CN1114619C - Liver cancer murine monoclone antibody without endogenous immunoglobulin and its preparation and application - Google Patents
Liver cancer murine monoclone antibody without endogenous immunoglobulin and its preparation and application Download PDFInfo
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Abstract
The present invention relates to a liver cancer mouse source monoclonal antibody without endogenous immune globulins. Culture solutions without blood serum and proteins are taken as culture substrates, and cell bio-reactor high-density hybridoma cells are continuously cultured, separated and purified for preparing the liver cancer mouse source monoclonal antibody. The cell density of the liver cancer mouse source monoclonal antibody is as high as 10<6>/ml, the concentration of the antibody is as high as 1 mg/ml, and composition ratio in antibody proteins is 100%. The liver cancer mouse source monoclonal antibody can be used as raw medicinal medicines for preparing preparations for treating the liver cancer and can avoid the safety hidden danger caused by using ascitic fluids or culture solutions comprising blood serum to produce liver cancer monoclonal antibodies and the medicine effect instability brought by the composition ratio of the monoclonal antibody in medicine proteins.
Description
The present invention relates to the monoclonal antibody field, relate in particular to a kind of preparation method of mouse hybridoma cell strains Hepama-1 excretory liver cancer murine resource monoclonal antibody.
Koehler in 1975 and Milstein (Nature Vol.256, p495-497) prepared first mouse resource monoclonal antibody in the world with hybridoma technology, for biotechnology has been opened a brand-new field, along with development of biology, monoclonal antibody technique has also had very big development, and is various whole molecules, micromolecular, humanized, cocktail, bifunctional so that all appearances successively of anti-unique monoclonal antibody; Various monoclonal antibodies have various purposes, that curative is used, diagnosis is medicinal, it is usefulness, that purifying is used to analyze, check usefulness etc.That Chinese Academy of Sciences's cell in 1985 is thanked is great, Yao Xin etc. succeeds in developing human liver cancer monoclonal antibody Hepama-1, and (p263-270), this is one of mouse resource monoclonal antibody of the liver cancer reported the earliest in the world for Journal of Experimental Biology, Vol.18.Wherein, this mouse hybridoma cell strains Hepama-1 number of patent application be in 95111623.1 the patent application by preservation, its preserving number is CCTCCNO:C95013.
The mouse resource monoclonal antibody is by the hybridoma excretory, and the growth of hybridoma and survival must have nutrient solution to keep (Gillis S, Henney CS, J Immunol 1981 May, 126 (5): 1978-1984), and foetal calf serum or new-born calf serum are the necessary compositions of nutrient solution.
Complicated component in view of serum, the quality difference of every batch of serum, if the important source material when being used as bio-pharmaceutical and producing can be brought problem to the safety of medicine, for many years people to wish to set up a kind of be the cell culture processes of matrix with the serum-free protein-free medium always.After nineteen sixty-five, through Matsuya, people's such as Fuve research, the serum-free culture of cell is initial success (MatsuyaY, Tohoku J Exp Med 1965 Jun25,86 (1): 84-92 finally; Fuve RM et.al, J Bacteriol1966 Oct, 92 (4): 1150-1153).And the composition that has found the most critical of serum free medium is a Regular Insulin, Transferrins,iron complexes, thanomin, selenium etc., and after the nineties, formation with DME/F12 or RDF be basic medium with Regular Insulin, Transferrins,iron complexes, thanomin, selenium and various somatomedins etc. are multiple serum free medium series (the Ozturk SS of main component, et al., J Biotechnol.1990Nov; 16 (3-4): 259-278; Shinohara K, et al., Agric Biol Chem.1990 Oct, 54 (10): 2599-2603).But Regular Insulin, Transferrins,iron complexes, somatomedins etc. still are protein, and Regular Insulin still is a kind of hormone, this still brings certain difficulty to separate targets albumen.Tsitologiia in 1976 etc. at first report with a kind of serum-free protein-free medium based on inorganic components and cultivate lymphocyte achieving success (Voitenok, Tsitologiia, 1976 Mar, 18 (3): 356-360), afterwards, Pinchuk in 1984 etc. cultivate the hybridoma achieving success with the serum-free protein-free medium, and (Pinchuk GV et.al, Eksp Onkol 1984,6 (5): 74-75).
