KR20180127930A - Cell culture method at low temperature applicable to mass production of protein - Google Patents

Abstract

The present application discloses relates to a method used usefully for mass production of proteins using microorganisms by increasing the concentration of a nitrogen source at a low temperature of not less than 20°C and less than 30°C. The process according to the present application can be usefully used for the expression of high yield of active protein suitable for mass production while inhibiting the production of unnecessary insoluble protein from the microorganisms.

Description

[0001] The present invention relates to a method for culturing cells at a low temperature,

The present application relates to a method which can be usefully used for mass production of a protein using microorganisms by increasing the concentration of a nitrogen source at a low temperature of 20 ° C or more and less than 30 ° C.

Currently, the method of producing a recombinant protein from a microorganism is problematic in that, when the recombinant protein is an animal cell-derived protein, inadequate folding occurs in most cases, or cohesion occurs frequently to produce an inert inclusion body. Furthermore, there is a problem in that it requires a secondary operation such as refolding which is very inefficient in order to use this protein.

Much research has been done to overcome this problem and to increase the production of properly folded active proteins. Basically, in order to produce an active protein from a microorganism, it is essential to induce movement to a position where a folding is possible by inserting a signal gene. Conventionally, a chaperone protein capable of blocking formation of an inclusion body and helping folding is expressed together with a target protein (Korean Patent Laid-Open No. 10-2009-0114422), the induction condition is changed to change the promoter (Champion et al. (2001) Proteomics , 1: 1133-1148), and changing fermentation conditions (Schein (1989) Bio / Technology , 7: 1141-1149).

In addition, attempts have been made to suppress the production of insoluble proteins by decreasing the rate of protein production by applying an expression temperature condition of 30 ° C or lower. However, since the optimum growth temperature of general microorganisms is 33 ° C to 37 ° C, low temperature conditions of 30 ° C or lower inevitably cause a decrease in microbial growth, resulting in a significant decrease in the amount of target protein expressed in microorganisms. Thus, a low yield of the product material can be very lethal in the production of large quantities of biological products using microorganisms.

In order to overcome this problem , researchers have been conducting research on Vasina and Baneyx (1996) Appl . Nano . Microbiol , 1444-1447 , which transforms the promoter into a cold resistant gene. However, this method has serious disadvantages that it can not be applied to all types of industrially common proteins because the low temperature resistance promoter must be used and the amount of the target protein can be changed depending on the combination of the target protein and the promoter.

In addition, there has been an attempt to increase the yield of the target protein from the microorganism by controlling the constituents of the medium. However, there is a risk that the growth of the protein and the expression of the protein may be decreased when the nitrogen source is excessively added (Popplewell et al. Methods in Molecular Biology, 308: 17-30).

Under these circumstances, the present inventors have made intensive efforts to increase the amount of protein produced. As a result, they have found that by increasing the concentration of the nitrogen source at a low temperature, it is possible to obtain a high- The present invention has been accomplished by discovering a method for producing a protein.

(A) fermenting a microorganism containing a recombinant vector at a temperature of 20 ° C or more and 30 ° C or less for 48 hours or more; And (b) adding a culture medium containing N-source before fermentation or during fermentation of the microorganism in step (a).

In the following, the present application will be described in more detail.

On the other hand, each description and embodiment disclosed in the present application can be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of the present application. In addition, the scope of the present application is not limited by the specific description to be described below.

In addition, those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described in this application. Such equivalents are also intended to be included in the present application.

According to one aspect of the present invention, there is provided a method for producing a recombinant vector, comprising: (a) fermenting a microorganism containing a recombinant vector at a temperature of 20 ° C or more and 30 ° C or less for 48 hours or more; And (b) adding a culture medium containing N-source before fermentation or during fermentation of the microorganism in step (a).

Generally, high temperature conditions or temperature shifting methods are attempted in order to accelerate growth while reducing impurities in the production of protein, and low temperature conditions are not generally tried since they are severe for growth. However, the present application is significant in that it is the first time that the present inventors have increased the amount of protein production not only on a lab scale but also on a mass scale, by adding an excess of nitrogen source under a low temperature condition.

In step (a), the microorganism containing the recombinant vector is fermented at a temperature of 20 ° C or higher and 30 ° C or lower for 48 hours or more.

For the purpose of the present application, the microorganism can be used for fermentation without limitation as long as it contains a recombinant vector capable of expressing a desired protein. Those skilled in the art can select and apply a microorganism containing an appropriate recombinant vector have.

