JP7214962B2 - Alkaline phosphatase high expression animal cells - Google Patents

Description

The present invention relates to a method for producing recombinant alkaline phosphatase.

Alkaline phosphatase (hereinafter abbreviated as ALP) is an enzyme that is often used along with horseradish peroxidase in detection systems such as antigen-antibody reactions. Among ALPs isolated from various animals, bovine small intestine-derived enzymes are mainly used due to their high specific activity. In addition, it is known that natural ALP has a highly active form (CIPII) and an intermediately active form (CIPI). was one of the issues.

In recent years, a method for producing highly active recombinant ALP in CHO cells using genetic engineering technology (Patent Document 1) and a method for expression in Pichia yeast have been reported (Patent Document 2).
The expression system in CHO cells (animal cells) trims the sugar chains added in the intracellular secretory pathway and corrects them into an appropriate shape, so it has the advantage of obtaining a sugar chain close to the natural type. It has been reported that there is a problem in economic efficiency due to the small amount (Patent Document 2).
Although an expression level sufficient for industrial use has been obtained in Pichia yeast, there is a strong tendency for excess N-glycans to be added when expressing proteins in yeast. This causes problems such as lack of flexibility (Non-Patent Document 1). These are considered to interfere with purification by gel filtration after ALP labeling in immunoassays. Moreover, it is apprehended that it may cause an allergenic problem in medical applications.
In Non-Patent Document 3, the heterogeneity of glycosylation in ALP expression in yeast is eliminated by introducing mutations into the amino acids of ALP using a genetic recombination technique. , the primary structure is different from that of natural ALP, and the sugar chain structure to be modified is also different.
Until now, it has been known that ALP closest to the natural form can be produced when ALP is expressed in animal cells, but this has not been implemented due to economic problems due to the low expression level.

Japanese Patent No. 4295386 Japanese Patent No. 3657895 JP 2008-5734 A

Dean, Biochim. Biophys. Acta, 1426, 309-322 (1999)

An object of the present invention is to provide an animal cell expression strain and production method capable of producing ALP at an economical expression level.

The present invention, which has been made in view of the above problems, includes the following aspects.
[1]
A method for producing alkaline phosphatase comprising the following steps (A) to (C), wherein the production amount of alkaline phosphatase is 100 mg/L or more:
(A) introducing into mammalian cells a gene encoding an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 3;
(B) selecting mammalian cells into which the gene has been introduced using drug resistance;
(C) A step of amplifying the gene introduced in (B) above using drug resistance.
[2]
The method according to [1], wherein the production of alkaline phosphatase is 300 mg/L or more.
[3]
The method of [1] or [2], wherein the mammalian cells are myeloma cells.
[4]
The method according to any one of [1] to [3], wherein the gene amplification using drug resistance in step (C) is a dhfr-MTX gene amplification method.
[5]
The method of [4] using mutant dhfr.
[6]
[1] to [5], further comprising the step of culturing the mammalian cells with increased alkaline phosphatase production obtained in step (C) in a medium, wherein the medium contains 10 to 250 μM of zinc ions. Any method described.
[7]
The method according to [6], wherein the cell culture substrate in the culturing step is a hollow fiber.
[8]
A gene encoding an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 3 was amplified by the dhfr-MTX gene amplification method using mutant dhfr, and the alkaline phosphatase production amount was 100 mg/ Myeloma cells that are ≧mL.
[9]
A method for producing alkaline phosphatase-producing mammalian cells comprising the following steps (A) to (C), wherein the cells have an alkaline phosphatase-producing capacity of 100 mg/L or more:
(A) introducing into mammalian cells a gene encoding an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 3;
(B) selecting mammalian cells into which the gene has been introduced using drug resistance;
(C) A step of amplifying the gene introduced in (B) above using drug resistance.

