CN108017689A - A kind of centrifuge tube and the method that antibody protein is obtained using the centrifuge tube - Google Patents
A kind of centrifuge tube and the method that antibody protein is obtained using the centrifuge tube Download PDFInfo
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- CN108017689A CN108017689A CN201610962807.2A CN201610962807A CN108017689A CN 108017689 A CN108017689 A CN 108017689A CN 201610962807 A CN201610962807 A CN 201610962807A CN 108017689 A CN108017689 A CN 108017689A
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 82
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 81
- 238000000034 method Methods 0.000 title claims abstract description 26
- 239000012528 membrane Substances 0.000 claims abstract description 73
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 claims abstract description 46
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 34
- 239000004695 Polyether sulfone Substances 0.000 claims abstract description 27
- 229920006393 polyether sulfone Polymers 0.000 claims abstract description 27
- 239000000463 material Substances 0.000 claims abstract description 21
- 239000004033 plastic Substances 0.000 claims abstract description 20
- 239000004627 regenerated cellulose Substances 0.000 claims abstract description 12
- 230000010148 water-pollination Effects 0.000 claims abstract description 11
- 239000007788 liquid Substances 0.000 claims description 26
- 239000000470 constituent Substances 0.000 claims description 14
- 238000005119 centrifugation Methods 0.000 claims description 12
- 239000012930 cell culture fluid Substances 0.000 claims description 10
- 238000001042 affinity chromatography Methods 0.000 claims description 9
- 210000000988 bone and bone Anatomy 0.000 claims description 8
- 238000001179 sorption measurement Methods 0.000 claims description 8
- 241000826860 Trapezium Species 0.000 claims description 7
- 230000014759 maintenance of location Effects 0.000 claims description 7
- NJPPVKZQTLUDBO-UHFFFAOYSA-N novaluron Chemical compound C1=C(Cl)C(OC(F)(F)C(OC(F)(F)F)F)=CC=C1NC(=O)NC(=O)C1=C(F)C=CC=C1F NJPPVKZQTLUDBO-UHFFFAOYSA-N 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 6
- 230000000087 stabilizing effect Effects 0.000 claims description 6
- 230000006641 stabilisation Effects 0.000 claims description 2
- 238000011105 stabilization Methods 0.000 claims description 2
- 239000000835 fiber Substances 0.000 claims 2
- 239000001913 cellulose Substances 0.000 abstract description 10
- 229920002678 cellulose Polymers 0.000 abstract description 10
- 238000010276 construction Methods 0.000 abstract description 4
- 239000000243 solution Substances 0.000 description 17
- 238000006073 displacement reaction Methods 0.000 description 9
- 238000011160 research Methods 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 238000013461 design Methods 0.000 description 6
- 229940125644 antibody drug Drugs 0.000 description 5
- 238000004587 chromatography analysis Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000003480 eluent Substances 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 239000003513 alkali Substances 0.000 description 4
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 4
- 239000000945 filler Substances 0.000 description 4
- 229920000570 polyether Polymers 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 150000003457 sulfones Chemical class 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 239000012531 culture fluid Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000012510 hollow fiber Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 101100230376 Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) celI gene Proteins 0.000 description 2
- 241000699802 Cricetulus griseus Species 0.000 description 2
- 229960000074 biopharmaceutical Drugs 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- -1 Ether sulfone Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000012501 chromatography medium Substances 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940126534 drug product Drugs 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 230000028023 exocytosis Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Water Supply & Treatment (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
A kind of method that the present invention is provided centrifuge tube and antibody protein is obtained using the centrifuge tube, wherein centrifuge tube are the built-in pipe embedded with a two film construction in the plastic centrifuge tube；The lower section of the built-in pipe sets affine adsorbed film, and ultrafiltration concentration mwco membrane is also set up below the affine adsorbed film；The plastic centrifuge tube is additionally provided with lid, and the spiral screwing structure with being set at plastic centrifuge tube upper opening cooperates, to make lid and spiral screwing structure are spiral mutually to tighten.Using the above scheme, it is that normal operating does not have risk more than 30 times in this film Reusability to stabilize affinity membrane of the regenerated cellulose element as skeleton, can so ensure security, the reusability of film.Ultrafiltration mwco membrane is using cellulose and its derivates polyether sulfone as skeleton, and membrane material is polyether sulfone, this film has the characteristics that the anti-corruption of the strong anti-pollution of hydrophily is wear-resistant, future can industrial amplificationization, safely, meet GMP needs.
