CN1180080C - An improved preparation method of liver cancer mouse monoclonal antibody - Google Patents

Abstract

The present invention relates to an improved preparation method of liver cancer mouse-derived monoclonal antibody. The method uses the improved DXL serum-free culture medium as the culture substrate, that is, various growth factors are added to the DXL serum-free and protein-free culture medium. Such as nerve cell growth factor, epidermal growth factor, fibroblast growth factor or embryonic growth factor, etc., each with a content of 0.01-0.1 mg/L. High-density hybridoma cells are continuously cultured in a packed bed bioreactor. The process conditions are: temperature 30-40°C, rotation speed 300-480RPM, pH 6.8-7.25, PO ₂ 0.5-1.95 bar, PCO ₂ 0.5 -1.95 bar, PN ₂ is 0.5-1.95 bar, air flow is 0.1-2/hour, pressure is 2-5 bar. The cell density is as high as 108/ml, and the monoclonal antibody yield per liter of culture medium reaches 1000-1500 mg.

Description

An improved preparation method of liver cancer mouse monoclonal antibody

technical field

The present invention relates to the field of monoclonal antibody production, in particular to a preparation method of liver cancer mouse-derived monoclonal antibody by changing the working mode and process of a bioreactor and improving the medium formula.

Background technique

The applicant of the present invention and Shanghai Meishan Biotechnology Co., Ltd. have applied for a patent on the product, method and application of endogenous immunoglobulin-free liver cancer monoclonal antibody on June 25, 1999. The patent application number is 99113824.4 , the name of the invention is: liver cancer mouse monoclonal antibody without endogenous immunoglobulin, preparation method and application thereof.

At that time, the mouse-derived monoclonal antibodies produced by various countries in the world, whether they were whole molecules or small molecules, if the serum-free and protein-free culture medium was not used as the culture substrate, no matter what production method was used, the obtained products contained different levels of Such endogenous immunoglobulins (mouse or bovine), this product containing endogenous immunoglobulins is not only difficult to control and standardize in quality, but also contains several unsafe factors.

Patent application No. 99113824.4 successfully provided a method for preparing mouse liver cancer monoclonal antibody without endogenous immunoglobulin by high-density culture of mouse hybridoma cells in a bioreactor using serum-free and protein-free culture medium as a substrate. The obtained cell density was 10 ^4-6 /ml, the antibody concentration was as high as 1 mg/ml and the composition ratio in antibody protein was 100%. It can be used as a raw material for preparing preparations for treating liver cancer. It can avoid the potential safety hazards of producing liver cancer monoclonal antibody with ascites or the production of serum-containing culture fluid and the instability of drug efficacy caused by the inconstant composition ratio of monoclonal antibody in the drug protein. As far as we know, this It is the first mouse liver cancer monoclonal antibody without endogenous immunoglobulin successfully obtained by bioreactor technology.

The present invention has been improved on the basis of the previous patent, and a more suitable reactor has been selected, and the reaction conditions have been optimized at the same time. In addition, we have added several cell growth factors in the culture medium, the purpose of which is to further increase the cell density and Antibody Yield. The goal of any in vitro culture is to simulate the in vivo environment of organisms, so that cells can grow to the maximum extent, but the in vivo environment is complex and changeable, and various factors are interlocking, so it is difficult to completely replicate, which is difficult to achieve in vitro cell culture. One of the most critical causes of cell density in the body. Growth factors are originally a key factor unique to the in vivo environment. Adding them in the in vitro environment can significantly increase the survival rate of cells, improve cell morphology, and increase cell density and antibody yield. In the past, people used less because cell growth factor is also a kind of protein, and when it is finally separated, there is an additional concern. But now, these protein factors can be completely separated through the purification process of Protein A or G, therefore, it is necessary and feasible to add these cell growth factors.

Contents of the invention

The present invention inherits the technical route of the No. 99113824.4 patent application, and aims to provide a method for producing liver cancer mouse monoclonal antibody with higher yield without endogenous immunoglobulin.

Therefore, the inventor improved on the basis of the 99113824.4 patent by adopting a packed bed continuous culture reactor and correspondingly changing the process conditions of the reactor. Temperature is 30-40°C, 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 is 0.1-2 /hour at a pressure of 2-5 bar. The most important thing is that the present invention adopts the improved DXL serum-free culture medium, that is, one or several growth factors are added to the formula of DXL serum-free and protein-free culture medium of the previous patent, including nerve cell growth factor, epidermal growth factor Factors, fibroblast growth factors, embryonic growth factors, etc., the content of various growth factors is 0.01-0.1 mg/L, and these growth factors can be completely separated in the subsequent purification process of Protein A or G.