The present invention will be described in more detail below.
(1) ALP gene sequence Since the 3′ region of the native alkaline phosphatase gene contains a sequence encoding a GPI anchor, when expressed as a recombinant protein containing this region, It is expressed as a protein on the cell membrane. Since expression on the cell membrane complicates the purification process, it is preferable to shorten the 3' amino acid within a range that does not affect ALP activity. A sequence shorter than the 500th amino acid shown in SEQ ID NO: 1 is preferred, and a sequence shorter than the 490th amino acid is more preferred.
(2) Mammalian Cells Mammalian cells used for expression are not particularly limited, and cells generally used for recombinant protein production may be used. Examples include CHO cells, HEK293 cells, and myeloma cells. Among them, myeloma cells deficient in HGPRT (hypoxanthine/guanine phosphoribosyltransferase) have only a DNA synthesis system by the de novo pathway. It is preferable because there is no need to consider DNA synthesis by cytotoxicity, and a serum-containing medium or HT (hypoxanthine-thymidine)-containing medium can be used.
(3) Gene Introduction Method Gene introduction into mammalian cells is not particularly limited as long as it allows efficient gene introduction. Examples include lipofection, calcium phosphate coprecipitation, electroporation, and the like. Among them, gene transfer by electroporation is preferred from the viewpoint of transfer efficiency.
(4) Cell Selection Based on Drug Resistance Cell selection based on drug resistance may be performed by introducing a drug resistance gene into an expression vector. Neomycin, hygromycin, puromycin resistance genes, etc. may be used as drug resistance genes. For example, selection may be performed by introducing a neomycin resistance gene onto the protein expression vector and adding G418 to the cell culture medium.
(5) Gene Amplification The gene amplification method in the present invention is not particularly limited as long as it is a method for increasing the target expression protein gene in mammalian cells. For example, IR/MAR (mammalian replication initiation region/nuclear matrix attachment region) gene amplification method, dhfr/MTX gene amplification method, and the like can be exemplified. When the dhfr/MTX gene amplification method is performed using myeloma cells, the dhfr gene to be introduced into the expression vector is preferably a mutant with low sensitivity to MTX. When performing gene amplification using MTX, the cells acquired resistance to MTX by amplifying a mutant dhfr gene with low drug sensitivity, compared to amplifying the dhfr gene originally possessed by myeloma cells. Gene amplification is advantageous because it is easy to

Mutant dhfr may be those described in Document 1 (Proc. Natl. Acad. Sci USA 80 (1983) 2495), or those described in Document 2 (Proc. Natl. Acad. Sci USA 90 (1993) 11797). You may use the thing of description. In particular, in the present invention, it is preferable to use a mutant in which the 22nd position of the dhfr amino acid sequence described in Document 1 is substituted with arginine.
(6) Culture Since ALP requires zinc ions in its active center, it is preferable to add a zinc salt to the extent that it does not show toxicity to the cells to be cultured. The concentration of zinc salt added is preferably 10 μM to 250 μM, more preferably 15 μM to 30 μM. Examples of zinc salts include, but are not limited to, zinc sulfate, zinc acetate, and zinc chloride. A preferable example is zinc chloride.

The culture substrate is also not particularly limited. Static culture may be performed using a culture petri dish, or shaking culture using a flask may be performed to increase the cell density. In addition, in order to control the nutritional state in the culture medium, culture may be performed using a jar fermenter, or in order to concentrate ALP in the medium, culture may be performed using hollow fibers. Especially in the present invention, it is preferable to carry out concentration culture using hollow fibers, in order to facilitate subsequent purification steps. The medium may be one commonly used for culturing animal cells, and E-RDF (Kyokuto Pharmaceutical Co., Ltd.) can be exemplified, but is not limited to these.
(7) Production volume
As described in JP-A-2017-192381, evaluation of the production amount may be performed by sandwich ELISA using two anti-ALP antibodies for ALP in the culture supernatant, or the activity value of ALP in the culture ( U/mL) may be measured and calculated together with the specific activity (U/mg) measured after purification.
(8) Purification Purification of ALP may be performed using mammalian cell culture supernatants by methods known to those skilled in the art. For example, as described in JP-A-2017-192381, a chromatographic separation method, particularly a method using a hydrophobic column and an anion exchange column, may be used.

According to the method of the present invention, it is possible to construct mammalian cells that produce highly active ALP at an economical expression level and to obtain purified ALP.

FIG. 4 shows ALP expression levels by cloned cells at each stage of MTX gene amplification. FIG. 10 is a chromatogram separated by a Phenyl-5PW column obtained in Example 9. FIG. FIG. 10 is a chromatogram separated by a DEAE-5PW column obtained in Example 9. FIG. FIG. 10 shows the results of gel filtration analysis obtained in Example 10. FIG.

[Example 1] Preparation of a gene encoding ALP Based on the ALP amino acid sequence (CIPII-CIPI chimera protein, SEQ ID NO: 1) described in JP-A-2017-192381, the signal sequence described in Japanese Patent No. 6131074 A gene (SEQ ID NO: 3) encoding the amino acid sequence of SEQ ID NO: 2 in which the signal sequence was substituted and the C-terminus was D at position 487 was synthesized and incorporated into an animal cell expression vector having a neomycin-resistant gene sequence.

[Example 2] Preparation of Mutant dhfr GeneJ. Biochem. , 108, 673; 1990. Based on the gene sequence of pECE-dhfr used in Proc. Natl. Acad. Sci USA 80 (1983) 2495 to the mutated dhfr gene sequence. The generated mutated dhfr gene sequence was incorporated into the expression vector described in Example 1.

