CN108586606A - One kind is for removing endotoxic method in antibody protein - Google Patents
One kind is for removing endotoxic method in antibody protein Download PDFInfo
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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Abstract
The present invention provides one kind for removing the endotoxic method of antibody protein, is that pending protein sample is carried out a step affinity chromatography, steps are as follows：(1) equilibrium liquid rinse affinity column, disinfection, rebalancing, loading are used；The equilibrium liquid is the phosphate buffer containing NaCl, and pH is 7.2~7.4, and conductivity is 16.0~18.0mS/cm；(2) chromatographic column is rinsed with flushing liquor, chromatographic column is rinsed with equilibrium liquid；The flushing liquor is the equilibrium liquid for being added to Arg；(3) elution is used, eluent is collected；The eluent is sodium acetate buffer, and pH is 3.3~3.5, and conductivity is 0.24~0.44mS/cm.The present invention can be under conditions of not changing antibody protein property, the effective endotoxin removed in protein sample, and does not influence the purity of albumen in the process, and the rate of recovery is high.Processing method of the present invention is simple, of low cost, takes short, is less than 0.1EU/mg using this method treated sample endotoxin content, safety is good, and potential applicability in clinical practice is good.
Description
Technical field
The present invention relates to biological product technical fields, and in particular to monoclonal antibody endotoxin removal method.
Background technology
A kind of lipopolysaccharides that endotoxin comes from Gram-negative bacteria outer membrane is otherwise known as by being discharged after cellular lysate
" heat source ".Their relative molecular mass generally 1~2.5 ten thousand, forms associated matter in aqueous solution, and molecular mass can reach
50~1,000,000.Its chemical composition is mainly lipopolysaccharide-protein complex, and toxic ingredient is mainly lipoids A.
Endotoxin is not albumen, therefore very heat-resisting, and also there is characteristics, the removals such as small, chemical stability is strong to have one
Determine difficulty.Strong acid, highly basic or strong can also be used by only heating 2~4h at a temperature of 160 DEG C and either heating 1h at a temperature of 240
30min is boiled in oxidant heating can just destroy its bioactivity.A small amount of endotoxin can cause organism fever, can when serious
It can cause disseminated intravascular coagulation (DIC), shock, fever and inflammatory reaction.
For monoclonal antibody as biopharmaceutical macromolecular drug, culture and purifying process are complicated, have many links that may draw
Enter endotoxin, therefore remove the endotoxin in sample, ensures that its safety is particularly important.To in injection product in National Pharmacopeia
Endotoxin content regulation should be less than 1EU/mL.Various countries' drug administration department also has stringent want to the endotoxic content of biological products
It asks.
Currently, mainly having for endotoxic minimizing technology：Phase separation method, ultrafiltration, active carbon adsorption, chemical degradation
Method, thin layer chromatography method etc..Phase separation method treating capacity is small, and there be the variation of temperature centre, unfavorable to some unstable albumen；It is super
Material and hole of the filter method for when removing endotoxin, needing relative molecular weight and characteristic selection ultrafiltration membrane according to processing sample
Diameter does not have versatility；Activated carbon method is adsorbing endotoxic while often adsorbing some active ingredients, the recycling to product
Rate is affected；Chemical degradation method carrys out induced by endotoxin by using strong acid and strong base strong oxidizer and degrades, this is by sample
The limitation of physicochemical property；Chromatography includes ionization, hydrophobic effect, gelatification chromatography, the different samples property of itself
Difference, chromatography effect is also just different, and screening takes time and effort.
Therefore, realize that endotoxic efficient removal will have great importance in antibody protein.
Invention content
The present invention is directed in view of the drawbacks of the prior art, provide a kind of endotoxic method of removal, to solve existing endogenous toxic material
The difficult problem of plain minimizing technology screening.
Another technical problem that the present invention solves is to provide a kind of endotoxin removal method not influencing antibody protein property.
The present invention another technical problem be to provide it is a kind of be substantially reduced protein endotoxins removal time and cost problem,
Improve efficiency.
