfection reagent is added into a mixture of the carrier and the culture medium, the adding time is about 0.5-2.0 minutes, the mixture is continuously mixed for about 1.0-2.0 minutes, and then the obtained mixture is injected into a reaction tank for transfection; preferably, the transfection mixture is added for a period of time not exceeding 15 minutes.
After transfection, fed-batch culture was performed. The time of feeding may be 2, 4, 6 and 8 days after transfection. The supplementary materials can be determined according to the actual culture conditions, on the premise that the cells in the reaction tank are maintained to grow normally and express the protein of interest. For example, in some embodiments, if the sugar content of the culture medium in the reaction tank is less than 2g/L after transfection, glucose can be supplemented at a concentration of about 2-3g/L so that the sugar content of the culture medium is 2g/L or more. Typically, the transient expression process is terminated 6-10 days after transfection. The culture conditions during this culture can be set as follows: the temperature is about 35.5-36.5 ℃, the pH is about 6.7-7.3, preferably 7.0 +/-0.3, the rotating speed is about 50-100 rpm, and the Dissolved Oxygen (DO) is about 30-50%; preferably, the temperature is about 36.0 ℃, the pH is about 7.0, the rotating speed is about 60rpm, and the dissolved oxygen is about 40%; more preferably, the deep air flow rate is about 1.0lpm and the superficial air flow rate is about 1.0 lpm.
In a particularly preferred embodiment of the invention, the invention provides a method for transient transfection of cells, preferably CHO cells, which method comprises:
(1) cell recovery;
(2) shake flask culture, wherein the revived cells are inoculated into a shake flask with the volume of 50mL-500mL, preferably 125mL or 250mL for the first shake flask culture, and when the VCD of the cells reaches (2.0-6.0) × 106Transferring the cells/mL and the cell viability rate of more than or equal to 90.0 percent into a shake flask with larger volume for subsequent shake flask culture, wherein the shake flask amplification is carried out for three times in sequence, the volume is expanded to 5L shake flasks, and the inoculation density of each shake flask culture is 0.2 × 106～0.6×106Each cell/mL culture medium, the conditions of each shaking culture are 35-38 ℃, 100-2The concentration is preferably 36-37 deg.C, 110-2Wherein, when the VCD of the cells in the culture of the last shake flask culture reaches (2.0-6.0) × 106Expanding the WAVE bioreactor when the cell per mL and the cell survival rate are more than or equal to 90.0 percent;
(3) amplification of WAVE reactor, wherein the inoculation density is 0.2 × 106～0.6×106Individual cells/mL medium; the reaction conditions in the bioreactor were: temperature of about 35-37 deg.C, CO2Concentration of about 0-5%, aeration flow rate of 0.2-0.6lpm, and angle of about 6-8 deg., preferably, the temperature is about 35.5-36.5 deg.C, the swing speed of the bioreactor can be 15-25rpm, preferably 18-22rpm, and the cell density in the bioreactor is about 2.0 × 106～6.0×106Individual cells/mL, preferably 4.0 × 106～6.0×106Individual cellPerforming amplification in a reaction tank when the cell viability is more than or equal to 90.0%; the volume of the WAVE reactor is about 10-100L, preferably about 20-80L, preferably about 40-60L, preferably about 50L;
(4) amplifying the reaction tank, wherein the inoculation density of the amplification of the reaction tank is 0.7 × 106～1.1×106The temperature of the reaction tank is about 35.5-36.5 deg.C, pH is about 6.7-7.3, rotation speed is about 50-100 rpm, and Dissolved Oxygen (DO) is about 30-50%, preferably, the temperature is about 36.0 deg.C, pH is about 7.0, rotation speed is about 60rpm, dissolved oxygen is about 40%, and when the cell density in the reaction tank is about 3.0 × 106～5.0×106Transient transfection is carried out at individual cells/mL; the volume of the reaction tank is about 100L-500L, preferably about 200-400L, preferably about 200L; and
(6) transient transfection; preferably, transient transfection is performed using any of the transient transfection methods described above.
By using the transient transfection method of the present invention, after the cell suspension is amplified several times, the cell culture volume is enlarged to about 150L, and after adding the transfection reagent and the plasmid mixture containing the target DNA to the cells, the pH level and sugar content are adjusted by feeding, and the expression level of about 300mg/L of the monoclonal antibody protein is reached under the culture for about 8 to 12 days.
In some embodiments, the present invention also provides a cell transient transfection device comprising:
(1) a reaction tank;
(2) a sealed stirred vessel in fluid communication with the reaction tank;
(3) a reservoir vessel in fluid communication with the sealed stirred vessel.
