CN1367258A - Preprartion method of improved hepatoma murine monoclonal antibody - Google Patents
Preprartion method of improved hepatoma murine monoclonal antibody Download PDFInfo
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- CN1367258A CN1367258A CN 02110583 CN02110583A CN1367258A CN 1367258 A CN1367258 A CN 1367258A CN 02110583 CN02110583 CN 02110583 CN 02110583 A CN02110583 A CN 02110583A CN 1367258 A CN1367258 A CN 1367258A
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Abstract
The present invention relates to an improved preparation method of hepatoma murine monoclonal antibody. This invented method uses the improved DXL serum-free medium as culture medium, i.e. various growth factors, such as neural cell growth factor, epidermal growth factor, fibroblast grwoth factor or embyro growth factor are added in the DXL serum-free proteinless medium, and the content is 0.01-0 mg/L. This invention adopts packed bed bioreactor to make high-density hybridoma cell continuous culture, and its technological conditions are as follows: temp. is 30-40 deg.c, rotating speed is 300-480 rpm, pH is 6.8-7.25, PO2 is 0.5-1.95 bar, PCO2 is 0.5-1.95 bar, PN2 is 0.5-1.95 bar, air flow rate is 0.1-2 hr. and pressure is 2-5 bar. Its cell density is upt o 10 to the eight power/ml, and the yield of monoclonal antibody of each litre of culture medium can be up to 1000-1500 mg.
Description
Technical field
What the present invention relates to is the monoclonal antibody production field, relate in particular to a kind of by changing bio-reactor mode of operation and the preparation method of the liver cancer murine resource monoclonal antibody of technology and improved culture medium prescription.
Background technology
Applicant of the present invention and Shanghai Mei Shan Bioisystech Co., Ltd have applied for a about the product of no endogenous immunoglobulin hepatoma monoclonal antibody, the patent of methods and applications on June 25th, 1999, number of patent application is 99113824.4, and denomination of invention is: liver cancer murine source monoclonal antibody, preparation method and the application thereof of no endogenous immunoglobulin.
At that time, the mouse resource monoclonal antibody that countries in the world produced, no matter be whole molecule or micromolecular, if not serum-free, protein-free medium as culture medium, no matter adopt which kind of production method, the product that is obtained all contains the endogenous immunoglobulin (mouse or ox) of varying degree, this not only difficult quality control and the stdn of product that contains endogenous immunoglobulin, but also containing some unsafe factors.
99113824.4 it is matrix that number patent application successfully provides with the serum-free protein-free medium, prepare the method for the mouse source hepatoma monoclonal antibody of no endogenous immunoglobulin in bio-reactor by the high-density culture mouse hybridoma cell, the cell density of gained is 10
4-6/ ml, antibody concentration is 100% up to the ratio of components in 1mg/ml and the antibody protein.Can be used as the bulk drug of preparation treatment liver cancer preparation.Can avoid with ascites production or with the unstable on the non-constant drug effect of being brought that contains hidden danger in the security of serum nutrient solution production liver cancer monoclonal antibody and monoclonal antibody ratio of components in pharmaceutical protein, as far as our knowledge goes, this is the mouse source hepatoma monoclonal antibody of the no endogenous immunoglobulin that successfully obtains with bioreactor technology for the first time.
The present invention has done improvement on the basis of preceding patent, selected the reactor that is fit to more, has optimized reaction conditions simultaneously, and we have also added some kinds of cell growth factors in nutrient solution in addition, its objective is further raising cell density and antibody yield.The target of any vitro culture all is the biological internal milieu of simulation, cell is grown to greatest extent, and internal milieu is complicated and changeable, various factors is all linked with one another, be difficult to duplicate fully, this is the cell in vitro reason of cultivating the most critical be difficult to reach cells in vivo density just.Somatomedin was the distinctive key factor of a kind of internal milieu originally, added in external environment, can obviously improve the survival rate of cell, and cellular form is improved, and then improved cell density and antibody yield.In the past, it is fewer that people use, and is because cell growth factor also is a kind of protein, in the time of isolating to the end with regard to many one heavy misgivings.But now, can be by separating these rho factors in the purge process of Protein A or G fully, therefore, it is necessary and feasible adding these cell growth factors.