[Example 3] Gene transfer into mammalian cells and drug screening The ALP expression vector prepared in Examples 1 and 2 was introduced into myeloma cells by electroporation using the Neon Transfection system (manufactured by Thermo Fisher). bottom. The myeloma cells after the gene transfer were cultured in E-RDF medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.) containing 10% bovine serum in a petri dish for suspension culture (manufactured by Sumitomo Bakelite Co., Ltd.) for one day, and then G418 (manufactured by Thermo Fisher Co., Ltd.) was added. It added so that it might become 500 micrograms/mL, and culture|cultivated for 1 week. After culturing in a G418-containing medium for one week, cells were counted and seeded in a 384-well culture plate (manufactured by Greiner) at 0.5 cells/well.

[Example 4] Screening of ALP-expressing strains by immunoassay Among the cells seeded on a 384-well culture plate as in Example 3, screening for which wells have a high expression level can be performed by the immunoassay method shown below. gone.
(1) Immobilization buffer (12 mM Na ₂ CO ₃ , 38 mM NaHCO ₃ , pH 9.6), anti-ALP antibody (APA05.3 antibody described in JP 2017-192381) to 1 μg / ml , 50 μL was added to a 384-well ELISA plate, incubated at room temperature for 1 hour, and APA05.3 was immobilized on the ELISA plate.
(2) After washing the ELISA plate three times with a washing buffer (20 mM tris(hydroxymethyl)aminomethane, 150 mM sodium chloride, 0.1% Tween 20), PBS containing 3% BSA was added for 1 hour. The ELISA plate was blocked by incubating at room temperature.
(3) After that, the supernatant of the 384-well culture plate prepared in Example 3 was dispensed at 50 µL/well with PBS containing 0.3% BSA-PBS using a replicator described in JP-A-2008-79517. It was transferred to an ELISA plate and allowed to react for 1 hour.
(4) After washing with washing buffer, 4-MUP solution (1 M diethanolamine, 0.5 mM magnesium chloride, 1 mM 4-methylumbelliferyl phosphate (4-MUP)) was added and incubated at room temperature for 30 minutes. . The fluorescence intensity of the ELISA plate (excitation wavelength 360 nm, emission wavelength 465 nm) was measured with a plate reader.

Cells were recovered from the upper wells exhibiting high fluorescence intensity, and gene amplification was performed.

[Example 5] Gene amplification using drug resistance (dhfr-MTX gene amplification method)
The clones with high expression levels obtained in Example 4 were subjected to increased expression levels by the dhfr-MTX gene amplification method. E-RDF medium containing 25 μM zinc chloride and 10% bovine serum was used as the medium. First, the clones obtained in Example 4 were cultured in the medium containing 4 μM MTX. The culture was continued for 3 to 4 weeks until the cell growth rate reached the same level as before the addition of MTX. Then, the cells were seeded on a 384-well culture plate (manufactured by Greiner) at 0.5 cells/well. After 1 to 2 weeks, clones with amplified ALP expression levels were selected by the immunoassay described in Example 4.

Based on the selected clones, clones with amplified ALP expression levels were selected by the same method as above using a medium containing 40 μM MTX.

FIG. 1 shows the ALP expression level by the cloned cells at each stage of MTX addition.
[Example 6] Cultivation of ALP-expressing cells in petri dishes Static culture was performed in a 37°C, 5% CO ₂ incubator for cell culture using a suspension culture petri dish. E-RDF medium containing 10% calf serum and 25 μM ZnCl ₂ was used as the culture medium. When measuring the expression level, the cell concentration was adjusted to 1×10 ⁵ Cells/mL, and after static culture for 10 days, the ALP activity of the culture supernatant was measured.
[Example 7] Measurement of activity of ALP in culture supernatant ALP activity (U/mL) in the culture supernatant was measured by the method shown below.
(1) The culture supernatant was diluted with ALP diluent (20 mM Tris-HCl pH 7.5, 10 mM MgCl ₂ , 10 μM ZnCl ₂ , 2 mg/mL BSA, 0.1% NaN ₃ ) to prepare a dilution series.
(2) Then add (1) to 500 μL of activity measurement buffer (391 mg of paranitrophenyl phosphate (PNPP) in 100 mL of 0.1 M glycine buffer (pH 9.6, 1 mM MgCl2, 0.1 mM ZnCl2, 1 mg/mL BSA)). 20 μL was added.
(3) Absorbance at 405 nm was measured after standing at 25°C for 10 minutes.
(4) By comparing a diluted measurement solution with an absorbance of around 1.0 and a standard sample with a known activity concentration (0.597 U / mL, bovine small intestine ALP ALPI-13G Lot 1706AA, Biozyme), Activity of ALP in the serum was measured.
Table 1 shows the ALP activity in the culture supernatant of the cloned cells at each stage of MTX addition.

