CN111057800A - Method for producing gene therapy plasmid by adopting starvation fermentation process and fermentation medium - Google Patents

Method for producing gene therapy plasmid by adopting starvation fermentation process and fermentation medium Download PDF

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CN111057800A
CN111057800A CN201911375992.5A CN201911375992A CN111057800A CN 111057800 A CN111057800 A CN 111057800A CN 201911375992 A CN201911375992 A CN 201911375992A CN 111057800 A CN111057800 A CN 111057800A
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曾杰
梁焯
曹曦
姚树元
辛晨浩
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Abstract

The invention discloses a method for producing gene therapy plasmids by adopting a starvation fermentation process, which comprises the following steps: (1) preparing a fermentation tank: inserting a disposable pH electrode, a disposable DO electrode and a cooling pipeline into corresponding positions of a disposable tank body; sterilizing the disposable tank body after the preparation of the fermentation medium; completing the connection of pipelines of the fermentation tank, setting the temperature of a temperature control system (TCU) and operating; (2) setting parameters; (3) inoculating; (4) and (4) controlling the hunger fermentation. The method combines the starvation fermentation process and the one-time totally-enclosed process, and can obviously reduce the residual content of the bacterial host protein HCP in the plasmid product. The invention also discloses a fermentation medium used for the method, which can effectively improve the plasmid fermentation yield.

Description

Method for producing gene therapy plasmid by adopting starvation fermentation process and fermentation medium
Technical Field
The invention belongs to a fermentation method for gene therapy plasmids in the field of biotechnology, and particularly relates to a method for producing gene therapy plasmids by adopting a starvation fermentation process and a safe and high-yield fermentation culture medium.
Background
Gene therapy has become an effective treatment for a number of major diseases, with major advances in The treatment of hemophilia, sickle cell disease, blindness, a variety of severe hereditary neurodegenerative diseases, and multiple cancers of The bone marrow and lymph nodes (Gene therapy community of age,2018, Science). currently, The European drug administration (EMA) and The U.S. Food and Drug Administration (FDA) have approved 6 Gene therapy products, 2 chimeric antigen receptor T cell products (Car-T) for B cell cancer and 4 chimeric antigen receptor T cell products (Car-T) for severe monogenic diseases, such as β -thalassemia, a rare vision loss, spinal muscular atrophy, and a rare primary immunodeficiency (Gene therapy,2019, The New England journal of Medicine).
Gene therapy mainly takes the form of both viral and non-viral vectors. In the long run, the non-viral vector has the advantages of low immune prototype, low cost, easy scale production and the like, thereby having better clinical application prospect. The vectors currently used in most cell and gene therapy programs are viral vectors. The most commonly used viral vectors include Adeno-associated virus (AAV), Lentivirus (LV), Adenovirus (AdV), and Retrovirus (Retrovirus, RV). These viral vectors can be produced by packaging plasmids, and thus plasmid DNA is the basis of gene therapy in both the near and long term, and is in great demand.
Plasmids, as a biological product for human use, have stringent requirements for their production and quality control. Although a large-scale plasmid DNA fermentation process meeting pharmaceutical specifications is established in patents (CN1914330B, 2012; CN 101213294B, 2013), the processes are all traditional fermentation methods and do not relate to a one-time full-closed production process. The one-time totally-enclosed production process is a development trend of the gene therapy industry, the process is more stable and controllable, and the product quality is safer and more effective.
It is known that plasmids can be obtained by bacterial fermentation and alkaline lysis (A rapid alkaline lysis process for screening recombinant plasmid DNA,1979, Nucleic Acids Res). However, the current patents (CN1914330B, 2012; CN 101213294B, 2013; CN 103396975B, 2016; CN108148831A, 2018; CN 109337834A, 2019) do not relate to the production of plasmids by a starvation fermentation method. The above fermentation method has a disadvantage of high HCP residue in the plasmid product and how to reduce HCP residue is not examined, for example, in patent CN108148831a (2018), the HCP residue of the host protein is 0.001 μ g/μ g plasmid DNA, which is reduced to 1000ng/mg, and the HCP residue in the plasmid product is high. In the existing literature (Industrial Manufacturing of Plasmid-DNA Products for genetic vaccination and Therapy, 2012) it is reported that the amount of residual host protein for DNA vaccines is < 1% and 10. mu.g/mg after conversion, and therefore the amount of residual HCP in the produced gene Therapy Plasmid product should meet the requirements of the DNA vaccine. No guidance is given in the prior art on how to overcome quality problems of plasmid products (e.g.HCP residues) by upstream fermentation processes.
