CN102179237A - Affinity chromatographic packing for separating and purifying monoclonal antibody and antibodyglobulin and preparation method thereof - Google Patents
Affinity chromatographic packing for separating and purifying monoclonal antibody and antibodyglobulin and preparation method thereof Download PDFInfo
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- CN102179237A CN102179237A CN 201010155469 CN201010155469A CN102179237A CN 102179237 A CN102179237 A CN 102179237A CN 201010155469 CN201010155469 CN 201010155469 CN 201010155469 A CN201010155469 A CN 201010155469A CN 102179237 A CN102179237 A CN 102179237A
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Abstract
The invention discloses an affinity chromatographic packing for separating and purifying monoclonal antibody and antibodyglobulin and a preparation method thereof. The affinity chromatographic packing consists of a monodisperse porous microsphere solid phase carrier and a protein A ligand. The preparation method comprises: bonding the protein A onto the surface of the solid phase carrier through amino acids containing a nitrogen group; and thus obtaining the affinity chromatographic packing. Concretely, the method comprises three steps, namely connecting an activated spatial arm, coupling the ligand and sealing residual active groups. Meanwhile the invention also discloses an affinity chromatographic column made of the affinity chromatographic packing. The affinity chromatographic packing and the affinity chromatographic column made of the affinity chromatographic packing can be used in the production process of monoclonal antibody medicines and the separation and purification of antibodyglobulin (IgG), and have high separation and purification efficiency.
Description
Technical field
The present invention relates to bio-separation purifying field, particularly, relate to a kind ofly be used to separate, the affinity chromatograph filling of purification monoclonal antibody and antibody globulin and preparation method thereof.
Background technology
Monoclonal antibody (Monoclonal antibodies) is a class biomolecule, and its molecular structure is unique Y shape, the polysaccharide structures that contains complexity that has, and molecular weight is about 150～160kD.Monoclonal antibody can be by hybridoma or gene engineering research preparation.Antibody globulin (IgG) is present in human and animal's the body fluid, and has the important physical function.Strictly say, monoclonal antibody belongs to a kind of special antibody globulin, recent two decades comes, and the monoclonal antibody drug development is swift and violent, is widely used in the illnesss such as rejection that produce in the treatment carcinoma of the rectum, breast cancer, lymph cancer, congenital rheumatism and organ transplant.
The main method of separation, purification monoclonal antibody and antibody globulin is a liquid chromatography at present.Obtained extensive use in the preparation that is combined in monoclonal antibody and antibody globulin laboratory scale and commercial production scale of liquid-phase chromatography methods such as affine, ion-exchange and gel filtration.
Affinity chromatography is a kind of in the liquid chromatogram various modes, is used for the isolation and purification of large biological molecule more, and its basic principle is the single-minded high-affinity of height that is had between large biological molecule and part.The advantage of affinity chromatography is the selectivity of its height.In affinity chromatography, at first aglucon must be connected on the insoluble solid phase carrier (also claiming matrix) with covalent bond, obtain having the affinity chromatograph filling of immobilization aglucon, this affinity chromatograph filling is filled to affinity chromatographic column.If the mixture that will contain target protein is added on the post, then to have only and can be retained with the protein that the immobilization aglucon shows obvious affinity interaction, other component that can not discern the immobilization aglucon then flows through the chromatographic column bed in the clear.Then, the composition that changes solvent just can dissociate target protein and elute from the immobilization aglucon.
Compare with other method for protein isolation, affinity chromatography has many outstanding advantages: affinity chromatography almost can be applicable to any two kinds of separation that special interactional biomolecule is arranged; Affinity chromatography is the highest chromatogram mode of selectivity, and under many occasions, affinity chromatography is the highest operation of purifying multiple, can significantly reduce the step and the time of separation and purification process; Its operating condition gentleness helps the structure and the activity of stable protein, improves the activity of proteins rate of recovery; Be particularly suited for the protein composition of low concentration in the separate complex living things system.
