CN113588361A - Isoelectric point standard substance for free flow electrophoresis - Google Patents
Isoelectric point standard substance for free flow electrophoresis Download PDFInfo
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- CN113588361A CN113588361A CN202110695878.1A CN202110695878A CN113588361A CN 113588361 A CN113588361 A CN 113588361A CN 202110695878 A CN202110695878 A CN 202110695878A CN 113588361 A CN113588361 A CN 113588361A
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- 239000000126 substance Substances 0.000 title claims abstract description 71
- 238000001997 free-flow electrophoresis Methods 0.000 title claims abstract description 20
- 239000003086 colorant Substances 0.000 claims abstract description 10
- 125000000623 heterocyclic group Chemical group 0.000 claims abstract description 6
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims abstract description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 6
- 239000012498 ultrapure water Substances 0.000 claims abstract description 6
- 230000031700 light absorption Effects 0.000 claims description 16
- -1 4-carboxypiperazinyl Chemical group 0.000 claims description 8
- RMPQXSQYCGZHIS-UHFFFAOYSA-N 2,4-bis[(4-methylpiperazin-1-yl)methyl]-6-nitrophenol Chemical compound C1CN(C)CCN1CC1=CC(CN2CCN(C)CC2)=C(O)C([N+]([O-])=O)=C1 RMPQXSQYCGZHIS-UHFFFAOYSA-N 0.000 claims description 5
- SDGNNLQZAPXALR-UHFFFAOYSA-N 3-sulfophthalic acid Chemical compound OC(=O)C1=CC=CC(S(O)(=O)=O)=C1C(O)=O SDGNNLQZAPXALR-UHFFFAOYSA-N 0.000 claims description 5
- HEDOSLKDSIARIA-UHFFFAOYSA-N C1=C(CN2CCCCC2)C=C([N+]([O-])=O)C(O)=C1CN1CCCCC1 Chemical compound C1=C(CN2CCCCC2)C=C([N+]([O-])=O)C(O)=C1CN1CCCCC1 HEDOSLKDSIARIA-UHFFFAOYSA-N 0.000 claims description 5
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 5
- BCWYGXAEDYPTLE-UHFFFAOYSA-N 2,4-bis(morpholin-4-ylmethyl)-6-nitrophenol Chemical compound C1=C(CN2CCOCC2)C=C([N+]([O-])=O)C(O)=C1CN1CCOCC1 BCWYGXAEDYPTLE-UHFFFAOYSA-N 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 6
- 238000002360 preparation method Methods 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 238000010586 diagram Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- JYOHRRFJXDIFMZ-UHFFFAOYSA-N 2-methyl-4-(morpholin-4-ylmethyl)phenol Chemical compound C1=C(O)C(C)=CC(CN2CCOCC2)=C1 JYOHRRFJXDIFMZ-UHFFFAOYSA-N 0.000 description 4
- 238000009826 distribution Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 230000005684 electric field Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention provides an isoelectric point standard for free flow electrophoresis, which comprises: the device comprises 8 isoelectric point substances, wherein each isoelectric point substance corresponds to different isoelectric points, the isoelectric point substances have colors which can be directly observed by naked eyes, the isoelectric points of the isoelectric point substances are between 3.8 and 10.1, the isoelectric point substances are composed of small molecular substances diluted by ultrapure water, and the isoelectric point substances have molecular weights between 200 and 600KDa and are benzene rings and heterocyclic substances. The isoelectric point standard meets the system standards of pH3-10, pH7-10 and pH5-8, can position the pH in the process of separating and preparing isomers, greatly improves the positioning of the isomers and shortens the preparation time of the isomers.
Description
Technical Field
The invention belongs to the technical field of free flow electrophoresis, and particularly relates to an isoelectric point standard product for free flow electrophoresis.
Background
Charge isomer analysis is a regulatory requirement for biological therapeutic proteins. These large heterogeneous molecules undergo various enzymatic post-translational modifications during production, such as glycosylation and lysine cleavage. In addition, various chemical modifications, such as oxidation or deamination, can occur during purification and storage. Free flow electrophoresis is the best method for preparing protein drug charge isomers.