Patent CN 103396975B (2016) discloses a fermentation medium formula of DNA vaccine, each 1L of fermentation medium contains: the reagent contains peptone 16.5g, yeast powder 5g, NaCl 10g, glycerol 0.65g, and Na2HPO412.8g，K2HPO43g，MgSO40.24g，NH4Cl 3.1g and trace elements 1 mL. The formula contains trace metal elements, heavy metal impurities are introduced in the process, and the DNA vaccine for human injection has certain safety risk. The fermentation culture medium is adopted in the patent, the yield of plasmid fermentation (5L) reaches 180 mg/L-220 mg/L, and the yield is lower.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for producing gene therapy plasmids, which can obviously reduce the residual content of bacterial host protein HCP in plasmid products.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a method for producing gene therapy plasmids by adopting a starvation fermentation process comprises the following steps:
(1) preparing a fermentation tank: inserting a disposable pH electrode, a disposable DO electrode and a disposable cooling pipeline into corresponding positions of a disposable tank body; sterilizing the disposable tank body after the preparation of the fermentation medium; completing the connection of pipelines of the fermentation tank, setting the temperature of a temperature control system (TCU) and operating;
(2) setting parameters: setting the pressure of a process air reducing valve to be 0.50 bar; setting fermentation tank parameters: the temperature is 37.0 ℃, the ventilation volume is 3.0L/min, and the rotating speed is 200 rpm; setting the pH value to be 7.20 +/-0.02, and starting an automatic control alkali supplement bottle to supplement alkali; adjusting the zero point of DO (dissolved oxygen) to be 0% when removing the dissolved oxygen electrode wire, inserting the electrode wire, setting the maximum rotating speed, and adjusting the DO fullness to be 95-100%;
(3) inoculation: inoculating through an inoculation pipeline, namely connecting a disposable transfer cover pipeline of a shake flask with a fermentation tank through an aseptic tube connecting machine, pumping the bacterial liquid in the shake flask into the fermentation tank, and sealing the pipeline by using the aseptic tube sealing machine;
(4) and (3) controlling the hunger fermentation: performing associated control on DO and the rotating speed, controlling the DO to be more than or equal to 20%, setting the minimum rotating speed as the initial rotating speed, and starting DO automatic control; defining the inoculation time as 0 hour, feeding when the DO value begins to rise after the culture, and setting the speed of a feeding pump to be 0.5% -2% of the fermentation volume/h; when the DO value begins to rise again, the speed of the feed pump is increased to 1% -4% of the fermentation volume/h; stopping feeding when the culture reaches a plateau stage; after the DO rises to above 40%, the fermentation is finished after the starvation fermentation culture is continued for 20 min-2 h.
Specifically, in the step (1), the connection of each pipeline of the fermentation tank comprises the connection of a material supplementing pipeline, an alkali supplementing pipeline, an inoculation pipeline, a fermentation tank air inlet pipeline, a fermentation tank air outlet pipeline and a cooling pipeline. When the pipelines are connected, the aseptic pipe connecting machine and the aseptic pipe sealing machine are used for connection operation. Wherein the feeding pipeline is connected to the fermentation tank through a feeding pump; the alkali supplement pipeline is connected to the fermentation tank through an alkali supplement pump; the inoculation pipeline connects the disposable switching cover pipeline of the shake flask with the fermentation tank through an aseptic tube connecting machine, the bacteria liquid in the shake flask is pumped into the fermentation tank through a peristaltic pump, and the pipeline is sealed by the aseptic tube sealing machine; the fermentation tank air inlet pipeline is connected with a workshop air supply pipeline through a fermentation controller; the exhaust pipeline of the fermentation tank is exhausted through a steam cooling device and a 0.22 mu m filter; the cooling pipeline is connected with the disposable cooling pipeline through a temperature controller.