But these substratum major parts can only be used as laboratory study.As is generally known during the modern biotechnology industrialization, biological product all must be by obtaining in the bio-reactor, at present, bio-reactor is divided into two classes, and a class is used for the expression product of mass production bacterium, promptly so-called fermentor tank; The another kind of secretion product that then is used for the mass production cell, promptly so-called cell biological reactor.From technical standpoint, the technical difficulty of cell biological reactor is bigger, and successful report is few, and Antoine etc. once produce insect cell Sf-9 with the cell biological reactor of 4L and succeed, and its cell density reaches 4～15 * 10
6/ ml (Antoine et.al., Biotechnology and Bioengineering, Vol.43, p881-891,1994).Monoclonal antibody is also produced with the cell biological reactor, China Taiwan's scholars Meng MH in 1988 etc. at first produce anti-hepatitis B virus surface antigen monoclonal antibodies achieving success (Meng MH with the tubular fibre filling type bioreactor, et al., Chung Hua MinKuo Wei Sheng Wu Chi Mien I Hsueh Tsa Chih.1988 Aug, 21 (3): 125-140).American scholar Heifetz in 1989 at first is matrix with the serum-free medium, in the tubular fibre filling type bioreactor, produce anti-people's Zeta protein mouse resource monoclonal antibody achieving success (HeifetzAH et.al., Biotechniques 1989 Feb, 7 (2): 192-199), but the foreign protein of its product accounts for 5%.Czech scholar Franek was a matrix with serum-free, protein-free medium at first in 1991, reported monoclonal antibody success (the Franek Fet.al. that in a kind of breadboard bio-reactor, prepares, Cytotechnology 1991 Sep, 7 (1): 33-38), but all these reports not only only are Laboratary type, also all are intermittently to cultivate type.So far, not retrieving with serum-free, protein-free medium is matrix, in type of production cell biological reactor with the report of the mode scale production mouse resource monoclonal antibody of cultured continuously.
The mouse source monoclonal antibody that present countries in the world are produced, no matter be whole molecule or micromolecular, if not using serum-free, no albumen conditioned 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.But, be matrix with serum-free, no albumen conditioned medium, in bio-reactor, do not see the report of success because of technical difficulty with the mouse resource monoclonal antibody of the no endogenous immunoglobulin of preparation by the high-density culture mouse hybridoma cell.
The purpose of this invention is to provide a class does not have the liver cancer murine resource monoclonal antibody of endogenous immunoglobulin, it is the product that in type of production cell biological reactor, makes in the mode of cultured continuously, the ratio of components of monoclonal antibody in antibody protein can reach 100%, can be used for preparing the medicine for the treatment of liver cancer.
The liver cancer murine resource monoclonal antibody of no endogenous immunoglobulin of the present invention is meant the liver cancer murine resource monoclonal antibody that contains ox endogenous immunoglobulin and mouse endogenous immunoglobulin anything but.(Fig. 1 is the detected result (sELISA) of anti-ox IgG to hybridoma Hepama-1 culture supernatant in DXL serum-free, protein-free medium, proves in the hybridoma Hepama-1 culture supernatant not have bovine anteserum IgG).
One class does not have the liver cancer murine source monoclonal antibody Hepama-1 of endogenous immunoglobulin, Hepama-9403, Hepama-9501 etc. are to be culture medium with DXL serum-free protein-free medium, adopt cell biological reactor high-density cultured continuously hybridoma to make, the whole molecule monoclonal antibody of the liver cancer murine source monoclonal antibody of no endogenous immunoglobulin and by the small molecule segment monoclonal antibody that constitutes whole molecule for preparing through conventional enzyme cutting method all can be used for preparing the medicine for the treatment of liver cancer.
The production technique of no endogenous immunoglobulin liver cancer murine resource monoclonal antibody was divided into for two steps, earlier with DXL serum-free, protein-free medium mass production hybridoma in bio-reactor, again with special chromatographic process separation, purifying hepatoma monoclonal antibody.All technology is all carried out under aseptic condition.
