CN109336969A - A kind of purification process of antibody - Google Patents
A kind of purification process of antibody Download PDFInfo
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- CN109336969A CN109336969A CN201811332129.7A CN201811332129A CN109336969A CN 109336969 A CN109336969 A CN 109336969A CN 201811332129 A CN201811332129 A CN 201811332129A CN 109336969 A CN109336969 A CN 109336969A
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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
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Abstract
The present invention provides a kind of purification process of antibody.A kind of purification process of antibody, including protein A affinity chromatography step；The method of the protein A affinity chromatography are as follows: after loading and with before elution target antibody, first with intermediate cleaning buffer solution column scrubber bed；The intermediate cleaning buffer solution are as follows: pH value 5-6, conductivity are the buffer of 20-100mS/cm.The step of present invention among increasing during protein A affinity chromatography by cleaning improves the removal ability to HCP, DNA, and then improves the ability of anion-exchange chromatography virus removal.
Description
Technical field
The present invention relates to antibody purification fields, more particularly, to a kind of purification process of antibody.
Background technique
In pharmaceuticals industry, there are many drug for deriving from biotechnology, people are also more dense to the interest of bio-pharmaceuticals
It is thick.The features such as less toxic side effect, high specific curative effect, production technology of longer half-life period and hardware and software platform, monoclonal antibody is made to exist
Treatment field of biological product maintains the leading position, and is a most successful kind biological product.So far, most monoclonal antibody is all by feeding
Newborn animal cell expression, this is because it is complicated glycosylation modified to be that mammalian cell can be completed, and it is glycosylation modified
The stability and solubility of albumen can be increased, to reach longer half-life period；And most common monoclonal antibody expression system is
The IgG of CHO cell line, level of glycosylation closest to the people of the monoclonal antibody medicine generated is glycosylation modified.When treatment albumen by
When animal cell line is expressed, need to consider the viral pollution risk from cell line and culture medium.In order to ensure the peace of product
Full property and the requirement for meeting regulation, should design more purification steps, not only to removal product/technique related impurities, also for
Removal/inactivation of viruses.Typical monoclonal antibody purification step includes separation of solid and liquid, protein A affinity chromatography capture, low ph value incubation disease
Poison inactivation, two steps chromatography are consummate, nanofiltration virus removal and ultrafiltration and etc..
Anion-exchange chromatography (AEX) is a kind of technique for removing virus in antibody purification and generally using, and is mainly adopted
With flowing through operation mode.The mechanism of AEX removal virus is electrostatic interaction, conductivity value, the impurity content (such as HCP, DNA) of feed liquid
It is larger to virus removal capacity.It reduces the impurity content of feed liquid and AEX virus can be promoted using suitable conductivity
Scavenging activity.
In monoclonal antibody purifying technique, the upstream treatment step of anion-exchange chromatography removal virus is usually that albumin A is affine
Chromatography.Protein A affinity chromatography be in the widest just pure step of monoclonal antibody purification application, can be special from complicated cell culture fluid
Opposite sex capture monoclonal antibody.Protein A affinity chromatography has certain HCP, DNA removal ability, thus protein A affinity chromatography to HCP,
Key factor one of of the removal effect of DNA as anion-exchange chromatography removal virus capable, however the affine layer of routine protein A
The virus removal ability for analysing technique is very steady, and the variation of technological parameter does not have tangible influence to its virus removal ability.
In view of this, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of purification process of antibody, this method passes through in protein A affinity chromatography process
The step of cleaning, improves the removal ability to HCP, DNA among middle increase.
In order to solve the above technical problems, the present invention provides following technical schemes:
A kind of purification process of antibody, including protein A affinity chromatography step；
The method of the protein A affinity chromatography are as follows: after loading and in front of elution target antibody, first using
Between cleaning buffer solution column scrubber bed；
The intermediate cleaning buffer solution are as follows: pH value 5-6, conductivity are the buffer of 20-100mS/cm.
Above method utilizes: pH value 5-6, and conductivity is the buffer solution for cleaning sample of 20-100mS/cm, removes therein
HCP,DNA.Compared to the chromatography technique without intermediate cleaning step, the present invention has a significant improvement to the removal rate of HCP, DNA, and gained is washed
HCP content is lower than 1000ppm in de- liquid, and the content of residual DNA is lower than 100pg/mg.
In the present invention, the pH value and conductivity of intermediate cleaning buffer solution make the key parameter for removing HCP, DNA, and pH can be used
Arbitrary value within the scope of 5-6, such as 5.1,5.2,5.3,5.4,5.5,5.6,5.7,5.8,5.9,6.0 etc., conductivity can be used
Arbitrary value within the scope of 20-100mS/cm, such as 20mS/cm, 25mS/cm, 30mS/cm, 35mS/cm, 40mS/cm, 45mS/
cm、50mS/cm、55mS/cm、60mS/cm、65mS/cm、70mS/cm、75mS/cm、80mS/cm、85mS/cm、90mS/cm、
95mS/cm, 100mS/cm etc..
The present invention can also carry out following improve on the basis of intermediate cleaning added above:
Preferably, the intermediate cleaning buffer solution is that acetate salt buffer system, phosphate buffer or citrate are slow
Rush system.
These buffers will not attract Adventitious impurities, and raw material is easy to get.
There is no specific restriction, such as 50mM NaAc-HAc or 1M NaAc-HAc for the concentration of intermediate cleaning buffer solution
Deng.