Specifically, in the step (1), the disposable pH electrode is calibrated before being used.
Specifically, in the step (1), the sterilization is carried out under the condition of 121.0 ℃ for 20 min. Sterilization was performed in a pulse vacuum sterilizer.
Specifically, in the step (1), the temperature of the temperature control system (TCU) is set to be 4-10 ℃.
Specifically, in the step (1), the formula of the fermentation medium is as follows: each 1L of the fermentation medium contained: soybean peptone 10-20 g, yeast powder 5-10 g, (NH)4)2SO42g～6g，Na2HPO4·7H2O 12g～24g，KH2PO42g to 4g, 15g to 30g of glycerol and MgSO40.4g to 0.8 g. The fermentation medium also contains a proper amount of defoaming agent, and the amount of the defoaming agent is about 0.3-1 g.
Preferably, the formula of the fermentation medium is as follows: each 1L of the fermentation medium contained: soybean peptone 20g, yeast powder 10g, (NH)4)2SO44g，Na2HPO4·7H2O 17.8g，KH2PO43g, glycerol 25g, MgSO40.6g。
Preferably, in the step (4), the initial feeding rate is 1.2% -1.5% fermentation volume/h, and then the feeding rate is increased to 2.4% -3.0% fermentation volume/h.
Preferably, in the step (4), feeding is stopped when the fermentation is cultured for 14 h.
Preferably, in the step (4), the feeding is stopped, and after the DO is increased to above 40%, starvation fermentation culture is carried out for 20min to 60 min.
Specifically, in the step (4), when the culture is carried out for 3.5 hours, feeding is started, and the feeding pump speed is set to be 0.2ml/min (the feeding speed range is 4 g/kg/H3 kg/60min-5 g/kg/H3 kg/60min, and ml/min is approximately equal to g/min); increasing to 0.4ml/min when the culture is carried out for 5.5h (feeding speed range is 8g/kg/h 3kg/60min-10g/kg/h 3kg/60 min); stopping feeding after culturing for 14h, and continuing culturing for 20min after DO rises to more than 40% to finish fermentation;
the plasmid for gene therapy is used for packaging lentivirus, adeno-associated virus, DNA vaccine for injection and the like. In some embodiments, the plasmid contains a recombinant transgene, Gag-Pol gene, Rev gene, VSV-G gene, AAV serotype 1-9 gene, AAV serotype DJ/DJ8/Rh10 gene, and the like.
The invention provides a simple and effective starvation fermentation process, which can obviously reduce the residual content of bacterial host protein HCP in a plasmid product. No influence of the starvation fermentation process on HCP residues of the plasmid product is reported in the prior literature.
The method for producing gene therapy plasmid combines the starvation fermentation process and the disposable totally-enclosed process, and adopts a disposable fermentation tank body, a disposable pH electrode, a disposable DO electrode, a disposable pipeline, a disposable liquid preparation bag, a liquid storage bag and the like in the whole process. The totally enclosed plasmid fermentation production is realized by applying an aseptic sealing machine and a tube sealing machine. The existing plasmid fermentation patents are all traditional fermentations and do not relate to a one-time totally-enclosed plasmid fermentation process.
In a second aspect, the invention also provides a safe and high-yield fermentation medium, which can effectively improve the plasmid fermentation yield.
The formula of the fermentation medium provided by the invention is as follows: each 1L of the fermentation medium contained: soybean peptone 10-20 g, yeast powder 5-10 g, (NH)4)2SO42g～6g，Na2HPO4·7H2O 12g～24g，KH2PO42g to 4g, 15g to 30g of glycerol and MgSO40.4g to 0.8 g. The fermentation medium also contains a proper amount of defoaming agent, and the amount of the defoaming agent is about 0.3-1 g.