Production Flow Chart step (table 1):
Table 1. Production Flow Chart step
Process description clean environment degree
Produce cell bank and take 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 bio-reactors
↓
Collect centrifugal 100,000 grades of training liquid training liquid
↓
Concentrating low-temperature is vaporized or is distilled 100,000 grades
↓
The separation and purification chromatography
↓
Concentrate 100,000 grades of vacuum low-pressure low temperature
↓
Concentration, purity
100,000 grades of quality control mouse source DNAs
Intracellular toxin, exogenous factor
↓
Filter to divide and change 100 grades
Preparation DXL serum-free protein-free medium:
Be characterized in that the nutrient solution that is used for culturing cell does not contain any serum and albumen; That is to say that serum and proteic content are zero in the nutrient solution.DXL serum-free protein-free medium prescription: 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
Cultivate the hybridoma that to secrete the liver cancer murine resource monoclonal antibody with the above-mentioned DXL serum-free protein-free medium for preparing.Fig. 2 is the count value of hybridoma Hepama-1 in DXL serum-free, protein-free medium, and the density that shows hybridoma in DXL serum-free, protein-free medium is not less than and is containing in the serum nutrient solution.
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
4/ mL), move into the cell biological reactor and cultivate, monitoring process condition (temperature: 30～40 ℃, rotating speed: 100-500RPM, pH:6.5～7.5, PO
2: 0.1-2 crust, PCO
2: 0.1-2 crust, PN
2: 0.1-2 crust, air flow quantity: the 1-5 liter/hour, pressure: 1-5 crust), treat in the cell biological reactor cell long be A * 10 to density
6About/ml, when antibody concentration is about 0.5-1mg/ml, 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.
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.
The quality evalution of liver cancer murine resource monoclonal antibody that the present invention does not have endogenous immunoglobulin is as follows:
The anti-liver cancer hybridoma cell strain that monoclonal antibody product of the present invention system obtains through screening secreted monoclonal antibody in the serum-free protein-free medium for aseptic virus-free pyrogen-free solution, contains the 80.0-120.0% that monoclonal antibody concentration is the sign value.
[proterties] colourless clear liquid.
[inspection] pH value 6.5-7.5 (two appendix VIH of Chinese Pharmacopoeia nineteen ninety-five version).
Asepticly test by biological products rules (nineteen ninety-five version one one), up to specification.
It is an amount of that bacterial endotoxin is got this product, dilute 10 times after in accordance with the law (two appendix XIE of Chinese Pharmacopoeia nineteen ninety-five version) check that this product contains bacterial endotoxin less than 5Eu for every milliliter.
Mycoplasma is checked through directly culture method inspection, and is up to specification.
Mouse source virus checking is checked according to " preparation of human mouse monoclonal antibody and key Quality Control ", and is up to specification.
Remaining mouse source DNA content in the nutrient solution is measured through the mouse gene group DNA probe spot hybridization of digoxigenin labeled, and the remaining mouse source DNA of this product content is no more than 100pg for every dose.
[calibrating of no ox endogenous immunoglobulin]
1. through ellipsometry imaging biology microscope technology determination: with no endogenous immunoglobulin Hepama-1 monoclonal antibody is solid phase, is liquid phase with the liver cancer cell lysate, the monoclonal antibody product of the present invention (see figure 3) that is positive.
2. through ellipsometry imaging biology microscope technology determination: with anti-ox IgG is solid phase, and monoclonal antibody product of the present invention is negative, and the sample that contains the ox endogenous immunoglobulin is positive.
[calibrating of no mouse endogenous immunoglobulin]
1. through ellipsometry imaging biology microscope technology determination: with no endogenous immunoglobulin Hepama-1 monoclonal antibody is solid phase, is liquid phase with the liver cancer cell lysate, and monoclonal antibody product of the present invention is positive.
2. judge through horseradish peroxidase histochemical staining method: the section of human lymphoid nodal tissue is dyeed, with PBS dyeing feminine gender is prerequisite, the no endogenous immunoglobulin Hepama-1 monoclonal antibody negative (see figure 4) that dyes, and the positive (see figure 5) of mouse IgG in contrast.
3. through ellipsometry imaging biology microscope technology determination: with no endogenous immunoglobulin Hepama-1 monoclonal antibody is solid phase, is liquid phase with lymphocytolysis liquid, and monoclonal antibody product of the present invention is negative.
4. through immunity combination test, do not contain any albumen in the saturated liver cancer cell supernatant liquor in conjunction with monoclonal antibody product of the present invention, do not have any immunocompetence to human liver cancer cell yet, then still contain albumen in the liver cancer cell supernatant liquor of the saturated sample that is combined with the mouse endogenous immunoglobulin, still have immunocompetence human liver cancer cell.