Albumin A (Protein A) is the protein on a kind of Staphylococcus of being present in aureue cell membrane, last century the seventies [J. that is separated
, J.Movitg, I.B, Johansson, H.Hjelm, Eur.J.Biochem.30 (1972) 190], and be found the single-minded interaction [J.J.Langone, Adv.Immunol.32 (1982) 157] that has with antibody globulin.Albumin A discharges antibody globulin under the condition of pH about 3 usually, the part conformation change may take place in the antibody globulin that is released, but in most cases, this conformation change does not influence its biologically active and function [S.Lofdahl, B.Guss, M.Uhlen, L.Philipson, M.Lindberg, Proc.Natl.Acad.Sci.USA 80 (1983) 697].Owing to have highly selectivity effect between albumin A and monoclonal antibody and the antibody globulin, one of present separation, purification monoclonal antibody and the most important means of antibody globulin have been become as the affinity chromatography of affinity chromatography aglucon with albumin A.At present, the albumin A affinity chromatography has become the first step of seriation chromatographic isolation, purifying flow process on all monoclonal antibody drug industrial-scale production lines.Over nearly more than 20 years, replaced the albumin A that from S.aureus, extracts with the albumin A of genetic engineering law technology production.
From solid phase carrier, early stage albumin A affinity chromatograph filling is a solid phase carrier with the polysaccharide flexible glue, such material good hydrophilic property, and nonspecific interaction is low, but that shortcoming is a quality is very soft, can't under high pressure use.Though in the recent period the polysaccharide flexible glue solid phase carrier withstand voltage properties of producing makes moderate progress, still have gap with user's expectation.1978, people such as Ohlson [S.Ohlson, L.Hansson, P.O.Larsson and K.Mosbach; FEBSLett.1978,93,5～9] proposed the notion of high-effective affinity chromatography, replaced the flexible glue matrix of polysaccharide structures, improved the separative efficiency of affinity chromatography greatly with the efficient matrix of small particle diameter.This shows that the improvement of host material (solid phase carrier) has played crucial effects in the affinity chromatography development.After this, hard solid phase carrier such as controllable bore diameter glass, silica gel and high molecular polymer are come out one after another, these materials under high pressure performance to be better than with the flexible glue be the material of matrix, on affinity chromatography, be used widely.But the above-mentioned solid phase carrier is non-regular shape controllable bore diameter glass, non-single (particle diameter wider distribution) microballoon or single atresia microballoon etc. that disperses of disperseing, and has limited the further raising of affinity chromatography separation, purification efficiency.
Summary of the invention
The objective of the invention is at above-mentioned defective of the prior art, propose a kind ofly to be used to efficiently to separate, the affinity chromatograph filling of purification monoclonal antibody and antibody globulin and preparation method thereof.
The technical scheme that realizes above-mentioned purpose is as follows:
A kind ofly be used to separate, the affinity chromatograph filling of purification monoclonal antibody and antibody globulin, by solid phase carrier and forming with the aglucon that covalent bond is connected with it, described aglucon is an albumin A, described solid phase carrier is a porous mono-dispersion microsphere, and so-called porous mono-dispersion microsphere is meant the porous microsphere that granularity (diameter) is highly consistent.This type of porous mono-dispersion microsphere has possessed the total high-strength characteristic of all hard solid phase carriers, can use under high pressure, conditions of high flow rate.
Further, the material of described porous mono-dispersion microsphere is selected from any in high molecular polymer, silica gel, carborundum, aluminium oxide, chloride and the ceramic microsphere.
Further, the diameter of described porous mono-dispersion microsphere is 1～300 μ m, and the aperture is 10～3000nm.
Further, primary alcohol functional group is contained on described porous mono-dispersion microsphere surface after treatment.
Further, described albumin A is to be obtained by genetic engineering means.