Preferably, the formula of the fermentation medium is as follows: each 1L of the fermentation medium contained: soybean peptone 20g, yeast powder 10g, (NH)4)2SO44g，Na2HPO4·7H2O 17.8g，KH2PO43g, glycerol 25g, MgSO40.6g。
The invention provides a safe and high-yield fermentation medium formula for fermentation production of virus packaging plasmids, and compared with other existing patents and technologies, the fermentation medium formula has many advantages. The method is characterized in that volatile toxic ammonia water is avoided, and sodium hydroxide is adopted to replace alkali supplement; the trace elements are not contained, so that heavy metal impurities introduced by the process are avoided; and glycerol is adopted to replace glucose as a carbon source, so that acid production in the fermentation process is reduced. By adopting a starvation fermentation process, the HCP residual quantity of the plasmid is obviously reduced. By adopting the fermentation medium formula, the plasmid fermentation yield can reach 350 mg/L.
Drawings
FIG. 1 is a graph showing the effect of the fermentation medium formulation on the growth of microbial cells.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
EXAMPLE 1 basic procedure for one-time totally-enclosed plasmid fermentation Process
1. Primary equipment of disposable fermentation process
Device name Brand
Bioreactor Eppendorf
Aseptic pipe connecting machine Sartorius
Aseptic tube sealing machine Sartorius
2. Main consumable list of disposable fermentation process
Figure BDA0002340972840000041
Figure BDA0002340972840000051
3. Disposable fermentation process control parameter
1) Preparing a fermentation tank: and (3) calibrating the pH electrode, inserting the pH electrode, the DO electrode and the cooling pipeline into corresponding positions of the disposable tank body, installing a feeding pipeline and an inoculation pipeline, and sealing the outlet end by using a plug. After the fermentation medium is prepared, the tank body is put into a pulse vacuum sterilizer at the temperature of 121.0 ℃ for 20min for sterilization. Installing relevant pipelines for air intake, air exhaust and cooling of a fermentation tank after sterilization, connecting a pipe connecting machine with a material supplementing pipeline and an alkali supplementing pipeline, setting the temperature of a temperature control system (TCU) to be 6.0 ℃, and operating. And logging in at a computer control end, inputting a batch number and recording data.
2) Setting parameters: setting the pressure of a process air reducing valve to be 0.50bar, and setting fermentation tank parameters: the temperature is 37.0 ℃, the ventilation rate is 3.0L/min, and the rotating speed is 200 rpm. The alkali supplement bottle is connected into a fermentation tank through an alkali supplement pump, and the pH value is set to be 7.20 +/-0.02, and the automatic control is started. When the dissolved oxygen electrode wire is removed, the DO zero point is adjusted to be 0%, the electrode wire is inserted, the rotation speed is set to be 1200rpm, and the DO fullness is adjusted to be 95-100%. While the feed pump was calibrated with purified water.
3) Inoculation: connecting the disposable switching cover pipeline of the shake flask with the fermentation tank through an aseptic tube sealing machine, pumping the bacterial liquid in the shake flask into the fermentation tank, and sealing the inoculation pipeline by the aseptic tube sealing machine.
4) And (3) fermentation control: and (4) performing associated control on DO and the rotating speed, controlling the DO to be more than or equal to 20%, setting the minimum rotating speed to be 200rpm, and starting DO automatic control. When the culture is carried out for 3.5H, feeding is started, the speed of a feeding pump is set to be 0.2ml/min (the feeding speed ranges from 4 g/kg/H3 kg/60min to 5 g/kg/H3 kg/60min, and the ml/min is approximately equal to g/min), and the speed is increased to be 0.4ml/min after 5.5H (the feeding speed ranges from 8 g/kg/H3 kg/60min to 10 g/kg/H3 kg/60 min). After 14h, stopping feeding materials, after DO rises to be above 40%, continuing culturing for 20min to finish fermentation, stopping recording data, and collecting fermentation liquor by using a 20L liquid storage bag.