As used herein, the term " a microorganism comprising a recombinant vector " refers to a microorganism transformed with a vector having a gene encoding at least one target protein.

The term " vector " as used herein also refers to a DNA construct containing the nucleotide sequence of a polynucleotide encoding the desired protein operably linked to a suitable regulatory sequence so as to be capable of expressing the protein of interest in the appropriate host . The regulatory sequence may include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. The vector may be transcribed into an appropriate host cell and then cloned or functioned independently of the host genome and integrated into the genome itself.

The vector used in the present application is not particularly limited as long as it is replicable in the host cell, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A and Charon21A can be used as the phage vector or cosmid vector, and pBR, pUC, pBluescriptII , pGEM-based, pTZ-based, pCL-based, pET-based, and the like. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector and the like can be used but are not limited thereto.

The vector usable in the present application is not particularly limited, and known expression vectors can be used. In addition, a polynucleotide encoding a target protein can be inserted into a chromosome through a vector for intracellular chromosome insertion. The insertion of the polynucleotide into the chromosome can be accomplished by any method known in the art, for example, homologous recombination, but is not limited thereto. And may further include a selection marker for confirming whether or not the chromosome is inserted. Selection markers are used to select cells that are transfected with a vector, that is, to confirm the insertion of a target nucleic acid molecule, and are provided with selectable phenotypes such as drug resistance, resistance to nutritional requirement, tolerance to cytotoxic agents, May be used. In the environment treated with the selective agent, only the cells expressing the selectable marker survive or express different phenotypes, so that the transformed cells can be selected.

The term " selectable marker " used in the present application is a gene having resistance to an antibiotic. Since cells having this gene survive in the environment treated with the antibiotic, they are useful as selection markers in the process of obtaining a large amount of plasmid in E. coli do. The antibiotic resistance gene in the present application is not a factor that greatly influences the expression efficiency depending on the optimal combination of the vector, which is a key technology of the present application, so that the antibiotic resistance gene generally used as a selection marker can be used without limitation. Specific examples include resistance genes for ampicilin, tetracyclin, kanamycin, chloroamphenicol, streptomycin, or neomycin, and more specifically, May be, but are not limited to, a tetracycline resistance gene.

The term "transformation" in the present application means introducing a vector containing a gene encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. Transformed polynucleotides may include all of these, whether inserted into the chromosome of the host cell or located outside the chromosome, provided that the polynucleotide can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding a target protein. The polynucleotide may be introduced in any form as far as it is capable of being introduced into a host cell and expressed. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for its expression. The expression cassette can typically include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. Also, the polynucleotide may be introduced into the host cell in its own form and operatively linked to the sequence necessary for expression in the host cell, but is not limited thereto. Such a transformation method includes any method of introducing a nucleic acid into a cell, and may be carried out by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (Ca (H ₂ PO ₄ ) ₂ , CaHPO ₄ , or Ca ₃ (PO ₄ ) ₂ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, Polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method, but the present invention is not limited thereto.

Also, the term " operably linked " as used herein above means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the protein of interest of the present application. Operable linkages can be made using known recombinant techniques in the art, and site-specific DNA cleavage and linkage can be made using, but not limited to, cutting and linking enzymes in the art.

The term " mass production " as used in this application means a level at which the amount of protein produced can be produced so as to be industrially usable. For example, it can be used in a 5 L scale or more, 30 L scale or more, .

Specifically, the microorganism containing the recombinant vector may be fermented at a temperature of 20 ° C or higher and 30 ° C or lower for 48 hours or longer, but the present invention is not limited thereto. The temperature of 20 ° C or more and 30 ° C or less belongs to a low temperature in a general fermentation stage, and fermentation at such a low temperature is a fermentation temperature newly determined by the present inventors.

Such low temperature fermentation temperature includes about 20 캜 or higher and 30 캜 or lower. As used herein, the term " about " includes all values equal to or similar to those following the term, including ranges of ± 0.5, ± 0.4, ± 0.3, ± 0.2, ± 0.1, etc. It is not limited.

The low temperature may be 20 ° C or more and 30 ° C or less and 20 ° C or more and less than 30 ° C. Examples include 21 ° C to 29 ° C, 21 ° C to 29 ° C, 22 ° C to 28 ° C, 23 ° C to 27 ° C, 23 ° C to 27 ° C, 24 ° C to 26 ° C, 24 ° C to 26 ° C, or about 25 ° C.

The fermentation time may be 48 hours or more to 72 hours or less.