Above-mentionedly be used to separate, the preparation method of the affinity chromatograph filling of purification monoclonal antibody and antibody globulin, comprise the steps:
(1) the space arm of taking over a job: get a porous mono-dispersion microsphere that contains primary alcohol functional group after treatment, add 0.1～10 part, 0.1 the KOH of～3.0mol/L and 0.1～10 part contain epoxy-functional compounds, in 10 ℃～100 ℃ the vibration or stirring reaction 1～72h, afterproduct after filtration, the washing standby;
(2) coupling aglucon: get the product that 1 part of step (1) obtains, place the PBS buffer solution that contains 0.1～10 part of albumin A, pH 6～7, vibrate under 10 ℃～30 ℃ or stir 2～72h, and the afterreaction thing after filtration, washing is standby;
(3) sealing residual activity group: get 1 part of step (2) gained reactant, add 5～10 parts 0.05M HCl, at 10 ℃～30 ℃ vibration or stirring reaction 1～24h down, end-product after filtration, the preservation of washing back.
In addition, the invention also discloses a kind ofly be used to separate, affinity chromatographic column that the affinity chromatograph filling of purification monoclonal antibody and antibody globulin is made.
Affinity chromatograph filling of the present invention is made up of porous mono-dispersion microsphere solid phase carrier and connected albumin A aglucon, because porous mono-dispersion microsphere diameter height unanimity has guaranteed very low reverse post pressure.As the chromatographic column that the porous mono-dispersion microsphere of diameter 30 μ m is filled, it is similar that the buffer brake that it manifested and average diameter 50 μ m have the chromatographic column of non-porous mono-dispersion microsphere filling of Gaussian distribution.
Classical liquid chromatogram bands of a spectrum broadening theory thinks that the main factor that influences the bands of a spectrum broadening is the outer and interior two large divisions of chromatographic column of chromatographic column.For the effect that causes the outer bands of a spectrum broadening of chromatographic column, chromatogram circle majority think that by 6 kinds of factors be the variances sigma that sample causes
2 SThe variances sigma that sampling valve causes
2 VThe variances sigma that the tube connector of connection sampling valve and pillar and pillar and detector causes
2 tThe variances sigma that column cap causes
2 fThe variances sigma that detection cell shape and volume cause
2 dThe variances sigma that causes with electronic circuit and sensor response time
2 rThe outer bands of a spectrum broadening effect of post is to be difficult to eliminate fully, can only reduce their influence as best one can.For influencing bands of a spectrum broadening effect in the post (referring to the filled-type chromatographic column here), it is generally acknowledged that 4 kinds of principal elements are arranged, i.e. multipath effect (multipath effect); Longitudinal diffusion (longitudinal diffusion); Resistance to mass tranfer (the resistance to mass transfer in the mobile phase) in flowing mutually and fixing resistance to mass tranfer (theresistance to mass transfer in the stationary phase) in mutually.Following formula has provided quantitative description:
H
In=H
M+ H
L+ H
RM+ H
RL
H in the formula
InBe meant the contribution that the post internal effect is done the chromatographic peak height equivalent to a theoretical plate; H
MIt is the share that multipath effect is contributed height equivalent to a theoretical plate; H
LIt is the share that the longitudinal diffusion effect is contributed height equivalent to a theoretical plate; H
RMIt is the share that mobile phase resistance to mass tranfer is contributed height equivalent to a theoretical plate; H
RLIt is fixing phase resistance to mass tranfer share that height equivalent to a theoretical plate is contributed.
The liquid-phase chromatographic column that mono-dispersion microballoon is filled is to reducing H
MUseful.In chromatography column, the gap between microballoon or the particulate filler is the passage of circulation mutually that flows.The inhomogeneity of packing material size, shape and packed bed tightness degree all can make packed column produce multipath effect:
Multipath effect to the contribution of height equivalent to a theoretical plate is:
H
M＝2λd
p
λ is for characterizing the constant term of filler granularity inhomogeneity in the formula, and it is relevant with size distribution, and particle diameter distributes narrow more, and it is even more promptly to distribute, and then the λ value approaches 1 more, otherwise then the λ value is big more.d
pBe particle or microsphere diameter.Obviously, the chromatographic column of using mono-dispersion microballoon to fill compares to the chromatographic column of using size distribution particles wide or other shapes to fill, can reduce the λ value effectively.In addition, using the little microballoon of diameter or the filler of particle also is to reduce H
MA method.But particle diameter reduces to cause the packed column buffer brake to raise, and too high post pressure can cause device fabrication and operational difficulty.The chromatographic column of particularly large-scale production usefulness, the limit of bearing pressure is lower.When using particle diameter to distribute wide microballoon or particle; little microballoon or particle can embed in big microballoon or the granuloplastic gap; thereby cause higher post to press, and the diameter of mono-dispersion microballoon is very approaching, makes the gap length unanimity between the microballoon in the packed column; cause lower post to press; can inference, press under limit requires at same post, as using the mono-dispersion microballoon filler; can select the littler microballoon of particle diameter, thereby the assurance multipath effect is reduced to the contribution of plate height minimum.