Description
Technical field
The invention belongs to life science and field of biological pharmacy technical field, more particularly to a kind of centrifuge tube and
The method that antibody protein is obtained using the centrifuge tube.
Background technology
It is exactly antibody drug that life science and field of biological pharmacy are most promising at present, the sales volume of antibody drug
More than the 50% of drug product is accounted for, from the point of view of occupation rate is researched and developed, China just there are at least 500 being engaged in antibody drug with medicine-feeding enterprise
Research and development, wherein in antibody drug R＆D process experiment basis almost indispensable be exactly cultivate 293 cells (HEK293,293E,
293F) or Chinese hamster ovary celI (Chinese hamster ovary cell, Chinese hamster ovary cell) expresses antibody.And purify
A small amount of antibody methods general at present are exactly with chromatography media affinity chromatography acquisition albumen first, then by albumen hollow fiber column
Replace in the stabilizer that finally preserve antibody to us, this method not only needs two different experiments to complete, but also recycles effect
Rate is low, inconvenient, easily pollution.It is badly in need of that antibody protein but also displacement liquid and concentrated antibody egg can be captured currently on the market
White convenient and efficient product
Therefore, the prior art is defective, it is necessary to improve.
The content of the invention
The technical problems to be solved by the invention are in view of the deficiencies of the prior art, there is provided a kind of centrifuge tube and using should be from
The method that heart pipe obtains antibody protein.
The antibody protein liquid of preservation can be stablized and have concentration by being obtained the present invention seeks to quickness and high efficiency from cell culture fluid
Antibody protein acts on, and has reached capture, displacement liquid, the triple role of concentrated antibody albumen, can so substantially reduce enterprise's time
Cost, while process risk is also reduced, it is not easy to pollute, it is easy to amplify, there is good economy.
Technical scheme is as follows：
A kind of centrifuge tube, including a plastic centrifuge tube, wherein, embedded with two film construction in the plastic centrifuge tube
Built-in pipe；The lower section of the built-in pipe sets affine adsorbed film, and ultrafiltration concentration is also set up below the affine adsorbed film and is cut
Stay film；The plastic centrifuge tube is additionally provided with lid, with the spiral screwing structure phase interworking set at plastic centrifuge tube upper opening
Close, to make lid and spiral screwing structure are spiral mutually to tighten.
The centrifuge tube, wherein, the concrete structure of the affine adsorbed film is circular membrane, diameter 30mm, thickness 3mm,
Protein A/G aglucons on film.
The centrifuge tube, wherein, the constituent of the affine adsorbed film includes：A is plain to stabilize regenerated cellulose
For skeleton；B non-specific adsorptions are extremely low；Existing traditional affinity chromatography Protein A/G aglucons are linked on membrane bone frame by C.
The centrifuge tube, wherein, the concrete structure that mwco membrane is concentrated by ultrafiltration is open trapezium structure film, polyethers
Sulfone material, film thickness 1.5mm-2.5mm.
The centrifuge tube, wherein, the ultrafiltration concentration mwco membrane constituent includes：A is with cellulose and its derivates
Polyether sulfone is skeleton；Film pedestal of the membrane aperture of B non-specificity 30KD sizes as retention antibody protein；C membrane materials are hydrophilic
The property wear-resistant polyether sulfone of the strong anti-corruption of anti-pollution.
