CN103275961A - Temperature-sensitive immobilized papain, and application thereof in preparing monoclonal antibody - Google Patents
Temperature-sensitive immobilized papain, and application thereof in preparing monoclonal antibody Download PDFInfo
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- CN103275961A CN103275961A CN2013102215323A CN201310221532A CN103275961A CN 103275961 A CN103275961 A CN 103275961A CN 2013102215323 A CN2013102215323 A CN 2013102215323A CN 201310221532 A CN201310221532 A CN 201310221532A CN 103275961 A CN103275961 A CN 103275961A
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Abstract
The invention provides a temperature-sensitive immobilized papain, and an application thereof in preparing a monoclonal antibody. A temperature-sensitive poly(N-isopropylacrylamide) is used as a carrier for preparing the immobilized papain. The immobilized papain can be dissolved in water to form a homogeneous phase at a normal temperature and can be precipitated when the temperature exceeds the minimum critical solution temperature, so that an immobilized enzyme prepared by using as the carrier has a thermal response property and combines the advantages of reusability and the like of conventional immobilization and the advantages such as relatively little diffusional limitation and the like of a soluble enzyme. A preparation method which is simple in operations and sensitive to the temperature for the solution-precipitation immobilized enzyme is provided by the invention; and the immobilized enzyme is used for preparing segments of the monoclonal antibody.
Description
(1) technical field
The present invention relates to a kind of immobilized papain of temperature sensitive property, and the application in the preparation monoclonal antibody.
(2) background technology
Monoclonal antibody (Monoclonal Antibody) is the homogeneity antibody that is produced by the cell clone of identifying a kind of antigenic determinant.Its physicochemical character height homogeneous, biological activity high specificity single, that be combined with antigen, be convenient to artificial the processing and quality control, and the source easily.These advantages make monoclonal antibody be paid much attention to once coming out, and become the important means of many significant problems such as solving biology and medical science.
Although there is remarkable advantages aspects such as its diagnosis in disease of monoclonal antibody, treatment, but, conventional monoclonal antibody is used also has tangible limitation, be mainly reflected in the big of its antigenicity and molecular structure and complicacy, the former easily produces the side effects such as rejection in the treatment, the latter causes the penetration power of its tissue poor, and specificity is low, expresses complicated and difficult.Monoclonal antibody or F (ab ')
2Fragment is owing to removed antibody constant region (Fc), and immunogenicity and molecular weight reduce greatly, and penetrance is strong, can also keep the binding ability to Proantigen simultaneously, thereby pharmacokinetic property improves, and more is conducive to clinical application.In addition, in the R﹠D work of many bio-pharmaceuticals, often need to use certain monoclonal antibody fragment rather than whole immunoglobulin molecules.
Monoclonal antibody fragment enzymolysis process commonly used prepares at present.Papoid (EC.3.4.22.2) is a kind of halfcystine endopeptidase, is widely used in the preparation of antibody fragment.The papoid that is used for enzyme digestion reaction has free and immobilization two states.Digest antibody with resolvase, though the enzyme activity height of resolvase is difficult for separating with product, be unfavorable for follow-up product purification in hydrolytic process, and the difficult control of reaction, enzyme also is difficult to recycling.Along with the development of immobilization technology, people are applied to immobilized papoid antibody hydrolysis field gradually.The immobilized enzyme that present stage uses is immobilization, though this type of immobilized enzyme have immobilized enzyme be easy to separate with substrate, product, recycling in a long time, in most cases stability improves, the enzyme reaction process is advantage such as strict control in addition, but diffusional limitation is big in water, and catalytic efficiency is low.
(3) summary of the invention
The object of the invention provides a kind of immobilized papain of temperature sensitive property, and the application in the preparation monoclonal antibody.This material immobilized papoid is water-soluble formation homogeneous phase at normal temperatures, just solid phase is separated out when temperature surpasses its lowest critical solution temperature, had heat as the immobilized enzyme of preparing carriers and replied character, with advantages such as advantages such as conventional fixedization can reuse and the less diffusional limitation of lyoenzyme together, has better application prospect.