[monoclonal antibody purity] adopts the SDS-PAGE method under reduction and the non-reduced condition to measure monoclonal antibody purity, and immunoglobulin (Ig) (monomer and dimer) content is not less than 95.0%.
[monoclonal anti body burden] adopts the Bradford method, is reference substance with the ovalbumin, measures the monoclonal anti body burden.Every milliliter contains monoclonal antibody 2.5mg.
[immunocompetence] adopts indirect immunofluorescence, is antigen with BEL-7402 or SMMC-7721 liver cancer cell, and immunocompetence is not higher than 2 mcg/ml.
The present invention is no endogenous immunoglobulin liver cancer murine resource monoclonal antibody, the ratio of components in the antibody protein of monoclonal antibody the time as medicinal application not only every batch constant, and can reach 100%.Guaranteed the stability of drug quality.Removed the hidden danger of security aspect.The present invention is to be matrix with serum-free, protein-free medium, is prepared and produces with the method for high-density cells bio-reactor cultured continuously hybridoma.The liver cancer murine resource monoclonal antibody Hepama-1, Hepama-9403, Hepama-9501 etc., micromolecular liver cancer murine resource monoclonal antibody sHepama-1, sHepama-9403, the sHepama-9501 etc. that have now successfully prepared the whole molecule of no endogenous immunoglobulin.With these monoclonal antibodies is carrier, through mark iodine radioisotope-131 etc., makes the medicine that is used for the treatment of liver cancer, and obtains encouraging result, does not wherein have the radioactivity of the anti-liver cancer murine of endogenous source monoclonal antibody mediation
131Iodine is used for the treatment of primary massive hepatocarcinoma 32 examples in late period, and wherein the healthy survival of 13 examples is 8 years, and survival rate was up to 40.2% in 8 years.Through animal experiment, the radioactivity of the anti-liver cancer murine of no endogenous source small molecules monoclonal antibody mediation
131Iodine is respectively applied for the tumour that the liver cancer nude mice is carried in treatment with high, medium and low dosage, and treatment back gross tumor volume all obviously dwindles, and tumor control rate shows that all greater than 30% this medicine truly has the effect that suppresses tumor growth.This kind effect has tangible dose-dependently.Therefore not have the liver cancer murine resource monoclonal antibody (whole molecule or small molecules) of endogenous immunoglobulin be a kind of promising medicine material to this class.
Advantage of the present invention is that the raw material that is adopted is serum-free, protein-free medium, so do not contain any mouse endogenous immunoglobulin and any ox endogenous immunoglobulin in the monoclonal antibody that makes, also do not contain any foreign protein, and the ratio of components in the antibody protein of monoclonal antibody is 100%; Preparing monoclonal antibody with prior art compares, no matter prior art is whole molecule or small molecules, its raw material sources are nothing more than the ascites that contains hybridoma and contain two kinds of the conventional nutrient solutions (containing the serum nutrient solution) of hybridoma, all tool quality instability, the unsafe shortcoming of medication.And monoclonal antibody product of the present invention has just in time avoided these two to be fatal shortcoming in fact to medicine.It is matrix that its key problem in technology is to adopt serum-free, protein-free medium, in the cell biological reactor through high-density (10
6/ product (0.5-1mg/ml) that mL) cultured continuously obtained; With such combination technique, can prepare the liver cancer murine resource monoclonal antibody of no endogenous immunoglobulin with industrial scale; With these monoclonal antibodies is carrier, through mark iodine radioisotope-131 etc., can make the pharmaceutical preparation that is used for the treatment of liver cancer, and obtains encouraging result, so these monoclonal antibodies are a kind of promising bulk drugs.
Description of drawings:
Fig. 1: with the sELISA method to the DXL serum-free of hybridoma Hepama-1, protein free supernatant liquor, DXL serum-free, protein-free medium, contain the detected result of the supernatant liquor of 20% calf serum RPMI1640 nutrient solution
A left side: anti-ox IgG is negative to the detected result of the DXL serum-free of hybridoma Hepama-1, protein free supernatant liquor
In: anti-ox IgG is negative to the detected result of DXL serum-free, protein-free medium
Right: anti-ox IgG is positive to the detected result of the supernatant liquor that contains 20% calf serum RPMI RPMI-1640 of hybridoma.