Summary of the invention
The present invention has acted on the technological line of No. 99113824.4 patent applications, and purpose provides the method that the higher production of a kind of productive rate does not have the liver cancer murine source monoclonal antibody of endogenous immunoglobulin.
Therefore, the contriver improves on the basis of 99113824.4 patents, adopts filling bed type cultured continuously reactor, has correspondingly changed the processing condition of reactor.Temperature is 30～40 ℃, and rotating speed is 300-480RPM, and pH is 6.8～7.25, PO
2Be 0.5-1.95 crust, PCO
2Be 0.5-1.95 crust, PN
2Be the 0.5-1.95 crust, air flow quantity is that 0.1-2/ hour, pressure are the 2-5 crust.The most important thing is, the present invention adopts modified form DXL serum-free medium, promptly in the prescription of the DXL of preceding patent serum-free, protein-free medium matter, one or more somatomedins have been added, comprise the nerve growth factor, Urogastron, fibroblast growth factor, the embryo growth factor etc., the content of various somatomedins is the 0.01-0.1 mg/litre, and these somatomedins can be separated in the purge process of afterwards Protein A or G fully.
Production Flow Chart step of the present invention sees Table 1:
Table 1. Production Flow Chart steps flow chart explanation clean environment degree is produced cell bank and is taken out 100,000 grades in Hepama-1 cell
↓ CO in a small amount increases
2100,000 grades of incubators
100,000 grades of ↓ mass production filling bed type reactors
↓ collect training liquid to train centrifugal 100,000 grades of liquid
↓ concentrating low-temperature is vaporized or is distilled 100,000 grades
↓ separation and purification chromatography
100,000 grades of ↓ concentrated vacuum low-pressure low temperature
↓
Concentration, 100,00 grades of purity quality control mouse source DNAs
Intracellular toxin, exogenous factor
100 grades of ↓ filtration packing
Preparation modified form DXL serum-free medium:
Be characterized in having added on the basis of DXL nutrient solution one or more somatomedins, its content is the 0.01-0.1 mg/litre.Modified form DXL serum-free culture liquid formula: mg/litre
VITAMIN B4 0.1-0.5
L-Ala 5-10
Aluminum chloride 0.0005-0.001
Ammonium meta-vanadate 0.0005-0.001
Arginine 100-500
L-asparagine 10-50
Aspartic acid 5-30
Bariumchloride 0.001-0.005
Vitamin H 0.05-0.5
Calcium chloride 10-100
Choline chloride 60 10-100
Potassium chromium sulfate 0.0005-0.005
Citric acid 10-50
Citrulline 1-10
Cobalt chloride 0.001-0.005
Copper sulfate 0.001-0.01
Halfcystine 10-100
Dilinoleic acid Yelkin TTS 0.1-1
Two stearic acid Yelkin TTS 0.1-1
Thanomin 1-10
Edetate 5-10
Polyoxyethylene glycol 0.5-4
Ferrous sulfate 0.5-5
Atom iron 2-5
Flavin adenine dinucleotide 0.01-0.05
Folic acid 1-5
Germanium dioxide 0.0001-0.001
L-glutamic acid 10-50
Glutamine 100-500
Glycine 1-10
Glucose 2000-10000
Histidine 10-100
Xanthoglobulin 1-10
Isoleucine 100-500
Leucine 100-500
Linolic acid 0.01-0.1
Lithium chloride 5-50
Methionin 50-500
Magnesium chloride 50-500
Manganous chloride tetrahydrate 0.00005-0.0005
Methionine(Met) 10-50
Molybdic acid 0.00005-0.0005
MOPS 1000-10000
Inositol 10-50
Niacinamide 1-10
Nickelous nitrate 0.0001-0.0005
Ornithine 1-10
Oxaloacetic acid 1-10
Pantothenic acid 1-5
Phenol red 1-10
Phenylalanine 10-100
Potassium Bromide 0.00005-0.0005
Repone K 100-500
Potassiumiodide 0.00005-0.0005
Proline(Pro) 10-100
Progesterone 0.001-0.01
Putrescine 0.1-0.5
Benadon 0.1-0.5
Pyruvic acid 100-500
Riboflavin 0.01-0.05
Rubidium chloride 0.000005-0.00005
Serine 10-100