The technical solution used in the present invention is:
A kind of immobilized papain of temperature sensitive property is prepared by following method:
(1) with the DMF(dimethyl formamide) use molecular sieve drying, temperature sensitive property carrier to be dissolved among the dry DMF that crosses, the ratio of temperature sensitive property carrier and DMF is 1g:1～10mL, obtains carrier soln; Described temperature sensitive carrier is poly-(the N-N-isopropylacrylamide) of N-hydroxy-succinamide ester end-blocking, and molecular-weight average is 2000, and its structural formula is as follows:
(2) papoid is dissolved in the borate buffer solution of pH7.5～10.0 accent enzyme liquid concentration to 1～10mg/mL(〉=10U/mg), obtain enzyme solution;
(3) carrier soln is mixed with enzyme solution, in 4～55 ℃ of reaction 1～8h, reaction solution dilutes with the borate buffer solution of pH7.5～10.0;
In (4) 40 ℃ of airbaths, the centrifugal 20min of 11000r/min removes supernatant liquor, and precipitation is with the phosphate buffered saline buffer dissolving, and is centrifugal under the same terms again;
(5) operation of repeating step (4) is 2～3 times, and last gained precipitation is dissolved in the phosphate buffered saline buffer of newly joining, and 4 ℃ of preservations namely get the immobilized papain solution of temperature sensitive property.
The invention still further relates to the application of immobilized papain in the preparation monoclonal antibody fragment of described temperature sensitive property.
Concrete; the method of utilizing the temperature sensitive carrier immobilized papoid of the present invention to prepare antibody fragment is as follows: it is in 4.0～9.0 the phosphate buffered saline buffer that the immobilized papain of temperature sensitive property is dissolved in the pH that contains protective material (5mmol/L halfcystine and 2mmol/L EDTA); 30min is placed in 20～45 ℃ of water-baths, takes out.After antibody was dialysed with identical damping fluid, accent concentration was 1～10mg/mL, adds immobilized enzyme liquid, and enzyme is that 1:1～20(m:m), 20～45 ℃ of water-baths vibrations digest 1～22h with the ratio of antibody.40 ℃ then, the centrifugal 20min of 11000r/min, supernatant is postdigestive antibody fragment, and precipitation (immobilized enzyme) is used for the preparation of antibody fragment next time with the phosphate buffered saline buffer dissolving.The antibody fragment mixed solution of gained adopts albumin A-albumen L affinity chromatography purifying, obtains highly purified monoclonal antibody fragment.Albumin A is the surface protein of streptococcus aureus, and molecular weight is 42KD, and 6 different IgG binding sites are arranged; Protein L is a kind of immunoglobulin-binding proteins that derives from bacterium peptostreptococcus magnus (Peptostreptococcusmagnus), (for example pass through the immunoglobulin fc region territory with main, heavy chain) albumin A of combination is different with Protein G, and protein L is by taking place to interact with light chain and being combined with Igs.
Albumin A described above-albumen L affinity chromatography concrete grammar is as follows: the antibody fragment mixed solution is earlier through the albumin A affinity chromatography, balance liquid is 20mmol/L pH7.0 phosphate buffered saline buffer (containing 0.15mol/L NaCl), elutriant is 0.1mol/L pH3.0 sodium citrate buffer solution, collect elution peak, transfer pH to neutral; Again through albumen L affinity chromatography, balance liquid is 0.1mol/L pH7.0 phosphate buffered saline buffer (containing 0.15mol/L NaCl) with elution peak, and elutriant is 0.1mol/L pH3.0 glycine buffer, collects elution peak, transfers pH to neutral, obtains antibody fragment.
The present invention has prepared temperature sensitive property immobilized enzyme with temperature sensitive carrier and enzyme covalent attachment, with its digestion Antibody Preparation antibody fragment, has the following advantages:
(1) immobilized enzyme has kept the temperature sensitive property of carrier.Immobilized enzyme has water-solublely under the normal temperature, and enzymatic is sent out and should be carried out in homogeneous reaction system, has increased the contact probability of enzyme-to-substrate greatly, has reduced diffusional limitation, improves the catalytic efficiency of immobilized enzyme.By the rising temperature, centrifugal, can reclaim immobilized enzyme easily.
(2) utilize the covalent attachment legal system to be equipped with immobilized enzyme, carrier is combined with enzyme firmly, difficult drop-off.