Promptly compared with prior art, the present invention has following beneficial effect:
By adopting mono-dispersion microballoon to combine with the albumin A aglucon as solid phase carrier, press under the limit requirement at same post, can select littler microsphere supported of particle diameter, thereby guarantee that multipath effect reduces to minimumly to the contribution of plate height, separate to improve, purification efficiency.
Mono-dispersion microballoon of the present invention is a porous form simultaneously, the microballoon of porous form can improve the specific area of solid phase carrier, compare with single atresia microballoon that disperses, can make the binding capacity of aglucon albumin A and material to be separated the corresponding increase of post carrying capacity several times to tens times, improve the efficient of preparation and industrial-scale production greatly.
Below by drawings and Examples, technical scheme of the present invention is described in further detail.
Description of drawings
Accompanying drawing is used to provide further understanding of the present invention, and constitutes the part of specification, is used from explanation the present invention with embodiments of the invention one, is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the porous mono-dispersion microsphere sem photograph;
Fig. 2 A was the chromatogram of people's gamma Globulin of blank post;
Fig. 2 B was the chromatogram of people's gamma Globulin of affinity chromatographic column of the present invention;
Fig. 3 is the comparison diagram of different protease retention behavior on affinity chromatographic column;
Fig. 4 measures affinity chromatographic column post carrying capacity experimental result picture for the personnel selection gamma Globulin;
Fig. 5 affinity chromatography figure of serum solution on affinity chromatographic column that behave;
Fig. 6 is SDS～PAGE analysis result figure.
The specific embodiment
Below in conjunction with accompanying drawing the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein only is used for description and interpretation the present invention, and be not used in qualification the present invention.
The material of the solid phase carrier-porous mono-dispersion microsphere of of the present inventionly be used for separating, the affinity chromatograph filling of purification monoclonal antibody and antibody globulin is used can be selected from any in high molecular polymer, silica gel, carborundum, aluminium oxide, chloride and the ceramic microsphere, is that example describes with preferred high molecular polymer only below.
With the high molecular polymer polystyrene is that solid phase carrier, albumin A are the preparation of the affinity chromatograph filling and the affinity chromatographic column of aglucon:
1, (PS～DVB) porous polymer is a solid phase carrier to select an equal grain high-crosslinking-degree polystyrene.The about 30 μ m of microspherulite diameter, an aperture part is 10～130nm, and another part is 130～3000nm, and it has excellent mechanical intensity and chemical stability, and is surperficial through coating film treatment, enrichment primary alcohol functional group.This film covers all microsphere surfaces, can not washed off by any organic solvent, and guarantee and can use in pH1～14 scopes.Get this microballoon 10g, place 100mL triangle ground round-bottomed flask, in the middle of this flask mechanical agitator is housed, a side mouth is equipped with water condensing tube, and opposite side is equipped with the 50mL dropping funel.With 60mL 3mol/L KOH solution and 10mL chloropropylene oxide, room temperature stirring at low speed 2h injects dropping funel with the 20mL chloropropylene oxide, keeps slowly being added dropwise in the reaction flask under the stirring at low speed.Continue under agitation to react 16h, filter, wash with water, with acetone washing 3 times, dry under the vacuum at last.
2, the 50mg albumin A is placed the 100mL round-bottomed flask, add 10mL pH 7.0PBS buffer solution, with the solid carrier adding of oven dry, room temperature vibration 16h on shaking table.Suction filtration is also clean with distilled water.
3, the porous mono-dispersion microsphere (as shown in Figure 1) through step 2 preparation adds 20mL 0.05MHCl.Room temperature vibration 16h on shaking table, suction filtration is also clean with distilled water.Place 10mL 20% ethanol/water solution to preserve.
