KR102040926B1 - Cell culture method at low temperature applicable to mass production of protein - Google Patents

Abstract

The present application relates to a method that can be usefully used for mass production of proteins using microorganisms by increasing the concentration of the nitrogen source at a low temperature of 20 ° C or more and less than 30 ° C. The process method according to the present application can be usefully used for high yield of active protein expression, which is suitable for mass production while suppressing the production of unnecessary insoluble proteins from microorganisms.

Description

Cell culture method at low temperature applicable to mass production of protein}

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

Currently, methods for producing recombinant proteins from microorganisms, especially when recombinant proteins are proteins derived from animal cells, often cause problems that inadequate folding occurs or aggregates to frequently produce inactive inclusion bodies. Furthermore, there is a problem that the use of this protein requires a second operation such as a very inefficient refolding.

Many studies have been conducted to overcome this problem and to increase the production of properly folded, active protein. Basically, in order to produce an active protein in a microorganism, it is essential to insert a signal gene to induce a foldable position. Conventionally, the chaperone protein, which can prevent the formation of inclusion bodies and help folding, is expressed with the target protein (Korean Patent Publication No. 10-2009-0114422), or the induction conditions are changed to change the promoter (Champion et al. (2001) Proteomics, 1: 1133-1148 ), attempts have been made, such as changing the fermentation conditions (Schein (1989) Bio / Technology , 7: 1141-1149).

In addition, attempts have been made to inhibit the production of insoluble proteins by reducing the production rate of proteins by applying expression temperature conditions below 30 ° C. However, since the optimum growth temperature of general microorganisms is 33 ° C to 37 ° C, low temperature conditions below 30 ° C inevitably reduce the growth of microorganisms, resulting in a large decrease in the amount of target protein expressed in the microorganisms. As such, low yields of the production material can be very lethal in the production of large quantities of biological agents using microorganisms.

In order to solve this problem, researches such as changing a promoter into a low temperature resistant gene (Vasina and Baneyx (1996) Appl . Nviron . Microbiol , 1444-1447) have been conducted. However, this method has a serious disadvantage that 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.

On the other hand, attempts have been made to increase the yield of the target protein from microorganisms by controlling the constituents of the medium, but the risk of decreased growth and protein expression when excessive nitrogen sources are reported (Popplewell et al. (2005)). Methods in Molecular Biology, 308: 17-30.

Under this background, the present inventors have made intensive efforts to increase the production of protein, thereby increasing the concentration of the nitrogen source at low temperatures, thereby suppressing the production of unnecessary insoluble proteins from microorganisms, high yield active forms suitable for application in mass production. The present invention was completed by discovering a protein production method.

One object of the present application is to (a) fermenting a microorganism comprising a recombinant vector for at least 48 hours at a temperature of 20 ℃ to 30 ℃; And (b) adding a medium containing a nitrogen source (N-source) before or during fermentation of (a) the microorganism in step (a).

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

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

In addition, one of ordinary skill in the art can recognize or ascertain a number of equivalents to certain aspects of the present application described in this application using conventional experiments only. Also, such equivalents are intended to be included in this application.

One aspect of the present application for achieving the above object is, (a) fermenting a microorganism comprising a recombinant vector for at least 48 hours at a temperature of 20 ℃ 30 ℃ or less; And (b) adding a medium containing a nitrogen source (N-source) before or during fermentation of (a) the microorganism in step (a).

In general, in order to accelerate growth while reducing impurities in protein production, high temperature conditions or temperature shifting methods are attempted, and low temperature conditions are not generally tried because they are harsh to growth. However, this application is significant in that it was first identified that the protein production was increased not only on the lab scale but also on the mass production scale by over-adding a nitrogen source under low temperature conditions.

Specifically, step (a) is a step of fermenting a microorganism including a recombinant vector 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 may be used for fermentation without limitation as long as it includes a recombinant vector capable of expressing a protein of interest, and those skilled in the art can select and apply a microorganism including an appropriate recombinant vector according to the purpose. have.

As used herein, the term "microorganism comprising a recombinant vector" refers to a microorganism transformed with a vector having a gene encoding one or more target proteins.

In addition, the term "vector" as used herein refers to a DNA preparation containing a nucleotide sequence of a polynucleotide encoding said target protein operably linked to a suitable regulatory sequence such that the target protein can be expressed in a suitable host. . The regulatory sequence may comprise a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation. After being transformed into a suitable host cell, the vector can be replicated or function independent 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 can replicate in a host cell, and any vector known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as a phage vector or cosmid vector. , pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector and the like may be used, but are not limited thereto.

