CN108101987A - A kind of purification process for recombinating the full human monoclonal antibody of anti-tnf-alpha - Google Patents
A kind of purification process for recombinating the full human monoclonal antibody of anti-tnf-alpha Download PDFInfo
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- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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Abstract
The invention discloses a kind of purification process for recombinating the anti-full human monoclonal antibodies of TNF α, and including carrying out cation-exchange chromatography elution to antibody, process specifically includes：It after balancing loading, is eluted for the first time using level pad 1 first, then using 2 second elution of level pad, is then eluted using the mixed liquor of level pad 2 and level pad 3 third time, finally carry out elution and obtain antibody purification；Wherein, level pad 1 is phosphate buffer or acetate buffer, and level pad 2 is phosphate buffer or acetate buffer, and level pad 3 is phosphate buffer or acetate buffer.Present invention reduces the acidic components content recombinated in the anti-full human monoclonal antibodies of TNF α, and improve the purity for recombinating the anti-full human monoclonal antibodies of TNF α.
Description
Technical field
The present invention relates to antibody purification technical field more particularly to a kind of recombinate anti-tnf-alpha full human monoclonal antibody
Purification process.
Background technology
TNF-α is a kind of proinflammatory cytokine, is played an important role in autoimmune disease pathogenesis, rheumatoid
Arthritis (RA), juvenile idiopathic arthritis (JIA), psoriatic arthritis (PsA) and ankylosing spondylitis (AS) patient articular
Find that TNF-α level increases in synovia, psoriasis in plaques (PPs) patient's body it has also been found that TNF-α level increases, it is all this
All prove that TNF-α take part in many lysises a bit, into the important target of disease treatment, exploitation special target antibody drug into
One research hotspot, having obtained the TNF-α targeting antibodies of U.S. FDA approval listing at present has the adalimumab in full people source
(Adalimumab), new goli mumab (golimumab), Pegylation antibody fragment plug trastuzumab
(Certolizumab) etc..
The restructuring full human monoclonal antibody of anti-tnf-alpha is the IgG1 immunoglobulin like protein in full people source, complete by integration anti-tnf-alpha
CHO (Chinese hamster ovary cell) cultures of human monoclonal antibody gene obtain.Since IgG1 antibody-likes can be specifically
It combines, therefore can be captured by affinity chromatography with protein A.But during cell expression, have several factors meeting
The formation of charge alterations body is influenced, such as：The amputation of heavy chain C-terminal lysine, the cyclisation of N-terminal pyroglutamic acid, asparagine, paddy ammonia
The deamination of amide residues and different glycosylation modified, oxidations etc..Charge alterations body, particularly acidic charge variation
Know from experience the immunogenicity for causing human body, have become one of Key Quality attribute in monoclonal antibody drug technique.Due to these
The electrically charged branch in charge alterations body surface face is inhomogenous, can be removed by ion chromatography.But due to different acidic variants
There is similar physicochemical property, single elution mode is not notable to variant removal effect, it is difficult to meet normal production requirement.
The content of the invention
Technical problems based on background technology, the present invention propose a kind of restructuring full people's resource monoclonal of anti-tnf-alpha and resist
The purification process of body present invention reduces the acidic components content in the restructuring full human monoclonal antibody of anti-tnf-alpha, and improves
The purity of restructuring anti-tnf-alpha full human monoclonal antibody.
A kind of purification process for recombinating the full human monoclonal antibody of anti-tnf-alpha proposed by the present invention, will incorporate first
The Chinese hamster ovary celI of TNF-α Cloning Human Immunoglobulin Genes is expression system culture cell liquid, and cell liquid then is removed cell through in-depth filtration
Device and cell fragment, finally the cell liquid after filtering is splined on after Protein A affinity chromatographys the sun after balance from
In sub- chromatographic column；Wherein, the filler of the chromatographic column is sulfonic group (- SO3H) cation-exchange chromatography medium, loaded cells liquid
The concentration of the middle restructuring full human monoclonal antibody of anti-tnf-alpha is 6.88mg/mL, and the actual carrying capacity of filler is 15mg/mL；Balance loading
Afterwards, eluted for the first time using level pad 1 first, it is then slow using balance then using 2 second elution of level pad
The mixed liquor of fliud flushing 2 and level pad 3 third time elutes, and finally carries out elution and obtains antibody purification；Wherein, level pad
1 is phosphate buffer or acetate buffer, and level pad 2 is phosphate buffer or acetate buffer, and balance is slow
Fliud flushing 3 is phosphate buffer or acetate buffer.
