CN106198780B - Protein-modified open-column and its application in the separation of monoclonal antibody charge variants - Google Patents
Protein-modified open-column and its application in the separation of monoclonal antibody charge variants Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
本发明公开了一种蛋白固定相修饰的开管柱及其在单克隆抗体电荷异构体分离中的应用，该开管柱由以下步骤制得：将PDDA通过静电自组装方法固定在毛细管内壁，再将蛋白通过静电自组装吸附在PDDA表面，得到蛋白固定相修饰开管柱。本发明的开管柱对单克隆抗体的电荷异构体表现了特殊的选择性。西妥昔单抗的七种不同形式的电荷异构体获得成功分离，利妥昔单抗的两种碱性异构体和一种酸性异构体以及曲妥珠单抗的两种碱性电荷异构体和四种酸性异构体被成功地从主峰中分离。
The invention discloses an open column modified by a protein stationary phase and its application in the separation of monoclonal antibody charge isomers. The open column is prepared by the following steps: fixing PDDA on the inner wall of a capillary by an electrostatic self-assembly method , and then the protein was adsorbed on the surface of PDDA through electrostatic self-assembly to obtain a protein stationary phase modified open column. The open column of the present invention exhibits special selectivity for the charge variant of the monoclonal antibody. Seven different forms of charge isomers of cetuximab were successfully separated, two basic and one acidic isomer of rituximab and two basic isomers of trastuzumab The charge isomer and four acidic isomers were successfully separated from the main peak.
Description
Technical field
The invention belongs to instrument analysis fields, and in particular to the open tubular column of a kind of protein stationary phase modification and its in monoclonal
Application in the separation of antibody charge isomer.
Background technique
Monoclonal antibody is a kind of protein medicaments having a extensive future in biomedicine field, due to its good medicine
Effect, the advantages such as highly selective, side effect is low, are widely used in the treatment of cancer.Currently, the monoclonal of most of clinical applications
Antibody is immunoglobulin class, these monoclonal antibodies (~150kDa) are the glycoprotein of four poly- types, identical heavy by two
Chain and two identical light chains are constituted, and centre is connected by several disulfide bond.
During production, extraction, formation and storage, monoclonal antibody can after a series of translations modification, packet
The effects of including glycosylation, polymerization, being crushed, is deamidated.Although these are varied less, monoclonal antibody can be made to show
A plurality of types of inhomogeneities, and then different types of variant is generated, they are in physicochemical properties such as molecular weight, hydrophobicity, charges
On have differences.
It is the most extensive to the analysis and research of its charge isomer in the analysis of monoclonal antibody inhomogeneity.Charge isomery
Body usually according to its isoelectric point relative to main peak, is divided into acid or alkali isomerization body.Generally, heterogeneous acidic body has opposite
The lower isoelectric point of main peak, and alkali isomerization body then has the opposite higher isoelectric point of main peak.
The complexity of monoclonal antibody to carry out characterizing to its inhomogeneity to become relatively difficult.One has been developed at present
A little methods analyze the inhomogeneity of monoclonal antibody, such as ion-exchange chromatography, reversed-phase liquid chromatography, hydrophobic chromatography, body
Product exclusion chromatography, SDS- polyacrylamide gel electrophoresis, capillary isoelectric focusing, capillary zone electrophoresis, mass spectrum etc..At these
In technology, ion-exchange chromatography is widely used in the characterization of monoclonal antibody charge isomer.In ion-exchange chromatography,
Charge isomer is presented usually in the form of small peak, and these charge isomers extremely close the quality and stability of monoclonal antibody
It is important.Therefore, the key for developing monoclonal antibody charge isomer analysis method is exactly by these small variant forms from main peak
In distinguish.
Capillary electric chromatogram is a kind of efficient compound-split technology, and separating mechanism is mainly according to electrophoretic migration and chromatography
Retain, therefore it had not only had the highly selective of high performance liquid chromatography but also the high efficiency with Capillary Electrophoresis, in complex biological sample
The separation field great potential of product.Chromatographic column is that the separation core of capillary electric chromatogram is opened in different types of electric chromatographic column
The advantages that tubing string is simple and convenient to operate, generates without bubble because of its preparation, is more and more favored by people.