In the method for producing a protein by microbial fermentation according to the present application, the step (b) is a step of adding a medium containing an N-source before fermentation or fermentation of the microorganism in step (a).

In particular, the addition of the medium containing the nitrogen source in step (b) may vary depending on the culture method known in the art. Specifically, a medium containing a nitrogen source may be added before the start of fermentation or during fermentation of the microorganism.

In addition, the culture may be a fed-batch culture in addition to the batch culture, but is not limited thereto.

Specifically, the medium containing the nitrogen source is added before the fermentation of the microorganism is started by culturing the microorganism by inoculating a culture medium containing all the nutrients necessary for cell proliferation or material production by a batch culture, It is difficult to add. For the purpose of the present application may include inoculating a microorganism containing a recombinant vector into a medium containing a nitrogen source and fermenting it by a batch culture method.

In addition, adding a medium containing a nitrogen source at the time of fermentation of the microorganism may be accomplished by feeding a medium containing one or more nutrients in a fed-batch culture, immediately after the start of fermentation, or after the culture reaches a certain stage, When the nutrient is depleted from the culture, the culture is continuously supplied to the culture. In fed-batch culture, single nutrients and carbon sources are generally limiting factors for growth. Other types of nutrient limitations may be used, such as restriction by nitrogen sources, by oxygen, by sulfur, and by phosphorus. For the purpose of the present application, to continuously feed a medium containing a nitrogen source upon fermentation of the microorganism containing the recombinant vector.

For the purpose of this application, the medium includes all of the medium used for culturing the cells, the medium used for maximizing the production of the desired protein, and the like.

Specifically, " cell culture medium " or " culture medium " means a nutrient solution for maintenance, growth, proliferation or expansion of cells in an artificial in vitro environment that is outside the multicellular organism or tissue. The cell culture medium may be optimized for a specific cell culture, for example, a basic culture medium prepared for supporting cell growth, or a cell culture production medium prepared to promote the production of a target protein, Which may be a concentrated medium. Nutrients, and medium components refer to components constituting the medium, and are used interchangeably herein.

Specifically, the term " primary culture medium " or " primary culture medium " means a medium capable of supporting minimal cell growth. The base medium means a medium for supplying vitamins and inorganic salts in addition to a carbon source and a nitrogen source.

Specifically, the term " cell culture production medium " or " production medium " refers to a medium used for the purpose of maximizing the expression of a target protein in a bioreactor. The production medium can be the same as or different from the basic medium, and if different, it can be prepared by concentrating the basic medium itself or adding specific ingredients to the basic medium.

The term "feeding medium" and "additional culture medium" may be a culture medium composed of a specific nutrient or a plurality of nutrients, which may be a concentrated product of the entire basic medium, but is not limited thereto. Depending on a cell cultured by a person skilled in the art, It is possible to produce various constituent components and concentrations.

For the purpose of the present application, when the sugar in the medium is used in accordance with the growth of cells during fermentation, the fermentation time can be further increased by adding a sugar, wherein the amount of the nitrogen source added is 10 g / L to 80 g / L, more specifically from 15 g / L to 70 g / L, and more specifically from 20 g / L to 64.5 g / L, but is not limited thereto.

In addition, the medium containing the nitrogen source may be a medium containing no animal-derived material, but is not limited thereto. More specifically, the medium containing the nitrogen source may include any one or more components selected from the group consisting of ammonium sulfate, yeast extract, or a combination thereof, but is not limited thereto no.

Typically, the concentration of the nitrogen source in the medium is 2 to 14 g / L, but the concentration of the nitrogen source in the conventional medium is in the range of 30 g / L to 150 g / L, More specifically from 40 g / L to 120 g / L, and more specifically from 49.6 g / L to 106.7 g / L, but is not limited thereto.

In addition, the nitrogen source may have a weight ratio of 1: 0.15 to 1.24 based on carbon source, but is not limited thereto.

For the purpose of this application, the production of insoluble proteins and inclusion bodies is extremely suppressed while the microorganisms are fermented at low temperature, and the amount of nitrogen source is put in a range beyond the conventionally known method, thereby restoring the microorganism growth property at low temperature, The amount can be mass-produced to such an extent that it can be used industrially.

The microorganism capable of enhancing the growth or proliferation of microorganisms in the medium containing the nitrogen source of the present application may be Escherichia coli , but is not limited thereto.