Silver chloride 0.000001-0.00001
Sodium-chlor 5000-10000
Sodium Fluoride 0.001-0.01
Sodium Nitroprusside 1-10
Sodium phosphate dibasic 100-1000
Sodium Selenite 0.01-0.05
Spermine 0.1-1
Tin protochloride 0.00005-0.0005
Taurine 10-50
Thioctic Acid 0.08-0.4
VitB1 0.1-0.8
Threonine 8-18
Thymus pyrimidine 0.2-1.5
Titanium chloride 0.0005-0.004
Give birth to plain 0.05-4
Tryptophane 1-10
Tween 80 0.05-3
Tyrosine 25-45
Xie Ansuan 70-120
Vitamins B
122-15
Zinc sulfate 0.6-5
Add:
One or more each 0.01-0.1 mg/litre in the various somatomedins such as Urogastron, the nerve growth factor, fibroblast growth factor or the embryo growth factor.
Cultivate the hybridoma that to secrete the liver cancer murine resource monoclonal antibody with the above-mentioned modified form DXL nutrient solution for preparing.
Take out the Hepama-1 hybridoma through amplification in a small amount from produce cell bank, (inoculating cell density is A * 10 to cell before the phase of taking the logarithm
5/ ml) move into filling bed type cell biological reactor to cultivate monitoring process condition (temperature: 30～40 ℃, rotating speed: 300-480RPM, pH:6.8～7.25, PO
2: 0.5-1.95 crust, PCO
2: 0.5-1.95 crust, PN
2: 0.5-1.95 crust, air flow quantity: the 0.1-2 liter/hour, pressure: 2-5 crust), treat that it is A * 10 to density that antibody concentration is about in 1-1.5mg/ml or the cell biological reactor cell long
8During/ml the left and right sides, collect nutrient solution, the low-temperature distillation method is centrifugal to be concentrated, and obtains primary products.
With HPLC separation and purification primary products,, simultaneously product is carried out quality control again, at last through obtaining the whole molecule monoclonal antibody of the liver cancer murine source monoclonal antibody of no endogenous immunoglobulin after the sterile filtration with low-pressure low-temperature subliming method concentrated product.Use Protein G or Protein A pillar that the somatomedin of adding is separated fully in this step.
In case of necessity, available papoid, stomach en-etc. routinely the enzyme cutting method whole molecule monoclonal antibody that will not have a liver cancer murine source monoclonal antibody of endogenous immunoglobulin be prepared into the liver cancer murine source small molecules monoclonal antibody Fab of no endogenous immunoglobulin or (Fab)
237 ℃ of effects of liver cancer murine source whole molecule monoclonal antibody of the no endogenous immunoglobulin that contains enzymic digestion liquid and equal volume that will prepare earlier, after endonuclease reaction stops, again with 4 ℃ of dialysis of pH8.0 phosphoric acid buffer 12-20 hour, again with the HPLC separation and purification, obtain the liver cancer murine source small molecules monoclonal antibody of no endogenous immunoglobulin at last.
Owing to adopted technical scheme as above, therefore, to compare with preceding patent, this patent has following significant advantage:
1, adopts filling bed type cultured continuously reactor.Its maximum characteristics are, cell distributes in jar and concentrates, and density is big in the effective volume, have reduced cell poor growth or fully long risk when low density, and cell all is adsorbed on the microcarrier, and free considerably less in nutrient solution is convenient to the collection of product.Therefore, at all culturing cells, especially in the bio-reactor of zooblast and hybridoma, microcarrier filling bed type bio-reactor advantage is remarkable, more and more becomes the main flow in the cell culture apparatus.