In order to solve the above technical problems, the present invention uses following technical scheme：
One kind takes pending biological sample to pass through a step affinity chromatography i.e. for removing endotoxic method in antibody protein
Can, it is as follows：
One kind is for removing the endotoxic method of antibody protein, it is characterised in that：One step is carried out to pending protein sample
Affinity chromatography, steps are as follows：
(1) equilibrium liquid rinse affinity column, disinfection, rebalancing, loading are used；The equilibrium liquid is the phosphoric acid containing NaCl
Salt buffer, pH are 7.2~7.4, and conductivity is 16.0~18.0mS/cm；
(2) chromatographic column is rinsed with flushing liquor, chromatographic column is rinsed with equilibrium liquid；The flushing liquor is the balance for being added to Arg
Liquid；
(3) elution is used, eluent is collected；The eluent is sodium acetate buffer, pH is 3.3~
3.5, conductivity is 0.24~0.44mS/cm.
In step (1), the filler that the affinity column uses is MabSelect SuRe.
In step (1), in the equilibrium liquid, NaCl concentration 150mM, phosphate concn 20mM.
In step (1), the disinfection is carried out disinfection processing to chromatographic column with 0.5M NaOH.
In step (1), when loading, which is rinsed with equilibrium liquid to efflux 280nm UV absorptions, is back to baseline.
In step (2), in the flushing liquor, a concentration of 500mM of Arg.Chromatographic column about 30min is rinsed with flushing liquor.
In step (3), in the eluent, sodium acetate concentration 25mM.When with elution, it is purple to collect 280nm
Efflux at outer absorption peak is to get to elution samples.It takes a certain amount of neutralizer to be added in elution samples, adjusts sample
pH.Sample after neutralization is subjected to film process, as eliminates endotoxic product.
The method further includes regeneration and equilibrium step.
9, the biological products being prepared by the method described in claim 1~8 any one.
It is characterized in that：Endotoxin content is less than 0.1EU/mg.
It is characterized in that：The biological products are monoclonal antibodies.
It is characterized in that：The monoclonal antibody hypotype is hIgG1.
In step (1), the purpose of rebalancing is the alkali removed inside chromatographic column, and filler is made to be in the environment of equilibrium liquid.
In step (1), it is by one that when loading, which rinses to efflux 280nm UV absorptions the purpose for being back to baseline with equilibrium liquid,
A little foreign proteins being not coupled on chromatographic column all rinse out.
In step (2), the purpose for rinsing chromatographic column with equilibrium liquid is the Arg rinsed out inside chromatographic column, reduces the residual of Arg
It stays, and then avoids its influence to subsequent detection.
In step (3), used neutralizer is 1M Arg, 400mM succinic, pH8.5~9.5.
The present invention also provides a kind of method of monoclonal antibody purification, the monoclonal antibody hypotype is hIgG1.
The present invention can be under conditions of not changing antibody protein property, the effective endotoxin removed in protein sample,
And the purity of albumen is not influenced in the process, the rate of recovery is high.Processing method is simple, of low cost, time-consuming short, at this method
Sample endotoxin content after reason is less than 0.1EU/mg, and safety is good, and potential applicability in clinical practice is good.
Specific implementation mode
Clear, complete description will be carried out to technical scheme of the present invention below, it is clear that described embodiment is this hair
Bright a part of the embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art exist
The every other embodiment obtained under the premise of not making creative work, shall fall within the protection scope of the present invention.
The culture of 1 monoclonal antibody of embodiment and purifying process are complicated, have many links that may introduce endotoxin.It is existing
It is explained as an example.
The Protein A affinity chromatographys of step 1 cell supernatant
Chromatographic stuffing：A kind of MabSelect SuRe (alkaline-resisting protein A fillers) of GE companies.
Chromatographic flow rates：150~300cm/h.
Rinse：With equilibrium liquid 20mM phosphate, 150mM NaCl, pH7.2~7.4 balance chromatographic column.
Disinfection：Chromatographic column 15min is rinsed with 0.5M NaOH.
Balance：Chromatographic column is rinsed with equilibrium liquid, until pH drops to 7.2~7.4.
