CN1136317C - Process for preparing bisegmentum and monosegmentum of monoclone antibody - Google Patents
Process for preparing bisegmentum and monosegmentum of monoclone antibody Download PDFInfo
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- CN1136317C CN1136317C CNB991157303A CN99115730A CN1136317C CN 1136317 C CN1136317 C CN 1136317C CN B991157303 A CNB991157303 A CN B991157303A CN 99115730 A CN99115730 A CN 99115730A CN 1136317 C CN1136317 C CN 1136317C
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Abstract
The present invention discloses a method for preparing bisegmentum and monosegmentum of a monoclone antibody, which relates to the biological technique engineering. A main technique route comprises coarse extraction of the antibody, enzyme cutting, secondary purification and freeze drying, the purity of the prepared F(ab')2 and Fab is larger than 98%, and the recovery rate is larger than 50%. Impurities, such as pyrogen, nucleic acid, viruses, etc., can be effectively removed. The safety, the efficiency, the stability and the consistency of the bisegmentum and monosegmentum of a monoclone antibody all reach the quality standard of a human use mouse originated single antibody of the Chinese medicine biological product identification office.
Description
The present invention relates to the biotechnology engineering.
Konler in 1975 and Milstein have set up since the hybridoma technology, monoclonal antibody gets widespread use in basic medical research and clinical disease diagnosis and treatment, particularly aspect the in-vivo diagnostic and treatment of tumour, monoclonal antibody shows very tempting application prospect.Drugs approved by FDA CD in 1987
3Monoclonal antibody is used for clinical, and nineteen ninety has reached 1,000 ten thousand dollars annual value of production, in China
131Radio immuno imaging agent of I-HAb18 liver cancer monoclonal antibody and therapeutical agent have entered II phase clinical study, and the tumor imaging rate reaches 90.6%, and treating efficient is 69%, diagnosis aspect in vivo, the two sections of monoclonal antibody [F (ab ')
2] compare owing to removed the FC section with complete murine antibody IgG with single hop [Fab] fragment antibody, it is few to have a molecular weight, and it is rapid to arrive tumor locus, and blood background and non-tumor tissue are removed advantages such as fast; The F of treatment aspect in vivo (ab ')
2Fragment antibody has reduced the anti-mouse foreign protein of people rejection, has improved the repeat administration probability.Tens of kinds of F of U.S. FDA approved (ab ')
2Or put in the body of Fab fragment antibody and exempt from medicine and enter clinical study.
At present, relevant both at home and abroad F (ab ')
2, the Fab fragment antibody the preparation method more, with molecular sieve, ion-exchange or Protein A isochromatic spectrum purifying F (ab ')
2, Fab, the outmoded complex process of its method can't be amplified in pilot scale and the dog scale production, purity also can't reach the ideal standard.The KoichiMorimoto of Japan in 1992 and Kuniyo Inouye have reported with TSKgel Phenyl-5PW hydrophobic chromatography IgG purification 1 mouse monoclonal antibody F (ab ')
2Fragment antibody, purity reaches 98%, but TSKgel Phenyl-5PW purification column is the analysis mode post, is not suitable for pilot scale and amplifies research.At F (ab ')
2Quality control aspect with the Fab fragment antibody there is no report both at home and abroad.
F of the present invention (ab ')
2With the preparation method of Fab fragment antibody, means of purification adopts Phenyl SepharoseHigh Performance hydrophobic chromatography, F after secondarily purified (ab ')
2The purity of Fab all can be greater than 98%, and the rate of recovery is greater than 50%, and the aseptic no thermal source of whole process is controlled, product freeze-drying immediately behind the purifying, and room temperature validity period 2 years, its purity and activity do not change.Reach Chinese pharmaceutical biological product fully and identify institute's relevant human mouse source property monoclonal antibody quality control standard, the production of big rule film suits to do.