Preferably, the intermediate cleaning buffer solution is the buffer that conductivity is adjusted with villaumite, the preferred chlorination of villaumite
Sodium.
It is of course also possible to use the salt adjusting conductivity that other dissolubilities are good, and be not limited only to that a kind of salt, Huo Zheduo is added
It is added after kind combination.
Preferably, the target antibody is the antibody of Chinese hamster ovary cell expression.
Preferably, the filler of the protein A affinity chromatography is MabSelect Sure, MabSelect LX, Amsphere
A3 or Praesto Jetted A50.
Through investigating, the step of cleaning among increase in the chromatographic column of the above filler, the effect got twice the result with half the effort can reach, it is right
The removal ability of HCP, DNA significantly improve.
Preferably, the pH value of the intermediate cleaning buffer solution is 5.5-6, preferably 5.5-5.8.
Preferably, conductivity 50-100mS/cm, preferably 50-80mS/cm.
Preferably, the process of the protein A affinity chromatography is successively are as follows: disinfection, equilibrium liquid washing, loading, equilibrium liquid washing,
The intermediate cleaning buffer solution washing, equilibrium liquid washing, the elution target antibody.
Preferably, the equilibrium liquid be Tris-HCl+150~160mM NaCl, pH7.2~7.6 buffer, preferably 50
~55mM Tris-HCl.
Preferably, the target antibody be expressing cho cell monoclonal antibody when, the eluent be NaAc-HAc, pH3.0~
3.8 buffer, preferably 50~55mM NaAc-HAc.
Preferably, further include that anion-exchange chromatography is carried out to the eluent of collection after the protein A affinity chromatography:
Successively disinfection, equilibrium liquid washing, loading, equilibrium liquid washing.
Preferably, the equilibrium liquid when anion-exchange chromatography is the buffer of Tris-HCl, pH7.2~8.0.
Preferably, the filler of the anion-exchange chromatography be POROS 50HQ, Q Sepharose FF, Capto Q or
Eshmuno Q。
In addition, if be further improved on the basis of all of above scheme the filler of protein A affinity chromatography column, pillar height and
The parameters such as flow velocity can also improve yield when the multiple circulatory purification of chromatographic column single-column, i.e., under unit time unit packing volume
Treating capacity, it is specific as follows.
Preferably, the method for the protein A-sepharose affinity chromatography antibody purification is to recycle following protein A affinity chromatography column
Purification of target antibody:
Filler is MabSelect LX, Amsphere A3 or Praesto Jetted A50, and the dress pillar height degree of filler is 5
~15cm, process operation linear flow rate are 200~500cm/h.
Above method is the purifying process that single-column repeatedly recycles, and main by selecting, carrying capacity is high, mass transfer ability is good, operation stream
The fast affine filler of albumin A of speed, to improve the utilization rate of the affine filler of albumin A；Also by reduction pillar height, linear flow rate is improved,
To shorten the single cycle time；The continuous capture process to realize protein A affinity chromatography is repeatedly recycled finally by single-column, is both dropped
Low medium cost, and improve purification efficiency.
In addition, the program is filled out further advantage is that unit time unit can be improved without increasing additional technical steps i.e.
The treating capacity of Material product, the cost for substituting prior art is low, does not influence on the production chain of antibody, and technique is steady, reliable.
To sum up, compared with prior art, process above of the invention has filler utilization rate height, unit time unit filler
The advantages that treating capacity of volume is high, at low cost, technique is steadily and surely reliable.
In the present invention, MabSelect LX, Amsphere A3, Praesto Jetted A50 filler are usually fixed
Producer, respectively GE Health Care company, JSR company and Purolite company.
In the present invention, the dress pillar height degree of filler arbitrarily selects within the scope of 5~15cm, for example, 5cm, 5.5cm, 6.0cm,
6.5cm、7.0cm、7.5cm、8.0cm、8.5cm、9.0cm、9.5cm、10.0cm、10.5cm、11.0cm、11.5cm、12.0cm、
12.5cm, 13.0cm, 14.0cm, 14.5cm or 15.0cm etc., preferred range have a 5~12cm, preferably 5~10cm, and more preferable 6
~10cm.
In the present invention, process operation linear flow rate arbitrarily selects within the scope of 200~500cm/h, such as 200cm/h,
250cm/h, 300cm/h, 350cm/h, 400cm/h, 450cm/h or 500cm/h etc., preferred range have 300~500cm/h,
More preferable 300~400cm/h.
Purification process of the invention is primarily adapted for use in the purifying of the purifying of antibody, especially monoclonal antibody, especially Chinese hamster
The antibody of gonad cell expression.
To sum up, compared with prior art, invention achieves following technical effects:
(1) increase intermediate cleaning step when protein A affinity chromatography purification antibody, improve the removal ability to HCP, DNA,
And there is substantive raising compared to tradition chromatography, remove virus for anion-exchange chromatography and provide advantageous prerequisite；
(2) it by other technological parameters in optimization protein A affinity chromatography and anion-exchange chromatography, further improves
Removal ability of the purifying process to HCP, DNA；
(3) under the premise of not increasing additional technical steps, by selecting, carrying capacity is high, mass transfer ability is good, operation flow velocity is fast
The affine filler of albumin A, to improve the utilization rate of the affine filler of albumin A；Also by reducing pillar height, linear flow rate is improved, to contract
The short single cycle time；The continuous capture process to realize protein A affinity chromatography is repeatedly recycled finally by single-column, is both reduced
Medium cost, and improve purification efficiency.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with specific embodiment, but ability
Field technique personnel will be understood that following described embodiments are some of the embodiments of the present invention, instead of all the embodiments,
It is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.Based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.The person that is not specified actual conditions in embodiment, carries out according to conventional conditions or manufacturer's recommended conditions.Agents useful for same or instrument
Production firm person is not specified, is the conventional products that can be obtained by commercially available purchase.