Example 2 study of the influence of starvation fermentation Process on plasmid HCP
1. Adopting a fermentation medium 1 formula: each 1L of fermentation medium contains 10g of soybean peptone, 5g of yeast powder, 0g of ammonium sulfate, 7.7g of disodium hydrogen phosphate heptahydrate, 1.5g of potassium dihydrogen phosphate, 16g of glycerol, 0.6g of magnesium sulfate and 0.3g of defoaming agent.
The formula of the supplemented medium comprises 100g of peptone, 50g of yeast powder, 150g of glycerol and 1g of antifoaming agent in 1L of supplemented medium.
The formula of the supplemented medium comprises 100g of peptone, 50g of yeast powder, 150g of glycerol and 1g of antifoaming agent in 1L of supplemented medium.
The formula of the supplementary alkali liquor comprises 120g of sodium hydroxide per 1L of supplementary alkali liquor.
2. The starvation fermentation method comprises the following steps: and (4) performing associated control on DO and the rotating speed, controlling the DO to be more than or equal to 20%, and starting DO automatic control when the rotating speed is 200rpm at minimum and 1200rpm at maximum. The inoculation time was defined as 0 hour, and when the culture was carried out for 3.5 hours, feeding was started, and the feed pump speed was set to 0.2mL/min (feed speed range: 4mL/L/h 3L/60min), and increased to 0.4mL/min after 5.5 hours (feed speed range: 8mL/L/h 3L/60 min). After 14 hours, stopping feeding materials, allowing the escherichia coli to consume the nutrients remained in the culture medium, after DO is increased to more than 40%, allowing the escherichia coli to be in a starvation state, stopping growth, allowing the plasmids to still keep a replication state, continuing to consume the host proteins of the escherichia coli to maintain life, continuing to culture for 20min, ending fermentation (the fermentation time is 16 hours), and harvesting.
3. Fermentation batch and sampling: 3 different lentivirus packaging plasmids were used for fermentation, respectively, for a total of 3 batches. The samples are shown in Table 1:
table 1: sampling
Figure BDA0002340972840000061
4. Plasmid extraction: the 3 batches of fermentation liquor samples are diluted to be within the linear range of a plasmid extraction kit, the OD600 is diluted to be 1, 1mL of extracted plasmid DNA is taken, and extraction is carried out according to the kit operation. The extracted plasmid samples were tested for bacterial host protein residues (HCP) as in example 2.
5. As a result: the effect of the starvation fermentation process on plasmid HCP residues is shown in table 2. From the fermentation results of 3 different packaged plasmids in table 2, it can be seen that after the starvation fermentation treatment, the bacterial HCP was reduced to 70.8%, 78.5%, 58.9% before starvation, respectively, and the starvation fermentation method could significantly reduce the residual amount of HCP in the plasmid.
Table 2: effect of starvation fermentation Process on plasmid HCP residues
Figure BDA0002340972840000062
Figure BDA0002340972840000071
Example 3: method for detecting escherichia coli host protein residue
1. Preparing a standard substance: the detection kit is an E.coli HCP enzyme linked immunosorbent assay kit (CYGNUS company), and the standard substances 0, 1, 3, 12, 40 and 100ng/mL carried by the kit are taken out and balanced to room temperature.
2. Diluting a test sample: a1 XPBST wash was prepared and the samples were serially diluted to a standard curve range of 1-100ng/mL using sample dilutions (CYGNUS).
3. Plate distribution: the coated plate is taken out, and a standard sample and a test sample with 25 mu L/hole are respectively added into the coated plate, and each hole is repeated for 2 times.
4. Adding an enzyme-labeled antibody: adding HRP-labeled antibody to 100. mu.L/well, sealing, placing in a constant temperature oscillator at 28 deg.C and 600rpm, and incubating for 90 min.
5. Washing the plate: discard the sample solution in each well, add 1 XPBST wash solution at 350. mu.l/well, without soaking, wash 4 times. After the last washing, the washing liquid in the holes is discarded, and the reaction plate is reversely buckled on the water absorption paper to suck the washing liquid.
6. Substrate color development: TMB substrate solution was added at 100. mu.l/well, and the plates were sealed with a membrane plate and incubated at room temperature for 30 min.