A kind of method that centrifuge tube described above obtains antibody protein, wherein, comprise the following steps：
Step 1：Cell culture fluid first passes through centrifugation, on affine adsorbed film plus Deproteinated solution elution is washed, after centrifugation
Antibody protein, which is eluted out to lower floor, is concentrated by ultrafiltration mwco membrane；
Step 2：This protein eluate is replaced with stablizing albumen and preserving liquid 3-5 time, final antibody protein will be stored in surely
Determine in liquid.
The method, wherein, the concrete structure of the affine adsorbed film is circular membrane, diameter 30mm, thickness 3mm, film
Upper Protein A/G aglucons.
The method, wherein, the constituent of the affine adsorbed film includes：A using stabilize regenerated cellulose element as
Skeleton；B non-specific adsorptions are extremely low；Existing traditional affinity chromatography Protein A/G aglucons are linked on membrane bone frame by C.
The method, wherein, the concrete structure that mwco membrane is concentrated by ultrafiltration is open trapezium structure film, polyether sulfone
Material, film thickness 1.5mm-2.5mm.
The method, wherein, the ultrafiltration concentration mwco membrane constituent includes：A is gathered with cellulose and its derivates
Ether sulfone is skeleton；Film pedestal of the membrane aperture of B non-specificity 30KD sizes as retention antibody protein；C membrane materials are hydrophily
The wear-resistant polyether sulfone of the anti-corruption of strong anti-pollution.
Using the above scheme, structural advantages of the invention：1. using the design of centrifuge tube, do not change scientific research and enterprise uses
Custom, does not increase extra equipment investment for enterprise.2. built-in pipe 2 has the space of 10-20ml cell culture fluids, so set
Meter can meet the needs of most antibody research laboratories, operate very simple, and be easy to amplification technique.3. affine absorption
Film 3, be to stabilize affinity membrane of the regenerated cellulose element as skeleton, can so ensure security, the reusability of film,
Normal operating does not have risk more than 30 times in this film Reusability.4. ultrafiltration mwco membrane 4, is with cellulose and its derivates polyethers
Sulfone is skeleton, and membrane material is polyether sulfone, this film has the characteristics that the anti-corruption of the strong anti-pollution of hydrophily is wear-resistant, and future industrial can amplify
Change, safety, meets GMP needs.
And the beneficial effects of the present invention are：It is of the invention to break through traditional two steps even three step experimental methods, by affinity membrane and surpass
Filter concentration film is incorporated into the 50ml centrifuge tubes of a portable, realizes capture antibody protein and energy displacement liquid and concentration are anti-
The triple role of body protein, general cell culture fluid volume is between 10-20ml, and nutrient solution turbidity is under 10NTU, it is possible to
Operated with this convenient and efficient centrifuge tube, obtain the antibody protein of storage-stable.
Brief description of the drawings
Fig. 1 is the structure diagram of the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.
Embodiment 1
As shown in Figure 1, affine adsorbed film 3, its concrete structure is circular membrane, diameter 30mm, thickness 3mm, Protein on film
A/G aglucons, can capture antibody protein：The constituent of specific affine adsorbed film, including：1) it is plain to stabilize regenerated cellulose
For skeleton；2) non-specific adsorption is extremely low；3) existing traditional affinity chromatography Protein A/G aglucons are linked on membrane bone frame.
Mwco membrane 4 is concentrated by ultrafiltration：Constituent：1) using cellulose and its derivates polyether sulfone as skeleton；2) it is non-specific
Film pedestal of the membrane aperture of 30KD sizes as retention antibody protein；3) membrane material is the wear-resistant polyethers of the anti-corruption of the strong anti-pollution of hydrophily
Sulfone；The concrete structure of mwco membrane is concentrated by ultrafiltration：Open trapezium structure film, polyether sulfone material, alkali resistance is fabulous, can tolerate pH13
Alkalies, film thickness about 2mm.
The constituent of centrifuge tube is polyacrylic plastic centrifuge tube.