The vector usable in the present application is not particularly limited and known expression vectors may be used. In addition, a polynucleotide encoding a target protein may be inserted into a chromosome through a vector for intracellular chromosome insertion. Insertion of the polynucleotide into the chromosome can be made by any method known in the art, such as, but not limited to, homologous recombination. The method may further include a selection marker for checking whether the chromosome is inserted. Selection markers are used to select cells transformed with a vector, i.e., to confirm the insertion of a nucleic acid molecule of interest, and confer a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface proteins. Markers can be used. In an environment in which a selective agent is treated, only cells expressing a selection marker survive or exhibit different expression traits, so that transformed cells can be selected.

As used herein, the term "selection marker" is a gene that is resistant to antibiotics, and since the cells having the cells survive in the environment treated with the antibiotic, they are useful as a selection marker in the process of obtaining a large amount of plasmid from E. coli. do. Since the antibiotic resistance gene in the present application is not a factor that greatly affects the expression efficiency according to the optimal combination of vectors, which is the core technology of the present application, antibiotic resistance genes generally used as selection markers can be used without limitation. As specific examples, resistance genes for ampicillin, tetratracyclin, kanamycin, kanamycin, chloroamphenicol, streptomycin, or neomycin may be used, and more specifically, May be a tetracycline resistance gene, but is not limited thereto.

The term "transformation" in the present application means introducing a vector containing a gene encoding a protein of interest into the host cell to allow the protein encoded by the polynucleotide in the host cell. Transformed polynucleotides can include all of them, as long as they can be expressed in the host cell, either inserted into or located outside the chromosome of the host cell. The polynucleotide also includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be expressed by being introduced into a host cell. 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 elements necessary for self-expression. The expression cassette may include a promoter, a transcription termination signal, a ribosomal binding site, and a translation termination signal, which are typically operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self replication. In addition, the polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto. The transformation method may include any method of introducing a nucleic acid into a cell, and may be performed 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, lithium acetate-DMSO method and the like, but are not limited thereto.

In addition, the term "operably linked" means that the polynucleotide sequence is functionally linked with a promoter sequence for initiating and mediating the transcription of a polynucleotide encoding a target protein of the present application. Operable linkages can be prepared using known genetic recombination techniques, and site-specific DNA cleavage and ligation can be made using, but are not limited to, cleavage and ligation enzymes in the art.

In addition, "mass production" as used in the present application means a level that can be produced so that the amount of protein produced industrially available, for example, can be used in more than 5L scale, 30L scale, 50L scale or more. Means.

Specifically, the microorganism including the recombinant vector may be fermented for 48 hours or more at a temperature of 20 ° C or more and 30 ° C or less, but 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 step, and the fermentation at such a low temperature is a fermentation temperature newly identified by the present inventors.

Such low temperature fermentation temperature includes about 20 ° C or more and 30 ° C or less. As used herein, the term "about" includes all of the values of ± 0.5, ± 0.4, ± 0.3, ± 0.2, ± 0.1, etc., and includes all values in the range equivalent to or similar to those following the term about, It is not limited.

The low temperature may be 20 ° C or more and 30 ° C or less, more than 20 ° C and less than 30 ° C, for example, 21 ° C or more and 29 ° C or less, 21 ° C or more and less than 29 ° C, 22 ° C or more and 28 ° C or less, or 22 ° C or more and 28 ° C. Less than 23 ° C. and less than 27 ° C., more than 23 ° C. and less than 27 ° C., more than 24 ° C. and less than 26 ° C., more than 24 ° C. and less than 26 ° C., or about 25 ° C., but is not limited thereto.

In addition, the fermentation time may be more than 48 hours to less than 72 hours.

In the protein production method according to the microorganism fermentation of the present application, step (b) is a step of adding a medium containing a nitrogen source (N-source) before or during the fermentation of the step (a) microorganisms.

In particular, adding 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 or during fermentation of microorganisms.

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

Specifically, before the start of fermentation of microorganisms, the addition of a medium containing a nitrogen source is a batch culture in which a medium containing all the nutrients necessary for cell growth or material production is inoculated to culture the microorganisms. Hard to add For the purpose of the present application may include inoculating a microorganism containing a recombinant vector in a medium containing a nitrogen source and fermentation in a batch culture method.

In addition, the addition of a medium containing a nitrogen source during fermentation of the microorganism may be fed-batch culture to supply a medium containing one or more nutrients immediately after the start of fermentation or after the culture reaches a certain stage, or When depleted nutrients are depleted from the culture, it means culture in a manner that is continuously fed to the culture. In fed-batch cultures, a single nutrient, carbon source, is usually the limiting factor for growth. Other kinds of nutrient limitations may also be used, for example, by nitrogen sources, by oxygen, by sulfur and by phosphorus. For the purpose of the present application may include continuously supplying a medium containing a nitrogen source during fermentation of the microorganism containing the recombinant vector.