Embodiment 1
MabSelect SuRe LX, chromatographic column Vantage 11/250 (Millipore), diameter 1.1cm, pillar height 20cm,
Column volume 19.0mL operates flow velocity 220cm/h.Sample source is the monoclonal antibody (number Mab1) of expressing cho cell.With 3CV's
The sodium hydroxide solution of 0.2mol/L rinses chromatographic column, then with the equilibrium liquid of 5CV (50mM Tris-HCl+150mM NaCl,
PH7.4 chromatographic column) is balanced；Cell culture harvest liquid (HCCF) after clarification is loaded to chromatographic column, loading carrying capacity is 50g/L；
Column bed is cleaned with the equilibrium liquid of 3CV, with the intermediate cleaning buffer solution (50mM of 3CV
NaAc-HAc+1M NaCl, pH5.5, conductivity 88.5mS/cm) column scrubber bed, then column is cleaned with 3CV equilibrium liquid
Bed；It is eluted again with the eluent (50mM NaAc-HAc, pH3.5) of 3.5CV, collects eluent；The 0.2mol/ of 3CV is finally used again
The sodium hydroxide solution of L rinses chromatographic column, after balancing chromatographic column with the equilibrium liquid of 3CV, finally saves chromatography with the preservation liquid of 3CV
Column.The HCP content of eluent is 900ppm, and DNA residual quantity is 85pg/mg.
POROS 50HQ, chromatographic column BenchMark 06/25 (Omnifit), diameter 0.66cm, pillar height 20cm, column volume
6.8mL operates flow velocity 220cm/h.Sample source is upper step affinity elution liquid, adjusts pH to 7.5, and through 0.22 μm of filter membrane mistake
Filter, the additive amount of X-MuLV are 1% (v/v).Chromatographic column is rinsed with the sodium hydroxide solution of the 0.5mol/L of 3CV, then with 3CV's
Equilibrium liquid (50mM Tris-HCl, pH7.5) balances chromatographic column；Sample after pH is adjusted and clarified is loaded to chromatographic column, loading
Carrying capacity is 100g/L；The equilibrium liquid column scrubber bed of 3CV is used again, and collection flows through liquid；Last spent regeneration solution (50mM Tris- again
HCl+1M NaCl, pH7.5) to column regeneration；Chromatographic column is rinsed with the sodium hydroxide solution of the 0.5mol/L of 3CV, is finally used
The preservation liquid of 3CV saves chromatographic column.The step can achieve 5.1log10 to the Scavenging activity of X-MuLV.
Comparative example 1
MabSelect SuRe LX, chromatographic column Vantage 11/250 (Millipore), diameter 1.1cm, pillar height 20cm,
Column volume 19.0mL operates flow velocity 220cm/h.Sample source is the monoclonal antibody (number Mab1) of expressing cho cell.With 3CV's
The sodium hydroxide solution of 0.2mol/L rinses chromatographic column, then with the equilibrium liquid of 5CV (50mM Tris-HCl+150mM NaCl,
PH7.4 chromatographic column) is balanced；Cell culture harvest liquid (HCCF) after clarification is loaded to chromatographic column, loading carrying capacity is 50g/L；
Column bed is cleaned with the equilibrium liquid of 3CV；It is eluted again with the eluent (50mM NaAc-HAc, pH3.5) of 3.5CV, collects eluent；
Chromatographic column finally is rinsed with the sodium hydroxide solution of the 0.2mol/L of 3CV again, after balancing chromatographic column with the equilibrium liquid of 3CV, finally
Chromatographic column is saved with the preservation liquid of 3CV.The HCP content of eluent is 3600ppm, and DNA residual quantity is 400pg/mg.
POROS 50HQ, chromatographic column BenchMark 06/25 (Omnifit), diameter 0.66cm, pillar height 20cm, column volume
6.8mL operates flow velocity 220cm/h.Sample source is upper step affinity elution liquid, adjusts pH to 7.5, and through 0.22 μm of filter membrane mistake
Filter, the additive amount of X-MuLV are 1% (v/v).Chromatographic column is rinsed with the sodium hydroxide solution of the 0.5mol/L of 3CV, then with 3CV's
Equilibrium liquid (50mM Tris-HCl, pH7.5) balances chromatographic column；Sample after pH is adjusted and clarified is loaded to chromatographic column, loading
Carrying capacity is 100g/L；The equilibrium liquid column scrubber bed of 3CV is used again, and collection flows through liquid；Last spent regeneration solution (50mM Tris- again
HCl+1M NaCl, pH7.5) to column regeneration；Chromatographic column is rinsed with the sodium hydroxide solution of the 0.5mol/L of 3CV, is finally used
The preservation liquid of 3CV saves chromatographic column.The Scavenging activity removing solid capacity without cleaning step among affinity chromatography is 4.0log10.
Comparing embodiment 1 and comparative example 1, it may be seen that affine intermediate cleaning step can effectively improve the removing energy of X-MuLV
Power.