Production process step of the present invention is shown in Table 1:

Table 1. Production process steps

Process Process Description Environmental Cleanliness

Production cell bank Take out Hepama-1 cells Level 100,000

↓

Small scale expansion CO ₂ incubator 100,000 class

↓

Mass production Packed bed reactor Class 100,000

↓

Collect culture fluid Culture fluid centrifugation Level 100,000

↓

Concentration Low temperature vaporization or sublimation 100,000 grade

↓

Separation and Purification Chromatography

↓

Concentration Vacuum low pressure low temperature Class 100,000

↓

Concentration, purity

Quality Control Mouse DNA Grade 100,00

Endotoxin, exogenous factor

↓

Filtration and packaging Class 100

Preparation of modified DXL serum-free medium:

Its characteristic is that one or several growth factors are added on the basis of DXL culture medium, the content of which is 0.01-0.1 mg/L.

Improved DXL serum-free medium formula: mg/L

Adenine 0.1-0.5

Alanine 5-10

Aluminum Chloride 0.0005-0.001

Ammonium metavanadate 0.0005-0.001

Arginine 100-500

Asparagine 10-50

Aspartic Acid 5-30

Barium chloride 0.001-0.005

Biotin 0.05-0.5

Calcium chloride 10-100

Choline Chloride 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

Cysteine 10-100

Dilinoleic acid lecithin 0.1-1

Lecithin distearate 0.1-1

Ethanolamine 1-10

EDTA 5-10

Polyethylene glycol 0.5-4

Ferrous Sulfate 0.5-5

Atomic Iron 2-5

Flavin adenine dinucleotide 0.01-0.05

Folic Acid 1-5

Germanium dioxide 0.0001-0.001

Glutamic acid 10-50

Glutamine 100-500

Glycine 1-10

Glucose 2000-10000

Histidine 10-100

Hypoxanthine 1-10

Isoleucine 100-500

Leucine 100-500

Linoleic acid 0.01-0.1

Lithium chloride 5-50

Lysine 50-500

Magnesium chloride 50-500

Manganese chloride 0.00005-0.0005

Methionine 10-50

Molybdic acid 0.00005-0.0005

MOPS 1000-10000

Inositol 10-50

Niacinamide 1-10

Nickel nitrate 0.0001-0.0005

Ornithine 1-10

Oxaloacetate 1-10

Pantothenic acid 1-5

Phenol red 1-10

Phenylalanine 10-100

Potassium Bromide 0.00005-0.0005

Potassium chloride 100-500

Potassium iodide 0.00005-0.0005

Proline 10-100

Progesterone 0.001-0.01

Putrescine 0.1-0.5

Pyridoxine 0.1-0.5

Pyruvate 100-500

Riboflavin 0.01-0.05

Rubidium Chloride 0.000005-0.00005

Serine 10-100

Silver chloride 0.000001-0.00001

Sodium chloride 5000-10000

Sodium fluoride 0.001-0.01

Sodium nitroprusside 1-10

Disodium hydrogen phosphate 100-1000

Sodium selenite 0.01-0.05

Spermine 0.1-1

Stannous chloride 0.00005-0.0005

Taurine 10-50

Lipoic acid 0.08-0.4

Thiamine 0.1-0.8

Threonine 8-18

Thymine 0.2-1.5

Titanium chloride 0.0005-0.004

Tocopherin 0.05-4

Tryptophan 1-10

Twain 800.05-3

Tyrosine 25-45

Valine 70-120

Vitamin B ₁₂ 2-15

Sulfuric acid 0.6-5

Add to:

One or more of various growth factors such as epidermal growth factor, nerve cell growth factor, fibroblast growth factor or embryonic growth factor, each 0.01-0.1 mg/L.

The hybridoma cells that can secrete liver cancer mouse monoclonal antibody were cultured with the improved DXL medium prepared above.

Hepama-1 hybridoma cells were taken out from the production cell bank and amplified in a small amount, and the cells before the logarithmic phase (inoculation cell density was A×10 ⁵ /ml) were transferred to a packed bed cell bioreactor for cultivation, and the process conditions were monitored ( Temperature: 30～40℃, Speed: 300-480 RPM, pH: 6.8～7.25, PO ₂ : 0.5-1.95 bar, PCO ₂ : 0.5-1.95 bar, PN ₂ : 0.5-1.95 bar, Air flow: 0.1-2 L/hour, pressure: 2-5 bar), when the antibody concentration is about 1-1.5mg/ml or the cells in the cell bioreactor grow to a density of about A×10 ⁸ /ml, collect the culture medium, and use the low-temperature sublimation method Concentrate by centrifugation to obtain the primary product.