Technical scheme of the present invention:
Monoclonal antibody ascites is slightly carried secondary with 50% saturated sulfuric acid amine, and after the antibody protein throw out fully dissolved with 0.1M citrate buffer solution (PH3.5), levelling concentration was 10-20mg/ml, centrifugal again removal insolubles.F (ab ')
2Preparation is 1: 100 with stomach en-and the IgG enzyme ratio of cutting, and 37 ℃ of reaction 2-3h are with about 3MTris levelling PH7, with termination reaction; The Fab papoid, the ratio of enzyme and IgG 1: 100,37 ℃ of reaction 1-2h, 0.25% (W/V) iodo-acid amide termination reaction.HPLC hydrophobic liquid phase chromatography purifying F (ab ')
2, the Fab fragment antibody, divide secondarily purified with Phenyl Sepharose High Performance hydrophobic chromatography post (Pharmacia): one time purifying A liquid is 1～1.4M sulfuric acid amine 0.05M PH5.0 sodium-acetate buffer, and B liquid is 0.05M PH5.0 sodium-acetate buffer.Secondarily purified A liquid is with 0.7～1.0M sulfuric acid amine 0.05M PH5.0 sodium-acetate buffer, and B liquid is 0.05M PH5.0 sodium-acetate buffer, 30-40 minute linear gradient elution time.Freeze-drying immediately behind the purifying adds 10% low molecular dextran and does the renaturation agent.
Embodiments of the invention:
One, the ascites of monoclonal antibody is handled
The hybridoma 1 * 10 of secrete monoclonal antibody
61ml, inoculation cleaning level BABL/c mouse peritoneal; Aseptic collection ascites after 8-12 days, centrifugal 3000g * 20min removes throw out; Supernatant with 0.8 μ membrane filtration after; Phosphate buffered saline buffer (PBS) equal-volume dilution with 0.01M PH7.4; Slowly add 50% saturated sulfuric acid amine (solid), after static 2 hours, centrifugal 10000g * 20min, precipitation adds 50% saturated sulfuric acid amine after dissolving with 0.01M PH7.4PBS again, after static 2 hours, centrifugal 10000g * 20min.
Two, F (ab ')
2Preparation with Fab
1. the stomach en-enzyme is cut F (ab ')
2Fragment antibody
Antibody throw out behind the thick purifying of saturated sulfuric acid amine, after fully temperature was spared dissolving with 100mM citrate buffer solution (PH3.5), adjusting concentration was 10-20mg/ml, centrifugal 10000g * 10min removes not solute.Stomach en-(pepsin) (the lot No.P6887 of Sigma company, active 3200-4500 unit) enzyme is cut ratio pepsin (mg): IgG (protein content mg in the ascites)=1: 100, put into 37 ℃ of water baths 2 hours, termination reaction adds the 3M Tris of 10% volume, makes it PH about 7.
2. the papoid enzyme is cut the Fab fragment antibody
Antibody throw out and papoid behind the thick purifying of saturated sulfuric acid amine, contain 2mM EDTA with 0.05M Tris-HCL respectively, the dialysis of 1mM DTT damping fluid, enzyme and antibody additional proportion are 1: 100, hatched 1 hour 0.25% (W/V) iodo-acid amide termination reaction behind the mixing for 37 ℃.
3. hydrophobic chromatography purifying F (ab ')
2And Fab
HPLC liquid chromatographic system (Water 650), Protein Detection ultraviolet spectrometry 280mm wavelength, purifying A liquid of PhenylSepharose High Performance hydrophobic chromatography post (Pharmacia Biotech): 1.2M sulfuric acid amine 0.05M PH5.0 sodium-acetate buffer, B liquid: 0.05M PH5.0 sodium-acetate buffer, with 4-5 column volume of A liquid balance, flow velocity 4ml/min.Enzyme is cut after product and is added 60% saturated sulfuric acid amine, and centrifugal 10000g * 20min abandons supernatant, and throw out is with A liquid dissolving upper prop, after waiting to pass the peak and flowing out, last linear gradient, sulfuric acid amine concentration from 1.2M to OM, 30 minutes time.F (ab ')
2Between 20-30 minute, the Fab appearance time is greatly between 15-25 minute greatly for appearance time.Behind the purifying, F (ab ')
2, Fab purity greatly about about 90%, F (ab ')
2The rate of recovery is about 70%, and the Fab rate of recovery is about 50%.