Preferably, in eluting for the first time, the concentration of phosphate anion or acetate ion is 20- in level pad 1
40mmol/L。
Preferably, in eluting for the first time, the pH value of level pad 1 is 4.8-5.2.
Preferably, in eluting for the first time, sodium chloride is also contained in level pad 1.
Preferably, the concentration of the sodium chloride is 30-70mmol/L.
Preferably, in second of elution, the concentration of phosphate anion or acetate ion is 30- in level pad 2
70mmol/L。
Preferably, in second of elution, the pH value of level pad 2 is 5.8-6.6.
Preferably, during third time elutes, the concentration of phosphate anion or acetate ion is 30- in level pad 2
70mmol/L。
Preferably, during third time elutes, the pH value of level pad 2 is 5.8-6.6.
Preferably, during third time elutes, the concentration of phosphate anion or acetate ion is 30- in level pad 3
70mmol/L。
Preferably, during third time elutes, the pH value of level pad 3 is 6.8-7.6.
Preferably, during third time elutes, the volume ratio of level pad 2 and level pad 3 is 3-7：2-3.
Preferably, the elution volume of elution is no less than 3 column volumes for the first time.
Preferably, the elution volume of second of elution is no less than 3 column volumes.
Preferably, third time elution is eluted by the way of isocratic elution.
Preferably, during third time elution is using eluting by the way of isocratic elution, elution volume is 5-9 column volume.
Preferably, elution is carried out using level pad 3 and obtains antibody purification.
Preferably, during being eluted using level pad 3, phosphate anion or vinegar in level pad 3
The concentration of acid ion is 30-70mmol/L.
Preferably, during being eluted using level pad 3, the pH value of level pad 3 is 6.8-7.6.
Preferably, the starting point of elution is after being eluted using the mixed liquor of level pad 2 and level pad 3.
Preferably, the terminal of elution reaches 10-40mAU/m for UV detector detected value.
The present invention is first eluted for the first time during elution using level pad 1, then using level pad 2
Secondary elution is finally eluted using the mixed liquor of level pad 2 and level pad 3 third time, after carrying out third time elution
Elution is carried out again and collects antibody-solutions, significantly reduces the acidic components content in the restructuring full human monoclonal antibody of anti-tnf-alpha,
And improve the purity of the restructuring full human monoclonal antibody of anti-tnf-alpha.
Description of the drawings
Fig. 1 is that the UV of the embodiment of the present invention 7 monitors collection of illustrative plates.
Fig. 2 is that the UV of test example 1 of the present invention monitors collection of illustrative plates.
Specific embodiment
In the following, technical scheme is described in detail by specific embodiment.
Embodiment 1
A kind of purification process for recombinating the full human monoclonal antibody of anti-tnf-alpha, including carrying out cation exchange color to antibody
Spectrum elution, process specifically include：After balancing loading, eluted for the first time using level pad 1 first, it is then slow using balance
2 second elution of fliud flushing, is then eluted using the mixed liquor of level pad 2 and level pad 3 third time, is finally washed
It is de- to obtain antibody purification；Wherein, level pad 1 is phosphate buffer, and level pad 2 is phosphate buffer, is balanced
Buffer solution 3 is acetate buffer.