The primary product is separated and purified by HPLC, the product is concentrated by low-pressure low-temperature sublimation method, and the quality control of the product is carried out at the same time. Finally, the whole molecular monoclonal antibody of liver cancer mouse monoclonal antibody without endogenous immunoglobulin is obtained after sterile filtration. In this step, use Protein G or Protein A columns to completely separate the added growth factors.

If necessary, papain, pepsin, etc. can be used to prepare whole molecular monoclonal antibodies of liver cancer mouse-derived monoclonal antibodies without endogenous immunoglobulins into endogenous immunoglobulin-free liver cancer mouse-derived small molecule monoclonal antibodies according to conventional enzymatic digestion methods. Anti-Fab or Fab) ₂ . First, the prepared enzyme-containing digestion solution was reacted with the same volume of mouse-derived monoclonal antibody against liver cancer without endogenous immunoglobulin at 37°C. After the enzyme digestion reaction was terminated, it was dialyzed with pH8.0 phosphate buffer solution at 4°C for 12 -20 hours, and finally separated and purified by HPLC to obtain the mouse-derived small molecule monoclonal antibody to liver cancer without endogenous immunoglobulin.

Due to the adoption of the above technical solution, compared with the previous patent, this patent has the following significant advantages:

1. A packed bed continuous culture reactor is adopted. Its biggest feature is that the cells are concentrated in the tank, and the effective volume has a high density, which reduces the risk of slow or no growth of cells at low densities, and the cells are all adsorbed on the microcarriers, and it is very easy to dissociate into the culture medium. Less, easy to collect products. Therefore, among all bioreactors for culturing cells, especially animal cells and hybridoma cells, microcarrier packed bed bioreactors have significant advantages and are increasingly becoming the mainstream of cell culture devices.

2. The improved DXL serum-free culture medium is adopted, and growth factors are added on the basis of the DXL culture medium, which helps to improve the yield. Moreover, since these growth factors can be completely purified in the subsequent purification process of Protein A or G Isolation, therefore, does not affect the properties of the product antibody.

3. The process conditions of the bioreactor were improved.

Generally speaking, the monoclonal antibody produced by this method does not contain any mouse endogenous immunoglobulin and any bovine endogenous immunoglobulin, the composition ratio of the antibody protein of the monoclonal antibody is 100%, and The yield is high, the cell density is 10 ^6-8 /ml, and the yield of the monoclonal antibody in each liter of culture medium reaches 1000-1500 mg, meeting the requirements of large-scale production.

A class of liver cancer mouse-derived monoclonal antibodies Hepama-1, Hepama-9403, Hepama-9501 (whole molecule and small molecule) etc. without endogenous immunoglobulins of the present invention, the preparation method is similar, below with no endogenous immunity The preparation of globulin hepatocarcinoma mouse monoclonal antibody Hepama-1 is an example to further illustrate the present invention, but does not limit the scope of the present invention.

Description of drawings

Figure 1 is the determination of bovine IgG by indirect immunoenzyme-linked adsorption method (sELISA method).

As shown, the CUT-OFF value is 0.5. The left side of the figure is the monoclonal antibody produced by the reactor, the middle is the improved DCL culture solution, and the right side is the 1640 culture solution containing 20% newborn bovine serum. The reactor product monoclonal antibody does not contain bovine IgG (endogenous globulin).

Figure 2 is the activity test (indirect immunofluorescence method) of the monoclonal antibody produced by the reactor.

Figure 2(1) is the activity of the monoclonal antibody produced by the reactor, Figure 2(2) is the negative control of immunoglobulin, and Figure 2(3) is the positive control of polyclonal antibody.

Figure 3 The test results of the improved DXL culture medium without serum.

The left side of the picture shows negative staining of the modified DXL culture medium; the right side of the picture shows the 1640 culture medium containing 10% newborn bovine serum, which is strongly positive for Coomassie Brilliant Blue G250 staining.

