CN111349614B - Method for improving culture density of CHO cells - Google Patents

Method for improving culture density of CHO cells Download PDF

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CN111349614B
CN111349614B CN201811579340.9A CN201811579340A CN111349614B CN 111349614 B CN111349614 B CN 111349614B CN 201811579340 A CN201811579340 A CN 201811579340A CN 111349614 B CN111349614 B CN 111349614B
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郭清城
徐进
杨立敏
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Shanghai Maitai Junao Biotechnology Co ltd
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12Y101/01027—L-Lactate dehydrogenase (1.1.1.27)
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01028—D-Lactate dehydrogenase (1.1.1.28)
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    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

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Abstract

The invention provides a method for improving the culture density of CHO cells, which is characterized in that exogenous lactate dehydrogenase genes are transferred into CHO cells and expressed in the fermentation process, so that the lactic acid produced in the culture process can be reduced, and the culture density of CHO cells in a bioreactor is improved.

Description

Method for improving culture density of CHO cells
Technical Field
The invention relates to the technical field of biological medicines, and in particular discloses a method for improving the culture density of CHO cells.
Background
CHO (Chinese Hamster Ovary) cells are a chinese hamster ovary fibroblast cell line constructed by Puck in 1957, and CHO cells have been the most important expression or production system for biotechnology drugs to date. Along with the development of serum-free suspension culture technology, genetic engineering technology, bioreactor design amplification and reinforcement technology, large-scale high-density fed-batch and continuous perfusion culture technology and the like, the CHO cell system is widely applied to research and development and industrial production of biotechnology products such as antibodies, gene recombinant protein drugs, virus vaccines and the like. CHO cells are the first system for current recombinant glycoprotein production, because of its accurate post-transcriptional modification function, the expressed protein is closer to the native protein molecule in terms of molecular structure, physicochemical properties and biological functions, and more than 60% of the currently marketed antibody drugs are expressed by CHO cells.
Biotechnology products are widely used in the medical field, and the annual sales of antibody drugs exceeds 1000 hundred million dollars only, and the annual sales of single-drug sales champion adalimumab exceeds 100 hundred million dollars. In industrialized culture fermentation, the protein expression level of the unit volume of the bioreactor is a main determinant of production cost, and the high expression level can obviously reduce the requirement on the scale of the bioreactor, reduce the consumption of culture medium, improve the purification efficiency and reduce the purification cost, so that the improvement of the protein expression level of the unit volume has important significance for the biomedical industry.
The protein expression level per unit volume in the bioreactor can be decomposed into two factors, namely, the density of living cells and the protein secretion level of single cells. Lactic acid accumulation is a significant cause of limiting cell density during CHO cell growth, particularly at the later stages of the bioreactor fermentation culture process. Since lactic acid causes a decrease in pH of the culture medium, and adding alkaline substances to adjust pH in the bioreactor causes an increase in osmotic pressure and thus adversely affects cell growth, and finally causes a decrease in cell viability and protein yield, it is a difficult problem to solve the effect of lactic acid accumulation in the bioreactor on CHO cell density.
The LDH-C (lactate dehydrogenase C) gene is present in the mitochondria of sperm cells, testis cells and certain tumor cells, and as a subtype of lactate dehydrogenase, LDH-C preferentially uses lactate as a substrate to catalyze the reaction of lactate in the direction of pyruvate as compared to lactate dehydrogenases A and B. Earlier-stage researches show that CHO cells transfected and over-expressing exogenous human LDH-C genes have higher cell density and cell activity rate under the condition of adding lactic acid and sodium pyruvate, and the lactic acid concentration of the CHO cells is obviously reduced compared with that of a control group; in addition, cells overexpressed by LDH-C have faster proliferation and greater resistance to apoptosis.
Disclosure of Invention
In order to further improve the culture density of CHO cells in a bioreactor, the invention provides a novel method, and the technical scheme is as follows:
exogenous lactate dehydrogenase gene was transferred into CHO cells and expressed during fermentation.
Preferably, the cells are CHO/dhFr - Cells (ATCC accession No. CRL-9096).
Preferably, the protein encoded by the exogenous lactate dehydrogenase gene has the amino acid sequence of SEQ ID NO. 1.
Preferably, the exogenous lactate dehydrogenase gene has the nucleotide sequence of SEQ ID NO. 2.
The invention has the beneficial effects that the culture density of CHO cells in the bioreactor can be greatly improved, thereby improving the yield of recombinant protein in unit volume in the bioreactor, reducing the requirement of the production process of biological products on the scale of the bioreactor and the consumption of cell culture medium, improving the production efficiency and finally reducing the production cost of biological products.