(3) preparation process of immobilized enzyme is simple, mild condition, and the immobilized enzyme that makes is stable high.
(4) immobilized enzyme of preparation gained is compared with resolvase, and pH stability all is significantly improved with thermostability; Operational stability and stability in storage are good, are convenient to recycle, are easy to storage.
(5) antibody fragment preparation process mild condition, the antagonist vigor keeps high, and immobilized enzyme is easily removed, and is beneficial to the separation and purification of fragment.
(6) adopt albumin A-albumen L affinity chromatography antibody purification fragment, step is simple, and the fragment purity of gained can reach 98%.
(4) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
(1) preparation of temperature sensitive property immobilized papain: with DMF 3A molecular sieve drying; Poly-(N-N-isopropylacrylamide) (sigma company, M of temperature sensitive property carrier N-hydroxy-succinamide ester end-blocking
n2000) be dissolved among the DMF of drying processing, the ratio of carrier and DMF is 1g:10mL.With papoid (sigma company, enzyme is lived and be 〉=10U/mg) to be dissolved in the borate buffer solution of 0.1mol/L, pH10.0, papoid concentration is 10mg/mL.Carrier soln is mixed by the 1:6 volume ratio with papoid solution, in 55 ℃ of reaction 1h.Reaction solution dilutes 4 times with the borate buffer solution of pH10.0,40 ℃, the centrifugal 20min of 11000r/min, remove supernatant liquor, precipitation is dissolved with the phosphate buffered saline buffer of pH7.0, and in 40 ℃ centrifugal (condition is the same), this process repeats 3 times, precipitation is dissolved in the 0.1mol/L phosphate buffered saline buffer of newly joining at last, 4 ℃ of preservations namely get immobilized enzyme solution (enzyme activity is 55U/mg, and carrying the enzyme amount is 0.25mg/mg).
(2) preparation of monoclonal antibody fragment: it is in 4.0 phosphate buffered saline buffers that above-mentioned temperature sensitive property immobilized papain solution is dissolved in the 0.1mol/L, the pH that contain 5mmol/L halfcystine and 2mmol/L EDTA, and 30min is placed in 20 ℃ of water-baths, takes out.After monoclonal antibody (β-HCG monoclonal antibody, Hangzhou Bo Lin Bioisystech Co., Ltd) is the dialysis of 4.0,0.1mol/L phosphate buffered saline buffer with pH, accent concentration is 1mg/mL, adds immobilized enzyme liquid, and the mass ratio of enzyme and antibody is 1:20,20 ℃ of water-bath vibrations, digestion 12h.Through the SDS-PAGE electrophoresis detection, digestibility is 82%.Digestion back solution is in 40 ℃, and 11000r/min, centrifugal 20min, supernatant liquor transfer pH extremely neutral, are postdigestive antibody fragment mixed solution, and precipitation (immobilized enzyme) is used for the preparation of antibody fragment next time with the phosphate buffered saline buffer dissolving.
(3) purifying of monoclonal antibody fragment: the antibody fragment mixed solution is earlier through the albumin A affinity chromatography, balance liquid is the phosphate buffered saline buffer (containing 0.15mol/L NaCl) of 20mmol/L, pH7.0, elutriant is the sodium citrate buffer solution of 0.1mol/L, pH3.0, collect elution peak, transfer pH to neutral; With elution peak again through albumen L affinity chromatography, balance liquid is the phosphate buffered saline buffer (containing 0.15mol/L NaCl) of 0.1mol/L, pH7.0, elutriant is the glycine buffer of 0.1mol/L, pH3.0, collect elution peak, transfer pH to neutral, be monoclonal antibody fragment, purity is 98% after measured.
Embodiment 2:
(1) preparation of temperature sensitive property immobilized papain: with DMF 3A molecular sieve drying; Poly-(the N-N-isopropylacrylamide) of temperature sensitive property carrier N-hydroxy-succinamide ester end-blocking is dissolved among the dry DMF that crosses, and the ratio of carrier and DMF is 1g:1mL.Papoid is dissolved in the borate buffer solution of 0.1mol/L, pH8.5, papoid concentration is 1mg/mL.Carrier soln is mixed by the 1:4 volume ratio with papoid solution, in 4 ℃ of reaction 8h.Reaction solution dilutes with the borate buffer solution of pH8.5, in 40 ℃ of airbaths, the centrifugal 20min of 11000r/min removes supernatant liquor, and precipitation is dissolved with the phosphate buffered saline buffer of pH7.0, in 40 ℃ centrifugal (condition is the same), this process repeats 3 times, and precipitation is dissolved in the 0.1mol/L phosphate buffered saline buffer of newly joining 4 ℃ of preservations at last, namely get immobilized enzyme solution (enzyme activity is 87U/mg, and carrying the enzyme amount is 0.28mg/mg).
