WO2018018613A1 - 一种提高抗体纯度的细胞培养基和培养方法 - Google Patents

一种提高抗体纯度的细胞培养基和培养方法 Download PDF

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WO2018018613A1
WO2018018613A1 PCT/CN2016/092322 CN2016092322W WO2018018613A1 WO 2018018613 A1 WO2018018613 A1 WO 2018018613A1 CN 2016092322 W CN2016092322 W CN 2016092322W WO 2018018613 A1 WO2018018613 A1 WO 2018018613A1
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medium
self
culture
day
antibody
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PCT/CN2016/092322
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English (en)
French (fr)
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程习文
鄢成伟
杨彬
孙文正
翁源灿
邓崇飞
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广东东阳光药业有限公司
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Priority to PCT/CN2016/092322 priority Critical patent/WO2018018613A1/zh
Priority to US15/405,327 priority patent/US10196665B2/en
Priority to EP17151743.6A priority patent/EP3275998A1/en
Priority to AU2017200458A priority patent/AU2017200458B2/en
Publication of WO2018018613A1 publication Critical patent/WO2018018613A1/zh
Priority to HK18106578.7A priority patent/HK1248754A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0043—Medium free of human- or animal-derived components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a cell culture medium and a culture method for increasing the purity of an antibody.
  • mammalian cells have obvious advantages as expression vectors for recombinant protein drugs, which can complete complex post-translational modification of recombinant proteins, and ensure the correct formation of complex protein structures such as disulfide bonds and glycosylation in products. Therefore, it is an important expression system to ensure the pharmacokinetics and efficacy of recombinant proteins.
  • the recombinant antibody as a biomacromolecule has various variations such as a polymer, a degradation, a glycosylation modification, an oxidation, a deacylation, an isomer, and a disulfide bond mismatch. Purity as the most important mass attribute of an antibody has a decisive effect on the molecular effector function of the antibody.
  • the existing methods for improving the purity of the antibody are mainly to shorten the cell culture cycle or the downstream purification process to sacrifice the yield, shorten the cell culture cycle to reduce the antibody expression, and other glycosylation levels will also change; the downstream purification process sacrifices the yield.
  • the method obtains high-purity antibodies, it increases production costs. Therefore, there is a need for a cell culture method that does not affect the amount of antibody expression and can significantly increase the purity of the antibody.
  • the present invention provides a self-made feed medium for mammalian cell culture, the self-feeding medium containing 90 mM to 500 mM cysteine, preferably, the concentration of cysteine is 100 mM to 400 mM More preferably, the concentration of cysteine is from 120 mM to 300 mM.
  • the self-feeding medium further contains tyrosine and tryptophan.
  • concentration of tyrosine is 200 mM to 400 mM
  • concentration of tryptophan is 50 mM to 150 mM.
  • the self-fed feed medium is a liquid medium having a pH of ⁇ 10, preferably a pH of 10-12.
  • the method for preparing the self-made feed medium comprises the steps of: taking tyrosine, cysteine, tryptophan, adding to water, adjusting the pH to 10 to 12, and forming a volume. To 1L. The entire formulation process is completed under agitation and protection from light.
  • the pH is adjusted with NaOH.
  • the concentration of cysteine is from 90 mM to 500 mM, preferably, the concentration of cysteine is from 100 mM to 400 mM, and more preferably, the concentration of cysteine is from 120 mM to 300 mM.
  • the concentration of tyrosine is from 200 mM to 400 mM, and the concentration of tryptophan is from 50 mM to 150 mM.
  • the present invention provides a cell culture method for expressing high-purity antibodies in mammalian cell culture, wherein the cell culture method is supplemented with the self-feeding medium after the cell culture enters a proliferative phase.
  • the cell culture method comprises the following steps:
  • the basal medium according to the step (1) is characterized in that: 1) serum-free and protein-free components; and 2) large-scale culture of mammalian cells.
  • the basal medium in step (1) is selected from one or more of the commercially available media, Hycell CHO Medium, CD FortiCHO TM AGT TM Medium, Dynamic TM AGT TM Medium , CDM4PERMAb, TFS-RDMP-1 , TFS-RDMP-9, preferably CD FortiCHO TM AGT TM Medium, Hycell CHO Medium.
  • the mammalian cell of step (1) is Chinese Hamster Overy (CHO).
  • the self-feeding medium contains 90 mM to 500 mM cysteine, preferably, the concentration of cysteine is 100 mM to 400 mM, and more preferably, the concentration of cysteine is 120 mM to 300 mM.
  • the self-feeding medium further contains tyrosine and tryptophan.
  • concentration of tyrosine is 200 mM to 400 mM
  • concentration of tryptophan is 50 mM to 150 mM.
  • the method for preparing the self-made feed medium comprises the steps of: taking tyrosine, cysteine, tryptophan, adding to water, adjusting the pH to 10 to 12, and forming a volume. To 1L. The entire formulation process is completed under agitation and protection from light.
  • the pH is adjusted with NaOH.
  • the concentration of cysteine is from 90 mM to 500 mM, preferably, the concentration of cysteine is from 100 mM to 400 mM, and more preferably, the concentration of cysteine is from 120 mM to 300 mM.
  • the concentration of tyrosine is from 200 mM to 400 mM, and the concentration of tryptophan is from 50 mM to 150 mM.
  • the addition medium is supplemented in addition to the supplemental culture medium.
  • the fed medium is selected from the group consisting of concentrated feed medium CHO CD Efficient Feed TM A, Efficient Feed TM A + AGT Supplement, Efficient Feed TM B, Efficient Feed TM B + AGT Supplement, Efficient Feed TM C, Efficient Feed TM C + AGT Supplement, Cellvento TM Feed 200 or Acti CHO Feed A, preferably Acti CHO Feed A CD.
  • the supplemental feed medium and the fed medium are added on the third day of the culture.
  • the homemade feed medium and the fed medium are supplemented daily or for a portion of the day from the start of the third day of culture to the end of the culture.
  • the self-feeding medium and the fed medium are added every other day from the third day of culture, such as adding the self-feeding medium on the 3rd, 5th, 7th, 9th, and 11th day. Add the medium.
  • each additional medium (including homemade feed medium and fed medium) is added in an amount from 1% to 10%, preferably 3%, of the total volume of the fermentation medium. 6%.
  • the total volume of the fermentation medium is the total volume of the medium already present in the fermentor or fermentor during cell culture.
  • the ratio of the self-fed feed medium to the fed medium is 1:10.
  • the culture is stopped when the cell density is below 90%.
  • the antibody of the present invention is an IgG1 antibody, preferably an anti-human tumor necrosis factor (TNF ⁇ ) antibody, an anti-human vascular endothelial growth factor (VEGF) antibody.
  • TNF ⁇ tumor necrosis factor
  • VEGF vascular endothelial growth factor
  • Cysteine is a common cell culture medium component, but the relationship between the amount of cysteine added and the purity of the antibody has not been reported.
  • the inventor developed a self-made feed medium through a lot of creative labor, and controlled the self-made supplement.