Drawings
FIG. 1 is a graph showing the results of immunohybridization of semi-quantitative analysis of lactate dehydrogenase expression levels in example 2;
FIG. 2 example 3 cell density versus time in high density culture of CHO cells overexpressing lactate dehydrogenase;
FIG. 3A graph of cell viability versus time in culture of CHO cells overexpressing lactate dehydrogenase in high density culture;
FIG. 4A plot of cell density versus time in culture of CHO cells overexpressing lactate dehydrogenase under 40 mmol/L lactic acid addition;
FIG. 5 is a schematic diagram showing the difference in amino acid sequence between SEQ ID NO. 1 and wild-type hamster lactate dehydrogenase C (Uniprot protein database No. Q06BU 8).
Detailed Description
Example 1 construction of expression cell lines
CHO cell lines overexpressing human lactate dehydrogenase C were constructed according to the method described in "construction of CHO cell lines overexpressing human LDH-C and study of the effects of the same on lactate metabolism and apoptosis" (university of Suzhou's university's treatise, ind. Jing Yu, 2014), cloning the human LDH-C gene (NM-002301.4), constructing eukaryotic expression vector pcDNA3.1/hLDH-C, and transfection of CHO-dhfr - Cells (ATCC No. CRL-9096), cells stably expressing human LDH-C were selected and designated as CHO/hLDH-C.
CHO cell lines CHO/cLDH-C, CHO/cLDH-Cm in which hamster (Cricetulus griseus) lactate dehydrogenase C (cLDH-C) and its mutants (cLDH-Cm) were overexpressed were constructed in a manner similar to that described above, i.e., cloning hamster LDH-C gene (NCBI accession No. NM-001244050.1) and autonomously designed mutant gene (SEQ ID NO: 2), adding HindIII (AAGCTT), xhoI (CTCGAG) cleavage sites upstream and downstream of the 5' end of the nucleic acid sequence, respectively, and inserting them into pcDNA3.1 eukaryotic expression vectors, and the liposome method transfecting CHO-dhfr - Cells, cells stably expressing cLDH-C (designated CHO/cLDH-C) and cLDH-Cm (CHO/cLDH-Cm) were selected.
The protein encoded by SEQ ID NO. 2 has the amino acid sequence shown in SEQ ID NO. 1, and the difference of the amino acid sequence compared with the protein Q06BU8 (Uniprot protein database number) encoded by NM_001244050.1 is shown in FIG. 5.
Example 2 measurement of lactate dehydrogenase expression level and enzyme Activity and specific Activity
1. Measurement method
The cells selected in example 1 were subjected to monoclonalization and 10 individual clones were selected for each, and expanded to 2X 10 6 cell/mL density, crude enzyme was then extracted and total protein, enzyme activity, specific activity were determined. The specific process is as follows:
(1) Preparation of crude enzyme extract
Taking 1×10 7 Centrifuging the individual cells at 1000 g for 5 min, removing the supernatant, extracting mitochondria by using a mitochondrial separation kit (product number: 89874) of ThermoFisher company, adding 1 mM PMSF (protease inhibitor), ultrasonically crushing the cells by using 30% W ultrasonic crusher, and centrifuging at 4 ℃ for 5 min at 1000 g, wherein the supernatant is the crude enzyme extract. The BCA protein quantification kit was used for the quantitative determination of total protein in the crude enzyme extract.
(2) Semi-quantitative analysis of lactate dehydrogenase expression levels
Taking part of crude enzyme extract to carry out polyacrylamide gel electrophoresis (PAGE), adopting gel concentration of 12%, loading 50 mug total protein, transferring the protein to PVDF membrane to carry out immune hybridization (Western Blot) after electrophoresis is finished, using rabbit anti-human LDH-C monoclonal antibody (Abcam company product No. ab222910, cross-binding with mice, rats and pigs) as primary antibody, and horseradish peroxidase-labeled mouse anti-rabbit IgG monoclonal antibody (Abcam company product No. ab 99702) as secondary antibody.
(3) Separation and purification of lactic dehydrogenase C
According to the method of separation and purification study of rat testis lactic dehydrogenase isozyme C4 (Nanjing university of medical science, 1994, volume 14, 2 nd phase), the lactic dehydrogenase C protein is purified from the crude enzyme extract by adopting a method mainly comprising DEAE-Sephadex anion chromatography and combining the methods of thermal denaturation at 72 ℃ and ammonium sulfate precipitation.
Quantitative determination of purified lactate dehydrogenase C protein was performed using BCA protein quantification kit.
(4) Enzyme activity determination of the direction of the production of lactic acid by pyruvic acid
A1:10 dilution of the substrate mixture 0.49 mL (containing 94.0 mmol/L potassium phosphate buffer pH7.2, 0.2 mmol/L NADH, 2.0 mmol/L pyruvic acid) and 0.01mL enzyme protein was mixed and rapidly transferred to a 0.5 mL cuvette, and the absorbance at 340 nm was measured at 25℃and read every 30 s. The change in absorbance with time was recorded as DeltaA/min.
(5) Enzyme activity determination of lactic acid to pyruvate
A mixture of 0.49. 0.49 mL substrate (containing 82.0 mmol/L potassium phosphate buffer pH7.6, 2.0 mmol/L NAD) + 70 mmol/L sodium lactate) and 0.01mL of a 1:10 dilution of the enzyme protein were mixed and quickly transferred to a 0.5 mL cuvette, and the absorbance at 340 nm was measured at 25℃with every 30 s readings. The change in absorbance with time was recorded as DeltaA/min.
(6) Calculating specific activity