(2) preparation of monoclonal antibody fragment: above-mentioned temperature sensitive property immobilized papain is dissolved in the phosphate buffered saline buffer of the 0.1mol/L, the pH7.0 that contain 5mmol/L halfcystine and 2mmol/L EDTA,, 30min is placed in 30 ℃ of water-baths.After β-HCG monoclonal anti body and function pH was the dialysis of 7.0,0.1mol/L phosphate buffered saline buffer, accent concentration was 2mg/mL, added immobilized enzyme liquid, and the mass ratio of enzyme and antibody is 1:10,30 ℃ of water-bath vibrations, digestion 22h.Through the SDS-PAGE electrophoresis detection, digestibility is 95%.Digestive system is in 40 ℃, the centrifugal 20min of 11000r/min, and supernatant liquor transfers pH to neutral, is postdigestive antibody fragment, and precipitation (immobilized enzyme) is used for the preparation of antibody fragment next time with the phosphate buffered saline buffer dissolving.
(3) purifying of monoclonal antibody fragment: the antibody fragment mixed solution is earlier through the albumin A affinity chromatography, balance liquid is the phosphate buffered saline buffer (containing 0.15mol/L NaCl) of 20mmol/L, pH7.0, elutriant is the sodium citrate buffer solution of 0.1mol/L, pH3.0, collect elution peak, transfer pH to neutral; Again through albumen L affinity chromatography, balance liquid is the phosphate buffered saline buffer (containing 0.15mol/L NaCl) of 0.1mol/L, pH7.0 with elution peak, and elutriant is the glycine buffer of 0.1mol/L, pH3.0, collect elution peak, transfer pH to neutral, be antibody fragment, purity is 98% after measured.
Embodiment 3:
(1) preparation of temperature sensitive property immobilized papain: with DMF 3A molecular sieve drying; Poly-(the N-N-isopropylacrylamide) of temperature sensitive property carrier N-hydroxy-succinamide ester end-blocking is dissolved among the dry DMF that crosses, and the ratio of carrier and DMF is 1g:5mL.Papoid is dissolved in the borate buffer solution of 0.1mol/L, pH7.5, papoid concentration is 10mg/mL.Carrier soln is mixed by the 1:5 volume ratio with papoid solution, in 20 ℃ of reaction 4h.Reaction solution dilutes with the borate buffer solution of pH7.5,40 ℃, the centrifugal 20min of 11000r/min, remove supernatant liquor, precipitation is dissolved with the phosphate buffered saline buffer of pH7.0, and in 45 ℃ centrifugal (condition is the same), this process repeats 3 times, precipitation is dissolved in the 0.1mol/L phosphate buffered saline buffer of newly joining at last, 4 ℃ of preservations namely get immobilized enzyme solution (enzyme activity is 30U/mg, and carrying the enzyme amount is 0.11mg/mg).
(2) preparation of monoclonal antibody fragment: temperature sensitive property immobilized papain is dissolved in the phosphate buffered saline buffer of the 0.1mol/L, the pH9.0 that contain 5mmol/L halfcystine and 2mmol/L EDTA, and 30min is placed in 45 ℃ of water-baths, takes out.After β-HCG monoclonal anti body and function pH was the dialysis of 9.0,0.1mol/L phosphate buffered saline buffer, accent concentration was 5mg/mL, added immobilized enzyme liquid, and the mass ratio of enzyme and antibody is 1:1,45 ℃ of water-bath vibrations, digestion 1h.Through the SDS-PAGE electrophoresis detection, digestibility is 55%.40 ℃, the centrifugal 20min of 11000r/min, supernatant is postdigestive antibody fragment, and precipitation (immobilized enzyme) is used for the preparation of antibody fragment next time with the phosphate buffered saline buffer dissolving.