For the purposes of the present application, the medium includes both the medium used to culture the cells, the medium used to maximize the production of the protein of interest, and the like.

Specifically, "cell culture medium" or "culture medium" means a nutrient solution for the maintenance, growth, proliferation or expansion of cells in an artificial in vitro environment outside of a multicellular organism or tissue. Cell culture media can be optimized for specific cell cultures, such as basal culture media formulated to support cell growth, or cell culture production media, nutrients formulated to promote production of the desired protein, to high concentrations. May be made of concentrated medium. The terms nutrients and media components refer to components that constitute the media, and are used interchangeably herein.

Specifically, the term "basal culture medium" or "basal medium" refers to a medium capable of supporting minimal growth of cells. The basal medium means a medium for supplying vitamins and inorganic salts in addition to carbon and nitrogen sources.

Also specifically, the term "cell culture production medium" or "production medium" refers to a medium used for the purpose of maximizing expression of a target protein in a bioreactor. The production medium may be the same as or different from the base medium, and if it is different, it may be produced by concentrating the base medium itself or adding specific ingredients to the base medium.

In addition, the "feeding medium" and "addition culture medium" is a medium consisting of a specific nutrient or a plurality of nutrients may be all concentrated products of the base medium, but is not limited thereto, the feeding medium according to the cells cultured by those skilled in the art Various components and concentrations can be produced.

In addition, when all the sugar in the medium is used as the cell growth during fermentation for the purpose of the present application can further increase the fermentation time by adding more sugar, wherein the amount of nitrogen source added is 10 g / L to 80 It may be in the range of g / L, specifically may be in the range of 15 g / L to 70 g / L, more specifically in the range of 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.

The concentration of the nitrogen source in the conventional medium is 2 ~ 14g / L, but the excess amount is added to the concentration of the nitrogen source in the conventional medium, specifically, the concentration of the nitrogen source may be in the range of 30 g / L to 150 g / L, More specifically, it may range 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 mass ratio of 1: 0.15 to 1.24 relative to the carbon source, but is not limited thereto.

For the purposes of the present application, while fermenting microorganisms at low temperature, the production of insoluble proteins and inclusion bodies is extremely suppressed, and the amount of nitrogen source is put in a range beyond conventionally known methods to recover microbial growth at low temperatures and The quantities can be mass produced to industrial use.

The microorganism that can improve the growth or proliferation of the microorganism with a medium containing a 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 of interest. The protein may be the same as 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 the protein may be chimeric in that some contain the same or similar to a protein normally expressed in the microorganism, while others contain an amino acid sequence that is exogenous to the microorganism. For the purposes of the present application, the protein is not particularly limited thereto, but may be an antibody or a fragment thereof. For the purposes of the present application, any protein produced by the method of the present application using a microorganism including a recombinant vector may be included.

In general, a protein produced using a microorganism including a recombinant vector may be mixed with various types of proteins such as active proteins, truncated proteins, inactive proteins, and protein aggregates. However, for the purposes of the present application, the protein may mean an active protein, and the active protein may mean an active form, and specifically, may mean a L-H form protein.

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

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

In addition, the antibody is a concept including both the full-length antibody and antibody fragments, the type of the antibody fragment includes all Fv, Fab, Fab ', F (ab') ₂ , Fd and the like. The Fv includes both double disulfide Fv (dsFv) and short chain Fv (scFv) forms. Fd means the heavy chain portion included in the Fab fragment.

The protein prepared by the method of the present application can be used as a therapeutic protein. The term "therapeutic protein" as used in the present application is a general term for a protein commonly used in biomedical medicine, and means various biological activities. The physiological activity is to adjust the genetic expression and physiological function to correct the abnormal condition due to the deficiency or excessive secretion of substances involved in the function control in vivo, it may include a general protein therapeutics .

The therapeutic protein in the present application includes without limitation, as long as it has a physiological activity in vivo, such as insulin, insulin secretion peptide, growth factor hormone (GH) luteinizing hormone (LTH), follicle stimulating hormone (FSH), Blood coagulation factor 8 (Factor,), blood coagulation factor (Factor,), erythropoietin, adiponectin, antibody (Antibody), antibody fragment scFv, Fab, Fab 'or F (ab') 2 have.

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

1 is a result of measuring the cell growth rate at each temperature.
Figure 2 shows the SDS-PAGE, Western blot results at each temperature.
3 shows the results of confirming the expression rate of the active protein per cell at each temperature.
4 is a result of measuring the cell growth rate according to the nitrogen source concentration at low temperature (25 ℃).
Figure 5 shows the SDS-PAGE, Western blot results according to the nitrogen source concentration at low temperatures.
Figure 6 is a graph confirming the active protein expression amount and the active protein% compared to the total protein amount with increasing concentration of nitrogen source at low temperature.
Figure 7 is the result of measuring the cell growth rate.