Detailed ways

Example 1

Preparation of Hepama-1 mouse monoclonal antibody against liver cancer without endogenous immunoglobulin:

Configure 10 liters of modified DXL serum-free culture medium:

Adenine 3 mg, alanine 70 mg, aluminum chloride 0.007 mg, ammonium metavanadate 0.007 mg, arginine 3 g, asparagine 300 mg, aspartic acid 150 mg, barium chloride 0.025 mg, Biotin 2.5mg, Calcium Chloride 500mg, Choline Chloride 500mg, Chromium Potassium Sulfate 0.025mg, Citric Acid 300mg, Citrulline 50mg, Cobalt Chloride 0.025mg, Copper Sulfate 0.05mg, Cysteine 500mg, Lecithin Dilinoleate 5mg, Lecithin Distearate 5mg, Ethanolamine 50mg, EDTA 70mg, Macrogol 20mg, Ferrous Sulfate 25mg, Atomic Iron 25mg, flavin adenine dinucleotide 0.25mg, folic acid 25mg, germanium dioxide 0.005mg, glutamic acid 250mg, glutamine 2.5g, glycine 50mg, glucose 60g, histidine 500mg, Hypoxanthine 50mg, Isoleucine 3g, Leucine 3g, Linoleic Acid 0.5mg, Lithium Chloride 250mg, Lysine 2.5g, Magnesium Chloride 2.5g, Manganese Chloride 0.0025mg, Methionine Molybdic acid 250 mg, molybdic acid 0.0025 mg, MOPS 50 mg, inositol 300 mg, nicotinamide 50 mg, nickel nitrate 0.0025 mg, ornithine 50 mg, oxaloacetic acid 50 mg, pantothenic acid 25 mg, phenol red 50 mg, benzene Alanine 500mg, potassium bromide 0.0025mg, potassium chloride 3g, potassium iodide 0.0025mg, proline 500mg, progesterone 0.05mg, putrescine 2.5mg, pyridoxine 2.5mg, pyruvate 2.5g, nucleus Flavin 0.25mg, Rubidium Chloride 0.00025mg, Serine 500mg, Silver Chloride 0.00005mg, Sodium Chloride 70g, Sodium Fluoride 0.05mg, Sodium Nitroprusside 50mg, Disodium Hydrogen Phosphate 5g, Sodium Selenite 0.25 mg, spermine 5 mg, stannous chloride 0.0025 mg, taurine 250 mg, lipoic acid 1.5 mg, thiamine 4 mg, threonine 130 mg, thymine 7 mg, titanium chloride 0.02 mg, fertility Vitamin 20 mg, tryptophan 50 mg, Tween 80 15 mg, tyrosine 350 mg, valine 1 g, vitamin B ₁₂ 80 mg, zinc sulfate 28 mg, epidermal growth factor 300 micrograms, nerve cell growth factor 300 μg, make it fully dissolved in deionized water, and set the volume to 10 liters. Sterile filter for later use.

The Hepama-1 cells were taken out from the production cell bank, and transferred into a 10-liter packed bed cell bioreactor to continuously culture hybridoma cells Hepama-1 in large quantities after a small amount of expansion. According to the following reactor process conditions, temperature: 37 ° C; rotating speed: 420 RPM; pH: 7.0; PO ₂ : 1.25 bar; PCO ₂ : 1.25 bar; PN ₂ : 1.25 bar; Continuous high-density hybridoma cell culture. The cell density in the reactor is as high as about 1.1×10 ⁸ /ml. Antibody concentrations up to 1.2mg/ml.

Then use HPLC (BECKMAN Biosys 2000) to separate and purify the primary product, use Protein A column to separate the added growth factor, and use vacuum low pressure low temperature concentration (AES2010 Automatic Environmental SpeedVac) product, test according to the quality standard, meet the standard, and finally obtain the sterile product after sterile filtration Whole molecule monoclonal antibody Hepama-1 of endogenous immunoglobulin hepatocellular carcinoma mouse monoclonal antibody.

Example 2

Using the liver cancer murine monoclonal antibody Hepama-1 without endogenous immunoglobulins prepared in Example 1 as a raw material, the small molecule monoclonal antibody sHepama-1 (Fab) was prepared by conventional enzyme digestion method:

First prepare a digestive solution containing 0.1 mg/ml papain (0.02 mol edetate disodium, 0.02 mol cysteine), and mix the newly prepared digestive solution containing 0.1 mg/ml papain with the same volume of Integral monoclonal antibody of endogenous immunoglobulin to hepatic carcinoma was reacted at 37°C for 8 hours, then iodoacetamide was added to a final concentration of 0.3 moles to terminate the enzyme digestion reaction, and then dialyzed with pH 8.0 phosphate buffer at 4°C for 15 hours , and finally separated and purified by HPLC to obtain the small molecule monoclonal antibody sHepama-1 (Fab) without endogenous immunoglobulin.