The term " protein " in the present application means a protein expressed in a microorganism genetically engineered to express the protein. The protein may be the same or similar to a protein normally expressed in a microorganism, and includes a recombinant protein. In addition, the protein may be heterologous to a protein normally expressed in a microorganism. Or may be chimeric in that some of these proteins are the same or similar to proteins normally expressed in microorganisms while others contain chimeric sequences that are exogenous to the microorganism. For the purpose of the present application, the protein is not particularly limited, but may be an antibody or a fragment thereof. For the purpose of the present application, all of the proteins produced by the method of the present application using microorganisms containing the recombinant vector can be included.

In general, proteins produced using microorganisms containing a recombinant vector may contain various types of proteins such as active protein, truncated protein, inactive protein, and protein aggregate. However, for the purposes of the present application, the protein may refer to an active protein, and the active protein may have an active form, and may specifically refer to a L-H protein.

Specifically, in the present application, the protein may be an antibody, more specifically a monoclonal antibody.

The term " monoclonal antibody " as used herein refers to an antibody that a single antibody-forming cell can produce, and recognizes one antigenic determinant. The antibody of the present application is not limited thereto, and may include all therapeutic antibodies conventionally used in the art.

In addition, the antibody includes both the full-length antibody and the antibody fragment. The antibody fragments include Fv, Fab, Fab ', F (ab') ₂ , Fd and the like. The Fv comprises both double disulfide Fv (dsFv) and short chain Fv (scFv) forms. Fd refers to the heavy chain portion contained in the Fab fragment.

The protein produced by the production method of the present application can be used as a therapeutic protein. The term " therapeutic protein " as used in the present application is generally used to refer to proteins used in biomedicine, and refers to having various physiological activities. The physiological activity regulates genetic expression and physiological function and corrects abnormal conditions caused by deficiency or excessive secretion of substances involved in function regulation in vivo, and may include a general protein therapeutic agent .

In the present application, the therapeutic protein includes, but is not limited to, a protein having physiological activity in vivo, such as insulin, insulin secretion peptide, growth hormone hormone (GH) luteinizing hormone (LTH), follicle stimulating hormone (FSH) Factor VIII, Factor VII, Erythropoietin, Adiponectin, Antibody, antibody fragments scFv, Fab, Fab 'or F (ab') 2 days have.

The protein production method according to the present application can be usefully used for the expression of high yielding active protein, which is suitable for mass production while suppressing the production of unnecessary insoluble protein from microorganisms.

Fig. 1 shows the result of measuring the growth rate of the cells at each temperature.
Figure 2 shows SDS-PAGE and Western blot results at each temperature.
FIG. 3 shows the result of confirming the expression ratio of active protein per cell at each temperature.
FIG. 4 shows the result of measuring the growth rate of cells according to the nitrogen source concentration at low temperature (25 ° C.).
FIG. 5 shows the results of SDS-PAGE and Western blotting according to the nitrogen source concentration at low temperature.
FIG. 6 is a graph showing the amount of active protein expressed as a function of the concentration of nitrogen source at low temperature and the percent active protein relative to the total protein amount.
Fig. 7 shows the result of measuring the growth rate of the cells.

Hereinafter, preferred embodiments are presented to facilitate understanding of the present application. However, the following embodiments are provided to more easily understand the present invention, and the contents of the present application are not limited by the embodiments.

Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Condition Temperature 25 ℃ 25 ℃ 25 ℃ 37 ℃ 37 ℃ 37 ℃ The concentration of N (g / L) 18.9 49.6 74.8 18.9 49.6 106.7 62.2 74.8 106.7 Concentration of C (g / L) 70 40 40 70 40 40 Initial working volume (L) 0.05 2 30 0.05 2 2 result No single chain band
(Fig. 2) Growth (Fig. 4)
ELISA results (Table 3) Growth (Fig. 6)
ELISA results (Table 4) insoluble form insoluble form insoluble form

In preparing the medium for the process of the present application, there are various sources for each required substance, and may be purchased and used from the source if necessary. The reagent suppliers include Merck, Sigma-Aldrich, Wako, BD, and Dae-Jung. Further, the ratio of the composition to the above can be appropriately changed according to the embodiment.

Experimental Example  1. Protein production on flask scale

In order to determine the expression pattern of protein on the flask scale according to the temperature, the following procedure was performed. Experiments were carried out at 37 ° C, a temperature that is known to be suitable for microbial growth, and the control group was carried out at 18 ° C, 25 ° C and 33 ° C in each experimental group. Fermentation was carried out by a batch culture process and the growth was confirmed by measuring the absorbance at 562 nm. When the absorbance was 70% of the maximum absorbance, the fermentation broth was harvested and the amount of each protein was measured by ELISA.