  • the concentration of cysteine in the feed medium and the timing and amount of addition can significantly increase the purity of the antibody.
  • the inventors found that after supplementing the self-feeding medium, the correct assembly of the disulfide bond of the antibody is enhanced, and By increasing the purity of the antibody, the amount of antibody expression and the normal level of glycosylation can be maintained, and the efficacy of the antibody is ensured.
  • the method is simple in operation, easy to implement, and suitable for industrial production.
  • Figure 1 is a graph showing the trend of viable cell density in the cell culture process of Examples 1-3.
  • Figure 2 is a graph showing the cell viability trend of the cell culture process of Example 1-3.
  • Figure 3 is a graph showing the trend of viable cell density in the cell culture process of Examples 4-6.
  • Figure 4 is a graph showing the cell viability trend of the cell culture process of Example 4-6.
  • Figure 5 is a schematic view of the sugar type structure.
  • Figure 6 is a comparison of the results of glycosylation detection of Examples 1-3 and standards (adalimumab).
  • Figure 7 is a comparison of the results of glycosylation detection of Examples 4-6 and standards (bevacizumab).
  • the base medium, the fed medium, and the standards used in the examples are all commercially available.
  • Tyrosine, cysteine, and tryptophan were purchased from sigma.
  • CHO (Chinese hamster ovary cells) cell line was purchased from lonza.
  • Self-made feed medium 300 mmol of tyrosine (181.2 g/mol), 280 mmol of cysteine (121.15 g/mol), 100 mmol of tryptophan (204.23 g/mol), added with stirring and protected from light
  • 700 ml of ultrapure water stir for 40 min and add NaOH (32.1 g/L).
  • the pH was adjusted to 10.95 to 11.2 with 5M NaOH, stirring was continued for 20 min, the turbidity was measured, and the turbidity should be less than 10 NTU. 1000 ml, stirred for more than 10 min, filtered through a 0.22 ⁇ m filter to a sterile reagent bottle.
  • Addition medium commercially available medium Acti CHO FeedA CD (purchased from GE)
  • Cell line a CHO cell line containing a nucleic acid encoding an anti-human tumor necrosis factor (TNF ⁇ ) antibody (refer to WO1997029131 patent)
  • TNF ⁇ tumor necrosis factor
  • the cells were cultured in a 500 ml shake flask, and the cell line was inoculated at 0.85 ⁇ 10 6 cells/mL.
  • the initial culture volume was 100 ml
  • the culture temperature was 37 ° C
  • the CO 2 concentration was 8%
  • the shaker speed was 130 rpm
  • the daily test was started from the first day of the culture.
  • the glucose concentration was controlled at 6-8 g/L and the glutamic acid monosodium salt concentration was 4-7 mM according to the results of the NOVA biochemical analyzer. From the third day, the addition medium and the self-feeding medium were added, and the days after the next day were added.
  • the third day supplemented medium (including the self-feeding medium and the fed medium) was added to the fermentation culture. 3.5% of the total volume of the base, the addition medium (including the self-feeding medium and the fed medium) on the 5th, 7th, 9th, and 11th days was added in an amount of 5.5% of the total volume of the fermentation medium, and the self-feeding culture was carried out.
  • the ratio of base to fed medium was 1:10.
  • the fermentation was stopped on the 13th day of the cell culture, and the supernatant was collected, and the relative expression amount and purity of the antibody were detected by a Protein A column by high performance liquid chromatography (HPLC). The results are shown in Table 1. Glycosylation was detected by LC/MS analysis. The results are shown in Fig. 6.
  • the standard product was adalimumab (trade name, Xiumele).
  • the viable cell density see Figure 1
  • cell viability (see Figure 2) during cell culture were detected by trypan blue (0.2%) staining.
  • Self-made feed medium 300 mmol of tyrosine (181.2 g/mol), 210 mmol of cysteine (121.15 g/mol), 100 mmol of tryptophan (204.23 g/mol), added with stirring and protected from light
  • 700 ml of ultrapure water stir for 40 min and add NaOH (32.1 g/L).
  • the pH was adjusted to 10.95-11.2 with 5M NaOH
  • stirring was continued for 20 min
  • the turbidity was measured, the turbidity should be less than 10 NTU
  • the volume was adjusted to 1000 ml
  • the mixture was stirred for 10 min or more, and the 0.22 ⁇ m filter was filtered to a sterile reagent bottle.
  • Addition medium commercially available medium Acti CHO FeedA CD (purchased from GE)
  • Cell line a CHO cell line containing a nucleic acid encoding an anti-human tumor necrosis factor (TNF ⁇ ) antibody (refer to WO1997029131 patent)
  • TNF ⁇ tumor necrosis factor
  • the cells were cultured in a 500 ml shake flask, and the cell line was inoculated at 0.85 ⁇ 10 6 cells/mL.
  • the initial culture volume was 100 ml
  • the culture temperature was 37 ° C
  • the CO 2 concentration was 8%
  • the shaker speed was 130 rpm
  • the daily test was started from the first day of the culture.
  • the glucose concentration was controlled at 6-8 g/L and the glutamic acid monosodium salt concentration was 4-7 mM according to the results of the NOVA biochemical analyzer. From the third day, the addition medium and the self-feeding medium were added, and the days after the next day were added.
  • the third day supplemented medium (including the self-feeding medium and the fed medium) was added to the fermentation culture. 3.5% of the total volume of the base, the addition medium (including the self-feeding medium and the fed medium) on the 5th, 7th, 9th, and 11th days was added in an amount of 5.5% of the total volume of the fermentation medium, and the self-feeding culture was carried out.
  • the ratio of base to fed medium was 1:10.
  • the fermentation was stopped on the 13th day of the cell culture, and the supernatant was collected, and the relative expression amount and purity of the antibody were detected by a Protein A column by high performance liquid chromatography (HPLC). The results are shown in Table 1. Glycosylation was detected by LC/MS analysis. The results are shown in Fig. 6.
  • the standard product was adalimumab (trade name, Xiumele).
  • the viable cell density see Figure 1
  • cell viability (see Figure 2) during cell culture were detected by trypan blue (0.2%) staining.
  • Self-made feed medium 300 mmol of tyrosine (181.2 g/mol), 140 mmol of cysteine (121.15 g/mol), 100 mmol of tryptophan (204.23 g/mol), added with stirring and protected from light
  • 700 ml of ultrapure water stir for 40 min and add NaOH (32.1 g/L).
  • the pH was adjusted to 10.95-11.2 with 5M NaOH
  • stirring was continued for 20 min
  • the turbidity was measured, the turbidity should be less than 10 NTU
  • the volume was adjusted to 1000 ml
  • the mixture was stirred for 10 min or more, and the 0.22 ⁇ m filter was filtered to a sterile reagent bottle.
  • Addition medium commercially available medium Acti CHO FeedA CD (purchased from GE)
  • Cell line a CHO cell line containing a nucleic acid encoding an anti-human tumor necrosis factor (TNF ⁇ ) antibody (refer to WO1997029131 patent)
  • TNF ⁇ tumor necrosis factor
  • the cells were cultured in a 500 ml shake flask, and the cell line was inoculated at 0.85 ⁇ 10 6 cells/mL.