Embodiment 2
MabSelect SuRe, chromatographic column Vantage 11/250 (Millipore), diameter 1.1cm, pillar height 20cm, column
Volume 19.0ml operates flow velocity 200cm/h.Sample source is the monoclonal antibody (Mab2) of expressing cho cell.With the 0.1mol/L of 3CV
Sodium hydroxide solution rinse chromatographic column, then chromatographed with the equilibrium liquid (20mM Tris-HCl+1M NaCl, pH7.4) of 5CV balance
Column；Cell culture harvest liquid (HCCF) after clarification is loaded to chromatographic column, loading carrying capacity is 30g/L；It is clear with the equilibrium liquid of 3CV
Column bed is washed, with intermediate cleaning buffer solution (1M NaAc-HAc, pH5.8, conductivity 47.3mS/cm) column scrubber bed of 3CV, then is used
3CV equilibrium liquid cleans column bed；It is eluted again with the eluent (100mM HAc, pH3.0) of 3.5CV, collects eluent；Finally use again
The sodium hydroxide solution of the 0.1mol/L of 3CV rinses chromatographic column, after balancing chromatographic column with the equilibrium liquid of 3CV, finally uses the guarantor of 3CV
Liquid storage saves chromatographic column.The HCP content of eluent is 960ppm, and DNA residual quantity is 90pg/mg.
Q Sepharose FF, chromatographic column BenchMark 06/25 (Omnifit), diameter 0.66cm, pillar height 20cm, column
Volume 6.8ml operates flow velocity 200cm/h.Sample source is upper step affinity elution liquid, adjusts pH to 8.0, and filter through 0.22 μm
Film filtering, the additive amount of MVM are 1% (v/v).Chromatographic column is rinsed with the sodium hydroxide solution of the 0.5mol/L of 3CV, then uses 3CV
Equilibrium liquid (50mM Tris-HCl, pH8.0) balance chromatographic column；Sample after pH is adjusted and clarified is loaded to chromatographic column, on
Sample carrying capacity is 60g/L；The equilibrium liquid column scrubber bed of 3CV is used again, and collection flows through liquid；Last spent regeneration solution (50mM Tris- again
HCl+1M NaCl, pH8.0) to column regeneration；Chromatographic column is rinsed with the sodium hydroxide solution of the 0.5mol/L of 3CV, is finally used
The preservation liquid of 3CV saves chromatographic column.The step can achieve 4.5log10 to the Scavenging activity of MVM.
Comparative example 2
MabSelect SuRe, chromatographic column Vantage 11/250 (Millipore), diameter 1.1cm, pillar height 20cm, column
Volume 19.0ml operates flow velocity 200cm/h.Sample source is the monoclonal antibody (Mab2) of expressing cho cell.With the 0.1mol/L of 3CV
Sodium hydroxide solution rinse chromatographic column, then chromatographed with the equilibrium liquid (20mM Tris-HCl+1M NaCl, pH7.4) of 5CV balance
Column；Cell culture harvest liquid (HCCF) after clarification is loaded to chromatographic column, loading carrying capacity is 30g/L；It is clear with the equilibrium liquid of 3CV
Column bed is washed, then cleans column bed with 3CV equilibrium liquid；It is eluted again with the eluent (100mM HAc, pH3.0) of 3.5CV, collects elution
Liquid；Chromatographic column finally is rinsed with the sodium hydroxide solution of the 0.1mol/L of 3CV again, after balancing chromatographic column with the equilibrium liquid of 3CV, most
Chromatographic column is saved with the preservation liquid of 3CV afterwards.The HCP content of eluent is 3710ppm, and DNA residual quantity is 524pg/mg.
Q Sepharose FF, chromatographic column BenchMark 06/25 (Omnifit), diameter 0.66cm, pillar height 20cm, column
Volume 6.8ml operates flow velocity 200cm/h.Sample source is upper step affinity elution liquid, adjusts pH to 8.0, and filter through 0.22 μm
Film filtering, the additive amount of MVM are 1% (v/v).Chromatographic column is rinsed with the sodium hydroxide solution of the 0.5mol/L of 3CV, then uses 3CV
Equilibrium liquid (50mM Tris-HCl, pH8.0) balance chromatographic column；Sample after pH is adjusted and clarified is loaded to chromatographic column, on
Sample carrying capacity is 60g/L；The equilibrium liquid column scrubber bed of 3CV is used again, and collection flows through liquid；Last spent regeneration solution (50mM Tris- again
HCl+1M NaCl, pH8.0) to column regeneration；Chromatographic column is rinsed with the sodium hydroxide solution of the 0.5mol/L of 3CV, is finally used
The preservation liquid of 3CV saves chromatographic column.The Scavenging activity removing solid capacity of cleaning step is 3.3log10 among no affinity chromatography.
Comparing embodiment 2 and comparative example 2, it may be seen that affine intermediate cleaning step can effectively improve the Scavenging activity of MVM.
Embodiment 3
MabSelect SuRe LX, chromatographic column Vantage 11/250 (Millipore), diameter 1.1cm, pillar height 20cm,
Column volume 19.0ml operates flow velocity 220cm/h.Sample source is the monoclonal antibody (Mab1) of expressing cho cell.With the 0.2mol/ of 3CV
The sodium hydroxide solution of L rinses chromatographic column, then is balanced with the equilibrium liquid (50mM Tris-HCl+150mM NaCl, pH7.4) of 5CV
Chromatographic column；Cell culture harvest liquid (HCCF) after clarification is loaded to chromatographic column, loading carrying capacity is 50g/L；With the balance of 3CV
Liquid cleans column bed, with the intermediate cleaning buffer solution (50mM NaAc-HAc+1M NaCl, pH5.0, conductivity 91.3mS/cm) of 3CV
Column scrubber bed, then column bed is cleaned with 3CV equilibrium liquid；It is eluted, is received with the eluent (50mM NaAc-HAc, pH3.5) of 3.5CV again
Collect eluent；Chromatographic column finally is rinsed with the sodium hydroxide solution of the 0.2mol/L of 3CV again, balances chromatography with the equilibrium liquid of 3CV
After column, chromatographic column finally is saved with the preservation liquid of 3CV.The HCP content of eluent is 810ppm, and DNA residual quantity is 78pg/mg.