4, cut-off directly is 4.6mm, and length is that 50mm stainless steel chromatogram void column is cleaned standby.About 0.6g is carrier, is the affinity chromatograph filling of aglucon with the albumin A with PS～DVB, is suspended in the 20mMPBS buffer solution of pH 7, with homogenate method dress post, prepares affinity chromatographic column under 15Mpa pressure.To be equipped with blank affinity chromatographic column with legal system.
The affinity chromatograph filling of above-mentioned preparation and affinity chromatographic column can be used for separating, purification monoclonal antibody and antibody globulin, only describe with the typical example that is separated into of people's gamma Globulin below.
Embodiment 2
The separation of people's gamma Globulin:
1, the chromatogram retention behavior of people's gamma Globulin on blank post and affinity chromatographic column
Affinity chromatographic column or blank post are connected on the high performance liquid chromatograph, and sample behaviour gamma Globulin (Human γ～Globulin).Mobile phase A liquid is 20mM kaliumphosphate buffer (pH 7.5)+150mM NaCl, and Mobile phase B liquid is 0.1M glycine hydrochloride (pH 2.5).People's gamma Globulin is dissolved in mobile phase A, makes the gamma Globulin solution of 1.0mg/mL, flow velocity: 1mL/min; Gradient condition: 0～10min A liquid, 10～20min B liquid; 20～30min A liquid, sample size 20 μ L detect wavelength 254nm.Shown in Fig. 2 A, show that people's gamma Globulin does not keep on blank post, shown in Fig. 2 B, show that people's gamma Globulin keeping on the affinity chromatographic column and being eluted when being changed to B liquid mutually flowing, illustrates that the A albumen aglucon of bonding and people's gamma Globulin have single-minded effect.
2, different proteins retention behavior on affinity chromatographic column
Affinity chromatographic column is connected on the high performance liquid chromatograph, (Human γ～Globulin) distinguish sample introduction wash-out to affinity chromatographic column and under identical elution requirement observes its affinity chromatography behavior with cromoci (CytochromeC), bovine serum albumin(BSA) (Bovine Serum Albumin), ribonuclease A (Ribonuclease A), lysozyme (Lysozyme), soybean trypsin inhibitor (TrypsinInhibitor from Soybean) and people's gamma Globulin.Protein concentration is 1% (w/v), and sample size is 20 μ L, and other chromatographic conditions are with step 1.The result shows that affinity chromatographic column carries in the above-mentioned dissimilar protein as shown in Figure 3, only people's gamma Globulin has been shown specific absorption, and this is very favourable for separation and purification people gamma Globulin from the actual sample of complexity.
3, the mensuration of affinity chromatographic column post carrying capacity
Affinity chromatographic column is connected on the high performance liquid chromatograph, and (Human γ～Globulin), concentration is 5% (w/v) to sample solution behaviour gamma Globulin.Same steps as 1 flows, experiment flow is as follows: 0～5min mobile phase A 0.2mL/min, 5～70min sample solution 0.2mL/min, 70～80min A liquid 1mL/min, 80～90min B liquid 1mL/min, investigate the post carrying capacity of chromatographic column, the result is 54mg/ml by calculating the post carrying capacity as shown in Figure 4.
4, utilize affinity chromatographic column of the present invention separation of human gamma Globulin from human serum
Get Freshman serum before the experiment, be mixed with 5% (v/v) human serum solution with mobile phase A liquid, same steps as 1 flows.Last sample and wash-out flow process are as follows: 0～5min mobile phase A (0.2mL/min), 5～55min human serum solution (0.2mL/min), 55～65min line of flow A (1mL/min), 65～75min Mobile phase B (1mL/min).The result as shown in Figure 5, specific adsorption takes place in the people's gamma Globulin in the human serum on chromatographic column, other compositions then do not take place to keep and by A liquid wash-out, final people's gamma Globulin obtains enrichment under B liquid wash-out.