  • the initial culture volume was 100 ml
  • the culture temperature was 37 ° C
  • the CO 2 concentration was 8%
  • the shaker speed was 130 rpm
  • the daily test was started from the first day of the culture.
  • the glucose concentration was controlled at 6-8 g/L and the glutamic acid monosodium salt concentration was 4-7 mM according to the results of the NOVA biochemical analyzer. From the third day, the addition medium and the self-feeding medium were added, and the days after the next day were added.
  • the third day supplemented medium (including the self-feeding medium and the fed medium) was added to the fermentation culture. 3.5% of the total volume of the base, the addition medium (including the self-feeding medium and the fed medium) on the 5th, 7th, 9th, and 11th days was added in an amount of 5.5% of the total volume of the fermentation medium, and the self-feeding culture was carried out.
  • the ratio of base to fed medium was 1:10.
  • the fermentation was stopped on the 13th day of the cell culture, and the supernatant was collected, and the relative expression amount and purity of the antibody were detected by a Protein A column by high performance liquid chromatography (HPLC). The results are shown in Table 1. Glycosylation was detected by LC/MS analysis. The results are shown in Fig. 6.
  • the standard product was adalimumab (trade name, Xiumele).
  • the viable cell density see Figure 1
  • cell viability (see Figure 2) during cell culture were detected by trypan blue (0.2%) staining.
  • Basic medium commercially available medium Forti CHO AGT (purchased from Gibco)
  • Self-made feed medium 300 mmol of tyrosine (181.2 g/mol), 280 mmol of cysteine (121.15 g/mol), 100 mmol of tryptophan (204.23 g/mol), added with stirring and protected from light
  • 700 ml of ultrapure water stir for 40 min and add NaOH (32.1 g/L).
  • the pH was adjusted to 10.95-11.2 with 5M NaOH
  • stirring was continued for 20 min
  • the turbidity was measured, the turbidity should be less than 10 NTU
  • the volume was adjusted to 1000 ml
  • the mixture was stirred for 10 min or more, and the 0.22 ⁇ m filter was filtered to a sterile reagent bottle.
  • Addition medium commercially available medium Acti CHO FeedA CD (purchased from GE)
  • Cell line a CHO cell line comprising an anti-human vascular endothelial growth factor (VEGF) antibody nucleic acid (refer to WO1998045331 patent)
  • VEGF vascular endothelial growth factor
  • the cells were cultured in a 500 ml shake flask, and the cell line was inoculated at 1 ⁇ 10 6 cells/mL.
  • the initial culture volume was 100 ml
  • the culture temperature was 37 ° C
  • the CO 2 concentration was 8%
  • the shaker speed was 130 rpm
  • the daily test was started from the first day of the culture.
  • the glucose concentration was controlled at 6-8 g/L and the glutamic acid monosodium salt concentration was 4-7 mM according to the results of the NOVA biochemical analyzer. From the third day, the addition medium and the self-feeding medium were added, and the days after the next day were added.
  • the third day supplemented medium (including the self-feeding medium and the fed medium) was added to the fermentation culture. 3.5% of the total volume of the base, the addition medium (including the self-feeding medium and the fed medium) on the 5th, 7th, 9th, and 11th days was added in an amount of 5.5% of the total volume of the fermentation medium, and the self-feeding culture was carried out.
  • the ratio of base to fed medium was 1:10.
  • the fermentation was stopped on the 13th day of the cell culture, and the supernatant was collected, and the relative expression amount and purity of the antibody were detected by a Protein A column by high performance liquid chromatography (HPLC). The results are shown in Table 1. Glycosylation was detected by LC/MS analysis. The results are shown in Figure 7.
  • the standard was bevacizumab (trade name, Avastin).
  • the viable cell density (see Figure 3) and cell viability (see Figure 4) during cell culture were detected by trypan blue (0.2%) staining.
  • Basic medium commercially available medium Forti CHO AGT (purchased from Gibco)
  • Self-made feed medium 300 mmol of tyrosine (181.2 g/mol), 210 mmol of cysteine (121.15 g/mol), 100 mmol of tryptophan (204.23 g/mol), added with stirring and protected from light
  • 700 ml of ultrapure water stir for 40 min and add NaOH (32.1 g/L).
  • the pH was adjusted to 10.95-11.2 with 5M NaOH
  • stirring was continued for 20 min
  • the turbidity was measured, the turbidity should be less than 10 NTU
  • the volume was adjusted to 1000 ml
  • the mixture was stirred for 10 min or more, and the 0.22 ⁇ m filter was filtered to a sterile reagent bottle.
  • Addition medium commercially available medium Acti CHO FeedA CD (purchased from GE)
  • Cell line a CHO cell line comprising an anti-human vascular endothelial growth factor (VEGF) antibody nucleic acid (refer to WO1998045331 patent)
  • VEGF vascular endothelial growth factor
  • the cells were cultured in a 500 ml shake flask, and the cell line was inoculated at 1 ⁇ 10 6 cells/mL.
  • the initial culture volume was 100 ml
  • the culture temperature was 37 ° C
  • the CO 2 concentration was 8%
  • the shaker speed was 130 rpm
  • the daily test was started from the first day of the culture.
  • the glucose concentration was controlled at 6-8 g/L and the glutamic acid monosodium salt concentration was 4-7 mM according to the results of the NOVA biochemical analyzer. From the third day, the addition medium and the self-feeding medium were added, and the days after the next day were added.
  • the third day supplemented medium (including the self-feeding medium and the fed medium) was added to the fermentation culture. 3.5% of the total volume of the base, the addition medium (including the self-feeding medium and the fed medium) on the 5th, 7th, 9th, and 11th days was added in an amount of 5.5% of the total volume of the fermentation medium, and the self-feeding culture was carried out.
  • the ratio of base to fed medium was 1:10.
  • the fermentation was stopped on the 13th day of the cell culture, and the supernatant was collected, and the relative expression amount and purity of the antibody were detected by a Protein A column by high performance liquid chromatography (HPLC). The results are shown in Table 1. Glycosylation was detected by LC/MS analysis. The results are shown in Figure 7.
  • the standard was bevacizumab (trade name, Avastin).
  • the viable cell density (see Figure 3) and cell viability (see Figure 4) during cell culture were detected by trypan blue (0.2%) staining.
  • Basic medium commercially available medium Forti CHO AGT (purchased from Gibco)
  • Self-made feed medium 300 mmol of tyrosine (181.2 g/mol), 140 mmol of cysteine (121.15 g/mol), 100 mmol of tryptophan (204.23 g/mol), added with stirring and protected from light
  • 700 ml of ultrapure water stir for 40 min and add NaOH (32.1 g/L).
  • the pH was adjusted to 10.95-11.2 with 5M NaOH
  • stirring was continued for 20 min
  • the turbidity was measured, the turbidity should be less than 10 NTU
  • the volume was adjusted to 1000 ml
  • the mixture was stirred for 10 min or more, and the 0.22 ⁇ m filter was filtered to a sterile reagent bottle.