7. And (4) terminating: stop solution was added at 100. mu.l/well.
8. Reading a plate: the plate reading was completed within 10min by reading at a dual wavelength of 450nm/650nm according to the instructions of a microplate reader (company: MD, model: i3 x).
Example 4: fermentation Medium formulation study
1. The fermentation medium of the invention is characterized in that: the glycerol is used as a carbon source to replace glucose, so that acid production of the thalli is reduced, growth of the thalli and accumulation of plasmids are facilitated, and the thalli is promoted to grow rapidly by containing an inorganic nitrogen source ammonium sulfate. The sodium hydroxide is adopted to replace ammonia water for alkali supplement, so that the risk of respiratory tract damage to people caused by volatilization of volatile ammonia water in the alkali supplement process is avoided. The other difference from the traditional plasmid fermentation is that the method does not contain trace elements, and avoids the introduction of heavy metal ion residues in the process.
2. Fermentation medium formula 1: each 1L of fermentation medium contains 10g of soybean peptone, 5g of yeast powder, 0g of ammonium sulfate, 7.7g of disodium hydrogen phosphate heptahydrate, 1.5g of potassium dihydrogen phosphate, 16g of glycerol, 0.6g of magnesium sulfate and 0.3g of defoaming agent.
The formula of the supplemented medium comprises 100g of peptone, 50g of yeast powder, 150g of glycerol and 1g of antifoaming agent in 1L of supplemented medium.
The formula of the supplemented medium comprises 100g of peptone, 50g of yeast powder, 150g of glycerol and 1g of antifoaming agent in 1L of supplemented medium.
The formula of the supplementary alkali liquor comprises 120g of sodium hydroxide per 1L of supplementary alkali liquor.
3. The fermentation medium 2 comprises the following components: each 1L of fermentation medium contains 20g of soybean peptone, 10g of yeast powder, 4g of ammonium sulfate, 17.8g of disodium hydrogen phosphate heptahydrate, 3g of potassium dihydrogen phosphate, 25g of glycerol, 0.6g of magnesium sulfate and 1g of defoaming agent.
The formulas of the feed-batch culture medium and the feed-batch alkali liquor are the same as those of the fermentation culture medium 1.
4. Fermentation: fermentation was carried out as in example 1.
5. Analysis of fermentation samples: and detecting the OD600 value of the fermentation liquid, using the fermentation culture liquid as a blank control, and diluting the fermentation liquid by using the fermentation culture liquid to ensure that the OD600 value is between 0.2 and 0.8. And detecting the weight of wet bacteria, taking an empty 15mL centrifuge tube, weighing the empty tube, sucking 5mL fermentation liquid into the 15mL centrifuge tube, centrifuging at 8000rpm for 10min, carefully discarding the supernatant, weighing the weight, and calculating to obtain the weight (g/L) of the wet bacteria.
6. As a result: the formula of the fermentation medium is shown in Table 3, and the influence of the formula of the fermentation medium on the growth of the thallus is shown in FIG. 1.
Table 3: fermentation Medium formulation study
Figure BDA0002340972840000081
In FIG. 1, [ 1 ] and [ 2 ] represent fermentation medium formulations 1 and 2, respectively. As can be seen from fig. 1: the content of the carbon source and the nitrogen source in the fermentation medium formula 2 is 1-2 times of that in the formula 1, and the growth of the thalli can be remarkably promoted.