2, adopt modified form DXL serum-free medium, on the basis of DXL nutrient solution, increased somatomedin, helped to improve yield, and, because these somatomedins can be separated in the purge process of afterwards Protein A or G fully, therefore, do not influence the character of product antibody.
3, the processing condition of bio-reactor have been improved.
Generally speaking, the monoclonal antibody that present method is produced does not wherein contain any mouse endogenous immunoglobulin and any ox endogenous immunoglobulin, and the ratio of components in the antibody protein of monoclonal antibody is 100%, and the yield height, and cell density is 10
6-8/ ml, every liter of yield of training the monoclonal antibody in the liquid reaches 1000-1500mg, meets the requirement of scale operation.
The present invention's one class does not have liver cancer murine resource monoclonal antibody Hepama-1, Hepama-9403, Hepama-9501 (whole molecule and small molecules) or the like of endogenous immunoglobulin, the preparation method is similar, making of liver cancer murine resource monoclonal antibody Hepama-1 with no endogenous immunoglobulin is embodiment below, further illustrate the present invention, but do not limit the scope of the invention.
Description of drawings
Fig. 1 measures ox IgG for indirect immunoperoxidase connection absorption method (sELISA method).
As shown in the figure, the CUT-OFF value is 0.5.The left side of figure is the reactor product monoclonal antibody, and the centre is a modified form DXL nutrient solution, and the right is for containing 20% new-born calf serum RPMI-1640.The reactor product monoclonal antibody does not contain ox IgG (endogenous sphaeroprotein).
Fig. 2 is the active testing (indirect immunofluorescence) of reactor product monoclonal antibody.
Fig. 2 (1) is the activity of reactor product monoclonal antibody, and Fig. 2 (2) is the negative control of immunoglobulin (Ig), and Fig. 2 (3) is the positive control of polyclonal antibody.
The test result of the DXL nutrient solution serum-free of Fig. 3 improvement.
A figure left side is negative for the DXL nutrient solution dyeing of improvement; Figure is right for containing the RPMI-1640 of 10% new-born calf serum, the Coomassie Brilliant Blue G250 strong positive that dyes.
Embodiment
The liver cancer murine resource monoclonal antibody Hepama-1 of the no endogenous immunoglobulin of embodiment 1 preparation: 10 liters of configuration modified form DXL serum-free mediums: need 3 milligrams of VITAMIN B4; 70 milligrams of L-Ala; 0.007 milligram in aluminum chloride; 0.007 milligram of ammonium meta-vanadate; arginine 3 grams; 300 milligrams of l-asparagines; 150 milligrams of aspartic acids; 0.025 milligram of bariumchloride; 2.5 milligrams of vitamin Hs; 500 milligrams in calcium chloride; 500 milligrams of choline chloride 60s; 0.025 milligram of potassium chromium sulfate; 300 milligrams of citric acids; 50 milligrams of citrulline; 0.025 milligram of cobalt chloride; 0.05 milligram in copper sulfate; 500 milligrams of halfcystines; 5 milligrams in dilinoleic acid Yelkin TTS; 5 milligrams in two stearic acid Yelkin TTS; 50 milligrams of thanomins; 70 milligrams of edetates; 20 milligrams of polyoxyethylene glycol; 25 milligrams in ferrous sulfate; 25 milligrams of atom iron; 0.25 milligram of flavin adenine dinucleotide; 25 milligrams in folic acid; 0.005 milligram of germanium dioxide; 250 milligrams in L-glutamic acid; glutamine 2.5 grams; 50 milligrams of glycine; glucose 60 grams; 500 milligrams of Histidines; 50 milligrams of xanthoglobulin; Isoleucine 3 grams; leucine 3 grams; 0.5 milligram of linolic acid; 250 milligrams of lithium