Embodiment 4
MabSelect SuRe LX, chromatographic column Vantage 11/250 (Millipore), diameter 1.1cm, pillar height 20cm,
Column volume 19.0ml operates flow velocity 220cm/h.Sample source is the monoclonal antibody (Mab1) of expressing cho cell.With the 0.2mol/ of 3CV
The sodium hydroxide solution of L rinses chromatographic column, then is balanced with the equilibrium liquid (50mM Tris-HCl+150mM NaCl, pH7.4) of 5CV
Chromatographic column；Cell culture harvest liquid (HCCF) after clarification is loaded to chromatographic column, loading carrying capacity is 50g/L；With the balance of 3CV
Liquid cleans column bed, is washed with the intermediate cleaning buffer solution (1M NaAc-HAc+NaCl, pH6.0, conductivity 85.1mS/cm) of 3CV
Column bed, then column bed is cleaned with 3CV equilibrium liquid；It is eluted again with the eluent (50mM NaAc-HAc, pH3.5) of 3.5CV, collection is washed
De- liquid；Chromatographic column finally is rinsed with the sodium hydroxide solution of the 0.2mol/L of 3CV again, after balancing chromatographic column with the equilibrium liquid of 3CV,
Finally chromatographic column is saved with the preservation liquid of 3CV.The HCP content of eluent is 970ppm, and DNA residual quantity is 90pg/mg.
Embodiment 5
MabSelect SuRe LX, chromatographic column Vantage 11/250 (Millipore), diameter 1.1cm, pillar height 20cm,
Column volume 19.0mL operates flow velocity 220cm/h.Sample source is the monoclonal antibody (number Mab1) of expressing cho cell.With 3CV's
The sodium hydroxide solution of 0.2mol/L rinses chromatographic column, then with the equilibrium liquid of 5CV (50mM Tris-HCl+150mM NaCl,
PH7.4 chromatographic column) is balanced；Cell culture harvest liquid (HCCF) after clarification is loaded to chromatographic column, loading carrying capacity is 50g/L；
Column bed is cleaned with the equilibrium liquid of 3CV, with intermediate cleaning buffer solution (50mM NaAc-HAc+0.2M NaCl, pH5.5, the electricity of 3CV
Conductance 20mS/cm) column scrubber bed, then column bed is cleaned with 3CV equilibrium liquid；Again with the eluent of 3.5CV (50mM NaAc-HAc,
PH3.5 it) elutes, collects eluent；Chromatographic column finally is rinsed with the sodium hydroxide solution of the 0.2mol/L of 3CV again, with putting down for 3CV
It weighs after liquid balance chromatographic column, finally saves chromatographic column with the preservation liquid of 3CV.The HCP content of eluent is 990ppm, DNA residual
Amount is 98pg/mg.
Embodiment 6
MabSelect SuRe LX, chromatographic column Vantage 11/250 (Millipore), diameter 1.1cm, pillar height 20cm,
Column volume 19.0mL operates flow velocity 220cm/h.Sample source is the monoclonal antibody (number Mab1) of expressing cho cell.With 3CV's
The sodium hydroxide solution of 0.2mol/L rinses chromatographic column, then with the equilibrium liquid of 5CV (50mM Tris-HCl+150mM NaCl,
PH7.4 chromatographic column) is balanced；Cell culture harvest liquid (HCCF) after clarification is loaded to chromatographic column, loading carrying capacity is 50g/L；
Column bed is cleaned with the equilibrium liquid of 3CV, with intermediate cleaning buffer solution (50mM NaAc-HAc+1.2M NaCl, pH5.5, the electricity of 3CV
Conductance 100mS/cm) column scrubber bed, then column bed is cleaned with 3CV equilibrium liquid；Again with the eluent of 3.5CV (50mM NaAc-HAc,
PH3.5 it) elutes, collects eluent；Chromatographic column finally is rinsed with the sodium hydroxide solution of the 0.2mol/L of 3CV again, with putting down for 3CV
It weighs after liquid balance chromatographic column, finally saves chromatographic column with the preservation liquid of 3CV.The HCP content of eluent is 890ppm, DNA residual
Amount is 90pg/mg.
Embodiment 7
Praesto Jetted A50, chromatographic column Vantage 11/250 (Millipore), diameter 1.1cm, pillar height
20cm, column volume 19.0ml operate flow velocity 300cm/h.Sample source is the monoclonal antibody (Mab1) of expressing cho cell.With 3CV's
The sodium hydroxide solution of 0.2mol/L rinses chromatographic column, then with the equilibrium liquid of 5CV (50mM Tris-HCl+150mM NaCl,
PH7.4 chromatographic column) is balanced；Cell culture harvest liquid (HCCF) after clarification is loaded to chromatographic column, loading carrying capacity is 60g/L；
Column bed is cleaned with the equilibrium liquid of 3CV, with the intermediate cleaning buffer solution of 3CV (1M NaAc-HAc+NaCl, pH5.5, conductivity
88.5mS/cm) column scrubber bed, then column bed is cleaned with 3CV equilibrium liquid；Again with the eluent of 3.5CV (50mM NaAc-HAc,
PH3.5 it) elutes, collects eluent；Chromatographic column finally is rinsed with the sodium hydroxide solution of the 0.2mol/L of 3CV again, with putting down for 3CV
It weighs after liquid balance chromatographic column, finally saves chromatographic column with the preservation liquid of 3CV.The HCP content of eluent is 980ppm, DNA residual
Amount is 97pg/mg.