Preferably, the transient cell transfection apparatus further comprises a driving device, including a driving device for driving the liquid in the sealed stirring container into the reaction tank and a driving device for driving the solution in the liquid storage container into the sealed stirring container.
Preferably, the transient cell transfection equipment further comprises a bioreactor and a pipeline for communicating the bioreactor with the sealed stirring container; optionally including a drive means to drive the contents of the bioreactor into the sealed stirred vessel.
Typically, the drive means is a peristaltic pump.
Preferably, a filter is further arranged between the liquid storage container and the sealed stirring container and used for filtering liquid entering the sealed stirring container from the liquid storage container.
Preferably, the reaction tank and bioreactor are as hereinbefore described.
The following examples are provided to demonstrate and further illustrate some preferred embodiments and aspects of the present invention and should not be construed as limiting its scope.
The transient production process flow is shown in figure 1.
Example 1: cell resuscitation
30ml of CD CHO medium (Gibco, Life Technologies) were placed in 125ml shake flasks, which were preheated for more than 15min on a carbon dioxide shaker. Taking out a CHO 18 cell (modified from CHO-K1 cell) freezing tube from liquid nitrogen tank, and thawing in 37 deg.C constant temperature water bath for no more than 2 min. After thawing, the outer wall of the cryopreservation tube is sprayed with 75% ethanol and placed in a centrifuge at 1000rpm for 2 min. The supernatant was removed from the vial, and a small amount of pre-warmed medium was taken to resuspend the CHO 18 cells, which were then transferred to a 125ml shake flask. After mixing well, 0.5ml of cell sap was taken, and VCD (viable cell density), VIA (viable cell rate) and DIA (cell diameter) were measured using a cell viability analyzer (Beckman Co. mu. Lter, ViCell XR). Placing the shake flask into a carbon dioxide shaking table, and setting parameters: 36.5 +/-0.5 ℃, 120 +/-10 rpm and 7.0% +/-1.0% CO2. 0.5ml was sampled on days 3-6 of culture, and VCD, VIA and DIA were measured by a cell viability analyzer.
Example 2: shake flask amplification
When the VCD of the recovered cells reaches (2.0-6.0) × 106The VCD is estimated to be (0.2-0.6) × 10 after the cells are amplified6The amount of medium required was calculated per mL of cells. A calculated volume of CD CHO medium was transferred to a 2L sterile shake flask, seed fluid was added, mixed well, and VCD, VIA and DIA were measured using a cell viability analyzer (Beckman Co. mu. Lter, ViCell XR). Placing the shake flask into a carbon dioxide shaking table, and setting parameters: 36.5 +/-0.5 ℃ and 120 +/-10rpm、7.0％±1.0％CO2. After amplification, the seed liquid was shaken and tested for VCD, VIA and DIA using a cell viability analyzer. 0.5ml of the sample was taken on days 2-4 of the culture, and VCD, VIA and DIA were measured by a cell viability analyzer. The cells were expanded twice more as above and finally into 3 5L shake flasks, 2.5L/flask.
Example 3: WAVE bioreactor (Sartorius Stedim, Biostat RM) amplification
3.1. When the VCD of the seed reaches (2.0-6.0) × 106The cells can be inoculated to a WAVE bioreactor when the cell activity rate is more than or equal to 90.0 percent per mL.
3.2. VCD is (0.2-0.6) × 10 after inoculation according to WAVE6The amount of seed required for WAVE inoculation, as well as the amount of media to be supplemented, was calculated per mL of cells and the cells were transferred to seed flasks in a clean bench.
3.3. The seed flask drain and WAVE bag access tubes were welded together using a tube adapter (Sartorius Stedim, Biowelder).
3.4. The seed cells were inoculated into the culture bag by a peristaltic pump.
3.5. After inoculation, the inlet of the WAVE bag was sealed with a tube sealing machine (Sartorius Stedim, Biosealer) and the seal was checked for complete sealing.
3.6. Opening the WAVE bioreactor, starting culture, switching the WAVE bioreactor to a Sample Position mode after the WAVE bioreactor swings for 5-10 min, sampling, and measuring VCD and cell viability rate by using a cell viability analyzer, DIA and performing microscopic examination.
3.7. Sampling and counting after continuing to culture for 2-4 days, and when the viable cell density reaches (4.0-6.0) × 106The cell activity rate is more than or equal to 90 percent per mL, and then the next inoculation is carried out.
WAVE bioreactor parameters:
rocking speed 18-22rpm
Swing angle 6-8°
Flow rate of ventilation 0.2-0.6lpm
Temperature of 36±0.5℃
CO2Concentration of 0-5％
Example 4: 200L retort amplification (Sartorius Stedim, Biostat STR)
4.1. When the cell density in the WAVE reactor reaches (2-6) × 106Inoculating when the cell per mL and the survival rate is more than or equal to 90 percent.