Badge components Amount (g / L) Glucose 70 KH ₂ PO ₄ 0.75 MgSO ₄ .7H 2 O One MnSO ₄ .4H ₂ O 0.04 FeSO ₄ .7H 2 O 0.75 ZnSO ₄ .7H ₂ O 0.05 Nitrogen source 18.9 L-met 0.3

As a result, it was confirmed that the microbial growth remained at a very low level even after 24 hours from the initiation of the culture in the batch culture process at the incubation temperatures of 18 캜 and 25 캜 (Fig. 1). As a result, it was found that the growth rate was significantly decreased at a growth temperature below 25 ° C, and there was a difference in growth rate depending on the temperature.

2 and 3, it was confirmed that the ratio of the insoluble protein was greatly reduced and the ratio of the active protein was increased at a temperature of 20 ° C or more and less than 30 ° C.

Experimental Example  2. Production of protein in 5L class fermenter

In order to confirm the expression pattern of protein according to the amount of nitrogen source at the low temperature culture, the experiment was performed in the following order on the 5L scale. Specifically, the nitrogen source was added in an amount of 49.6 g / L to 106.7 g / L, and the degree of protein expression was checked after fermentation for 40 hours or more at a temperature of 20 ° C or more and less than 30 ° C. Fermentation was carried out by Fed-batch culture and the growth was confirmed by measuring the absorbance at 562 nm. When the absorbance was about 70% of the maximum absorbance, the fermentation broth was harvested and the amount of each protein was measured by ELISA.

Badge components Amount (g / L) Glucose 40.0 MgSO ₄ -7H ₂ O 1.8 KH ₂ PO ₄ 3.00 Citrate monohydrate 1.10 Potassium chloride 1.00 Amm. Fe (III) citrate 0.13 MnSO ₄ -H ₂ O 0.023 CuSO ₄ -5H ₂ O 0.024 H ₃ BO ₃ 0.009 ZnSO ₄ -7H ₂ O 0.013 CoCl ₂ -6H ₂ O 0.019 Na ₂ MoO ₄ 2H ₂ O 0.031 antifoam 5 N-source 49.6, 62.2, 74.8, 106.7

As a result, it was confirmed that as the concentration of nitrogen source increases at low temperature, the growth rate increases and the maximum cell number arrival time is shortened and the maximum cell number is also increased (FIG. 4). In addition, it was confirmed that the reduction in productivity at low temperature can be overcome by adding a nitrogen source in excess (FIG. 6).

This suggests that fermentation conditions through low temperature fermentation and the addition of excess nitrogen source overcome low production rates in the production of therapeutic proteins or antibodies through existing microorganisms.

Experimental Example  3. Protein production in a 50 L class fermenter

The amount of nitrogen source was fixed at 74.8 g / L, and the fermentation was carried out by Fed-batch culture and the growth was confirmed by measuring the absorbance at 562 nm. The fermentation broth was harvested when the absorbance was 70% or more relative to the maximum absorbance, and the amount of each protein was measured by ELISA.

Batch number Target protein production (mg / L) Batch 1 330.600 Batch 2 407.934

The reproducibility of the fermentation was confirmed (Fig. 7) and the ELISA measurement showed that the target protein was secured at 200 mg / L per batch (Table 4). This confirms that patent conditions can be secured at a level that is industrially available.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present application is to be interpreted as being within the scope of the present application, all changes or modifications derived from the meaning and scope of the appended claims and from their equivalents rather than the detailed description.

Claims (8)

(a) fermenting a microorganism containing a recombinant vector at a temperature of 20 ° C or more and 30 ° C or less for 48 hours or more; And
(b) adding a medium containing an N-source before or during the fermentation of the microorganism in step (a).
The method according to claim 1,
Wherein the medium containing the nitrogen source comprises one or more components selected from the group consisting of ammonium sulfate, yeast extract or a combination thereof.
The method according to claim 1,
Wherein the medium containing the nitrogen source is a medium containing no animal-derived material.
The method according to claim 1,
Wherein the concentration of the nitrogen source is from 49.6 g / L to 106.7 g / L.
The method according to claim 1,
Wherein the nitrogen source has a mass ratio to carbon source of 1: 0.15 to 1.24.
The method according to claim 1,
The microorganism is Escherichia coli ). < / RTI >
The method according to claim 1,
Wherein the protein is an antibody or a fragment thereof.
The method according to claim 1,
Wherein the protein is a therapeutic protein.

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