Claims (2)

1. A preparation method of an improved liver cancer mouse source monoclonal antibody is characterized in that, using the improved DXL serum-free culture fluid as a culture substrate, adopting a packed bed bioreactor, and a high-density hybridoma continuous culture method followed by Prepared by separation and purification, in which: The improved DXL serum-free culture medium is based on the DXL serum-free and protein-free culture medium, adding one or more of nerve cell growth factor, epidermal growth factor, fibroblast growth factor or embryonic growth factor , the content of each growth factor is 0.01-0.1 mg/L; The DXL serum-free, protein-free culture medium is as follows: Adenine 0.1-0.5 mg/L Alanine 5-10 mg/L Aluminum Chloride 0.0005-0.001 mg/L Ammonium metavanadate 0.0005-0.001 mg/L Arginine 100-500 mg/L Asparagine 10-50 mg/L Aspartic acid 5-30 mg/L Barium Chloride 0.001-0.005 mg/L Biotin 0.05-0.5 mg/L Calcium chloride 10-100 mg/L Choline Chloride 10-100mg/L Potassium chromium sulfate 0.0005-0.005 mg/L Citric acid 10-50 mg/L Citrulline 1-10 mg/L Cobalt chloride 0.001-0.005 mg/L Copper sulfate 0.001-0.01 mg/L Cysteine 10-100 mg/L Dilinoleic acid lecithin 0.1-1 mg/L Lecithin distearate 0.1-1 mg/L Ethanolamine 1-10 mg/L EDTA 5-10 mg/L Polyethylene glycol 0.5-4 mg/L Ferrous Sulfate 0.5-5 mg/L Atomic Iron 2-5 mg/L Flavin adenine dinucleotide 0.01-0.05 mg/L Folic acid 1-5 mg/L Germanium dioxide 0.0001-0.001 mg/L Glutamic acid 10-50 mg/L Glutamine 100-500 mg/L Glycine 1-10 mg/L Glucose 2000-10000 mg/L Histidine 10-100 mg/L Hypoxanthine 1-10 mg/L Isoleucine 100-500 mg/L Leucine 100-500 mg/L Linoleic acid 0.01-0.1 mg/L Lithium chloride 5-50 mg/L Lysine 50-500 mg/L Magnesium chloride 50-500 mg/L Manganese chloride 0.00005-0.0005 mg/L Methionine 10-50 mg/L Molybdic acid 0.00005-0.0005 mg/L MOPS 1000-10000 mg/L Inositol 10-50 mg/L Niacinamide 1-10mg/L Nickel nitrate 0.0001-0.0005 mg/L Ornithine 1-10 mg/L Oxaloacetate 1-10 mg/L Pantothenic acid 1-5 mg/L Phenol red 1-10 mg/L Phenylalanine 10-100 mg/L Potassium Bromide 0.00005-0.0005 mg/L Potassium chloride 100-500 mg/L Potassium iodide 0.00005-0.0005 mg/L Proline 10-100 mg/L Progesterone 0.001-0.01 mg/L Putrescine 0.1-0.5 mg/L Pyridoxine 0.1-0.5 mg/L Pyruvate 100-500 mg/L Riboflavin 0.01-0.05 mg/L Rubidium Chloride 0.000005-0.00005 mg/L Serine 10-100 mg/L Silver Chloride 0.000001-0.0000 1 mg/L Sodium chloride 5000-10000 mg/L Sodium fluoride 0.001-0.01 mg/L Sodium nitroprusside 1-10 mg/L Disodium hydrogen phosphate 100-1000 mg/L Sodium selenite 0.01-0.05 mg/L Spermine 0.1-1 mg/L Stannous chloride 0.00005-0.0005 mg/L Taurine 10-50 mg/L Lipoic acid 0.08-0.4 mg/L Thiamine 0.1-0.8 mg/L Threonine 8-18 mg/L Thymine 0.2-1.5 mg/L Titanium chloride 0.0005-0.004 mg/L Tocopherin 0.05-4 mg/L Tryptophan 1-10 mg/L Tween 80 0.05-3 mg/L Tyrosine 25-45 mg/L Valine 70-120 mg/L Vitamin B ₁₂ 2-15 mg/L Zinc sulfate 0.6-5 mg/L. 2. the preparation method of liver cancer murine source monoclonal antibody as claimed in claim 1 is characterized in that, the processing condition of described packed bed bioreactor high-density hybridoma cell continuous culture method is, temperature is 30～40 ℃, speed 300-480RPM, pH 6.8-7.25, PO ₂ 0.5-1.95 bar, PCO ₂ 0.5-1.95 bar, PN ₂ 0.5-1.95 bar, air flow 0.1-2/hour, pressure 2 -5 bar.

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