4.2. According to the cell density after inoculation of (0.7-1.1) × 106The amount of seeds and the amount of medium used were calculated per cell/mL.
4.3. A tube connecting machine is used for connecting a WAVE reactor and a 200L reaction tank pipeline, and a peristaltic pump is used for inoculating the seed cells into the 200L reaction tank from the WAVE reactor.
4.4. And (3) 0.5-1 hr after inoculation, taking 30mL of sample from a sampling port of the 200L reaction tank, discarding the sample, taking 30mL of sample again, and detecting the cell density, the cell viability and the cell diameter.
A schematic diagram of the amplification of the reaction tank is shown in FIG. 2.
Example 5: transient transfection
5.1.200L reactor culturing cells for 44-52h, sampling, and counting cells with cell viability analyzer until cell density reaches (3.0-5.0) × 106Individual cells/mL can be transfected. Before transfection, a PEI liquid storage bag/bottle and a 50L sterile sealed stirring bag are welded together, and then the 50L sterile sealed stirring bag and a 200L reaction tank are welded together.
5.2 to a 50L solution mixing bag adding 13.9-23.9kg CD CHO culture medium, then CB6 (anti 2019-nCOV monoclonal antibody) heavy and light chain plasmid each 165mg (plasmid skeleton see figure 12) into the above culture medium, after mixing, plasmid mixture sterile filtration to 5.1 in the 50L sterile sealed mixing bag.
5.3 preparing 3mg/mL PEI solution (weighing 2310mg PEI powder and 693g ultrapure water, mixing and stirring until PEI is completely dissolved, adjusting the pH value to 6.8-7.2, and fixing the volume to 770mL), filtering and sterilizing into the PEI liquid storage bag/bottle, after the plasmid mixture is completely injected into the stirring bag, injecting the PEI solution into the PEI solution, timing, injecting the PEI solution while stirring, wherein the injection time of the PEI solution is 30s-2min, and after the mixture is continuously stirred for 1-2min, injecting the PEI solution into a 200L reaction tank.
5.4. Respectively and aseptically welding Cell Boost 7a and Cell Boost 7b (Hyclone, GEHealthcare) feed culture media to a 200L reactor by using an aseptic welding machine, and checking whether pipelines are damaged or not after the operation is finished. The culture medium is added at 2, 4, 6 and 8 days after transfection, the addition amount of the Cell Boost 7a is 3-6% of the transfection volume (the total liquid volume in the reaction tank), and the addition amount of the Cell Boost 7b is 0.3-0.6% of the transfection volume.
5.5. The cell viability rate is higher than 70% when the cells are placed in the tank 6-10 days after transfection.
During the transfection process, samples are taken every 24 hours, 30mL of samples are taken from the sampling port and discarded, and 30mL of samples are taken for analysis and sample retention. Viable cell density, viability, diameter, and metabolic parameters were measured. After the residual sample is centrifuged at 4000rpm for 5 minutes, the sample is stored in a refrigerator at 4 ℃ for analysis of expression amount or other detection after the culture is finished.
According to the setting of the pH control range of 7.0 +/-0.3 in the process, acid and alkali are automatically added by equipment for adjustment. If the residual sugar content of the fermentation liquor is lower than 2g/L, supplementing sugar, and supplementing 2-3g/L glucose.
The detection of each index during transient production is shown in fig. 3-8.
The detection shows that the expression level of the protein reaches about 300 mg/L.
200L reactor parameters:
Figure BDA0002504266750000141
example 6: comparison of dynamic Medium and CD CHO Medium
6.1 cell count, density adjusted to 3.5 × 106Taking two 27mL of each cell/mL, respectively centrifuging, resuspending with dynamics and CD CHO respectively at 1000rpm and 3min, placing in a shaker for culturing, at 37 ℃, 110rpm and 7% CO2。
6.2 two portions of CD CHO culture medium are respectively taken, added with 30 mu g of CB6 plasmid heavy-light chain respectively, filtered by a 0.22 mu m filter, respectively added with 140 mu L of PEI solution (3mg/mL) and evenly mixed, and then kept stand for 15min at room temperature.
6.3 the mixture is slowly added to the treated cells by pipette and mixed evenly while adding. After transfection, the cells were shake-cultured at 37 ℃ and 110rpm with 7% CO2. Samples were taken during the culture to monitor the cell status and expression level, as shown in FIGS. 9-11.
The results show that both the culture media are suitable for transient transformation of the CB6 molecule, and the CD CHO culture medium is superior to dynamic culture media in terms of expression quantity (Titer) and cell Viability (VIA).