  • Addition medium commercially available medium Acti CHO FeedA CD (purchased from GE)
  • Cell line a CHO cell line comprising an anti-human vascular endothelial growth factor (VEGF) antibody nucleic acid (refer to WO1998045331 patent)
  • VEGF vascular endothelial growth factor
  • the cells were cultured in a 500 ml shake flask, and the cell line was inoculated at 1 ⁇ 10 6 cells/mL.
  • the initial culture volume was 100 ml
  • the culture temperature was 37 ° C
  • the CO 2 concentration was 8%
  • the shaker speed was 130 rpm
  • the daily test was started from the first day of the culture.
  • the glucose concentration was controlled at 6-8 g/L and the glutamic acid monosodium salt concentration was 4-7 mM according to the results of the NOVA biochemical analyzer. From the third day, the addition medium and the self-feeding medium were added, and the days after the next day were added.
  • the third day supplemented medium (including the self-feeding medium and the fed medium) was added to the fermentation culture. 3.5% of the total volume of the base, the addition medium (including the self-feeding medium and the fed medium) on the 5th, 7th, 9th, and 11th days was added in an amount of 5.5% of the total volume of the fermentation medium, and the self-feeding culture was carried out.
  • the ratio of base to fed medium was 1:10.
  • the fermentation was stopped on the 13th day of the cell culture, and the supernatant was collected, and the relative expression amount and purity of the antibody were detected by a Protein A column by high performance liquid chromatography (HPLC). The results are shown in Table 1. Glycosylation was detected by LC/MS analysis. The results are shown in Figure 7.
  • the standard was bevacizumab (trade name, Avastin).
  • the viable cell density (see Figure 3) and cell viability (see Figure 4) during cell culture were detected by trypan blue (0.2%) staining.
  • Self-made feed medium take 300mmol tyrosine (181.2g/mol), 100mmol tryptophan (204.23g/mol), add to 700ml ultrapure water with stirring and protection from light, stir for 40min, add NaOH (32.1g/L). Stirring was continued for 30 min, the pH was adjusted to 10.95-11.2 with 5M NaOH, stirring was continued for 20 min, the turbidity was measured, the turbidity should be less than 10 NTU, the volume was adjusted to 1000 ml, the mixture was stirred for 10 min or more, and the 0.22 ⁇ m filter was filtered to a sterile reagent bottle.
  • Addition medium commercially available medium Acti CHO FeedA CD (purchased from GE)
  • Cell line a CHO cell line containing a nucleic acid encoding an anti-human tumor necrosis factor (TNF ⁇ ) antibody (refer to WO1997029131 patent)
  • TNF ⁇ tumor necrosis factor
  • the cells were cultured in a 500 ml shake flask, and the cell line was inoculated at 0.85 ⁇ 10 6 cells/mL.
  • the initial culture volume was 100 ml
  • the culture temperature was 37 ° C
  • the CO 2 concentration was 8%
  • the shaker speed was 130 rpm
  • the daily test was started from the first day of the culture.
  • the glucose concentration was controlled at 6-8 g/L and the glutamic acid monosodium salt concentration was 4-7 mM according to the results of the NOVA biochemical analyzer. From the third day, the addition medium and the self-feeding medium were added, and the days after the next day were added.
  • the third day supplemented medium (including the self-feeding medium and the fed medium) was added to the fermentation culture. 3.5% of the total volume of the base, the addition medium (including the self-feeding medium and the fed medium) on the 5th, 7th, 9th, and 11th days was added in an amount of 5.5% of the total volume of the fermentation medium, and the self-feeding culture was carried out.
  • the ratio of base to fed medium was 1:10.
  • the fermentation was stopped on the 13th day of the cell culture, and the supernatant was collected, and the relative expression amount and purity of the antibody were detected by a Protein A column by high performance liquid chromatography (HPLC). The results are shown in Table 1.
  • Basic medium commercially available medium Forti CHO AGT (purchased from Gibco)
  • Self-made feed medium take 300mmol tyrosine (181.2g/mol), 100mmol tryptophan (204.23g/mol), add to 700ml ultrapure water with stirring and protection from light, stir for 40min, add NaOH (32.1g/L). Stirring was continued for 30 min, the pH was adjusted to 10.95-11.2 with 5M NaOH, stirring was continued for 20 min, the turbidity was measured, the turbidity should be less than 10 NTU, the volume was adjusted to 1000 ml, the mixture was stirred for 10 min or more, and the 0.22 ⁇ m filter was filtered to a sterile reagent bottle.
  • Addition medium commercially available medium Acti CHO FeedA CD (purchased from GE)
  • Cell line a CHO cell line comprising an anti-human vascular endothelial growth factor (VEGF) antibody nucleic acid (refer to WO1998045331 patent)
  • VEGF vascular endothelial growth factor
  • the cells were cultured in a 500 ml shake flask, and the cell line was inoculated at 1 ⁇ 10 6 cells/mL.
  • the initial culture volume was 100 ml
  • the culture temperature was 37 ° C
  • the CO 2 concentration was 8%
  • the shaker speed was 130 rpm
  • the daily test was started from the first day of the culture.
  • the glucose concentration was controlled at 6-8 g/L and the glutamic acid monosodium salt concentration was 4-7 mM according to the results of the NOVA biochemical analyzer. From the third day, the addition medium and the self-feeding medium were added, and the days after the next day were added.
  • the third day supplemented medium (including the self-feeding medium and the fed medium) was added to the fermentation culture. 3.5% of the total volume of the base, the addition medium (including the self-feeding medium and the fed medium) on the 5th, 7th, 9th, and 11th days was added in an amount of 5.5% of the total volume of the fermentation medium, and the self-feeding culture was carried out.
  • the ratio of base to fed medium was 1:10.
  • the fermentation was stopped on the 13th day of the cell culture, and the supernatant was collected, and the relative expression amount and purity of the antibody were detected by a Protein A column by high performance liquid chromatography (HPLC). The results are shown in Table 1.