The preparation method of open tubular column mainly includes chemical bonding, sol-gal process, molecular imprinting method, porous layer at present
Method, physisorphtion and nano coating method.In these methods, the method for LBL self-assembly due to its it is easy, stable and it is low at
This advantages of, has been successfully applied to the preparation of the specific function film of Nano grade.
Summary of the invention
In order to overcome the defects of the prior art described above, the charge occurred in the form of small peak in monoclonal antibody is preferably separated
Isomers, the primary purpose of the present invention is that providing a kind of preparation method of the open tubular column of protein stationary phase modification, this method is logical
Different types of albumen is coated to by the mode for crossing electrostatic self-assembled to be repaired with diallyl dimethyl ammoniumchloride (PDDA) in advance
The open tubular column of different protein stationary phase modifications is obtained on the open tubular column of decorations, the preparation method is simple, mild condition, favorable reproducibility.
Another object of the present invention is to provide open tubular column prepared by the above method, the open tubular column surface is rich in
Action site can effectively inhibit the absorption of biological sample, improve separating effect, improve separation selectivity.
A further object of the present invention is to provide the open tubular columns of above-mentioned protein stationary phase modification in monoclonal antibody charge
Application in isomer separation.
The purpose of the invention is achieved by the following technical solution:
A kind of preparation method of the open tubular column of protein stationary phase modification, is to be fixed on PDDA by electrostatic self-assembled method
Capillary tube inner wall, then albumen is adsorbed on the surface PDDA by electrostatic self-assembled, obtain protein stationary phase modification open tubular column；
Specifically, the above method the following steps are included:
(1) PDDA and electrolyte are dissolved in Tris-HCl buffer and obtain PDDA solution, wash away capillary column 0.5~
3h, then be rinsed with water 0.5~2h and remove extra PDDA, obtain PDDA open tubular column；
(2) protein dissolution is made into protein solution, the PDDA that injection step (1) prepares in Tris-HCl buffer
In open tubular column, heating saves 10~14h, obtains protein stationary phase modification open tubular column；
In the present invention, stationary phase albumen can be acidic protein or basic protein；When stationary phase albumen is acidic protein
When, open tubular column obtained can be used for the separation of basic protein；When stationary phase albumen is basic protein, open tubular column obtained is available
In the separation of acidic protein；
The preferred bovine serum albumin(BSA) of the acidic protein, bovine hemoglobin；
The preferred lysozyme of the basic protein, cromoci, ribonuclease A；
Salt ion in PDDA solution described in step (1) can be by ionizing base on PDDA polyelectrolyte molecules chain
The shielding action of group, makes PDDA molecule be assembled into film with the state of rolling up, the thickness that film is adjusted by adjusting salt ionic concentration；
Salt ionic concentration in step (1) PDDA solution is 0.1~2.0M；
Tris-HCl buffer preferable ph described in step (1) is 8.3；
PDDA solution described in step (1), the concentration of PDDA preferably 5~20mg/mL；
The preferred sodium chloride of electrolyte and/or potassium chloride described in step (1)；
Protein solution described in step (2), protein concentration preferably 4~10mg/mL；
Tris-HCl buffer preferable ph described in step (2) is 7.4；
The preservation of heating described in step (2) is heated to 30~50 DEG C.
The open tubular column of protein stationary phase modification prepared by the above method can be applied to monoclonal antibody charge isomer, acid
In the separation of property albumen or basic protein, which can effectively inhibit albumen
Absorption improves separating effect, improves separation selectivity.
The open tubular column of protein stationary phase modification of the invention, is dependent on simple and practical electrostatic self-assembled method and completes open pipe
Column is constructed.The open tubular column of PDDA modification is prepared by electrostatic self-assembled method first, then by proteopexy to PDDA
On the open tubular column of modification, the open tubular column of protein stationary phase modification is obtained.