Embodiment 8
Use GE's25 tomographic system of Pure, using Praesto Jetted A50 filler, chromatographic column
Vantage 11/250 (Millipore), diameter 1.1cm, pillar height 6.5cm, column volume 6.2ml operate flow velocity 200cm/h.Sample
Product source is the monoclonal antibody (Mab1) of expressing cho cell, and target protein concentration is 3.5g/L, and albumen feed liquid uses the side of ice bag cooling
Formula is docked with tomographic system.Chromatographic column, then the equilibrium liquid (50mM with 3CV are rinsed with the sodium hydroxide solution of the 0.5mol/L of 3CV
Tris-HCl+150mM NaCl, pH7.4) balance chromatographic column；Cell culture harvest liquid (HCCF) after clarification is loaded to chromatography
Column, loading carrying capacity are 50g/L；Column bed is cleaned with the equilibrium liquid of 3CV, with intermediate cleaning buffer solution (the 50mM NaAc-HAc+ of 3CV
1M NaCl, pH5.5) column scrubber bed, then column bed is cleaned with 3CV equilibrium liquid；Again with the eluent of 3.5CV (50mM NaAc-HAc,
PH3.5 it) elutes, collects eluent.It is flat with the equilibrium liquid of 2CV finally with the acetum of the 1mol/L of 3CV to column regeneration
It weighs after chromatographic column, is recycled into next affinity chromatography；It is carried out continuously 10 circulations.When all affinity chromatographys after circulation terminates,
Chromatographic column finally is rinsed with the sodium hydroxide solution of the 0.5mol/L of 3CV again, after balancing chromatographic column with the equilibrium liquid of 3CV, finally
Chromatographic column is saved with the preservation liquid of 3CV.After tested, 77 minutes each Cyclical Theory used times, 90 minutes practical used times (tomographic system
Pump impulse is washed, and extra time can be consumed), 15 hour processing target albumen 3.1g, yield are as follows: 33.3g mAb/L resin/hr
(every liter of filler can handle 33.3g monoclonal antibody per hour).
And if each circulation needs 215 minutes time-consuming, yield are as follows: 9.8g using traditional handicraft (process such as comparative example 3)
MAb/L resin/hr (every liter of filler can handle 9.8g monoclonal antibody per hour).And the packing volume meeting that traditional handicraft needs
Much increase.Single-column continuous flow chromatography is with the obvious advantage.
Comparative example 3
Use GE's25 tomographic system of Pure, using MabSelect Sure filler, chromatographic column Vantage
11/250 (Millipore), diameter 1.1cm, pillar height 20.0cm, column volume 19.0ml operate flow velocity 200cm/h.Sample source
For the monoclonal antibody (Mab1) of expressing cho cell, target protein concentration is 3.5g/L, by the way of albumen feed liquid is cooled down using ice bag and layer
Analysis system docking.Chromatographic column, then equilibrium liquid (the 50mM Tris- with 3CV are rinsed with the sodium hydroxide solution of the 0.5mol/L of 3CV
HCl+150mM NaCl, pH7.4) balance chromatographic column；Cell culture harvest liquid (HCCF) after clarification is loaded to chromatographic column, on
Sample carrying capacity is 35g/L；Column bed is cleaned with the equilibrium liquid of 3CV, with intermediate cleaning buffer solution (the 50mM NaAc-HAc+1M of 3CV
NaCl, pH5.5) column scrubber bed, then column bed is cleaned with 3CV equilibrium liquid；Again with the eluent of 3.5CV (50mM NaAc-HAc,
PH3.5 it) elutes, collects eluent.It is flat with the equilibrium liquid of 2CV finally with the acetum of the 1mol/L of 3CV to column regeneration
It weighs after chromatographic column, is recycled into next affinity chromatography；It is carried out continuously 4 circulations.When all affinity chromatographys after circulation terminates, most
Chromatographic column is rinsed with the sodium hydroxide solution of the 0.5mol/L of 3CV again afterwards, after balancing chromatographic column with the equilibrium liquid of 3CV, is finally used
The preservation liquid of 3CV saves chromatographic column.After tested, 201 minutes each Cyclical Theory used times, 215 minutes practical used times (tomographic system
Pump impulse is washed, and extra time can be consumed), 14.3 hour processing target albumen 2.66g, yield are as follows: 9.8g mAb/L resin/hr
(every liter of filler can handle 9.8g monoclonal antibody per hour).