One example is the built-in pipe 2 embedded with a two film construction in polyacrylic 50ml plastic centrifuge tubes 6, built-in
Affine adsorbed film 3, the concrete structure of affine adsorbed film 3 are set in pipe 2：Circular membrane, diameter 30mm, thickness 3mm, on film
Protein A/G aglucons, can capture antibody protein；Ultrafiltration concentration is also set up in built-in pipe 2 in the lower section of affine adsorbed film 3 to cut
Film 4 is stayed, the concrete structure of mwco membrane 4 is concentrated by ultrafiltration：Open trapezium structure film, polyether sulfone material, alkali resistance is fabulous, can tolerate
The alkalies of pH13, film thickness 1.5mm-2.5mm；Best film thickness is 2mm；50ml plastic centrifuge tubes 6 are additionally provided with lid 1
Spiral screwing structure 5 with being set at 6 upper opening of 50ml plastic centrifuge tubes cooperates, to make lid 1 tighten knot with spiral
Structure 5 is spiral mutually to be tightened, in order to avoid leak during centrifugal liquid.
Using the present invention centrifuge tube carry out when operation principle be：Cultivate 293 cells (HEK293,293E, 293F)
Or Chinese hamster ovary celI (Chinese hamster ovary cell, Chinese hamster ovary cell) to express antibody when it is nearly all anti-
The nutrient solution that can all there be below 30ml in body laboratory needs purifying protein, and the culture supernatant after filtering, is directly placed into and designs
Chromatography change in liquid pipe, after centrifugation, discard bottom of the tube nutrient solution, then displacement required for adding on affine adsorbed film you is certain
The eluent of amount and the solution for stablizing albumen, centrifugation, can also replace more several times general 3-5 times it is complete to liquid is changed, reach final
Stablize the effect for preserving antibody protein.The present invention incorporates the traditional chromatography of bio-pharmaceutical industry and changes the technology of liquid, forms symbol
The system of GMP pharmaceutical standards is closed, safe and efficient efficiently to obtain the antibody protein liquid that stablize preservation, its time is approximately traditional handicraft
1/4, economic direct cost is saved about more than twice, while improves product economy and security.
The present invention technological process be：Cell culture fluid first passes through centrifugation, on affine adsorbed film plus washes Deproteinated molten
Liquid elutes, and antibody protein is eluted out to lower floor's mwco membrane after centrifugation, then replaces this egg with stablizing albumen and preserving liquid 3-5 times
White eluent, final antibody protein will be stored in stabilizing solution, have been reached affinity chromatography and have been obtained antibody protein and displacement stabilization
The triple role of liquid and protein concentrate, operation very easily and avoid cross contamination, and are easy to amplify, can greatly save the time,
Financial cost, plays the role of important.
Affine adsorbed film 3：To stabilize regenerated cellulose element as skeleton, and with affine combination functional group.Although in Fig. 1
Affine 3 shape of adsorbed film seems ordinary filter, but actually affine adsorbed film 3 has an effect of absorption antibody protein and not general
By filter, antibody protein can be combined so far chromatography protein A/G films, and affine adsorbed film 3 can be widely used in anti-
The capture of body.Compared with conventional filler column chromatography, affine 3 adsorption technology of adsorbed film has following many advantages：Easily operated place
Reason (without loading, plug and play)；High flow rate (improves 3-5 times) than conventional filler column chromatography；Non-specific adsorption is low；Hardware
Investment is less；Buffer solution consumption is less；It is easy to amplify.
Ultrafiltration mwco membrane 4：Using cellulose and its derivates polyether sulfone as skeleton, membrane material is anti-rotten anti-for the strong anti-pollution of hydrophily
The polyether sulfone of mill, film can be generally divided into a variety of grades from 35um to 3K by aperture point, and aperture is smaller, and separation rejection effect is got over
Clarification, but antibody protein generally uses 30K films more.The antibody protein of more high-recovery in order to obtain, general replacement protein are stablized
Liquid can be all changed during liquid repeatedly and reaches optimum protein storage condition 3-5 times.