The present invention has the following advantages and effects with respect to the prior art:
(1) open tubular column of the invention is using albumen as chromatographic stationary phases, surface action site abundant, effectively inhibition egg
White absorption improves separating effect, improves separation selectivity.
(2) open tubular column preparation condition of the invention is mild, easy to operate, and reproducibility and stability are preferable.
(3) open tubular column of the invention is demonstrated by special selectivity to the charge isomer of monoclonal antibody.Western appropriate former times is single
Seven kinds of anti-various forms of charge isomers succeed separation, two kinds of alkali isomerization bodies of Rituximab and a kind of acidity
The two kinds of alkaline charge isomers and four kinds of heterogeneous acidic bodies of isomers and Herceptin are successfully separated from main peak.
Detailed description of the invention
Fig. 1 is the capillary electric chromatogram spectrogram of basic protein separation: where 1 is lysozyme, and 2 be cromoci, and 3 be core
Ribonuclease T. A.
Fig. 2 is the capillary electric chromatogram spectrogram of western appropriate former times charge isomer separation: where 1~7 for Cetuximab not
Same charge isomer.
Fig. 3 is the capillary electric chromatogram spectrogram of Rituximab charge isomer separation: where b1, b2 are rituximab list
Alkali resistance charge isomer, a1 are Rituximab acidic charge isomers.
Fig. 4 is the capillary electric chromatogram spectrogram of Herceptin charge isomer separation: where b1, b2 are toltrazuril list
Alkali resistance charge isomer, a1~a4 are Herceptin acidic charge isomers.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment 1
The open tubular column of bovine serum albumin(BSA) (BSA) modification, is made by following steps:
(1) it the preparation of PDDA open tubular column: takes PDDA and sodium chloride to be dissolved into 20mM Tris-HCl (pH value 8.3) and obtains
PDDA concentration is the PDDA solution of 20mg/mL, and salt ionic concentration therein is 0.1M, then washes away hair with the flow velocity of 10 μ L/min
Capillary column 1h.Finally remove extra PDDA solution with deionized water.
(2) preparation of PDDA@BSA open tubular column: after PDDA open tubular column is successfully prepared, take BSA that 20mM Tris-HCl is added
It is made into the uniform solution of 5mg/mL in (pH value 7.4), it is then injected into the PDDA open pipe prepared with the flow velocity of 10 μ L/min
In column, is stood in 40 DEG C of baking oven and save 12h.Finally, removing extra BSA with deionized water, dried under 40 DEG C of nitrogen protections
It is dry, obtain PDDA@BSA open tubular column.
Embodiment 2
The open tubular column of bovine serum albumin(BSA) (BSA) modification, is made by following steps:
(1) it the preparation of PDDA open tubular column: takes PDDA and potassium chloride to be dissolved into 20mM Tris-HCl (pH value 8.3) and obtains
PDDA concentration is the PDDA solution of 10mg/mL, and salt ionic concentration therein is 1.0M, then washes away hair with the flow velocity of 10 μ L/min
Capillary column 0.5h.Finally remove extra PDDA solution with deionized water.
(2) preparation of PDDA@BSA open tubular column: after PDDA open tubular column is successfully prepared, take BSA that 20mM Tris-HCl is added
It is made into the uniform solution of 10mg/mL in (pH value 7.4), it is then injected into the PDDA open pipe prepared with the flow velocity of 10 μ L/min
In column, is stood in 40 DEG C of baking oven and save 12h.Finally, removing extra BSA with deionized water, dried under 40 DEG C of nitrogen protections
It is dry, obtain PDDA@BSA open tubular column.