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Abstract

提供了一种自制补料培养基，所述培养基含有90mM～500mM半胱氨酸。并提供了一种细胞培养方法，包括以下步骤：在基础培养基中接种包含编码抗体核酸的哺乳动物细胞；进入细胞增殖期后补加所述自制补料培养基；监测细胞密度调整培养时间。

Description

一种提高抗体纯度的细胞培养基和培养方法 技术领域
本发明涉及生物技术领域，具体涉及提高抗体纯度的细胞培养基和培养方法。
背景技术
在生物医药行业，哺乳动物细胞作为重组蛋白药物的表达载体具有明显的优势，其能够完成重组蛋白后期复杂的翻译后修饰，能够保证产品中二硫键、糖基化等复杂蛋白结构的正确形成，从而能够保证重组蛋白的药性和药效，是一类重要的表达***。
近年来，用于单抗药物生产的动物细胞大规模培养技术发展迅速。该领域的技术进展主要集中在个性化培养基开发和工艺条件优化等方面。作为生物大分子的重组抗体，同时具备聚体、降解、糖基化修饰、氧化、脱酰基化、异构体、二硫键错配等多种变异形式。纯度作为抗体最重要的质量属性对抗体的分子效应功能具有决定作用。现有的提高抗体纯度的方法主要是上游缩短细胞培养周期或者下游纯化工艺牺牲收率，缩短细胞培养周期会降低抗体表达量，其他的糖基化水平也会改变；下游纯化工艺牺牲收率的方式虽然获得高纯度抗体，但是会增加生产成本。因此，亟需一种既不影响抗体表达量又能显著提高抗体纯度的细胞培养方法。
发明内容
本发明的目的在于：提供一种用于细胞培养尤其是哺乳动物细胞培养中高纯度抗体表达的自制补料培养基和培养方法。
一方面，本发明提供一种用于哺乳动物细胞培养的自制补料培养基，所述自制补料培养基含有90mM～500mM半胱氨酸，优选地，半胱氨酸的浓度为100mM～400mM，更优选地，半胱氨酸的浓度为120mM～300mM。
在本发明的一些实施方式中，所述自制补料培养基中还含有酪氨酸和色氨酸，优选地，酪氨酸的浓度为200mM～400mM，色氨酸的浓度为50mM～150mM。
在本发明的一些实施方式中，所述自制补料培养基为液体培养基，培养基的pH值≥10，优选地pH为10～12。
在本发明的一些实施方式中，所述自制补料培养基的配制方法包括以下步骤：取酪氨酸、半胱氨酸、色氨酸，加入到水中，调节pH至10～12，定容至1L。整个配制过程在搅拌和避光的条件下完成。优选地，用NaOH调节pH值。半胱氨酸的浓度为90mM～500mM，优选地，半胱氨酸的浓度为100mM～400mM，更优选地，半胱氨酸的浓度为120mM～300mM。优选地，酪氨酸的浓度为200mM～400mM，色氨酸的浓度为50mM～150mM。
另一方面，本发明提供一种用于哺乳动物细胞培养中高纯度抗体表达的细胞培养方法，所述细胞培养方法为细胞培养进入增殖期后，补加所述自制补料培养基。
具体地，所述的细胞培养方法包括以下步骤：
(1)在基础培养基中接种包含编码抗体核酸的哺乳动物细胞；
(2)进入细胞增殖期后补加所述自制补料培养基；
(3)监测细胞密度调整培养时间。
具体的，步骤(1)所述的基础培养基，其特征在于具有如下特征：1)无血清、无蛋白成分；2)用于哺乳动物细胞大规模培养。
在本发明的一些实施例中，步骤(1)所述的基础培养基选自以下市售培养基中的一种或多种，Hycell CHO Medium、CD FortiCHOTMAGT TMMedium、DynamicTMAGTTMMedium、CDM4PERMAb、TFS-RDMP-1、TFS-RDMP-9，优选地为CD FortiCHOTMAGT TMMedium、Hycell CHO Medium。
在本发明的一些实施例中，步骤(1)所述的哺乳动物细胞为中国仓鼠卵巢细胞(Chinese Hamster Overy，CHO)。
所述自制补料培养基含有90mM～500mM半胱氨酸，优选地，半胱氨酸的浓度为100mM～400mM，更优选地，半胱氨酸的浓度为120mM～300mM。
在本发明的一些实施方式中，所述自制补料培养基中还含有酪氨酸和色氨酸，优选地，酪氨酸的浓度为200mM～400mM，色氨酸的浓度为50mM～150mM。
在本发明的一些实施方式中，所述自制补料培养基的配制方法包括以下步骤：取酪氨酸、半胱氨酸、色氨酸，加入到水中，调节pH至10～12，定容至1L。整个配制过程在搅拌和避光的条件下完成。优选地，用NaOH调节pH值。半胱氨酸的浓度为90mM～500mM，优选地，半胱氨酸的浓度为100mM～400mM，更优选地，半胱氨酸的浓度为120mM～300mM。优选地，酪氨酸的浓度为200mM～400mM，色氨酸的浓度为50mM～150mM。
在本发明的一些实施例中，进入细胞增殖期后，除补加自制培养基外还补充流加培养基，优选地，所述流加培养基选自浓缩补料培养基CHO CD Efficient FeedTM A、Efficient FeedTM A+AGT Supplement、Efficient FeedTM B、Efficient FeedTM B+AGT Supplement、Efficient FeedTM C、Efficient FeedTM C+AGT Supplement、CellventoTM Feed 200或Acti CHO Feed A，优选地为Acti CHO Feed A CD。
在本发明的一些实施例中，在培养的第三天开始补加自制补料培养基和流加培养基。
在本发明的一些实施例中，在培养第三天开始至培养结束每天或部分天数补加自制补料培养基和流加培养基。
在本发明的一些实施例中，在培养第三天开始隔天补加自制补料培养基和流加培养基，如第3、5、7、9、11天补加自制补料培养基和流加培养基。
在本发明的一些实施例中，每次补加培养基(包括自制补料培养基和流加培养基)加入的量为发酵培养基总体积的1％～10％，优选地为3％～6％。所述发酵培养基总体积为细胞培养过程中发酵罐或发酵瓶中已有培养基的总体积。
在本发明的一些实施例中，自制补料培养基和流加培养基加入的比例为1:10。
在本发明的一些实施例中，细胞密度低于90％时停止培养。
本发明所述的抗体为IgG1抗体，优选地为抗人肿瘤坏死因子(TNFα)抗体、抗人血管内皮生长因子(VEGF)抗体。
半胱氨酸是常见的细胞培养基成分，但是尚未见半胱氨酸的加入量与抗体纯度之间的关系的报道，发明人通过大量创造性劳动开发了自制补料培养基，通过控制自制补料培养基中半胱氨酸的浓度及加入时机、加入量，能显著提高抗体的纯度，发明人发现在补加自制补料培养基后，加强了抗体的二硫键的正确组装，且在提高抗体纯度的同时能够维持抗体表达量和正常的糖基化水平，保证了抗体的药效。本方法操作简单、便于实施，适合工业化生产的需要。
附图说明
图1为实施例1-3细胞培养过程的活细胞密度趋势图。
图2为实施例1-3细胞培养过程的细胞活力趋势图。
图3为实施例4-6细胞培养过程的活细胞密度趋势图。
图4为实施例4-6细胞培养过程的细胞活力趋势图。
图5糖型结构示意图。
图6实施例1-3与标准品(阿达木单抗)糖基化检测结果对比图。
图7实施例4-6与标准品(贝伐单抗)糖基化检测结果对比图。
具体实施方式
以下所述的是本发明的优选实施方式，本发明所保护的不限于以下优选实施方式。应当指出，对于本领域的技术人员来说在此发明创造构思的基础上，做出的若干变形和改进，都属于本发明的保护范围。
实施例中所用的基础培养基、流加培养基及标准品均可以通过商业途径获得。酪氨酸、半胱氨酸、色氨酸购自sigma。CHO(中国仓鼠卵巢细胞)细胞株购自lonza公司。
实施例1