It is commonly used in the prior art obtain antibody protein and substitutional solution two steps be respectively：The first step, affine packing layer
Analysis method, chromatography substantially step be cell culture fluid by the combination with filler in chromatographic column, then with elution, resisted
Body protein.Second step, with hollow fiber column, can stablize preservation to another by the albumen displacement in upper step albumen again eluent and resist
In the solution of body protein, this walks time generally all more than half an hour under laboratory scale, is also needed when in addition most after this step
Protein concentration is improved, the time to again when young when.In practice, scientific research enterprise obtains antibody protein and needs to replace to steady at once
The experimental implementation determined in liquid is very frequent, and consumptive material, time cost are all very high.
The structural advantages of the present invention：1. using the design of centrifuge tube, do not change scientific research and enterprise's use habit, be not enterprise
Increase extra equipment investment.2. built-in pipe 2 has the space of 10-20ml cell culture fluids, so design can meet big absolutely
The demand of most antibody research laboratories, operates very simple, and is easy to amplification technique.3. affine adsorbed film 3, is to stabilize
Regenerated cellulose element is the affinity membrane of skeleton, can so ensure security, the reusability of film, normal operating is in this film
Reusability does not have risk more than 30 times.4. ultrafiltration mwco membrane 4, is the membrane material using cellulose and its derivates polyether sulfone as skeleton
Matter is polyether sulfone, this film has the characteristics that the anti-corruption of the strong anti-pollution of hydrophily is wear-resistant, future can industrial amplificationization, safely, meet
GMP needs.The functional advantage of the present invention：Affine adsorbed film 3, ultrafiltration mwco membrane 4 in Fig. 1, each serve as capture antibody protein and
Change liquid, protein concentrate effect；Usage/technological process is as follows：Tube cover 1 is first opened, cell culture fluid is placed in built-in pipe 2, is passed through
When crossing affine adsorbed film 3, the antibody protein in cell liquid will be combined with affine adsorbed film 3, that is, destination protein can be fine
Ground is attached on affine adsorbed film 3, adds eluent centrifugation, and this type of elution is obtained with purpose antibody protein, this egg
It is typically all in vain 150KD, does not pass through the ultrafiltration mwco membrane 4 of the 30KD of lower floor, and ultrafiltration can be passed through to retain less than 30KD albumen
Film 4, but albumen is in elution solution at this time, and albumen is usually unstable or structure changes, so needing to put as early as possible
Change in stable solution, ultrafiltration mwco membrane 4 just plays the role of so easily, only needing stabilizing solution being added at this time affine
On adsorbed film 3, by centrifugation, original eluent is just displaced to new stabilizing solution and suffers, passed through at this time in plastic centrifuge tube 6
Liquid can discard, this step can pass through 3-5 time plus stabilizing solution and replace, and be required for centrifuging every time to replace, whole process
It can complete within 20 minutes, and destination protein will be trapped within the position of ultrafiltration mwco membrane 4, this design plays capture antibody egg
In vain but can displacement liquid and concentrated antibody albumen triple role, it is quick and efficient.
At present in bio-pharmaceuticals monoclonal antibody field, the development phase of antibody drug is all with 293 cells or expressing cho cell
, with the increment of cell, antibody protein can want that capture antibody protein replaces into stabilizing solution again in exocytosis, or even also
Need antibody protein to concentrate, can only be realized at present by affine filler chromatography and hollow fiber column filtration method, consumables cost, when
Between cost it is excessive.And the beneficial effects of the present invention are：The present invention breaks through traditional two steps even three step experimental methods, by affinity membrane and
Film is concentrated by ultrafiltration to be incorporated into the 50ml centrifuge tubes of a portable, realizes capture antibody protein and can displacement liquid and concentration
The triple role of antibody protein, general cell culture fluid volume is between 10-20ml, and nutrient solution turbidity is under 10NTU, so that it may
To be operated with this convenient and efficient centrifuge tube, the antibody protein of storage-stable is obtained.