chlorides; Methionin 2.5 grams; magnesium chloride 2.5 grams; 0.0025 milligram of Manganous chloride tetrahydrate; 250 milligrams of methionine(Met)s; 0.0025 milligram of molybdic acid; 50 milligrams of MOPS; 300 milligrams of inositols; 50 milligrams of niacinamide; 0.0025 milligram of nickelous nitrate; 50 milligrams of ornithines; 50 milligrams of oxaloacetic acids; 25 milligrams in pantothenic acid; 50 milligrams in phenol; 500 milligrams of phenylalanines; 0.0025 milligram of Potassium Bromide; Repone K 3 grams; 0.0025 milligram of potassiumiodide; 500 milligrams of proline(Pro); 0.05 milligram of Progesterone; 2.5 milligrams of putrescine; 2.5 milligrams of Benadons; pyruvic acid 2.5 grams; 0.25 milligram in riboflavin; 0.00025 milligram of rubidium chloride; 500 milligrams of Serines; 0.00005 milligram of silver chloride; sodium-chlor 70 grams; 0.05 milligram of Sodium Fluoride; 50 milligrams of Sodium Nitroprussides; Sodium phosphate dibasic 5 grams; 0.25 milligram of Sodium Selenite; 5 milligrams of spermine; 0.0025 milligram of tin protochloride; 250 milligrams of taurines; 1.5 milligrams of Thioctic Acids; 4 milligrams of VitB1s; 130 milligrams of Threonines; 7 milligrams of thymus pyrimidines; 0.02 milligram of titanium chloride; plain 20 milligrams of fertility; 50 milligrams of tryptophanes; 15 milligrams of tween 80s; 350 milligrams in tyrosine; Xie Ansuan 1 gram; vitamins B
1280 milligrams, 28 milligrams in zinc sulfate, Urogastron 300 micrograms, the nerve growth factor 300 micrograms make it fully to be dissolved in the deionized water, and constant volume is 10 liters.Sterile filtration is standby.
From produce cell bank, take out the Hepama-1 cell, by moving into a large amount of cultured continuously hybridoma Hepama-1 in 10 liters of filling bed type cell biological reactors after the amplification in a small amount.By following reactor process condition, temperature: 37 ℃; Rotating speed: 420RPM; PH:7.0; PO
2: 1.25 crust; PCO
2: 1.25 crust; PN
2: 1.25 crust; Air flow quantity: 0.8 liter/hour; Pressure: 3.5 crust, carry out high-density hybridoma cultured continuously.Cell density in the reactor is up to 1.1 * 10
8About/ml.Antibody concentration is up to 1.2mg/ml.
Again with HPLC (BECKMAN Biosys 2000) separation and purification primary products, adopt the ProteinA pillar to separate the somatomedin of adding, with vacuum low-pressure cryoconcentration (AES2010 AutomaticEnvironmental SpeedVac) product, by the quality standard calibrating, conformance with standard is at last through obtaining the whole molecule monoclonal antibody Hepama-1 of the liver cancer murine source monoclonal antibody of no endogenous immunoglobulin after the sterile filtration.
Embodiment 2
The liver cancer murine resource monoclonal antibody Hepama-1 of the no endogenous immunoglobulin that makes with embodiment 1 is a raw material, prepares small molecules monoclonal antibody sHepama-1 (Fab) with conventional enzyme cutting method:
Preparation earlier contains Digestive system (the 0.02 moles of ethylene diamine tetraacethyl disodium of 0.1 mg/ml papoid, 0.02 the mole halfcystine), with 37 ℃ of effects of liver cancer murine source whole molecule monoclonal antibody of the no endogenous immunoglobulin of the Digestive system that contains 0.1 mg/ml papoid of now joining and equal volume 8 hours, add iodo-acid amide again to 0.3 mole of final concentration, to stop endonuclease reaction, then with 4 ℃ of dialysis of pH8.0 phosphoric acid buffer 15 hours, with the HPLC separation and purification, obtain the small molecules monoclonal antibody sHepama-1 (Fab) of the liver cancer murine source monoclonal antibody of no endogenous immunoglobulin at last.