Embodiment 2
A kind of purification process for recombinating the full human monoclonal antibody of anti-tnf-alpha, including carrying out cation exchange color to antibody
Spectrum elution, process specifically include：After balancing loading, eluted for the first time using level pad 1 first, it is then slow using balance
2 second elution of fliud flushing, is then eluted using the mixed liquor of level pad 2 and level pad 3 third time, is finally washed
It is de- to obtain antibody purification；Wherein, level pad 1 is acetate buffer, and level pad 2 is acetate buffer, is balanced
Buffer solution 3 is acetate buffer；
Wherein, in eluting for the first time, the concentration of acetate ion is 20mmol/L in level pad 1；
In eluting for the first time, the pH value of level pad 1 is 4.8；
In eluting for the first time, also contain sodium chloride in level pad 1；
The concentration of the sodium chloride is 30mmol/L；
During second elutes, the concentration of acetate ion is 30mmol/L in level pad 2；
During second elutes, the pH value of level pad 2 is 5.8；
Third time elution is eluted by the way of isocratic elution；
During third time elution is using eluting by the way of isocratic elution, elution volume is 5 column volumes；
During third time elutes, the concentration of acetate ion is 30mmol/L in level pad 2；
During third time elutes, the pH value of level pad 2 is 5.8；
During third time elutes, the concentration of acetate ion is 30mmol/L in level pad 3；
During third time elutes, the pH value of level pad 3 is 6.8；
During third time elutes, the volume ratio of level pad 2 and level pad 3 is 3：2；
The elution volume of elution and second of elution is 3 column volumes for the first time；
Using level pad 3 carry out elution obtain antibody purification, wherein, the acetate ion of level pad 3 it is dense
It spends for 30mmol/L, pH value 6.8；
The starting point of elution is after being eluted using the mixed liquor of level pad 2 and level pad 3；
The terminal of elution reaches 10mAU/m for UV detector detected value.
Embodiment 3
A kind of purification process for recombinating the full human monoclonal antibody of anti-tnf-alpha, including carrying out cation exchange color to antibody
Spectrum elution, process specifically include：After balancing loading, eluted for the first time using level pad 1 first, it is then slow using balance
2 second elution of fliud flushing, is then eluted using the mixed liquor of level pad 2 and level pad 3 third time, is finally washed
It is de- to obtain antibody purification；Wherein, level pad 1 is acetate buffer, and level pad 2 is acetate buffer, is balanced
Buffer solution 3 is acetate buffer；
Wherein, in eluting for the first time, the concentration of acetate ion is 30mmol/L in level pad 1；
In eluting for the first time, the pH value of level pad 1 is 5.0；
In eluting for the first time, also contain sodium chloride in level pad 1；
The concentration of the sodium chloride is 50mmol/L；
During second elutes, the concentration of acetate ion is 50mmol/L in level pad 2；
During second elutes, the pH value of level pad 2 is 6.2；
Third time elution is eluted by the way of isocratic elution；
During third time elution is using eluting by the way of isocratic elution, elution volume is 5 column volumes；
During third time elutes, the concentration of acetate ion is 50mmol/L in level pad 2；
During third time elutes, the pH value of level pad 2 is 6.2；
During third time elutes, the concentration of acetate ion is 50mmol/L in level pad 3；
During third time elutes, the pH value of level pad 3 is 7.2；
During third time elutes, the volume ratio of level pad 2 and level pad 3 is 13：7；
The elution volume of elution and second of elution is 3 column volumes for the first time；
Using level pad 3 carry out elution obtain antibody purification, wherein, the acetate ion of level pad 3 it is dense
It spends for 50mmol/L, pH value 7.2；
The starting point of elution is after being eluted using the mixed liquor of level pad 2 and level pad 3；
The terminal of elution reaches 25mAU/m for UV detector detected value.