5, the sign of people's gamma Globulin of affinity chromatography purifying
Collect people's gamma Globulin eluting peak in the step 4, carry out SDS～PAGE and analyze, analysis result as shown in Figure 6, wherein swimming lane 1 is the standard molecular weight sample, swimming lane 2 is people's gamma Globulin standard items, swimming lane 3 is the human serum eluent, swimming lane 4 is last sample human serum solution.As seen from the figure, the band of the human serum eluent of affinity chromatographic column of the present invention is consistent with the standard items band excessively, shows that the people's gamma Globulin in the human serum after affinity chromatographic column of the present invention is purified has higher purity.
It should be noted that at last: the above only is the preferred embodiments of the present invention, be not limited to the present invention, although the present invention is had been described in detail with reference to previous embodiment, for a person skilled in the art, it still can be made amendment to the technical scheme that aforementioned each embodiment put down in writing, and perhaps part technical characterictic wherein is equal to replacement.Within the spirit and principles in the present invention all, any modification of being done, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (7)
- One kind be used to separate, the affinity chromatograph filling of purification monoclonal antibody and antibody globulin, by solid phase carrier and form with the aglucon that covalent bond is connected with it, described aglucon is an albumin A, it is characterized in that, described solid phase carrier is a porous mono-dispersion microsphere.
- 2. according to claim 1ly be used to separate, the affinity chromatograph filling of purification monoclonal antibody and antibody globulin, it is characterized in that the material of described porous mono-dispersion microsphere is selected from any in high molecular polymer, silica gel, carborundum, aluminium oxide, chloride and the ceramic microsphere.
- 3. according to claim 1ly be used to separate, the affinity chromatograph filling of purification monoclonal antibody and antibody globulin, it is characterized in that the diameter of described porous mono-dispersion microsphere is 1～300 μ m, the aperture is 10～3000nm.
- 4. according to claim 1ly be used to separate, the affinity chromatograph filling of purification monoclonal antibody and antibody globulin, it is characterized in that primary alcohol functional group is contained on described porous mono-dispersion microsphere surface after treatment.
- 5. according to claim 1ly be used to separate, the affinity chromatograph filling of purification monoclonal antibody and antibody globulin, it is characterized in that described albumin A is to be obtained by genetic engineering means.
- 6. according to claim 1ly be used to separate, the preparation method of the affinity chromatograph filling of purification monoclonal antibody and antibody globulin, it is characterized in that, comprise the steps:(1) the space arm of taking over a job: get 1 part of porous mono-dispersion microsphere that contains primary alcohol functional group after treatment, add 0.1～10 part, 0.1 the KOH of～3.0mol/L and 0.1～10 part contain epoxy-functional compounds, in 10 ℃～100 ℃ the vibration or stirring reaction 1～72h after, product after filtration, the washing standby;(2) coupling aglucon: get the product that 1 part of step (1) obtains, place the PBS buffer solution that contains 0.1～10 part of albumin A, pH 6～7, and after vibrating under 10 ℃～30 ℃ or stirring 2～72h, reactant after filtration, washing is standby;(3) the residual full active group of sealing: get 1 part of step (2) gained reactant, add 5～10 parts 0.05M HCl, at 10 ℃～30 ℃ down vibration or stirring reaction 1～24h, product after filtration, the preservation of washing back.
- One kind be used to separate, affinity chromatographic column that the affinity chromatograph filling of purification monoclonal antibody and antibody globulin is made.
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| CN105263947A (en) * | 2013-06-25 | 2016-01-20 | 卡迪拉保健有限公司 | Purification process for monoclonal antibodies |
| CN107486119A (en) * | 2017-08-02 | 2017-12-19 | 广饶县信和化工有限公司 | The hydro-thermal regenerating unit and method of dimerization acid catalyzed reaction spent bleaching clay catalyst |
| CN111879872A (en) * | 2020-07-29 | 2020-11-03 | 纳谱分析技术（苏州）有限公司 | Method for detecting immunoglobulin IgG based on monodisperse polymer and application thereof |
| CN115356425A (en) * | 2022-07-27 | 2022-11-18 | 上海奥浦迈生物科技股份有限公司 | NanoHPLC-Titer system applied to culture medium supernatant albumin quantification |
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Application publication date: 20110914 |