基础培养基：为市售培养基Hycell CHO Medium(购自Hyclone)
自制补料培养基：取300mmol酪氨酸(181.2g/mol)、280mmol半胱氨酸(121.15g/mol)、100mmol色氨酸(204.23g/mol)，在搅拌和避光的情况下加入到700ml超纯水中，搅拌40min，加入NaOH(32.1g/L)。继续搅拌30min，用5M NaOH调节pH至10.95～11.2，继续搅拌20min，测定浊度，浊度应小于10NTU，定容至 1000ml，搅拌10min以上，0.22μm滤膜过滤至无菌试剂瓶。
流加培养基：为市售培养基Acti CHO FeedA CD(购自GE)
细胞株：包含编码抗人肿瘤坏死因子(TNFα)抗体核酸的CHO细胞株(参照WO1997029131专利)
在500ml摇瓶中培养，细胞株以0.85×106cells/mL进行接种，初始培养体积100ml，培养温度37℃，CO2浓度8％，摇床转速130rpm，从培养的第1天开始每天检测细胞密度、活力和各种代谢参数。根据NOVA生化分析仪检测的结果控制葡萄糖浓度在6-8g/L，谷氨酸单钠盐浓度在4-7mM。从第三天开始补充流加培养基和自制补料培养基，以后天数隔天流加，第三天补加培养基(包括自制补料培养基和流加培养基)的加入量为发酵培养基总体积的3.5％，第5、7、9、11天补加培养基(包括自制补料培养基和流加培养基)的加入量为发酵培养基总体积的5.5％，自制补料培养基和流加培养基加入的比例为1:10。在细胞培养的第13天停止发酵，收取上清，用高效液相色谱(HPLC)的方法，通过Protein A柱检测抗体的相对表达量和纯度,结果见表1。糖基化采用LC/MS分析法检测，结果见图6，标准品为阿达木单抗(商品名，修美乐)。细胞培养过程中的活细胞密度(见图1)和细胞活力(见图2)采用台盼蓝(0.2％)染色法进行检测。
实施例2
基础培养基：为市售培养基Hycell CHO Medium(购自Hyclone)
自制补料培养基：取300mmol酪氨酸(181.2g/mol)、210mmol半胱氨酸(121.15g/mol)、100mmol色氨酸(204.23g/mol)，在搅拌和避光的情况下加入到700ml超纯水中，搅拌40min，加入NaOH(32.1g/L)。继续搅拌30min，用5M NaOH调节pH至10.95～11.2，继续搅拌20min，测定浊度，浊度应小于10NTU，定容至1000ml，搅拌10min以上，0.22μm滤膜过滤至无菌试剂瓶。
流加培养基：为市售培养基Acti CHO FeedA CD(购自GE)
细胞株：包含编码抗人肿瘤坏死因子(TNFα)抗体核酸的CHO细胞株(参照WO1997029131专利)
在500ml摇瓶中培养，细胞株以0.85×106cells/mL进行接种，初始培养体积100ml，培养温度37℃，CO2浓度8％，摇床转速130rpm，从培养的第1天开始每天检测细胞密度、活力和各种代谢参数。根据NOVA生化分析仪检测的结果控制葡萄糖浓度在6-8g/L，谷氨酸单钠盐浓度在4-7mM。从第三天开始补充流加培养基和自制补料培养基，以后天数隔天流加，第三天补加培养基(包括自制补料培养基和流加培养基)的加入量为发酵培养基总体积的3.5％，第5、7、9、11天补加培养基(包括自制补料培养基和流加培养基)的加入量为发酵培养基总体积的5.5％，自制补料培养基和流加培养基加入的比例为1:10。在细胞培养的第13天停止发酵，收取上清，用高效液相色谱(HPLC)的方法，通过ProteinA柱检测抗体的相对表达量和纯度，结果见表1。糖基化采用LC/MS分析法检测，结果见图6，标准品为阿达木单抗(商品名，修美乐)。细胞培养过程中的活细胞密度(见图1)和细胞活力(见图2)采用台盼蓝(0.2％)染色法进行检测。
实施例3
基础培养基：为市售培养基Hycell CHO Medium(购自Hyclone)
自制补料培养基：取300mmol酪氨酸(181.2g/mol)、140mmol半胱氨酸(121.15g/mol)、100mmol色氨酸(204.23g/mol)，在搅拌和避光的情况下加入到700ml超纯水中，搅拌40min，加入NaOH(32.1g/L)。继续搅拌30min，用5M NaOH调节pH至10.95～11.2，继续搅拌20min，测定浊度，浊度应小于10NTU，定容至1000ml，搅拌10min以上，0.22μm滤膜过滤至无菌试剂瓶。
流加培养基：为市售培养基Acti CHO FeedA CD(购自GE)
细胞株：包含编码抗人肿瘤坏死因子(TNFα)抗体核酸的CHO细胞株(参照WO1997029131专利)
在500ml摇瓶中培养，细胞株以0.85×106cells/mL进行接种，初始培养体积100ml，培养温度37℃，CO2浓度8％，摇床转速130rpm，从培养的第1天开始每天检测细胞密度、活力和各种代谢参数。根据NOVA生化分析仪检测的结果控制葡萄糖浓度在6-8g/L，谷氨酸单钠盐浓度在4-7mM。从第三天开始补充流加培养基和自制补料培养基，以后天数隔天流加，第三天补加培养基(包括自制补料培养基和流加培养基)的加入量为发酵培养基总体积的3.5％，第5、7、9、11天补加培养基(包括自制补料培养基和流加培养基)的加入量为发酵培养基总体积的5.5％，自制补料培养基和流加培养基加入的比例为1:10。在细胞培养的第13天停止发酵，收取上清，用高效液相色谱(HPLC)的方法，通过ProteinA柱检测抗体的相对表达量和纯度，结果见表1。糖基化采用LC/MS分析法检测，结果见图6，标准品为阿达木单抗(商品名，修美乐)。细胞培养过程中的活细胞密度(见图1)和细胞活力(见图2)采用台盼蓝(0.2％)染色法进行检测。
实施例4
基础培养基：为市售培养基Forti CHO AGT(购自Gibco)
自制补料培养基：取300mmol酪氨酸(181.2g/mol)、280mmol半胱氨酸(121.15g/mol)、100mmol色氨酸(204.23g/mol)，在搅拌和避光的情况下加入到700ml超纯水中，搅拌40min，加入NaOH(32.1g/L)。继续搅拌30min，用5M NaOH调节pH至10.95～11.2，继续搅拌20min，测定浊度，浊度应小于10NTU，定容至1000ml，搅拌10min以上，0.22μm滤膜过滤至无菌试剂瓶。
流加培养基：为市售培养基Acti CHO FeedA CD(购自GE)
细胞株：包含编码抗人血管内皮生长因子(VEGF)抗体核酸的CHO细胞株(参照WO1998045331专利)
在500ml摇瓶中培养，细胞株以1×106cells/mL进行接种，初始培养体积100ml，培养温度37℃，CO2浓度8％，摇床转速130rpm，从培养的第1天开始每天检测细胞密度、活力和各种代谢参数。根据NOVA生化分析仪检测的结果控制葡萄糖浓度在6-8g/L，谷氨酸单钠盐浓度在4-7mM。从第三天开始补充流加培养基和自制补料培养基，以后天数隔天流加，第三天补加培养基(包括自制补料培养基和流加培养基)的加 入量为发酵培养基总体积的3.5％，第5、7、9、11天补加培养基(包括自制补料培养基和流加培养基)的加入量为发酵培养基总体积的5.5％，自制补料培养基和流加培养基加入的比例为1:10。在细胞培养的第13天停止发酵，收取上清，用高效液相色谱(HPLC)的方法，通过ProteinA柱检测抗体的相对表达量和纯度，结果见表1。糖基化采用LC/MS分析法检测，结果见图7，标准品为贝伐单抗(商品名，阿瓦斯汀)。细胞培养过程中的活细胞密度(见图3)和细胞活力(见图4)采用台盼蓝(0.2％)染色法进行检测。