(3) purifying of monoclonal antibody fragment: the antibody fragment mixed solution is earlier through the albumin A affinity chromatography, balance liquid is 20mmol/L pH7.0 phosphate buffered saline buffer (containing 0.15mol/L NaCl), elutriant is the sodium citrate buffer solution of 0.1mol/L, pH3.0, collects elution peak, transfers pH to neutral; Again through albumen L affinity chromatography, balance liquid is the phosphate buffered saline buffer (containing 0.15mol/L NaCl) of 0.1mol/L, pH7.0 with elution peak, and elutriant is the glycine buffer of 0.1mol/L, pH3.0, collect elution peak, transfer pH to neutral, be antibody fragment, purity is 98% after measured.
Claims (2)
1. the immobilized papain of a temperature sensitive property is prepared by following method:
(1) with the DMF molecular sieve drying, temperature sensitive property carrier is dissolved among the dry DMF that crosses, and the ratio of temperature sensitive property carrier and DMF is 1g:1～10mL, obtains carrier soln; Described temperature sensitive carrier is poly-(the N-N-isopropylacrylamide) of N-hydroxy-succinamide ester end-blocking;
(2) papoid is dissolved in the borate buffer solution of pH7.5～10.0, transfers enzyme liquid concentration to 1～10mg/mL, obtain enzyme solution;
(3) carrier soln is mixed with 1:4～6 volume ratios with enzyme solution, in 4～55 ℃ of reaction 1～8h, reaction solution dilutes with the borate buffer solution of pH7.5～10.0;
In (4) 40 ℃ of airbaths, the centrifugal 20min of 11000r/min removes supernatant liquor, and precipitation is with the phosphate buffered saline buffer dissolving, and is centrifugal under the same terms again;
(5) operation of repeating step (4) is 2～3 times, and last gained precipitation is dissolved in the phosphate buffered saline buffer of newly joining, and 4 ℃ of preservations namely get the immobilized papain solution of temperature sensitive property.
2. the application of the immobilized papain of temperature sensitive property as claimed in claim 1 in the preparation monoclonal antibody fragment.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104313091A (en) * | 2014-09-27 | 2015-01-28 | 浙江工业大学 | Method for separating human chorionic gonadotropin monoclonal antibody segment |
| CN104498576A (en) * | 2014-12-05 | 2015-04-08 | 重庆乾德生物技术有限公司 | Immobilized enzyme digestion based preparation method of Fab antibody and application thereof |
| CN107586817A (en) * | 2016-07-08 | 2018-01-16 | 中国科学院过程工程研究所 | A kind of protein digestion method based on immobilised enzymes |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102417920A (en) * | 2011-12-07 | 2012-04-18 | 浙江工业大学 | Method for preparing anti-human chorionic gonadotrophin (hCG) monoclonal antibody fragment |
-
2013
- 2013-06-05 CN CN2013102215323A patent/CN103275961A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102417920A (en) * | 2011-12-07 | 2012-04-18 | 浙江工业大学 | Method for preparing anti-human chorionic gonadotrophin (hCG) monoclonal antibody fragment |
Non-Patent Citations (3)
| Title |
|---|
| ZHONGLI DING 等: "Synthesis and Purification of Thermally Sensitive Oligomer-Enzyme Conjugates of Poly(N-isopropylacrylamide)-Trypsin.", 《BIOCONJUGATE CHEM.》 * |
| 杨勇 等: "酶固定化技术用载体材料的研究进展", 《化学通报》 * |
| 王宪政: "固定化木瓜蛋白酶研究进展", 《山东化工》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104313091A (en) * | 2014-09-27 | 2015-01-28 | 浙江工业大学 | Method for separating human chorionic gonadotropin monoclonal antibody segment |
| CN104498576A (en) * | 2014-12-05 | 2015-04-08 | 重庆乾德生物技术有限公司 | Immobilized enzyme digestion based preparation method of Fab antibody and application thereof |
| CN107586817A (en) * | 2016-07-08 | 2018-01-16 | 中国科学院过程工程研究所 | A kind of protein digestion method based on immobilised enzymes |
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Application publication date: 20130904 |