First, the enzyme activity per unit volume (=Δa/min×test volume×dilution/absorption coefficient/optical path/enzyme solution volume) is calculated in U/mL, and then the specific activity (=enzyme activity per unit volume/enzyme protein concentration) is calculated in U/mg. In this test, the test volume was 0.5. 0.5 mL, dilution factor 20, optical path length 0.5. 0.5 cm, and enzyme solution volume 0.01. 0.01 mL.
Measurement results
(1) Results of semi-quantitative analysis of lactate dehydrogenase expression levels
The results of the immunohybridization of the crude enzyme extract of hLDH-C, cLDH-C, cLDH-Cm are shown in FIG. 1, the bands numbered 1-3 from left to right are hLDH-C, cLDH-C, cLDH-Cm in sequence, the expression level of hLDH-C, cLDH-C in CHO is equivalent to that of the concentration of the bands, and the expression level of cLDH-Cm is significantly higher than that of hLDH-C, cLDH-C.
(2) Specific activity measurement result of lactate dehydrogenase
The specific activity measurement results of the purified hLDH-C, cLDH-C, cLDH-Cm proteins are shown in Table 1, and the results show that the activities of the 3 enzyme proteins are mainly the activities of lactic acid in the direction of generating pyruvic acid, the specific activity of mutant cLDH-Cm in the direction of generating pyruvic acid by lactic acid is obviously improved by about 6.69 times compared with that of wild cLDH-C, and the specific activity of pyruvic acid in the direction of generating lactic acid is basically unchanged. The specific activities of wild-type cLDH-C and hLDH-C are approximately equivalent.
TABLE 1 results of specific Activity measurement of lactate dehydrogenase
Specific activity unit: u/mg
hLDH-C cLDH-C cLDH-Cm
Direction of pyruvic acid to lactic acid 5.32±0.26 5.44±0.22 8.69±0.35
Direction of lactic acid to produce pyruvic acid 54.31±2.71 56.82±1.47 380.45±12.71
EXAMPLE 3 measurement of expression level and enzyme Activity and specific Activity of CHO cells overexpressing lactate dehydrogenase
Original CHO-dhfr - Cells and CHO/hLDH-C cells, CHO/cLDH-Cm cells transformed with exogenous lactate dehydrogenase gene at 2X 10 6 The cells/mL were inoculated into a 1L bioreactor using Thermo Fisher Scientific Inc. company CD OptiCHO Medium (cat# 12681-029), L-glutamine (cat# 25030) was added to a final concentration of 5 mM as a basal medium, the initial volume was 60% of the bioreactor working volume, and the fed-batch cell culture process was started. CellsGlucose is supplemented daily during the culture process to maintain residual sugar in the tank at 2.5-3.5 g/L, and dissolved oxygen is controlled at 40-60 deg.C and 37 deg.C. The feed medium was fed daily starting from 3 days after inoculation, respectively, with the formula of feed being 300S medium as described in example 1 of patent CN102007207B, the daily feed dose being 3% of the initial tank volume.
Samples were taken from the bioreactor 1 time every 24 hours during fermentation and cell density in the medium was measured using a cytometer and trypan blue staining (x 10) 6 Per mL) and cell viability (%), and plotted against the culture time (days), the results are shown in fig. 2 and 3.
As can be seen from the results of FIG. 2, the density of CHO/cLDH-Cm cells reached 11X 10 by day 7 of culture 6 cell/mL, CHO/hLDH-C cell, CHO/cLDH-C cell density of 10X 10 6 cells/mL or so, whereas conventional CHO cells (CHO-dhfr - ) Density is only 8×10 6 The cell/mL shows that the exogenous cLDH-Cm coding gene can obviously improve the culture density of CHO cells.
As can be seen from the results of FIG. 3, when cultured to day 5, conventional CHO cells (CHO-dhfr - ) The viability of CHO/hLDH-C cells and CHO/cLDH-C cells was reduced to less than 90% from day 6, while the viability of CHO/cLDH-Cm was still more than 90% by day 7.
Example 4 test of lactic acid resistance of CHO cells overexpressing lactic acid dehydrogenase
Original CHO-dhfr - Cells and CHO/hLDH-C cells, CHO/cLDH-Cm cells transformed with exogenous lactate dehydrogenase gene at 2X 10 6 Inoculating cell/mL in cell culture flask, placing in shaking cell culture incubator, culturing in suspension, using Thermo Fisher Scientific Inc. CD OptiCHO Medium (product No. 12681-029), adding L-glutamine (product No. 25030) to final concentration of 5 mM, taking as basic culture medium, and adding sodium lactate to final concentration of 40 mmol/L (a group of original CHO-dhfr without lactic acid is provided) - Cell culture as control), culturing at 37deg.C for 3 days, sampling 1 time a day, and measuring with a cell counterDetermination of cell Density in Medium (. Times.10) 6 /mL) and plotted against incubation time (days), the results are shown in figure 4.
As can be seen from the results of FIG. 4, in the conventional CHO cell culture system without lactic acid addition, CHO cells grew rapidly and the number multiplication was achieved at day 2.3. Under the lactic acid environment of 40 mmol/L, the cell density of the common CHO cells is obviously reduced from the day 2, but the CHO/hLDH-C cells, the CHO/cLDH-C cells and the CHO/cLDH-Cm cells which are transferred with the exogenous lactate dehydrogenase genes still grow, wherein the growth speed of the CHO/cLDH-Cm cells loaded with the hamster lactate dehydrogenase C mutant genes is obviously faster than that of the CHO/hLDH-C cells and the CHO/cLDH-C cells. The experimental results show that the effect of the cLDH-Cm gene on the increase of cell density is related to lactic acid in the culture system.
Sequence listing
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<120> a method for increasing the culture Density of CHO cells
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Ala Leu Val Asp Val Met Glu Asp Lys Leu Lys Gly Glu Met Met Asp
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Claims (3)