Free Flow Electrophoresis (FFE) is a semi-preparative separation technology, has the advantages of continuous separation, multiple separation modes, no fixed support medium, mild separation conditions and the like, and is particularly suitable for separation, purification and preparation of biological materials. Free-flow isoelectric focusing electrophoresis (IEF-FFE) utilizes a continuous flow of ampholyte solution to create a linear pH gradient in the direction of the electric field within the separation chamber. The sample flows longitudinally along with the solution and is transversely deflected under the action of an electric field, does not move transversely when reaching the isoelectric point, is forcibly focused at the position, is not influenced by diffusion and convection, and only moves in the flow direction of the solution. Benign substances such as proteins or peptides having different isoelectric points can be separated by IEF-FFE. The isoelectric point standard products with different colors can accurately position the positions of different protein drug charge isomers during separation, but the existing isoelectric point standard products have poor effect of positioning and separating the protein drug charge isomers and hardly meet the requirements of different systems. Therefore, there is a need for a new isoelectric point standard for free-flow electrophoresis to solve the above problems.
Disclosure of Invention
The present invention is directed to providing an isoelectric point standard for free-flow electrophoresis to solve the problems set forth in the background art described above.
In order to achieve the purpose, the invention provides the following technical scheme: an isoelectric point standard for free-flow electrophoresis comprising: the device comprises 8 isoelectric point substances, wherein each isoelectric point substance corresponds to different isoelectric points, the isoelectric point substances have colors which can be directly observed by naked eyes, the isoelectric points of the isoelectric point substances are between 3.8 and 10.1, the isoelectric point substances are composed of small molecular substances diluted by ultrapure water, and the isoelectric point substances have molecular weights between 200 and 600KDa and are benzene rings and heterocyclic substances.
Preferably, the isoelectric point substances are 4, 6-diaminoazobenzene-2, 2-dicarboxylic acid, 3' -bis [ N- (4-carboxypiperazinyl) methyl ] -o-cresol sulfophthalein, 3', 5', 3' 5' -tetrakis (N- [ 4-carboxypiperazinyl ] methyl) -phenol sulfophthalic acid, 3' -bis (4-morpholinomethyl) -5', 5' -dichlorophenol sulfophthalein, 3' -bis (4-morpholinomethyl) -o-cresol sulfophthalein, 2, 4-bis- (4-morpholinomethyl) -6-nitrophenol, 2, 4-bis [ (4-methyl-1-piperazinyl) methyl ] -6-nitrophenol, respectively, 2-nitro-4, 6-bis (1-piperidinylmethyl) phenol.
Preferably, the isoelectric point of the isoelectric point substance is 3.0, 4.0, 4.7, 5.3, 6.4, 7.5, 8.5 or 10.1.
Preferably, the isoelectric point substances all have light absorption at 420nm, and the light absorption at 420nm can be directly detected by an instrument for detection.
The invention has the technical effects and advantages that: the isoelectric point standard meets the system standards of pH3-10, pH7-10 and pH5-8, can position the pH in the process of separating and preparing isomers, greatly improves the positioning of the isomers and shortens the preparation time of the isomers.
Drawings
FIG. 1 is a graph showing the light absorption at 420nm in example 1;
FIG. 2 is a distribution diagram of isoelectric point standards in example 1;
FIG. 3 is a graph showing the light absorption at 420nm in example 2;
FIG. 4 is a distribution diagram of isoelectric point standards in example 2;
FIG. 5 is a graph showing the light absorption at 420nm in example 3;
FIG. 6 is a distribution diagram of isoelectric point standards in example 3.