Embodiment 2
On the basis of above-described embodiment, the present invention, which provides one kind, can stablize preservation antibody protein centrifuge tube, including one
Plastic centrifuge tube, wherein, the built-in pipe embedded with a two film construction in the plastic centrifuge tube；The lower section of the built-in pipe is set
Affine adsorbed film, ultrafiltration concentration mwco membrane is also set up below the affine adsorbed film；The plastic centrifuge tube is additionally provided with
Lid, the spiral screwing structure with being set at plastic centrifuge tube upper opening cooperates, to make lid and spiral screwing structure mutual
It is mutually spiral to tighten.The concrete structure of the affine adsorbed film is circular membrane, diameter 30mm, thickness 3mm, Protein A/G on film
Aglucon.The constituent of the affine adsorbed film includes：A is to stabilize regenerated cellulose element as skeleton；B non-specific adsorptions pole
It is low；Existing traditional affinity chromatography Protein A/G aglucons are linked on membrane bone frame by C.It is described that the specific of mwco membrane is concentrated by ultrafiltration
Structure is open trapezium structure film, polyether sulfone material, film thickness 1.5mm-2.5mm.The ultrafiltration concentration mwco membrane composition
Component includes：A is using cellulose and its derivates polyether sulfone as skeleton；The membrane aperture of B non-specificity 30KD sizes is anti-as retention
The film pedestal of body protein；C membrane materials are the wear-resistant polyether sulfone of the anti-corruption of the strong anti-pollution of hydrophily.
On the basis of the above, it can stablize to preserve antibody protein centrifuge tube and obtain the present invention also provides a kind of use and resist
The method of body protein, wherein, comprise the following steps：
Step 1：Cell culture fluid first passes through centrifugation, on affine adsorbed film plus Deproteinated solution elution is washed, after centrifugation
Antibody protein, which is eluted out to lower floor, is concentrated by ultrafiltration mwco membrane；
Step 2：This protein eluate is replaced with stablizing albumen and preserving liquid 3-5 time, final antibody protein will be stored in surely
Determine in liquid.
In above-mentioned, the concrete structure of the affine adsorbed film is circular membrane, diameter 30mm, thickness 3mm, Protein on film
A/G aglucons.The constituent of the affine adsorbed film includes：A is to stabilize regenerated cellulose element as skeleton；B is non-specific to be inhaled
It is attached extremely low；Existing traditional affinity chromatography Protein A/G aglucons are linked on membrane bone frame by C.
In above-mentioned, the concrete structure that mwco membrane is concentrated by ultrafiltration is open trapezium structure film, polyether sulfone material, thickness
Spend for 1.5mm-2.5mm.The ultrafiltration concentration mwco membrane constituent includes：A is using cellulose and its derivates polyether sulfone as bone
Frame；Film pedestal of the membrane aperture of B non-specificity 30KD sizes as retention antibody protein；C membrane materials resist for the strong anti-pollution of hydrophily
Rotten wear-resistant polyether sulfone.
Using the above scheme, structural advantages of the invention：1. using the design of centrifuge tube, do not change scientific research and enterprise uses
Custom, does not increase extra equipment investment for enterprise.2. built-in pipe 2 has the space of 10-20ml cell culture fluids, so set
Meter can meet the needs of most antibody research laboratories, operate very simple, and be easy to amplification technique.3. affine absorption
Film 3, be to stabilize affinity membrane of the regenerated cellulose element as skeleton, can so ensure security, the reusability of film,
Normal operating does not have risk more than 30 times in this film Reusability.4. ultrafiltration mwco membrane 4, is with cellulose and its derivates polyethers
Sulfone is skeleton, and membrane material is polyether sulfone, this film has the characteristics that the anti-corruption of the strong anti-pollution of hydrophily is wear-resistant, and future industrial can amplify
Change, safety, meets GMP needs.