Embodiment 9
Technique amplification is carried out to embodiment 8, using the disposable layer analysis system of PALL, using Praesto Jetted A50
Filler, chromatographic column BPG140/50 (GE), diameter 14cm, pillar height 10cm, column volume 1.5L operate flow velocity 300cm/h.Sample comes
Source is the monoclonal antibody (Mab1) of expressing cho cell, and target protein concentration is 3.5g/L, and albumen feed liquid uses sterile docking mode and layer
Analysis system connection.Chromatographic column, then equilibrium liquid (the 50mM Tris- with 3CV are rinsed with the sodium hydroxide solution of the 0.5mol/L of 3CV
HCl+150mM NaCl, pH7.4) balance chromatographic column；Cell culture harvest liquid (HCCF) after clarification is loaded to chromatographic column, on
Sample carrying capacity is 60g/L；Column bed is cleaned with the equilibrium liquid of 3CV, with intermediate cleaning buffer solution (the 50mM NaAc-HAc+1M of 3CV
NaCl, pH5.5) column scrubber bed, then column bed is cleaned with 3CV equilibrium liquid；Again with the eluent of 3.5CV (50mM NaAc-HAc,
PH3.5 it) elutes, collects eluent.It is flat with the equilibrium liquid of 2CV finally with the acetum of the 1mol/L of 3CV to column regeneration
It weighs after chromatographic column, is recycled into next affinity chromatography；It is carried out continuously 10 circulations.When all affinity chromatographys after circulation terminates,
Chromatographic column finally is rinsed with the sodium hydroxide solution of the 0.5mol/L of 3CV again, after balancing chromatographic column with the equilibrium liquid of 3CV, finally
Chromatographic column is saved with the preservation liquid of 3CV.After tested, 77 minutes each Cyclical Theory used times, 77 minutes practical used times (tomographic system
No pump impulse is washed), 13 hour processing target protein 90 0g, yield are as follows: 46.1g mAb/L resin/hr (every liter of filler, per small
When can handle 46.1g monoclonal antibody).The bioreactor of 500L with regard to achievable affinity capture, and only needs filling out for 1.5L in 24 hours
Material.And if each circulation needs time-consuming 215 minutes using traditional handicraft (i.e. comparative example 4), amount of filler is 6.2L (BPG200/
50), it is also desirable to 24 hours or more.Single-column continuous flow chromatography is with the obvious advantage.
Embodiment 10
Use GE's25 tomographic system of Pure, using Amsphere A3 filler, chromatographic column Vantage 11/
250 (Millipore), diameter 1.1cm, pillar height 10cm, column volume 9.5ml operate flow velocity 300cm/h.Sample source is that CHO is thin
The monoclonal antibody (Mab2) of cellular expression, target protein concentration be 3.5g/L, albumen feed liquid using ice bag cool down by the way of and tomographic system
Docking.Chromatographic column, then equilibrium liquid (the 50mM Tris-HCl+ with 3CV are rinsed with the sodium hydroxide solution of the 0.2mol/L of 3CV
150mM NaCl, pH7.4) balance chromatographic column；Cell culture harvest liquid (HCCF) after clarification is loaded to chromatographic column, loading carries
Amount is 50g/L；Clean column bed with the equilibrium liquid of 3CV, with the intermediate cleaning buffer solution of 3CV (50mM NaAc-HAc+1M NaCl,
PH5.5) column scrubber bed, then column bed is cleaned with 3CV equilibrium liquid；It is washed again with the eluent (50mM NaAc-HAc, pH3.5) of 3.5CV
It is de-, collect eluent.Chromatographic column finally is balanced with the equilibrium liquid of 2CV to column regeneration with the acetum of the 1mol/L of 3CV
Afterwards, it is recycled into next affinity chromatography；It is carried out continuously 10 circulations.When all affinity chromatographys after circulation terminates, finally use again
The sodium hydroxide solution of the 0.2mol/L of 3CV rinses chromatographic column, after balancing chromatographic column with the equilibrium liquid of 3CV, finally uses the guarantor of 3CV
Liquid storage saves chromatographic column.After tested, 77 minutes each Cyclical Theory used times, 90 minutes practical used times, (tomographic system pump impulse was washed, meeting
Consume extra time), 15 hour processing target albumen 4.75g, yield are as follows: 33.3gmAb/L resin/hr (every liter of filler,
It can handle 33.3g monoclonal antibody per hour).
And if each circulation needs 215 minutes time-consuming, yield are as follows: 9.8g using traditional handicraft (see comparative example 4)
MAb/L resin/hr (every liter of filler can handle 9.8g monoclonal antibody per hour).And the packing volume meeting that traditional handicraft needs
Much increase.Single-column continuous flow chromatography is with the obvious advantage.
Comparative example 4
Use GE's25 tomographic system of Pure, using Eshmuno A filler, chromatographic column Vantage 11/250
(Millipore), diameter 1.1cm, pillar height 20.0cm, column volume 19.0ml operate flow velocity 200cm/h.Sample source is that CHO is thin
The monoclonal antibody (Mab1) of cellular expression, target protein concentration be 3.5g/L, albumen feed liquid using ice bag cool down by the way of and tomographic system
Docking.Chromatographic column, then equilibrium liquid (the 50mM Tris-HCl+ with 3CV are rinsed with the sodium hydroxide solution of the 0.5mol/L of 3CV
150mM NaCl, pH7.4) balance chromatographic column；Cell culture harvest liquid (HCCF) after clarification is loaded to chromatographic column, loading carries
Amount is 35g/L；Clean column bed with the equilibrium liquid of 3CV, with the intermediate cleaning buffer solution of 3CV (50mM NaAc-HAc+1M NaCl,
PH5.5) column scrubber bed, then column bed is cleaned with 3CV equilibrium liquid；It is washed again with the eluent (50mM NaAc-HAc, pH3.5) of 3.5CV
It is de-, collect eluent.Chromatographic column finally is balanced with the equilibrium liquid of 2CV to column regeneration with the acetum of the 1mol/L of 3CV
Afterwards, it is recycled into next affinity chromatography；It is carried out continuously 4 circulations.When all affinity chromatographys after circulation terminates, finally use again
The sodium hydroxide solution of the 0.5mol/L of 3CV rinses chromatographic column, after balancing chromatographic column with the equilibrium liquid of 3CV, finally uses the guarantor of 3CV
Liquid storage saves chromatographic column.After tested, 201 minutes each Cyclical Theory used times, 215 minutes practical used times (tomographic system pump impulse is washed,
Extra time can be consumed), 14.3 hour processing target albumen 2.66g, yield are as follows: (every liter is filled out 9.8g mAb/L resin/hr
Material, can handle 9.8g monoclonal antibody per hour).