Improve the purity of sample, need carry out secondarily purifiedly again, secondarily purified A liquid is with 0.8M sulfuric acid amine sodium-acetate buffer, and B liquid 0.05M PH5.0 sodium-acetate buffer sample is with the dissolving of A liquid, with sample on the identical method, gradient elution, collection F (ab ')
2The Fab elution peak.After secondarily purified, F (ab ')
2Fab purity is all greater than 98%, rate of recovery F (ab ')
2Greater than 50%, Fab is greater than 40%.
4. the freeze-drying of product behind the purifying
Fragment antibody solution behind the purifying adds 60% saturated sulfuric acid amine centrifugation, and the desalination of dialysing in PH7.4 0.01M PBS solution is adjusted protein concentration, adds 10% low molecular dextran and does the renaturation agent, lyophilize, 4 ℃ or room temperature preservation.
Safety evaluation:
Sterility is one of the most basic requirement to drug safety.Various pyrogens, interior toxicity, mouse borne virus and residual enzyme, though differ greatly on molecular weight size and chemical constitution, the overwhelming majority has the polar molecule that close and distant water electrode is rolled into a ball.We are at F (ab ')
2On the segmental purification process of Fab, adopt the HPHIC, and adopt secondarily purified method, can remove impurity such as pyrogen, nucleic acid, virus effectively.
Sterility test is carried out aerobe, order of succession, mould, microbial culture for 79 pages by 1995 editions two appendix of Chinese Pharmacopoeia, do not have any cell grow into qualified.
Bacterial endotoxin test is pressed 77 pages of two appendix of Chinese Pharmacopoeia, establishes 2Eu endogenous toxic material sex work standard substance and makes positive control, and carry out interference test simultaneously.The positive control positive, the interference test positive, the sample feminine gender is qualified.
The residual DNA assay: measure box (Boehringer Manhaim) with dna marker, the DNA residual volume is answered＜100pg in the sample.
Product purity is identified:
Adopt SDS-PEAG polyphenyl alkene acrylamide gel electrophoresis, F (ab ')
2Electrophoresis adopts reduction and non-reduced two kinds of conditions, and gel strength is 12.5% under the reductive condition, and gel strength is 7.5% under the non-reduced condition, molecular weight standard albumen (Promega) in using.Under reductive condition, F after the about 50KD of complete IgG heavy chain, the about 26KDa of light chain, enzyme cut (ab ')
2The about 30KDa of heavy chain, the about 28KDa of the about 26KDa Fab of light chain; The about 160KDa of the molecular weight of IgG under non-reduced condition, F (ab ')
2About 96KDa, the about 50KDa of Fab, electrophoresis result is calculated purity per-cent with day island proper Tianjin CS-9000 thin layer chromatography scanner scanning.
The product activity identification:
Determination of biological activity is the important means that guarantees the biological medicine validity of products, and is particularly important for monoclonal antibody drug.The tumour antibody activity identification adopts ABC immunohistochemical methods method or immunofluorescence technique, and antibody is done gradient dilution, sets positive criteria product and negative control, measures antibody mediated immunity and tires in conjunction with activity.
Study on the stability:
The stability of medicine is the important indicator that guarantees medicine validity and security, also is the main foundation of determining the expiration date of drug limit.Fragment antibody is the complete antibody poor stability relatively, and stored frozen is very easily degraded in general damping fluid, influences purity and activity.Therefore, freeze-drying immediately behind the fragment antibody purifying, validity period can continue 1-2.