Claims (19)

1. A method for transient transfection of mammalian cells for recombinant protein production, said method comprising the steps of:
shake flask culture of mammalian cell suspension;
the bioreactor expands the mammalian cell suspension;
the reaction tank expands the mammalian cell suspension in the bioreactor;
adding plasmids and transfection reagents into the reaction tank mammal cell suspension for transient transfection;
culturing the transfected mammalian cell.
2. The method according to claim 1, wherein the mammalian cells are CHO cells, preferably CHO 18 cells.
3. The method according to claim 2, wherein the culture medium used for the CHO cell culture is a CD CHO or Dynamics medium, preferably a CD CHO medium.
4. The method of claim 1, wherein the bioreactor is a WAVE bioreactor.
5. The method of any one of claims 1-4, wherein the shake flask culture is at about 3% -10% CO2Concentration, about 35-38 ℃ and about 100 and 140rpm for culture; preferably, it is between about 6% and 8% CO2Concentration, about 36-37 ℃ and about 110-.
6. The method according to claim 5, wherein the shake flask culture is performed 2 or more times, preferably by 2 or more than 2 stepwise amplifications to a 5L-10L shake flask, wherein the cell density of the culture is about (2.0-6.0) × 106When the cell per mL and the cell viability are more than or equal to 90.0 percent, the shake flask is replaced to carry out shake flask amplification, and preferably, the cell inoculation density of each shake flask culture is 0.2 × 106-0.6×106Individual cells/mL; preferably, the shake flask culture is performed at 35-38 deg.C, 100-2Under the condition of concentration, preferably at 36-37 deg.C, 110-130rpm and 6-8% CO2Under the condition of concentration.
7. The method of any one of claims 1-4, wherein a cell density of about 2.0 × 10 is used6～6.0×106Performing bioreactor amplification on the shake flask culture with each cell/mL and cell viability rate of more than or equal to 90.0%, preferably, the inoculation density is 0.2 × 10 during the bioreactor amplification6-0.6×106Individual cells/mL.
8. The method of any one of claims 1-4, wherein the bioreactor parameters are: temperature of about 35-37 deg.C, CO2About 0-5% concentration, about 0.2-0.6lpm aeration flow, about 6-8 ° angle; preferably, the temperature is about 35.5-36.5 ℃.
9. The method of claim 8, wherein a cell density of about 2.0 × 10 is used6-6.0×106The culture with cell/mL and cell activity rate of more than or equal to 90.0 percent is subjected to retort amplification, and preferably, the cells obtained by the bioreactor are expressed in a formula of (0.7-1.1) × 106The reaction tank was seeded with a seeding density of individual cells/mL.
10. The method of any one of claims 1 to 4, wherein the parameters of the reaction tank are: the temperature is about 35.5-36.5 ℃, the pH is about 6.7-7.3, the rotating speed is about 50-100 rpm, and the Dissolved Oxygen (DO) is about 30-50%; preferably, the temperature is about 36.0 ℃, the pH is about 7.0, the rotation speed is about 60rpm, and the dissolved oxygen is about 40%.
11. The process of any one of claims 1 to 4, wherein the reaction tank has a volume of about 100L to 500L, preferably about 200L to 400L, more preferably about 200L.
12. The method of claim 10, wherein the cell suspension in the reaction tank has a cell density of about 3.0 × 106～5.0×106At individual cells/mL, transient transfection was performed.
13. The method of any one of claims 1 to 4, wherein the plasmid concentration is not less than 0.5 μ g/μ L.
14. The method of any one of claims 1-4, wherein the transfection reagent is Polyethylenimine (PEI) and the concentration of PEI is about 1-3 mg/mL.
15. The method according to any one of claims 1 to 4, wherein the plasmid is added to the medium and mixed before the transient transfection, the transfection reagent is added to the plasmid mixture for about 0.5 to 2.0 minutes, and the mixture is injected into the reaction tank after the mixing is continued for about 1.0 to 2.0 minutes.
16. The method of any one of claims 1-4, wherein the feeding is performed on days 2, 4, 6, and 8 post-transfection.
17. The method of any one of claims 1-4, wherein the transient expression process is terminated 6-10 days after transfection.
18. The method according to any one of claims 1 to 17, wherein the recombinant protein is a monoclonal antibody, preferably a fully human anti 2019-nCOV monoclonal antibody.
19. A transient transfection device for cells implementing the method of any one of claims 1-17 comprising:
(1) a reaction tank; (2) a sealed stirred vessel in fluid communication with the reaction tank; (3) a reservoir vessel in fluid communication with the sealed stirred vessel; preferably, the transient cell transfection device further comprises a bioreactor and a pipeline for communicating the bioreactor with the sealed stirring container.
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