实施例5
基础培养基：为市售培养基Forti CHO AGT(购自Gibco)
自制补料培养基：取300mmol酪氨酸(181.2g/mol)、210mmol半胱氨酸(121.15g/mol)、100mmol色氨酸(204.23g/mol)，在搅拌和避光的情况下加入到700ml超纯水中，搅拌40min，加入NaOH(32.1g/L)。继续搅拌30min，用5M NaOH调节pH至10.95～11.2，继续搅拌20min，测定浊度，浊度应小于10NTU，定容至1000ml，搅拌10min以上，0.22μm滤膜过滤至无菌试剂瓶。
流加培养基：为市售培养基Acti CHO FeedA CD(购自GE)
细胞株：包含编码抗人血管内皮生长因子(VEGF)抗体核酸的CHO细胞株(参照WO1998045331专利)
在500ml摇瓶中培养，细胞株以1×106cells/mL进行接种，初始培养体积100ml，培养温度37℃，CO2浓度8％，摇床转速130rpm，从培养的第1天开始每天检测细胞密度、活力和各种代谢参数。根据NOVA生化分析仪检测的结果控制葡萄糖浓度在6-8g/L，谷氨酸单钠盐浓度在4-7mM。从第三天开始补充流加培养基和自制补料培养基，以后天数隔天流加，第三天补加培养基(包括自制补料培养基和流加培养基)的加入量为发酵培养基总体积的3.5％，第5、7、9、11天补加培养基(包括自制补料培养基和流加培养基)的加入量为发酵培养基总体积的5.5％，自制补料培养基和流加培养基加入的比例为1:10。在细胞培养的第13天停止发酵，收取上清，用高效液相色谱(HPLC)的方法，通过ProteinA柱检测抗体的相对表达量和纯度，结果见表1。糖基化采用LC/MS分析法检测，结果见图7，标准品为贝伐单抗(商品名，阿瓦斯汀)。细胞培养过程中的活细胞密度(见图3)和细胞活力(见图4)采用台盼蓝(0.2％)染色法进行检测。
实施例6
基础培养基：为市售培养基Forti CHO AGT(购自Gibco)
自制补料培养基：取300mmol酪氨酸(181.2g/mol)、140mmol半胱氨酸(121.15g/mol)、100mmol色氨酸(204.23g/mol)，在搅拌和避光的情况下加入到700ml超纯水中，搅拌40min，加入NaOH(32.1g/L)。继续搅拌30min，用5M NaOH调节pH至10.95～11.2，继续搅拌20min，测定浊度，浊度应小于10NTU，定容至1000ml，搅拌10min以上，0.22μm滤膜过滤至无菌试剂瓶。
流加培养基：为市售培养基Acti CHO FeedA CD(购自GE)
细胞株：包含编码抗人血管内皮生长因子(VEGF)抗体核酸的CHO细胞株(参照WO1998045331专利)
在500ml摇瓶中培养，细胞株以1×106cells/mL进行接种，初始培养体积100ml，培养温度37℃，CO2浓度8％，摇床转速130rpm，从培养的第1天开始每天检测细胞密度、活力和各种代谢参数。根据NOVA生化分析仪检测的结果控制葡萄糖浓度在6-8g/L，谷氨酸单钠盐浓度在4-7mM。从第三天开始补充流加培养基和自制补料培养基，以后天数隔天流加，第三天补加培养基(包括自制补料培养基和流加培养基)的加入量为发酵培养基总体积的3.5％，第5、7、9、11天补加培养基(包括自制补料培养基和流加培养基)的加入量为发酵培养基总体积的5.5％，自制补料培养基和流加培养基加入的比例为1:10。在细胞培养的第13天停止发酵，收取上清，用高效液相色谱(HPLC)的方法，通过ProteinA柱检测抗体的相对表达量和纯度，结果见表1。糖基化采用LC/MS分析法检测，结果见图7，标准品为贝伐单抗(商品名，阿瓦斯汀)。细胞培养过程中的活细胞密度(见图3)和细胞活力(见图4)采用台盼蓝(0.2％)染色法进行检测。
对比例1
基础培养基：为市售培养基Hycell CHO Medium(购自Hyclone)
自制补料培养基：取300mmol酪氨酸(181.2g/mol)、100mmol色氨酸(204.23g/mol)，在搅拌和避光的情况下加入到700ml超纯水中，搅拌40min，加入NaOH(32.1g/L)。继续搅拌30min，用5M NaOH调节pH至10.95～11.2，继续搅拌20min，测定浊度，浊度应小于10NTU，定容至1000ml，搅拌10min以上，0.22μm滤膜过滤至无菌试剂瓶。
流加培养基：为市售培养基Acti CHO FeedA CD(购自GE)
细胞株：包含编码抗人肿瘤坏死因子(TNFα)抗体核酸的CHO细胞株(参照WO1997029131专利)
在500ml摇瓶中培养，细胞株以0.85×106cells/mL进行接种，初始培养体积100ml，培养温度37℃，CO2浓度8％，摇床转速130rpm，从培养的第1天开始每天检测细胞密度、活力和各种代谢参数。根据NOVA生化分析仪检测的结果控制葡萄糖浓度在6-8g/L，谷氨酸单钠盐浓度在4-7mM。从第三天开始补充流加培养基和自制补料培养基，以后天数隔天流加，第三天补加培养基(包括自制补料培养基和流加培养基)的加入量为发酵培养基总体积的3.5％，第5、7、9、11天补加培养基(包括自制补料培养基和流加培养基)的加入量为发酵培养基总体积的5.5％，自制补料培养基和流加培养基加入的比例为1:10。在细胞培养的第13天停止发酵，收取上清，用高效液相色谱(HPLC)的方法，通过Protein A柱检测抗体的相对表达量和纯度,结果见表1。
对比例2
基础培养基：为市售培养基Forti CHO AGT(购自Gibco)
自制补料培养基：取300mmol酪氨酸(181.2g/mol)、100mmol色氨酸(204.23g/mol)，在搅拌和避光的情况下加入到700ml超纯水中，搅拌40min，加入NaOH(32.1g/L)。继续搅拌30min，用5M NaOH调节pH至10.95～11.2，继续搅拌20min，测定浊度，浊度应小于10NTU，定容至1000ml，搅拌10min以上，0.22μm滤膜过滤至无菌试剂瓶。
流加培养基：为市售培养基Acti CHO FeedA CD(购自GE)
细胞株：包含编码抗人血管内皮生长因子(VEGF)抗体核酸的CHO细胞株(参照WO1998045331专利)
在500ml摇瓶中培养，细胞株以1×106cells/mL进行接种，初始培养体积100ml，培养温度37℃，CO2浓度8％，摇床转速130rpm，从培养的第1天开始每天检测细胞密度、活力和各种代谢参数。根据NOVA生化分析仪检测的结果控制葡萄糖浓度在6-8g/L，谷氨酸单钠盐浓度在4-7mM。从第三天开始补充流加培养基和自制补料培养基，以后天数隔天流加，第三天补加培养基(包括自制补料培养基和流加培养基)的加入量为发酵培养基总体积的3.5％，第5、7、9、11天补加培养基(包括自制补料培养基和流加培养基)的加入量为发酵培养基总体积的5.5％，自制补料培养基和流加培养基加入的比例为1:10。在细胞培养的第13天停止发酵，收取上清，用高效液相色谱(HPLC)的方法，通过ProteinA柱检测抗体的相对表达量和纯度，结果见表1。
表1
Figure PCTCN2016092322-appb-000001

Claims (10)

  1. 一种自制补料培养基，其特征在于，含有90mM～500mM半胱氨酸。
  2. 根据权利要求1所述的培养基，其特征在于，所述半胱氨酸的浓度为100mM～400mM。
  3. 根据权利要求1所述的培养基，其特征在于，所述半胱氨酸的浓度为120mM～300mM。