And the beneficial effects of the present invention are：It is of the invention to break through traditional two steps even three step experimental methods, by affinity membrane and surpass
Filter concentration film is incorporated into the 50ml centrifuge tubes of a portable, realizes capture antibody protein and energy displacement liquid and concentration are anti-
The triple role of body protein, general cell culture fluid volume is between 10-20ml, and nutrient solution turbidity is under 10NTU, it is possible to
Operated with this convenient and efficient centrifuge tube, obtain the antibody protein of storage-stable..
It should be appreciated that for those of ordinary skills, can according to the above description be improved or converted,
And all these modifications and variations should all belong to the protection domain of appended claims of the present invention.
Claims (10)
1. a kind of centrifuge tube, including a plastic centrifuge tube, it is characterised in that embedded with a double film knot in the plastic centrifuge tube
The built-in pipe of structure；The lower section of the built-in pipe sets affine adsorbed film, and it is dense to also set up ultrafiltration below the affine adsorbed film
Contracting mwco membrane；The plastic centrifuge tube is additionally provided with lid, with the spiral screwing structure phase set at plastic centrifuge tube upper opening
Mutually coordinate, to make lid and spiral screwing structure are spiral mutually to tighten.
2. centrifuge tube as claimed in claim 1, it is characterised in that the concrete structure of the affine adsorbed film is circular membrane, directly
Footpath 30mm, thickness 3mm, Protein A/G aglucons on film.
3. centrifuge tube as claimed in claim 2, it is characterised in that the constituent of the affine adsorbed film includes：A is with stabilization
It is skeleton to change regenerated cellulose element；B non-specific adsorptions are extremely low；Existing tradition affinity chromatography Protein A/G aglucons are crosslinked by C
Onto membrane bone frame.
4. centrifuge tube as claimed in claim 3, it is characterised in that the concrete structure that mwco membrane is concentrated by ultrafiltration is open
Trapezium structure film, polyether sulfone material, film thickness 1.5mm-2.5mm.
5. centrifuge tube as claimed in claim 4, it is characterised in that the ultrafiltration concentration mwco membrane constituent includes：A is with fibre
Dimension element and its derivative polyether sulfone are skeleton；Film pedestal of the membrane aperture of B non-specificity 30KD sizes as retention antibody protein；
C membrane materials are the wear-resistant polyether sulfone of the anti-corruption of the strong anti-pollution of hydrophily.
6. a kind of method that centrifuge tube as claimed in claim 1 obtains antibody protein, it is characterised in that comprise the following steps：
Step 1：Cell culture fluid first passes through centrifugation, adds on affine adsorbed film and washes Deproteinated solution elution, antibody after centrifugation
Albumen, which is eluted out to lower floor, is concentrated by ultrafiltration mwco membrane；
Step 2：This protein eluate is replaced with stablizing albumen and preserving liquid 3-5 time, final antibody protein will be stored in stabilizing solution
In.
7. method as claimed in claim 6, it is characterised in that the concrete structure of the affine adsorbed film is circular membrane, diameter
30mm, thickness 3mm, Protein A/G aglucons on film.
8. the method for claim 7, it is characterised in that the constituent of the affine adsorbed film includes：A is to stabilize
Regenerated cellulose element is skeleton；B non-specific adsorptions are extremely low；Existing traditional affinity chromatography Protein A/G aglucons are linked to by C
On membrane bone frame.
9. method as claimed in claim 8, it is characterised in that the concrete structure that mwco membrane is concentrated by ultrafiltration is open ladder
Shape structural membrane, polyether sulfone material, film thickness 1.5mm-2.5mm.
10. method as claimed in claim 9, it is characterised in that the ultrafiltration concentration mwco membrane constituent includes：A is with fibre
Dimension element and its derivative polyether sulfone are skeleton；Film pedestal of the membrane aperture of B non-specificity 30KD sizes as retention antibody protein；
C membrane materials are the wear-resistant polyether sulfone of the anti-corruption of the strong anti-pollution of hydrophily.
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Application publication date: 20180511 |