Hereinafter, preferred examples are provided to aid in understanding the present application. However, the following examples are merely provided to more easily understand the present application, and the contents of the present application are not limited by the examples.

Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Condition Temperature 25 ℃ 25 ℃ 25 ℃ 37 ℃ 37 ℃ 37 ℃ 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
(Figure 2) Growth (Figure 4)
ELISA results (Table 3) Growth (Figure 6)
ELISA results (Table 4) large amount of insoluble form large amount of insoluble form large amount of insoluble form

In preparing the medium for the process of the present application, there are various sources for each of the necessary materials and can be purchased and used from the source as needed. Reagent supply sources include, but are not limited to, Merck, Sigma-Aldrich, Wako, BD, Dae-Jung, and others. In addition, the ratio of the composition to this can be suitably changed according to embodiment.

Experimental Example  1. Protein production on flask scale

In order to confirm the expression of the protein according to the temperature on the Flask scale, the experiment was performed in the following order. The control group was experimented at 37 ℃, a temperature known to be suitable for microbial growth, each experimental group proceeded to 18 ℃, 25 ℃, 33 ℃ and the rest of the conditions were performed in the same manner as Table 2. Fermentation was carried out in a batch culture process, and growth was confirmed by measuring absorbance at 562 nm. The fermentation broth was harvested when 70% of the absorbance was shown and the amount of each protein was measured by ELISA.

Badge component Volume (g / L) Glucose 70 KH ₂ PO ₄ 0.75 MgSO ₄ ㆍ 7H2O One MnSO ₄ ㆍ 4H2O 0.04 FeSO ₄ ㆍ 7H2O 0.75 ZnSO ₄ ㆍ 7H2O 0.05 Nitrogen source 18.9 L-met 0.3

As a result, in the batch culture process of the culture temperature of 18 ℃ and 25 ℃ it was confirmed that even after 24 hours after the start of the culture microbial growth stays at a very low level (Fig. 1). Through this, the growth rate is significantly reduced at the culture temperature of less than 25 ℃, it can be seen that there is a difference in growth rate according to the temperature.

In addition, the results of FIGS. 2 and 3 confirmed that the ratio of insoluble protein was significantly reduced and the ratio of active protein was increased at a temperature of 20 ° C. or more and less than 30 ° C.

Experimental Example  2. Protein Production in 5L Fermenter

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

Badge component Volume (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, the growth rate increases with increasing concentration of the nitrogen source at low temperature, it was confirmed that the maximum cell number reaching time is shortened and the maximum cell number is also increased (Fig. 4). In addition, it was confirmed that the excess of the nitrogen source to overcome the decrease in productivity at low temperatures (Fig. 6).

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

Experimental Example  3. Protein production in 50L fermenter

The amount of nitrogen source was fixed at 74.8 g / L, the fermentation was fed to a 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 higher compared to the highest absorbance, and the amount of each protein was measured by ELISA.

Batch Number Protein Production (mg / L) Batch 1 330.600 Batch 2 407.934

The growth of the two batches was very similar and the reproducibility of the fermentation was confirmed (FIG. 7). As a result of ELISA measurement, the target protein was confirmed to be more than 200 mg / L per batch (Table 4). Through this, it was confirmed that the patent condition can secure the industrially available level of process.

From the above description, those skilled in the art will appreciate that the present application can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present application should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present application.

Claims (8)

(a) fermenting Escherichia coli comprising the recombinant vector to a temperature of 20 ° C. or higher and 30 ° C. or lower for at least 48 hours; And
(b) adding a medium containing a nitrogen source (N-source) before or during fermentation of E. coli of step (a), the method for producing protein by E. coli fermentation, the concentration of the nitrogen source is 49.6 g / L to 106.7 g / L, protein production method by microbial fermentation.
The method of claim 1,
The medium containing the nitrogen source is ammonium sulfate (Ymmonium sulfate), yeast extract (Yeast extract) or a combination of any one or more components selected from the group consisting of a combination of them, E. coli fermentation protein production method.
The method of claim 1,
The medium containing the nitrogen source is a medium containing no animal-derived material, a method for producing protein by E. coli fermentation.
delete The method of claim 1,
The nitrogen source is a mass ratio of carbon source 1: 1: 0.15 to 1.24, E. coli fermentation protein production method.
delete The method of claim 1,
The protein is an antibody or fragment thereof E. coli fermentation protein production method.
The method of claim 1,
The protein is a therapeutic protein, E. coli fermentation protein production method.

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