Embodiment 11
Technique amplification is carried out to embodiment 10, using the disposable layer analysis system of PALL, using Amsphere A3 filler,
Chromatographic column BPG140/50 (GE), diameter 14cm, pillar height 10cm, column volume 1.5L operate flow velocity 300cm/h.Sample source is
The monoclonal antibody (Mab2) of expressing cho cell, target protein concentration are 3.5g/L, and albumen feed liquid is using sterile docking mode and chromatography
System connection.Chromatographic column, then equilibrium liquid (the 50mM Tris-HCl+ with 3CV are rinsed with the sodium hydroxide solution of the 0.5mol/L of 3CV
150mM NaCl, pH7.4) balance chromatographic column；Cell culture harvest liquid (HCCF) after clarification is loaded to chromatographic column, loading carries
Amount is 50g/L；Clean column bed with the equilibrium liquid of 3CV, with the intermediate cleaning buffer solution of 3CV (50mM NaAc-HAc+1M NaCl,
PH5.5) column scrubber bed, then column bed is cleaned with 3CV equilibrium liquid；It is washed again with the eluent (50mM NaAc-HAc, pH3.5) of 3.5CV
It is de-, collect eluent.Chromatographic column finally is balanced with the equilibrium liquid of 2CV to column regeneration with the acetum of the 1mol/L of 3CV
Afterwards, it is recycled into next affinity chromatography；It is carried out continuously 10 circulations.When all affinity chromatographys after circulation terminates, finally use again
The sodium hydroxide solution of the 0.5mol/L of 3CV rinses chromatographic column, after balancing chromatographic column with the equilibrium liquid of 3CV, finally uses the guarantor of 3CV
Liquid storage saves chromatographic column.After tested, 77 minutes each Cyclical Theory used times, 77 minutes practical used times, (tomographic system was without pump impulse
Wash), 13 hour processing target protein 75 0g, yield are as follows: (every liter of filler, per hour can be with by 38.4g mAb/L resin/hr
Handle 38.4g monoclonal antibody).The bioreactor of 500L with regard to achievable affinity capture, and only needs the filler of 1.5L in 30 hours.And
If each circulation needs time-consuming 215 minutes using traditional handicraft (see comparative example 4), amount of filler is 6.2L (BPG200/50),
It is also required to 29 hours.Single-column continuous flow chromatography is with the obvious advantage.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (10)
1. a kind of purification process of antibody, which is characterized in that including protein A affinity chromatography step；
The method of the protein A affinity chromatography are as follows: after loading and with before elution target antibody, first use centre clear
Wash buffer column scrubber bed；
The intermediate cleaning buffer solution are as follows: pH value 5-6, conductivity are the buffer of 20-100mS/cm.
2. purification process according to claim 1, which is characterized in that the intermediate cleaning buffer solution is acetate salt buffer body
System, phosphate buffer or citrate buffer system.
3. purification process according to claim 1, which is characterized in that the intermediate cleaning buffer solution is to adjust electricity with villaumite
The buffer of conductance, the preferred sodium chloride of villaumite.
4. purification process according to claim 1, which is characterized in that the target antibody is Chinese hamster ovary cell table
The antibody reached.
5. purification process according to claim 1, which is characterized in that the filler of the protein A affinity chromatography is
MabSelect Sure, MabSelect LX, Amsphere A3 or Praesto Jetted A50.
6. purification process according to claim 1, which is characterized in that the pH value of the intermediate cleaning buffer solution is 5.5-6,
It is preferred that 5.5-5.8.
7. purification process according to claim 1 or 6, which is characterized in that conductivity 50-100mS/cm, preferably 50-
80mS/cm。
8. purification process according to claim 1-6, which is characterized in that the process of the protein A affinity chromatography
Successively are as follows: disinfection, equilibrium liquid washing, loading, equilibrium liquid washing, the intermediate cleaning buffer solution washing, equilibrium liquid wash, are described
Elution target antibody；
Preferably, the equilibrium liquid be Tris-HCl+150~160mM NaCl, pH7.2~7.6 buffer, preferably 50~
55mMTris-HCl；
Preferably, when the target antibody is the monoclonal antibody of expressing cho cell, the eluent is NaAc-HAc, pH3.0~3.8
Buffer, preferably 50~55m MNaAc-HAc.
9. purification process according to claim 1-6, which is characterized in that after the protein A affinity chromatography
Further include that anion-exchange chromatography is carried out to the eluent of collection:
Successively disinfection, equilibrium liquid washing, loading, equilibrium liquid washing；
Preferably, the equilibrium liquid when anion-exchange chromatography is the buffer of Tris-HCl, pH7.2~8.0.
10. purification process according to claim 9, which is characterized in that the filler of the anion-exchange chromatography is POROS
50HQ, Q Sepharose FF, Capto Q or Eshmuno Q.
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