Loading：The supernatant that cell culture is obtained carries out upper prop, with equilibrium liquid rinse chromatographic column after completion of the sample until
Efflux 280nm UV absorptions return to baseline.
Rinse 1：Chromatographic column is rinsed with buffer solution 50mM NaOAc, 1M NaCl, pH5.4~5.6, until efflux 280nm
UV absorption returns to baseline.
Rinse 2：Chromatographic column is rinsed with buffer solution 50mM NaOAc, pH5.4~5.6, until with high salt above chromatographic column
All to rinsing out.
Elution and collection：With eluent 25mM NaOAc, pH3.3~3.5 pair antibody elutes.It is ultraviolet to collect 280nm
Absorption peak is used in combination neutralizer to adjust pH to 4.9~5.1, obtains eluent 1.
Regeneration：With regenerated liquid 50mM NaOAc, pH2.9~3.1 pair chromatographic column carries out regeneration treatment.
Balance：Chromatographic column is rinsed with equilibrium liquid, until pH is raised to 7.2~7.4.
Step 2 cation-exchange chromatography
Chromatographic stuffing：The POROS XS of Invitrogen companies.
Chromatographic flow rates：150~300cm/h.
Rinse：Chromatographic column is balanced with equilibrium liquid 50mM NaOAc, pH5.4~5.6.
Disinfection：Chromatographic column 15min is rinsed with 0.5M NaOH, suspends 45min.
Balance：Chromatographic column is rinsed with equilibrium liquid, until pH drops to 5.4~5.6.
Loading：The eluent 1 that cell supernatant is collected into through affinity chromatography, adjusts its pH to 4.9~5.1, with balance
Liquid adjusts its conductance to less than 5.0.Chromatographic column is rinsed until efflux 280nm UV absorptions return to equilibrium liquid after completion of the sample
Baseline.
Elution and collection：With eluent 50mM NaOAc, 1M NaCl, pH5.4~5.6 pair antibody carries out linear gradient and washes
It is de-.280nm ultraviolet absorption peaks are collected, eluent 2 is obtained.
Regeneration：Chromatographic column is handled with 0.5M NaOH, rinses out the stronger albumen of binding force.
Balance：Chromatographic column is rinsed with equilibrium liquid, until pH drops to 5.4~5.6.
Liquid is changed in step 3 dialysis
The pre-treatment of film packet：The flushing that water is first carried out to film packet, and then disappears to film packet and pipeline with the alkali of 0.1M
Poison processing 1h, then film and pipeline are rinsed with ultra-pure water, the remaining lye in the inside is flushed out, is finally used again
Formulation Buffer are rinsed film and pipeline.
Concentration：It uses molecular cut off to exchange cation the product (i.e. eluent 2) obtained for the film packet of 30kD to carry out
Slipstream concentrates, and the pressure at control inlet and end of flowing back is no more than 0.05MPa, and the two pressure summation is no more than 0.1MPa.With
This simultaneously, by magnetic stirring apparatus is slowly stirred sample.
Change liquid：It when sample concentration is to certain volume, is just carried out at the same time and changes liquid operation, until the buffer solution volume of outflow end is
6 times or more of sample liquid.Liquid is changed in the concentration for completing sample, the buffer solution of certain volume is finally squeezed into, by the pipeline of enrichment facility
The sample of dead volume is all gone out, and protein sample is obtained.
The post-processing of film packet：The flushing that water is first carried out to film packet, and then disappears to film packet and pipeline with the alkali of 0.1M
Poison processing 1h, then film and pipeline are rinsed with ultra-pure water, finally film packet is preserved with 0.05M NaOH.
Above-mentioned technical process includes that the affinity purification, cation-exchange chromatography and dialysis of cell supernatant change the processes such as liquid,
The culture of this field monoclonal antibody and the representative of purifying process are can be used as, endotoxin is can be controlled in the protein sample of production
＜ 0.5EU/mg reach the mean level of this field.But due to complex process and link is more, there are still infect endotoxic wind
Danger.Once having infected endotoxin, is once purified again if repeating above-mentioned technical process, it is too high to take too long and cost.