Embodiment 3
The open tubular column of bovine hemoglobin (BHb) modification, is made by following steps:
(1) it the preparation of PDDA open tubular column: takes PDDA and sodium chloride to be dissolved into 20mM Tris-HCl (pH value 8.3) and obtains
PDDA concentration is the PDDA solution of 20mg/mL, and salt ionic concentration therein is 2.0M, then washes away hair with the flow velocity of 10 μ L/min
Capillary column 3h.Finally remove extra PDDA solution with deionized water.
(2) preparation of PDDA@BHb open tubular column: after PDDA open tubular column is successfully prepared, take BHb that 20mM Tris-HCl is added
It is made into the uniform solution of 5mg/mL in (pH value 7.4), it is then injected into the PDDA open pipe prepared with the flow velocity of 10 μ L/min
In column, is stood in 40 DEG C of baking oven and save 12h.Finally, removing extra BHb with deionized water, dried under 40 DEG C of nitrogen protections
It is dry, obtain PDDA@BHb open tubular column.
Embodiment 4
The application of PDDA@BSA open tubular column made from embodiment 1
Lysozyme, cromoci, ribonuclease A are mixed and are dissolved in deionized water, is carried out by the method for electrochromatography
Separation, the result is shown in Figure 1.Separation condition are as follows: chromatography column internal diameter is 50 μm, overall length 63.5cm, effective length 37cm, mobile phase
For the phosphate buffer of 40mM (pH value 7.0), separation voltage 20kV, Detection wavelength 214nm, input mode be height into
Sample.
As seen from Figure 1, three kinds of basic proteins obtain good separating effect, peak type point on PDDA@BSA open tubular column
It is sharp, illustrate that the open tubular column can be effectively used for the separation of basic protein, greatly inhibit the absorption of albumen, improves separation effect
Fruit.
Embodiment 5
The application of PDDA@BSA open tubular column made from embodiment 1
Cetuximab after centrifugation, dilution is soluble in water, it is separated by the method for electrochromatography, as a result sees Fig. 2.
Separation condition are as follows: chromatography column internal diameter is 50 μm, overall length 63.5cm, effective length 37cm, and mobile phase is 40mM (pH value 6.0)
Phosphate buffer, separation voltage 20kV, Detection wavelength 214nm, input mode be height sample introduction.
From Figure 2 it can be seen that seven kinds of various forms of charge isomers of Cetuximab obtain on PDDA@BSA open tubular column
Good separating effect, peak type are sharp.Illustrate that the coating column can be effectively applied to Cetuximab charge isomer
Identification.
Embodiment 6
The application of PDDA@BSA open tubular column made from embodiment 2
Rituximab after centrifugation, dilution is soluble in water, it is separated by the method for electrochromatography, as a result sees Fig. 3.
Separation condition are as follows: chromatography column internal diameter is 50 μm, overall length 63.5cm, effective length 37cm, and mobile phase is 40mM (pH value 6.0)
Phosphate buffer, separation voltage 20kV, Detection wavelength 214nm, input mode be height sample introduction.
As seen from Figure 3, two kinds of alkali isomerization bodies of Rituximab and a kind of heterogeneous acidic body successfully divide from main peak
From illustrating that BSA coating has selectivity well to the charge isomer of Rituximab.
Embodiment 7
The application of PDDA@BSA open tubular column made from embodiment 2
Herceptin after centrifugation, dilution is soluble in water, it is separated by the method for electrochromatography, as a result sees Fig. 4.
Separation condition are as follows: chromatography column internal diameter is 50 μm, overall length 63.5cm, effective length 37cm, and mobile phase is 40mM (pH value 5.5)
Phosphate buffer, separation voltage 20kV, Detection wavelength 214nm, input mode be height sample introduction.
From fig. 4, it can be seen that two kinds of Herceptin alkaline charge isomers and four kinds of acidic charge isomers successfully from
It is separated in main peak, illustrates that BSA coating has selectivity well to the charge isomer of Herceptin.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (3)
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| CN104931628B (en) * | 2015-06-02 | 2017-03-22 | 华南师范大学 | Fe3O4‑COOH magnetic nanomaterial modified open-tubular column and its preparation method and application |
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2016
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