  4. 根据权利要求1所述的培养基，其特征在于，还含有酪氨酸和色氨酸，所述酪氨酸的浓度为200mM～400mM，所述色氨酸的浓度为50mM～150mM。
  5. 根据权利要求1至4其中任意一项所述的培养基，其特征在于，所述自制补料培养基pH为10～12。
  6. 一种使用权利要求1至5其中任意一项所述的培养基培养哺乳动物细胞的方法，其特征在于，包括以下步骤：
    1)在基础培养基中接种包含编码抗体核酸的哺乳动物细胞；
    2)进入细胞增殖期后补加所述自制补料培养基；
    3)监测细胞密度调整培养时间。
  7. 根据权利要求6所述的方法，其特征在于，进入细胞增殖期后，除补加自制培养基外还补充流加培养基。
  8. 根据权利要求7所述的方法，其特征在于，在培养第三天开始至培养结束每天或部分天数补加自制补料培养基和流加培养基。
  9. 根据权利要求7所述的方法，其特征在于，每次补加自制补料培养基和流加培养基的量为发酵培养基总体积的1％～10％，自制补料培养基和流加培养基加入的比例为1:10。
  10. 根据权利要求7所述的方法，其特征在于，补加自制补料培养基和流加培养基的时间为第3、5、7、9、11天。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008141207A1 (en) * 2007-05-11 2008-11-20 Amgen Inc. Improved feed media
CN101663391A (zh) * 2007-04-26 2010-03-03 中外制药株式会社 使用含高浓度氨基酸的培养基的细胞培养方法
CN102911958A (zh) * 2012-10-09 2013-02-06 暨南大学 编码重组人TNFR-Fc融合蛋白的基因及其应用
CN103361355A (zh) * 2013-07-29 2013-10-23 暨南大学 编码重组rhHER2-mAb人源化单克隆抗体的基因及其应用
CN104560882A (zh) * 2014-12-31 2015-04-29 广东东阳光药业有限公司 一种降低酸性变体含量的cho细胞培养工艺

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8516415D0 (en) * 1985-06-28 1985-07-31 Celltech Ltd Culture of animal cells
US5672502A (en) 1985-06-28 1997-09-30 Celltech Therapeutics Limited Animal cell culture
US5118618A (en) * 1989-10-05 1992-06-02 Foodscience Corporation Dimethylglycine enhancement of antibody production
US5593822A (en) * 1993-12-07 1997-01-14 John Wayne Cancer Institute IgG depleted serum preparations and methods for antibody production
CN1300173C (zh) 1996-02-09 2007-02-14 艾博特生物技术有限公司 结合人TNFα的人抗体
US6243372B1 (en) * 1996-11-14 2001-06-05 Omnipoint Corporation Methods and apparatus for synchronization in a wireless network
US20020012991A1 (en) * 1997-04-07 2002-01-31 Florence Chua Nee Ho Kit Fong Cell culture media for enhanced protein production
ES2236634T3 (es) 1997-04-07 2005-07-16 Genentech, Inc. Anticuerpos anti-vegf.
DE19735711C2 (de) 1997-08-18 2001-04-26 Aventis Pharma Gmbh Verfahren zur Herstellung eines Vorläufers von Insulin oder Insulinderivaten mit korrekt verbundenen Cystinbrücken
CN1238498C (zh) 2004-02-12 2006-01-25 陈志南 动物细胞无血清悬浮培养工艺过程控制参数的方法
KR101370253B1 (ko) 2004-10-22 2014-03-05 암젠 인크 재조합 항체의 재접힘 방법
US8911964B2 (en) 2006-09-13 2014-12-16 Abbvie Inc. Fed-batch method of making human anti-TNF-alpha antibody
NZ575328A (en) 2006-09-13 2012-06-29 Abbott Lab Cell culture improvements
AU2009296708A1 (en) 2008-09-26 2010-04-01 Merck Sharp & Dohme Corp. High titer antibody production
JP5749930B2 (ja) 2008-10-28 2015-07-15 中外製薬株式会社 ペプチド含有動物細胞培養用培地
PT3330370T (pt) * 2010-04-26 2021-05-11 Novartis Ag Processo para cultivo de células cho
US9133493B2 (en) 2011-04-21 2015-09-15 Amgen Inc. Method for culturing mammalian cells to improve recombinant protein production
AR093460A1 (es) 2012-11-14 2015-06-10 Merck Patent Ges Mit Beschränkter Haftung Medios de cultivo celular

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101663391A (zh) * 2007-04-26 2010-03-03 中外制药株式会社 使用含高浓度氨基酸的培养基的细胞培养方法
WO2008141207A1 (en) * 2007-05-11 2008-11-20 Amgen Inc. Improved feed media
CN102911958A (zh) * 2012-10-09 2013-02-06 暨南大学 编码重组人TNFR-Fc融合蛋白的基因及其应用
CN103361355A (zh) * 2013-07-29 2013-10-23 暨南大学 编码重组rhHER2-mAb人源化单克隆抗体的基因及其应用
CN104560882A (zh) * 2014-12-31 2015-04-29 广东东阳光药业有限公司 一种降低酸性变体含量的cho细胞培养工艺

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