In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (13)

1. A method for producing gene therapy plasmids by adopting a starvation fermentation process is characterized by comprising the following steps:
(1) preparing a fermentation tank: inserting a disposable pH electrode, a disposable DO electrode and a disposable cooling pipeline into corresponding positions of a disposable tank body; sterilizing the disposable tank body after the preparation of the fermentation medium; completing the connection of pipelines of the fermentation tank, setting the temperature of a temperature control system (TCU) and operating;
(2) setting parameters: setting the pressure of a process air reducing valve to be 0.50 bar; setting fermentation tank parameters: the temperature is 37.0 ℃, the ventilation volume is 3.0L/min, and the rotating speed is 200 rpm; setting the pH value to be 7.20 +/-0.02, and starting an automatic control alkali supplement bottle to supplement alkali; adjusting the zero point of DO (dissolved oxygen) to be 0% when removing the dissolved oxygen electrode wire, inserting the electrode wire, setting the maximum rotating speed, and adjusting the DO fullness to be 95-100%;
(3) inoculation: inoculating through an inoculation pipeline, namely connecting a disposable transfer cover pipeline of a shake flask with a fermentation tank through an aseptic tube connecting machine, pumping the bacterial liquid in the shake flask into the fermentation tank, and sealing the pipeline by using the aseptic tube sealing machine;
(4) and (3) controlling the hunger fermentation: carrying out correlation control on dissolved oxygen DO and the rotating speed, controlling the DO to be more than or equal to 20%, setting the minimum rotating speed as the initial rotating speed, and starting DO automatic control; defining the inoculation time as 0 hour, feeding when the DO value begins to rise after the culture, and setting the speed of a feeding pump to be 0.5% -2% of the fermentation volume/h; when the DO value begins to rise again, the speed of the feed pump is increased to 1% -4% of the fermentation volume/h; stopping feeding when the culture reaches a plateau stage; after the DO rises to above 40%, the fermentation is finished after the starvation fermentation culture is continued for 20 min-2 h.
2. The method of claim 1, wherein in step (1), the connection of the fermentation tank pipelines comprises the connection of a feed pipeline, an alkali supplement pipeline, an inoculation pipeline, a fermentation tank air inlet pipeline, a fermentation tank air outlet pipeline and a cooling pipeline.
3. The method of claim 2, wherein the connecting of the lines is performed using a sterile tube connector and a sterile tube sealer.
4. The method of claim 1, wherein in step (1), the disposable pH electrode is calibrated prior to use.
5. The method of claim 1, wherein in step (1), the sterilization is performed at 121.0 ℃ for 20 min.
6. The method of claim 1, wherein in step (1), the temperature of the temperature control system is set to 4-10 ℃.
7. The method of claim 1, wherein in step (1), the fermentation medium is formulated as: each 1L of the fermentation medium contained: soybean peptone 10-20 g, yeast powder 5-10 g, (NH)4)2SO42g～6g，Na2HPO4·7H2O 12g～24g，KH2PO42g to 4g, 15g to 30g of glycerol and MgSO40.4g to 0.8 g. The fermentation medium also contains a proper amount of defoaming agent, and the amount of the defoaming agent is about 0.3-1 g.
8. The method of claim 7, wherein the fermentation medium has a formulation of: each 1L of the fermentation medium contained: soybean peptone 20g, yeast powder 10g, (NH)4)2SO44g，Na2HPO4·7H2O 17.8g，KH2PO43g, glycerol 25g, MgSO40.6g。
9. The method of claim 1, wherein in step (4), the initial feeding rate is 1.2% to 1.5% fermentation volume/h, and then the feeding rate is increased to 2.4% to 3% fermentation volume/h.
10. The method of claim 1, wherein in step (4), feeding is stopped when the fermentation is cultured for 14 h.
11. The method according to claim 1, wherein in the step (4), the feeding is stopped, and after the DO level rises to 40% or more, the starvation fermentation culture is carried out for 20min to 60 min.
12. A safe and high-yield fermentation medium is characterized by comprising the following components in parts by weight: each 1L of the fermentation medium contained: soybean peptone 10-20 g, yeast powder 5-10 g, (NH)4)2SO42g～6g，Na2HPO4·7H2O 12g～24g，KH2PO42g to 4g of glycerol and 15g to 30g of glycerolg，MgSO40.4g to 0.8 g. The fermentation medium also contains a proper amount of defoaming agent, and the amount of the defoaming agent is about 0.3-1 g.
13. The fermentation medium of claim 12, wherein the fermentation medium has a formulation comprising: each 1L of the fermentation medium contained: soybean peptone 20g, yeast powder 10g, (NH)4)2SO44g，Na2HPO4·7H2O 17.8g，KH2PO43g, glycerol 25g, MgSO40.6g。
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