[Example 8] Cultivation of ALP-expressing cells using hollow fibers Cell culture using hollow fibers was performed using a FiberCell Systems cartridge (cellulose membrane MWCO; 5 kDa, FiberCell). The cells were expanded in a suspension culture petri dish, and 3.3×10 ⁸ cells were seeded on hollow fibers. E-RDF medium containing 10% bovine serum and 25 μM ZnCl ₂ was used as the medium for ECS (Extracapillary Space), and E-RDF medium containing 25 μM ZnCl ₂ for ICS (Inner Capillary Space) was used. Cell culture was performed in a 37° C., 5% CO ₂ incubator. Cell growth was confirmed by monitoring glucose consumption in the medium.
[Example 9] Purification of ALP ALP was purified by the following method.
(1) Ammonium sulfate was added to the culture supernatant to 55% saturation, allowed to stand at 4°C for 0.5 hours, and then centrifuged at 8000 rpm at 4°C for 20 minutes to obtain a salted-out ammonium sulfate precipitate as a precipitate fraction. rice field.
(2) The precipitated fraction was dissolved in PBS to ammonium sulfate concentration of 1 M, and separated by high performance liquid chromatography (HPLC) connected to a Phenyl-5PW column (21.5 mm×150 mm, Tosoh Corporation). Phenyl-5PW column chromatography was performed by using PBS containing 1M ammonium sulfate as an equilibration buffer and performing linear gradient elution from 1M to 0M ammonium sulfate at a flow rate of 5 ml/min for 60 minutes. The chromatogram at that time is shown in FIG.
(3) Collect the fraction shown in FIG. 2, add ammonium sulfate to 60% saturation, leave at 4° C. for 0.5 hours, then centrifuge at 4° C., 8000 rpm for 20 minutes to obtain a precipitated fraction. An ammonium sulfate precipitate was obtained. After that, it was equilibrated with 20 mM Tris-HCl buffer (pH 7.5, 1 mM MgCl ₂ , 10 μM ZnCl ₂ ) by dialysis.
(4) After that, after supplying DEAE-5PW (25 mm × 150 mm, Tosoh Corporation) equilibrated with 20 mM Tris-HCl pH 7.5 buffer, the sodium chloride concentration was linearly adjusted from 0 M to 0.25 M for 60 minutes. Gradient elution was performed. Fractions were taken into test tubes every minute, and the absorbance at 280 nm (A280) and the activity (A405) by the PNPP method were measured for each eluted fraction. A405 was measured by the method described in Example 6 using each fraction diluted 4000 times. The results are shown in FIG. The resulting high specific activity (A405/A280) fraction contains highly pure ALP. Fraction no. 21 to 26 were collected, ammonium sulfate was added to 60% saturation, allowed to stand at 4°C for 0.5 hours, and then centrifuged at 8000 rpm at 4°C for 20 minutes to obtain a salted-out ammonium sulfate precipitate as a precipitate fraction. rice field. After that, it was equilibrated with 20 mM Tris-HCl buffer (pH 7.5, 1 mM MgCl ₂ , 10 μM ZnCl ₂ ) by dialysis.
[Example 10] Confirmation of Purity and Specific Activity of Purified Mutants In order to confirm the purity of the purified ALP, the purity was confirmed using a gel filtration column (G3000swxl Tosoh Corporation). As a gel filtration control, commercially available highly active bovine intestinal ALP (ALPI-13G Lot 1706AA, Biozyme) was also analyzed at the same time. From this result, it was estimated that both purities were equivalent (Fig. 4).

Table 2 shows the results of comparing the specific activity of ALP by the PNPP method. Protein concentration was calculated using the A280 absorbance of 1 mg/ml ALP as 0.76.

Claims (2)

A method for producing alkaline phosphatase comprising the following steps (A) to (C),
(A) introducing a gene encoding the amino acid sequence represented by SEQ ID NO: 1 into mammalian cells;
(B) selecting mammalian cells into which the gene has been introduced using drug resistance;
(C) A step of amplifying the gene introduced in (B) above using drug resistance;
said mammalian cells are myeloma cells,
Gene amplification using drug resistance in step (C) is a dhfr-MTX gene amplification method, and a mutant dhfr gene that is less sensitive to methotrexate than the dhfr gene originally possessed by myeloma cells is expressed as an expression vector. using what was introduced in
further comprising the step of culturing the mammalian cells with increased alkaline phosphatase production obtained in step (C) in a medium, wherein the medium contains 10 to 250 μM of zinc ions;
Method. 2. The method according to claim 1 , wherein the cell culture substrate in the culturing step is hollow fiber.

Images