Embodiment 4
A kind of purification process for recombinating the full human monoclonal antibody of anti-tnf-alpha, including carrying out cation exchange color to antibody
Spectrum elution, process specifically include：After balancing loading, eluted for the first time using level pad 1 first, it is then slow using balance
2 second elution of fliud flushing, is then eluted using the mixed liquor of level pad 2 and level pad 3 third time, is finally washed
It is de- to obtain antibody purification；Wherein, level pad 1 is acetate buffer, and level pad 2 is acetate buffer, is balanced
Buffer solution 3 is acetate buffer；
Wherein, in eluting for the first time, the concentration of acetate ion is 40mmol/L in level pad 1；
In eluting for the first time, the pH value of level pad 1 is 5.2；
In eluting for the first time, also contain sodium chloride in level pad 1；
The concentration of the sodium chloride is 70mmol/L；
During second elutes, the concentration of acetate ion is 70mmol/L in level pad 2；
During second elutes, the pH value of level pad 2 is 6.6；
Third time elution is eluted by the way of isocratic elution；
During third time elution is using eluting by the way of isocratic elution, elution volume is 5 column volumes；
During third time elutes, the concentration of acetate ion is 70mmol/L in level pad 2；
During third time elutes, the pH value of level pad 2 is 6.6；
During third time elutes, the concentration of acetate ion is 70mmol/L in level pad 3；
During third time elutes, the pH value of level pad 3 is 7.6；
During third time elutes, the volume ratio of level pad 2 and level pad 3 is 7：3；
The elution volume of elution and second of elution is 3 column volumes for the first time；
Using level pad 3 carry out elution obtain antibody purification, wherein, the acetate ion of level pad 3 it is dense
It spends for 70mmol/L, pH value 7.6；
The starting point of elution is after being eluted using the mixed liquor of level pad 2 and level pad 3；
The terminal of elution reaches 40mAU/m for UV detector detected value.
Embodiment 5
A kind of purification process for recombinating the full human monoclonal antibody of anti-tnf-alpha, including carrying out cation exchange color to antibody
Spectrum elution, process specifically include：After balancing loading, eluted for the first time using level pad 1 first, it is then slow using balance
2 second elution of fliud flushing, is then eluted using the mixed liquor of level pad 2 and level pad 3 third time, is finally washed
It is de- to obtain antibody purification；Wherein, level pad 1 is phosphate buffer, and level pad 2 is phosphate buffer, is balanced
Buffer solution 3 is phosphate buffer；
Wherein, in eluting for the first time, the concentration of phosphate anion is 20mmol/L in level pad 1；
In eluting for the first time, the pH value of level pad 1 is 5.0；
In eluting for the first time, also contain sodium chloride in level pad 1；
The concentration of the sodium chloride is 50mmol/L；
During second elutes, the concentration of phosphate anion is 50mmol/L in level pad 2；
During second elutes, the pH value of level pad 2 is 6.2；
Third time elution is eluted by the way of isocratic elution；
During third time elution is using eluting by the way of isocratic elution, elution volume is 5 column volumes；
During third time elutes, the concentration of phosphate anion is 50mmol/L in level pad 2；
During third time elutes, the pH value of level pad 2 is 6.2；
During third time elutes, the concentration of phosphate anion is 50mmol/L in level pad 3；
During third time elutes, the pH value of level pad 3 is 7.6；
During third time elutes, the volume ratio of level pad 2 and level pad 3 is 13：7；
The elution volume of elution and second of elution is 3 column volumes for the first time；
Elution is carried out using level pad 3 and obtains antibody purification, wherein, the concentration of 3 phosphate anion of level pad
For 50mmol/L, pH value 7.6；
The starting point of elution is after being eluted using the mixed liquor of level pad 2 and level pad 3；
The terminal of elution reaches 25mAU/m for UV detector detected value.
Embodiment 6
A kind of purification process for recombinating the full human monoclonal antibody of anti-tnf-alpha, including carrying out cation exchange color to antibody
Spectrum elution, process specifically include：After balancing loading, eluted for the first time using level pad 1 first, it is then slow using balance
2 second elution of fliud flushing, is then eluted using the mixed liquor of level pad 2 and level pad 3 third time, is finally washed
It is de- to obtain antibody purification；Wherein, level pad 1 is phosphate buffer, and level pad 2 is phosphate buffer, is balanced
Buffer solution 3 is phosphate buffer；
Wherein, in eluting for the first time, the concentration of phosphate anion is 20mmol/L in level pad 1；
In eluting for the first time, the pH value of level pad 1 is 5.0；
In eluting for the first time, also contain sodium chloride in level pad 1；
The concentration of the sodium chloride is 50mmol/L；
During second elutes, the concentration of phosphate anion is 50mmol/L in level pad 2；
During second elutes, the pH value of level pad 2 is 6.2；
Third time elution is eluted by the way of isocratic elution；
During third time elution is using eluting by the way of isocratic elution, elution volume is 7 column volumes；
During third time elutes, the concentration of phosphate anion is 50mmol/L in level pad 2；
During third time elutes, the pH value of level pad 2 is 6.2；
During third time elutes, the concentration of phosphate anion is 50mmol/L in level pad 3；
During third time elutes, the pH value of level pad 3 is 7.6；
During third time elutes, the volume ratio of level pad 2 and level pad 3 is 13：7；
The elution volume of elution and second of elution is 3 column volumes for the first time；
Elution is carried out using level pad 3 and obtains antibody purification, wherein, the concentration of 3 phosphate anion of level pad
For 50mmol/L, pH value 7.6；
The starting point of elution is after being eluted using the mixed liquor of level pad 2 and level pad 3；
The terminal of elution reaches 25mAU/m for UV detector detected value.