Claims (4)
1. the preparation method of an improved liver cancer murine resource monoclonal antibody is characterized in that, is culture medium with modified form DXL serum-free medium, adopts the filling bed type bio-reactor, and high-density hybridoma continuous culture method continues and makes with separation and purification.
2. the preparation method of liver cancer murine resource monoclonal antibody as claimed in claim 1, it is characterized in that, the prescription of described modified form DXL serum-free culture matrix, be on the basis of the DXL of 99113824.4 patents serum-free, protein-free medium, added in the various somatomedins such as the nerve growth factor, Urogastron, fibroblast growth factor or the embryo growth factor one or more.
3. the preparation method of liver cancer murine resource monoclonal antibody as claimed in claim 2 is characterized in that, the content of the various somatomedins of interpolation is the 0.01-0.1 mg/litre.
4. as the preparation method of any described liver cancer murine resource monoclonal antibody of claim 1-3, it is characterized in that, the processing condition of described filling bed type bio-reactor high-density hybridoma continuous culture method are, temperature is 30～40 ℃, rotating speed is 300-480RPM, pH is 6.8～7.25, PO
2Be 0.5-1.95 crust, PCO
2Be 0.5-1.95 crust, PN
2Be the 0.5-1.95 crust, air flow quantity is that 0.1-2/ hour, pressure are the 2-5 crust.
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|---|---|---|---|
| CNB021105839A CN1180080C (en) | 2002-01-18 | 2002-01-18 | Preprartion method of improved hepatoma murine monoclonal antibody |
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|---|---|---|---|
| CNB021105839A CN1180080C (en) | 2002-01-18 | 2002-01-18 | Preprartion method of improved hepatoma murine monoclonal antibody |
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| CN1367258A true CN1367258A (en) | 2002-09-04 |
| CN1180080C CN1180080C (en) | 2004-12-15 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017773A (en) * | 2014-05-08 | 2014-09-03 | 南京工业大学 | Anti-Bel/fu monoclonal antibody and hybridoma cell line |
| CN104087558A (en) * | 2014-07-08 | 2014-10-08 | 西藏天虹科技股份有限责任公司 | Serum-free medium for hybridoma cells |
| CN105143444A (en) * | 2013-03-15 | 2015-12-09 | 瑞泽恩制药公司 | Serum-free cell culture medium |
| CN106029690A (en) * | 2013-10-18 | 2016-10-12 | 维也纳农业大学 | Purification of proteins |
| US10927342B2 (en) | 2015-08-04 | 2021-02-23 | Regeneran Pharmaceuticals, Inc. | Taurine supplemented cell culture medium and methods of use |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418330B (en) * | 2008-06-20 | 2012-01-04 | 华东理工大学 | Non protein culture medium adapted to large-scale culture of NSO cell and production of antibody |
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2002
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105143444A (en) * | 2013-03-15 | 2015-12-09 | 瑞泽恩制药公司 | Serum-free cell culture medium |
| US11332771B2 (en) | 2013-03-15 | 2022-05-17 | Regeneron Pharmaceuticals, Inc. | Serum-free cell culture medium |
| US11970724B2 (en) | 2013-03-15 | 2024-04-30 | Regeneron Pharmaceuticals, Inc. | Serum-free cell culture medium |
| CN106029690A (en) * | 2013-10-18 | 2016-10-12 | 维也纳农业大学 | Purification of proteins |
| CN104017773A (en) * | 2014-05-08 | 2014-09-03 | 南京工业大学 | Anti-Bel/fu monoclonal antibody and hybridoma cell line |
| CN104087558A (en) * | 2014-07-08 | 2014-10-08 | 西藏天虹科技股份有限责任公司 | Serum-free medium for hybridoma cells |
| US10927342B2 (en) | 2015-08-04 | 2021-02-23 | Regeneran Pharmaceuticals, Inc. | Taurine supplemented cell culture medium and methods of use |
| US11312936B2 (en) | 2015-08-04 | 2022-04-26 | Regeneron Pharmaceuticals, Inc. | Taurine supplemented cell culture medium and methods of use |
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