Fig. 2: after cultivating in 72 hours, hybridoma Hepama-1 at serum-free, protein-free medium, contain the cytometer numerical value in the 20% calf serum RPMI-1640
A left side: the count value of hybridoma Hepama-1 in DXL serum-free, protein-free medium is 7.0 * 10
5Individual/milliliter
Right: the count value of hybridoma Hepama-1 in containing 20% calf serum RPMI-1640 is 6.6 * 10
5Individual/milliliter.
Fig. 3: no endogenous immunoglobulin Hepama-1 monoclonal antibody through ellipsometry imaging biology microscope technology to liver cancer cell lysate specific immunity bonded distributed in three dimensions gray level image (height value corresponding grey scale value)
Among the figure: a: solid substrate
B: no endogenous immunoglobulin Hepama-1 monoclonal antibody
C: no endogenous immunoglobulin Hepama-1 monoclonal antibody+liver cancer cell lysate.
Fig. 4: no endogenous immunoglobulin Hepama-1 monoclonal antibody is the feminine gender demonstration figure to human lymphoid nodal tissue section microscopy through horseradish peroxidase histochemical staining method.
Fig. 5: mouse immuning ball protein IgG is the positive demonstration figure to human lymphoid nodal tissue section microscopy through horseradish peroxidase histochemical staining method.
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.
The liver cancer murine resource monoclonal antibody Hepama-1 of the no endogenous immunoglobulin of preparation:
10 liters of configuration DXL serum-free protein-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; the MOPS50 milligram; 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; phenol red 50 milligrams; 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
1228 milligrams of 80 milligrams, zinc sulfate 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 among 5 liters of cell biological reactor L1523 (Bioengineering) after the amplification in a small amount.By following reactor process condition, temperature: 37 ℃; Rotating speed: 400RPM; PH:7.0; PO
2: 0.2 crust; PCO
2: 0.4 crust; PN
2: 0.2 crust; Air flow quantity: 2 liters/hour; Pressure: 1 crust, carry out high-density hybridoma cultured continuously.Cell density in the reactor is up to 1.5 * 10
6About/ml.Antibody concentration is up to 0.7mg/ml.
Again with HPLC (BECKMAN Biosys 2000) separation and purification primary products, with vacuum low-pressure cryoconcentration (AES2010 Automatic Environmental 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.
By quality standard calibrating, conformance with standard.
Embodiment 3
The liver cancer murine resource monoclonal antibody Hepama-1 of the no endogenous immunoglobulin that makes with embodiment 1 is a carrier, and mark iodine radioisotope-131 makes the pharmaceutical preparation that is used for the treatment of liver cancer.
The one patient male sex is because of trouble primary massive hepatocarcinoma in late period was admitted to hospital in nineteen ninety, through CT examination, the lump diameter reaches 20 centimetres, can't perform the operation behind the exploratory laparotomy, declaration " not controlling " is under voluntary prerequisite, through radioactivity 131 iodine 25 millicurie one-time treatments with no endogenous liver cancer murine source monoclonal antibody mediation, lump is contracted to 6 centimetres, and residual tumor is through the pathological analysis total necrosis, and the patient immediately fully recovers, healthy survival does not have the recurrence sign so far.
The liver cancer murine resource monoclonal antibody Hepama-1 of the no endogenous immunoglobulin that makes with embodiment 1 is a carrier, and mark iodine radioisotope-131 makes the pharmaceutical preparation that is used for the treatment of liver cancer.
The one patient male sex is admitted to hospital in nineteen ninety because of trouble primary massive hepatocarcinoma in late period, and through CT examination, the lump diameter reaches 12 centimetres, can't perform the operation behind the exploratory laparotomy, and declaration " not controlling " is under voluntary prerequisite, through the radioactivity with no endogenous liver cancer murine source monoclonal antibody mediation
131Iodine 25 millicurie one-time treatments, healthy survival so far.