Embodiment 7
A kind of purification process for recombinating the full human monoclonal antibody of anti-tnf-alpha, including carrying out cation exchange color to antibody
Spectrum elution, process specifically include：After balancing loading, eluted for the first time using level pad 1 first, it is then slow using balance
2 second elution of fliud flushing, is then eluted using the mixed liquor of level pad 2 and level pad 3 third time, is finally washed
It is de- to obtain antibody purification；Wherein, level pad 1 is phosphate buffer, and level pad 2 is phosphate buffer, is balanced
Buffer solution 3 is phosphate buffer；
Wherein, in eluting for the first time, the concentration of phosphate anion is 20mmol/L in level pad 1；
In eluting for the first time, the pH value of level pad 1 is 5.0；
In eluting for the first time, also contain sodium chloride in level pad 1；
The concentration of the sodium chloride is 50mmol/L；
During second elutes, the concentration of phosphate anion is 50mmol/L in level pad 2；
During second elutes, the pH value of level pad 2 is 6.2；
Third time elution is eluted by the way of isocratic elution；
During third time elution is using eluting by the way of isocratic elution, elution volume is 9 column volumes；
During third time elutes, the concentration of phosphate anion is 50mmol/L in level pad 2；
During third time elutes, the pH value of level pad 2 is 6.2；
During third time elutes, the concentration of phosphate anion is 50mmol/L in level pad 3；
During third time elutes, the pH value of level pad 3 is 7.6；
During third time elutes, the volume ratio of level pad 2 and level pad 3 is 13：7；
The elution volume of elution and second of elution is 3 column volumes for the first time；
Elution is carried out using level pad 3 and obtains antibody purification, wherein, the concentration of 3 phosphate anion of level pad
For 50mmol/L, pH value 7.6；
The starting point of elution is after being eluted using the mixed liquor of level pad 2 and level pad 3；
The terminal of elution reaches 25mAU/m for UV detector detected value；Wherein, the UV monitoring collection of illustrative plates obtained is with reference to Fig. 1.
Test example 1
In cation-exchange chromatography elution is carried out to antibody, after balancing loading, first eluted using level pad 1, mistake
Column to the ultraviolet recurrence baseline of efflux walks to put down and elution volume is 3 column volumes, is then obtained using the directly elution of level pad 3
Obtain antibody purification；Wherein, level pad 1 and level pad 3 are acetate buffer；
Wherein, the concentration of acetate ion is 20mmol/L in the level pad 1；
The pH value of the level pad 1 is 4.8；
Also contain sodium chloride in the level pad 1；
The concentration of the sodium chloride is 30mmol/L；
The volume that the level pad 1 elutes is 3 column volumes；
The concentration of acetate ion is 50mmol/L in the level pad 3；
The pH value of the level pad 3 is 7.6；
The starting point of elution reaches 10mAU/mm for UV detector detected value；
The terminal of elution reaches 10mAU/m for UV detector detected value；Wherein, the UV monitoring collection of illustrative plates obtained is with reference to Fig. 2.
IEX-UPLC detections are carried out respectively to embodiment 2-7 and test example 1, acid is calculated respectively by areas of peak normalization method
Peak, alkali peak, main peak area ratio, testing result are as shown in the table：
It compares embodiment 2,3,4 in upper table and understands that three step rinsing steps can compared with common step elution with test example 1
With the content for more effectively removing acid heteroplasmon and improving destination protein.