The assurance of homogeneity of product:
The scale operation of monoclonal antibody, hybridoma will prevent cytometaplasia in the culturing process that goes down to posterity, chromosome elimination, problems such as antibody-secreting output reduction.Hybridoma must be produced cell bank, to guarantee the consistence of every batch process antibody through setting up the seed cell storehouse.On the source of composition, serum, additive and the enzyme of substratum and quality and preparation technology, all should guarantee to standardize as much as possible and consistence in addition, stringent condition control and quality arbitration, to guarantee each batch the finished product safety, effective, stable, consistent, reach Chinese pharmaceutical biological product and identify institute's human mouse source property monoclonal antibody quality standard.
Advantage of the present invention:
1. at F (ab ')2, the Fab fragment purification process on, adopt high pressure liquid phase Phenyl Sepharose High The secondarily purified method of Performance HC, purity can be greater than 98%, and the rate of recovery is greater than 50%, and can have The impurity such as pyrogen, nucleic acid, virus are removed on effect ground.
2. technology is simple, and flow process is short, the aseptic apyrogeneity control of whole process, and the immediately freeze-drying of purifying product, The room temperature term of validity 2 years, its purity and activity do not change. Reach Chinese pharmaceutical biological product fully and identify institute Relevant human mouse monoclonal antibody quality control standard, large-scale production suits to do.
Claims (1)
1. the preparation method of two sections of monoclonal antibody and single hop is characterized in that:
Monoclonal antibody ascites is slightly carried secondary with 50% saturated sulfuric acid amine, and after the antibody protein throw out fully dissolved with 0.1M citrate buffer solution (PH3.5), levelling concentration was 10-20mg/ml, centrifugal again removal insolubles; F (ab ')
2Preparation is 1: 100 with stomach en-and the IgG enzyme ratio of cutting, and 37 ℃ of reaction 2-3h are with about 3M Tris levelling PH7, with termination reaction; The Fab papoid, the ratio of enzyme and IgG 1: 100,37 ℃ of reaction 1-2h, 0.25% (W/V) iodo-acid amide termination reaction; HPLC hydrophobic liquid phase chromatography purifying F (ab ')
2The Fab fragment antibody, divide secondarily purified with Phenyl Sepharose High Performance hydrophobic chromatography post (Pharmacia): one time purifying A liquid is 1～1.4M sulfuric acid amine 0.05M PH5.0 sodium-acetate buffer, and B liquid is 0.05M PH5.0 sodium-acetate buffer; Secondarily purified A liquid is with 0.7～1.0M sulfuric acid amine 0.05M PH5.0 sodium-acetate buffer, and B liquid is 0.05M PH5.0 sodium-acetate buffer, 30-40 minute linear gradient elution time; Freeze-drying immediately behind the purifying adds 10% low molecular dextran and does the renaturation agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB991157303A CN1136317C (en) | 1999-03-12 | 1999-03-12 | Process for preparing bisegmentum and monosegmentum of monoclone antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB991157303A CN1136317C (en) | 1999-03-12 | 1999-03-12 | Process for preparing bisegmentum and monosegmentum of monoclone antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1266907A CN1266907A (en) | 2000-09-20 |
| CN1136317C true CN1136317C (en) | 2004-01-28 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB991157303A Expired - Fee Related CN1136317C (en) | 1999-03-12 | 1999-03-12 | Process for preparing bisegmentum and monosegmentum of monoclone antibody |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1908006B (en) * | 2006-08-07 | 2010-05-12 | 陈志南 | Method of fast purifying and preparing Fab fragment antibody |
| WO2013075740A1 (en) | 2011-11-23 | 2013-05-30 | Sanofi | Antibody purification method |
| CN102533911B (en) * | 2011-11-24 | 2013-08-21 | 中国人民解放军第四军医大学 | Preparation method for human thymosin beta 4 two-string body protein |
| CN103820515B (en) * | 2012-11-16 | 2017-07-04 | 财团法人农业科技研究院 | Prepare antibody F (ab ')2The method of fragment |
| TWI631132B (en) | 2013-05-06 | 2018-08-01 | 賽諾菲公司 | Continuous multistep process for purifying antibodies |
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1999
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| Publication number | Publication date |
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| CN1266907A (en) | 2000-09-20 |
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