Embodiment 5
The liver cancer murine source small molecules monoclonal antibody sHepama-1 (Fab) of the no endogenous immunoglobulin that makes with embodiment 2 is a carrier, mark iodine radioisotope-131, make the pharmaceutical preparation that is used for the treatment of liver cancer, treat the tumour of carrying the liver cancer nude mice, treatment back gross tumor volume obviously dwindles, and shows that this medicine truly has the effect that suppresses tumor growth.Every mouse is injected 50 microcuries, 100 microcuries, 500 microcuries
131The small molecules monoclonal antibody marker of I after 30 days, all can be seen clear and definite experimental effect (seeing Table 2), and the tumor control rate that carries the knurl nude mice is all greater than 30%.The radioactivity of liver cancer murine source small molecules monoclonal antibody sHepama-1 (Fab) mediation of the no endogenous immunoglobulin of table 2. injection
131Behind the iodine, carry the variation of the tumor average volume of liver cancer nude mice
| Injection back fate | Group (n=12/ group) |
| Dosage group high dose group is free in the low dose group 131I organizes control group (50 μ Ci) (100 μ Ci) (500 μ Ci) |
0 + + + + +
1 + + + + +
3 + + + 1.21±1.11 1.32±1.04
6 2.64±1.34 + + 2.83±1.72 3.44±0.96
9 244.09±122.33 43.62±11.66 52.38±12.34 388.62±174.44 540.66±133.21
12 432.52±169.38 378.46±189.37 339.41±178.43 488.96±212.38 612.97±167.44
15 588.96±178.77 407.35±236.43 378.52±169.33 604.08±244.34 688.24±233.23
18 645.25±212.32 578.64±268.72 471.34±172.32 1222.46±484.34 1388.78±344.78
21 832.41±386.62 734.56±242.38 686.72±312.17 1296.20±323.32 1496.93±588.32
24 878.94±277.17 793.45±343.51 741.33±327.24 1424.66±515.24 1588.92±464.66
27 1033.34±534.33 823.86±443.63 787.45±388.34 1893.32±777.82 1998.94±932.23
30 1427.32±767.74 1231.48±817.48 1123.48±787.63 2448.46±1233.42 2643.55±1323.34
Tumor control rate (%): the 12nd day the 21st day the 30th day
Low dose group 29.44 44.39 46.00
Middle dosage group: 38.26 50.93 53.42
High dose group: 44.63 54.12 57.50
Free
131The tumor control rate of I:20.23 each dosage group more than 13.41 7.38 is compared with physiological saline control group and free isotope group, and its P value is all less than 0.05.
Claims (2)
1. the preparation method of mouse hybridoma cell strains Hepama-1 excretory liver cancer murine resource monoclonal antibody, it is characterized in that with the serum-free protein-free medium be culture medium, adopt cell biological reactor high-density hybridoma continuous culture method to continue and make with separation and purification; Wherein, serum-free; the prescription of protein-free medium matter is as follows: VITAMIN B4 0.1-0.5 mg/litre; L-Ala 5-10 mg/litre; aluminum chloride 0.0005-0.001 mg/litre; ammonium meta-vanadate 0.0005-0.001 mg/litre; arginine 100-500 mg/litre; l-asparagine 10-50 mg/litre; aspartic acid 5-30 mg/litre; bariumchloride 0.001-0.005 mg/litre; vitamin H 0.05-0.5 mg/litre; calcium chloride 10-100 mg/litre; choline chloride 60 10-100 mg/litre; potassium chromium sulfate 0.0005-0.005 mg/litre; citric acid 10-50 mg/litre; citrulline 1-10 mg/litre; cobalt chloride 0.001-0.005 mg/litre; copper sulfate 0.001-0.01 mg/litre; halfcystine 10-100 mg/litre; dilinoleic acid Yelkin TTS 0.1-1 mg/litre; two stearic acid Yelkin TTS 0.1-1 mg/litre; thanomin 1-10 mg/litre; edetate 5-10 mg/litre; polyoxyethylene glycol 0.5-4 mg/litre; ferrous sulfate 0.5-5 mg/litre; atom iron 2-5 mg/litre; flavin adenine dinucleotide 0.01-0.05 mg/litre; folic acid 1-5 mg/litre; germanium dioxide 0.0001-0.001 mg/litre; L-glutamic acid 10-50 mg/litre; glutamine 100-500 mg/litre; glycine 1-10 mg/litre; glucose 