The column volume that embodiment 5,6,7 understands suitably to increase when third time elutes in comparison upper table can effectively remove acid
Property variant and increase destination protein purity.
Embodiment 7 is most preferred condition as seen from the above table, and destination protein purity can be made to improve 21.11%.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art in the technical scope disclosed by the present invention, technique according to the invention scheme and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (10)
1. it is a kind of recombinate the full human monoclonal antibody of anti-tnf-alpha purification process, which is characterized in that including to antibody carry out sun from
Sub- exchange chromatography elution, process specifically include：After balancing loading, eluted using level pad 1, then adopted for the first time first
With 2 second elution of level pad, then eluted using the mixed liquor of level pad 2 and level pad 3 third time, most
After carry out elution obtain antibody purification；Wherein, level pad 1 be phosphate buffer or acetate buffer, equalizing and buffering
Liquid 2 is phosphate buffer or acetate buffer, and level pad 3 is phosphate buffer or acetate buffer.
2. the purification process of the full human monoclonal antibody of anti-tnf-alpha is recombinated according to claim 1, which is characterized in that first
In secondary elution, the concentration of phosphate anion or acetate ion is 20-40mmol/L in level pad 1；Preferably, for the first time
In elution, the pH value of level pad 1 is 4.8-5.2.
3. the purification process of the restructuring full human monoclonal antibody of anti-tnf-alpha according to claim 1 or claim 2, which is characterized in that the
In once eluting, also contain sodium chloride in level pad 1；Preferably, the concentration of the sodium chloride is 30-70mmol/L.
4. according to the purification process of any one of the claim 1-3 restructuring full human monoclonal antibodies of anti-tnf-alpha, feature exists
During second elutes, the concentration of phosphate anion or acetate ion is 30-70mmol/L in level pad 2；It is preferred that
Ground, during second elutes, the pH value of level pad 2 is 5.8-6.6.
5. according to the purification process of any one of the claim 1-4 restructuring full human monoclonal antibodies of anti-tnf-alpha, feature exists
During third time elutes, the concentration of phosphate anion or acetate ion is 30-70mmol/L in level pad 2；It is preferred that
Ground, during third time elutes, the pH value of level pad 2 is 5.8-6.6；Preferably, during third time elutes, in level pad 3
The concentration of phosphate anion or acetate ion is 30-70mmol/L；Preferably, during third time elutes, the pH of level pad 3
It is worth for 6.8-7.6.
6. according to the purification process of any one of the claim 1-5 restructuring full human monoclonal antibodies of anti-tnf-alpha, feature exists
During third time elutes, the volume ratio of level pad 2 and level pad 3 is 3-7：2-3.
7. according to the purification process of any one of the claim 1-6 restructuring full human monoclonal antibodies of anti-tnf-alpha, feature exists
In the elution volume of elution is no less than 3 column volumes for the first time；Preferably, the elution volume of second of elution is no less than 3 columns
Volume.
8. according to the purification process of any one of the claim 1-7 restructuring full human monoclonal antibodies of anti-tnf-alpha, feature exists
In third time elution is eluted by the way of isocratic elution；Preferably, third time elution is eluted by the way of isocratic elution
In, elution volume is 5-9 column volume.
9. according to the purification process of any one of the claim 1-8 restructuring full human monoclonal antibodies of anti-tnf-alpha, feature exists
In, using level pad 3 carry out elution obtain antibody purification；Preferably, in the process eluted using level pad 3
In, the concentration of phosphate anion or acetate ion is 30-70mmol/L in level pad 3；Preferably, slow using balance
During fliud flushing 3 is eluted, the pH value of level pad 3 is 6.8-7.6.
10. according to the purification process of any one of the claim 1-9 restructuring full human monoclonal antibodies of anti-tnf-alpha, feature
It is, the starting point of elution is after being eluted using the mixed liquor of level pad 2 and level pad 3；Preferably, elution
Terminal reaches 10-40mAU/m for UV detector detected value.
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| CN114113450A (en) * | 2020-08-31 | 2022-03-01 | 华兰基因工程有限公司 | Novel charge heteroplasmon rapid separation method based on isocratic elution |
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