2 endotoxin removal method of embodiment：There is the possibility of infection endotoxin risk for prior art, the present invention provides
A kind of purification process rapidly and efficiently of the exceeded protein sample of induced by endotoxin.
1.Protein A affinity chromatographys
Chromatographic stuffing：The MabSelect SuRe of GE companies.
Chromatographic flow rates：150~300cm/h.
Rinse：With equilibrium liquid 20mM phosphate, 150mM NaCl, pH7.2~7.4 balance chromatographic column.
Disinfection：Chromatographic column 15min is rinsed with 0.5M NaOH.
Balance：Chromatographic column is rinsed with equilibrium liquid, until pH drops to 7.2~7.4.
Loading：A certain amount of endotoxic protein sample (endotoxin content is exceeded) will be contained and carry out upper prop, used after completion of the sample
Equilibrium liquid rinses chromatographic column until efflux 280nm UV absorptions return to baseline.
It rinses：With flushing liquor 20mM phosphate, 150mM NaCl, 500mM Arg, pH6.8~7.0 rinse chromatographic column
30min。
Balance：Chromatographic column is rinsed with equilibrium liquid, until the Arg in sample all to rinsing out.
Elution and collection：With eluent 25mM NaOAc, pH3.3~3.5 pair antibody elutes.It is ultraviolet to collect 280nm
Absorption peak is used in combination neutralizer to adjust pH to 4.9~5.1.
Regeneration：With regenerated liquid 50mM NaOAc, pH2.9~3.1 pair chromatographic column carries out regeneration treatment.
Balance：Chromatographic column is rinsed with equilibrium liquid, until pH is raised to 7.2~7.4.
2. determination of protein concentration (absorbance method)：
2000 operating system software A280 patterns of NanoDrop are used to detect the concentration of antibody protein samples.Lambert Bill
Law is associated with absorbance and concentration with concentration.Suitable extinction coefficient is selected, is combined with Beer law, can be counted
Calculate the concentration of sample.
A=E*b*C
A is absorbance value (A),
E is extinction coefficient, and unit is L/mol ﹒ cm
B is optical length, in centimeters,
C is sample concentration.
3. purity of protein measures (spatial exclusion chromatography)：
High performance liquid chromatograph (SEC-HPLC) is measured the purity of sample, and albumen detaches the only aperture with gel
Distribution is related to the molecular size of antibody, in sample to be tested in the case of all equal appearances of component, with the peak area of target protein/
The purity for the target protein that the summation of peak height divided by all peaks peak area/peak height is.
4. endotoxic detection (dynamic reduced turbidity method)：
Dynamic turbidimetric is to detect a certain preset required reaction of absorbance of turbidity arrival of reaction mixture
Time, or detect the method that turbidity is increased speed.The linear of double-log is established with standard endotoxin concentration and reaction time to return
Standard curve, unknown endotoxin concns is returned to calculate endotoxin concns by measuring its reaction time, by standard curve.
5. experimental result
1 testing result of table
As can be seen from Table 1, endotoxin content is in 10~50EU/mg before treatment for protein sample, purity 99.6%, but use
After this method processing, the endotoxin content of sample is less than 0.1EU/mg, and endotoxin removal rate is more than 99%, and sample purity does not have shadow
It rings, and the rate of recovery is also high, the rate of recovery is more than 91%.
Another comparative example is that small amount of albumen sample is added to the loading step of 1 step 1 of embodiment to be obtained
It flows through in liquid, and opening is stood overnight, the endotoxin content of sample reaches 2732EU/mL, obtained sample after film process
Product are to be obtained under natural conditions containing endotoxic protein sample.Embodiment 4 is used containing endotoxic protein sample for this
After method removes endotoxin, it is only 0.095EU/mg to obtain product endotoxin content, purity 99.4%, and endotoxin removal rate is big
In 99%, the rate of recovery is more than 91%.
The present invention can be under conditions of not changing antibody protein property, the effective endotoxin removed in protein sample,
And the purity of albumen is not influenced in the process, the rate of recovery is high.Processing method is simple, of low cost, time-consuming short, at this method
Sample after reason does content of toxins and is less than 0.1EU/mg, and safety is good, and potential applicability in clinical practice is good.