2000-10000 mg/litre; Histidine 10-100 mg/litre; xanthoglobulin 1-10 mg/litre; Isoleucine 100-500 mg/litre; leucine 100-500 mg/litre; linolic acid 0.01-0.1 mg/litre; lithium chloride 5-50 mg/litre; Methionin 50-500 mg/litre; magnesium chloride 50-500 mg/litre; Manganous chloride tetrahydrate 0.00005-0.0005 mg/litre; methionine(Met) 10-50 mg/litre; molybdic acid 0.00005-0.0005 mg/litre; MOPS 1000-10000 mg/litre; inositol 10-50 mg/litre; niacinamide 1-10 mg/litre; nickelous nitrate 0.0001-0.0005 mg/litre; ornithine 1-10 mg/litre; oxaloacetic acid 1-10 mg/litre; pantothenic acid 1-5 mg/litre; phenol red 1-10 mg/litre; phenylalanine 10-100 mg/litre; Potassium Bromide 0.00005-0.0005 mg/litre; Repone K 100-500 mg/litre; potassiumiodide 0.00005-0.0005 mg/litre; proline(Pro) 10-100 mg/litre; Progesterone 0.001-0.01 mg/litre; putrescine 0.1-0.5 mg/litre; Benadon 0.1-0.5 mg/litre; pyruvic acid 100-500 mg/litre; riboflavin 0.01-0.05 mg/litre; rubidium chloride 0.000005-0.00005 mg/litre; Serine 10-100 mg/litre; silver chloride 0.000001-0.00001 mg/litre; sodium-chlor 5000-10000 mg/litre; Sodium Fluoride 0.001-0.01 mg/litre; Sodium Nitroprusside 1-10 mg/litre; Sodium phosphate dibasic 100-1000 mg/litre; Sodium Selenite 0.01-0.05 mg/litre; mg/litre; spermine 0.1-1 mg/litre; tin protochloride 0.00005-0.0005 mg/litre; taurine 10-50 mg/litre; Thioctic Acid 0.08-0.4 mg/litre; VitB1 0.1-0.8 mg/litre; Threonine 8-18 mg/litre; thymus pyrimidine 0.2-1.5 mg/litre; titanium chloride 0.0005-0.004 mg/litre; give birth to plain 0.05-4 mg/litre; tryptophane 1-10 mg/litre; tween 80 0.05-3 mg/litre; tyrosine 25-45 mg/litre; Xie Ansuan 70-120 mg/litre; vitamins B
122-15 mg/litre, zinc sulfate 0.6-5 mg/litre.
2. the preparation method of liver cancer murine resource monoclonal antibody as claimed in claim 1 is characterized in that the processing condition of described cell biological reactor high-density hybridoma continuous culture method are temperature: 30～40 ℃, rotating speed: 100-500RPM, pH:6.5～7.5, PO
2: 0.1-2 crust, PCO
2: 0.1-2 crust, PN
2: 0.1-2 crust, air flow quantity: the 1-5 liter/hour, pressure: 1-5 crust.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 99113824 CN1114619C (en) | 1999-06-25 | 1999-06-25 | Liver cancer murine monoclone antibody without endogenous immunoglobulin and its preparation and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99113824 CN1114619C (en) | 1999-06-25 | 1999-06-25 | Liver cancer murine monoclone antibody without endogenous immunoglobulin and its preparation and application |
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| CN1241573A CN1241573A (en) | 2000-01-19 |
| CN1114619C true CN1114619C (en) | 2003-07-16 |
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| AR095196A1 (en) | 2013-03-15 | 2015-09-30 | Regeneron Pharma | SERUM FREE CELL CULTIVATION MEDIA |
| BR112016002622B1 (en) * | 2013-08-20 | 2023-04-11 | Lek Pharmaceuticals D.D | CELL CULTURE MEDIA AND USE OF SELENIUM |
| TWI797060B (en) | 2015-08-04 | 2023-04-01 | 美商再生元醫藥公司 | Taurine supplemented cell culture medium and methods of use |
| CN108623676B (en) * | 2018-03-20 | 2021-07-30 | 重庆中元汇吉生物技术有限公司 | Method for improving yield of monoclonal antibody and application thereof |
| CN109892320A (en) * | 2019-03-20 | 2019-06-18 | 浙江省人民医院 | A kind of cell-preservation liquid and its preparation method and application |
| CN112881578A (en) * | 2019-11-29 | 2021-06-01 | 中国科学院大连化学物理研究所 | Kit for liver cancer prognosis risk assessment and application |
| BR102019025965A2 (en) * | 2019-12-09 | 2021-06-22 | Cellavit Representação De Produtos Naturais Ltda | vitamin-mineral food supplement |
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