1. A method for improving the culture density of CHO cells is characterized in that a coding gene of exogenous hamster lactate dehydrogenase C is transferred into CHO cells and expressed in the culture process, and the coding gene codes for the amino acid sequence of SEQ ID NO. 1.
2. The method of claim 1, wherein the coding gene has the nucleic acid sequence of SEQ ID NO. 2.
3. The method of claim 1, wherein the CHO cells are CHO/dhFr - A type cell.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012075124A2 (en) * 2010-11-30 2012-06-07 Regents Of The University Of Minnesota Cell lines that overexpress lacatate dehydrogenase c
CN103305504A (en) * 2012-03-14 2013-09-18 江苏吉锐生物技术有限公司 Composition and method for site-specific recombination in hamster cells
CN106381287A (en) * 2015-07-28 2017-02-08 上海张江生物技术有限公司 Method for culturing animal cells for over expression of lactic dehydrogenase C

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012075124A2 (en) * 2010-11-30 2012-06-07 Regents Of The University Of Minnesota Cell lines that overexpress lacatate dehydrogenase c
CN103305504A (en) * 2012-03-14 2013-09-18 江苏吉锐生物技术有限公司 Composition and method for site-specific recombination in hamster cells
CN106381287A (en) * 2015-07-28 2017-02-08 上海张江生物技术有限公司 Method for culturing animal cells for over expression of lactic dehydrogenase C

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NCBI Reference Sequence: NM_001244050.1;Yip SS等;《Genbank》;20181223;第1-2页 *
Regulation of cell growth and apoptosis through lactate dehydrogenase C over-expression in Chinese hamster ovary cells;Tuo Fu等;《Applied Microbiology and Biotechnology》;20160203;第100卷;第5007-5016页 *

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