In conclusion the various embodiments described above are only presently preferred embodiments of the present invention, not limiting the present invention's
Protection domain, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should be all included in
In protection scope of the present invention.
Claims (12)
1. one kind is for removing the endotoxic method of antibody protein, it is characterised in that：Pending protein sample is subjected to step parent
And chromatography, steps are as follows：
(1) equilibrium liquid rinse affinity column, disinfection, rebalancing, loading are used；The equilibrium liquid is that the phosphate containing NaCl is slow
Fliud flushing, pH are 7.2~7.4, and conductivity is 16.0~18.0mS/cm；
(2) chromatographic column is rinsed with flushing liquor, chromatographic column is rinsed with equilibrium liquid；The flushing liquor is the equilibrium liquid for being added to Arg；
(3) elution is used, eluent is collected；The eluent is sodium acetate buffer, and pH is 3.3~3.5, electricity
Conductance is 0.24~0.44mS/cm.
2. according to the method described in claim 1, it is characterized in that：The filler that the affinity column uses is
MabSelectSuRe。
3. according to the method described in claim 1, it is characterized in that：In the equilibrium liquid, NaCl concentration 150mM, phosphoric acid
Salinity is 20mM.
4. according to the method described in claim 1, it is characterized in that：The disinfection is disappeared to chromatographic column with 0.5M NaOH
Poison processing.
5. according to the method described in claim 1, it is characterized in that：It is rinsed with equilibrium liquid when loading ultraviolet to efflux 280nm
Absorption is back to baseline.
6. according to the method described in claim 1, it is characterized in that：In the flushing liquor, a concentration of 500mM of Arg.
7. according to the method described in claim 1, it is characterized in that：In the eluent, sodium acetate concentration 25mM.
8. according to claim 1-7 any one of them methods, it is characterised in that：The method further includes regeneration and balance step
Suddenly.
9. the biological products being prepared by the method described in claim 1~8 any one.
10. biological products according to claim 9, it is characterised in that：Endotoxin content is less than 0.1EU/mg.
11. biological products according to claim 9, it is characterised in that：The biological products are monoclonal antibodies.
12. biological products according to claim 11, it is characterised in that：The monoclonal antibody hypotype is hIgG1.
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Cited By (4)
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| CN111778232A (en) * | 2020-07-10 | 2020-10-16 | 江苏尤里卡生物科技有限公司 | Method for removing endotoxin in refining process of human urokinase raw material |
| CN114230641A (en) * | 2021-12-29 | 2022-03-25 | 内蒙古必威安泰生物科技有限公司 | Method for removing endotoxin in foot-and-mouth disease antigen liquid |
| CN114452444A (en) * | 2022-01-27 | 2022-05-10 | 陕西巨子生物技术有限公司 | Preparation method of low endotoxin collagen |
| CN114602237A (en) * | 2022-03-10 | 2022-06-10 | 华兰生物工程股份有限公司 | Method for removing endotoxin from human plasma or human plasma derived raw material |
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| CN111778232A (en) * | 2020-07-10 | 2020-10-16 | 江苏尤里卡生物科技有限公司 | Method for removing endotoxin in refining process of human urokinase raw material |
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| CN114230641B (en) * | 2021-12-29 | 2024-03-26 | 内蒙古必威安泰生物科技有限公司 | Method for removing endotoxin in foot-and-mouth disease antigen liquid |
| CN114452444A (en) * | 2022-01-27 | 2022-05-10 | 陕西巨子生物技术有限公司 | Preparation method of low endotoxin collagen |
| CN114602237A (en) * | 2022-03-10 | 2022-06-10 | 华兰生物工程股份有限公司 | Method for removing endotoxin from human plasma or human plasma derived raw material |
| CN114602237B (en) * | 2022-03-10 | 2023-11-28 | 华兰生物工程股份有限公司 | Method for removing endotoxin from human plasma or human plasma derivative raw material |
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Application publication date: 20180928 |