Detailed Description
Example 1
An isoelectric point standard for free-flow electrophoresis comprising: the isoelectric point substances comprise 8 kinds of micromolecule substances diluted by ultrapure water, the molecular weight of the isoelectric point substances is between 200 and 600KDa, and the isoelectric point substances are benzene rings and heterocyclic substances, and the isoelectric point substances are respectively 4, 6-diaminoazobenzene-2, 2-dicarboxylic acid, 3' -bis [ N- (4-carboxypiperazinyl) methyl ] -o-cresol sulfophthalein, 3', 5', 3' 5' -tetra (N- [ 4-carboxypiperazinyl ] methyl) -phenol sulfophthalic acid, 3' -bis (4-morpholinomethyl) -5', 5' -dichlorophenol sulfophthalein, 3' -bis (4-morpholinomethyl) -o-cresol sulfophthalein, 2, 4-bis- (4-morpholinomethyl) -6-nitrophenol, 2, 4-bis [ (4-methyl-1-piperazinyl) methyl ] -6-nitrophenol and 2-nitro-4, 6-bis (1-piperidinylmethyl) phenol, wherein each isoelectric point substance corresponds to different isoelectric points and is respectively 3.0, 4.0, 4.7, 5.3, 6.4, 7.5, 8.5 and 10.1, the isoelectric point substances have colors which can be directly observed by naked eyes, and the isoelectric points of the isoelectric point substances are between 3.8 and 10.1.
The effect of the isoelectric point standard is verified by adopting a pH3-10 separation system, and the specific system parameters of the pH3-10 separation system are shown in the table I:
watch 1
The verification results are as follows: FIG. 1 is a 420nm light absorption diagram (isoelectric point substances in isoelectric point standard have light absorption at 420nm, and can be detected by directly detecting light absorption at 420nm with an instrument), and the standard substances with different isoelectric points are uniformly focused in different shunt holes; in fig. 2, it can be seen that the standard substances with different colors are uniformly dispersed in different shunting holes. In the process of separating and preparing protein isomers, the isomers can be enriched and prepared according to the position positioning of different standards.
Example 2
An isoelectric point standard for free-flow electrophoresis comprising: the isoelectric point substances comprise 8 kinds of micromolecule substances diluted by ultrapure water, the molecular weight of the isoelectric point substances is between 200 and 600KDa, and the isoelectric point substances are benzene rings and heterocyclic substances, and the isoelectric point substances are respectively 4, 6-diaminoazobenzene-2, 2-dicarboxylic acid, 3' -bis [ N- (4-carboxypiperazinyl) methyl ] -o-cresol sulfophthalein, 3', 5', 3' 5' -tetra (N- [ 4-carboxypiperazinyl ] methyl) -phenol sulfophthalic acid, 3' -bis (4-morpholinomethyl) -5', 5' -dichlorophenol sulfophthalein, 3' -bis (4-morpholinomethyl) -o-cresol sulfophthalein, 2, 4-bis- (4-morpholinomethyl) -6-nitrophenol, 2, 4-bis [ (4-methyl-1-piperazinyl) methyl ] -6-nitrophenol and 2-nitro-4, 6-bis (1-piperidinylmethyl) phenol, wherein each isoelectric point substance corresponds to different isoelectric points and is respectively 3.0, 4.0, 4.7, 5.3, 6.4, 7.5, 8.5 and 10.1, the isoelectric point substances have colors which can be directly observed by naked eyes, and the isoelectric points of the isoelectric point substances are between 3.8 and 10.1.
The effect of the isoelectric point standard is verified by adopting a pH7-10 separation system, and the specific system parameters of the pH7-10 separation system are shown in the following table II:
watch two
The verification results are as follows: FIG. 3 is a 420nm light absorption diagram (isoelectric point substances in isoelectric point standard have light absorption at 420nm, and can be detected by directly detecting light absorption at 420nm with an instrument), and the standard substances with different isoelectric points are uniformly focused in different shunt holes; in fig. 4, it can be seen that the standard substances with different colors are uniformly dispersed in different shunting holes. In the process of separating and preparing protein isomers, the isomers can be enriched and prepared according to the position positioning of different standards.
Example 3
An isoelectric point standard for free-flow electrophoresis comprising: the isoelectric point substances comprise 8 kinds of micromolecule substances diluted by ultrapure water, the molecular weight of the isoelectric point substances is between 200 and 600KDa, and the isoelectric point substances are benzene rings and heterocyclic substances, and the isoelectric point substances are respectively 4, 6-diaminoazobenzene-2, 2-dicarboxylic acid, 3' -bis [ N- (4-carboxypiperazinyl) methyl ] -o-cresol sulfophthalein, 3', 5', 3' 5' -tetra (N- [ 4-carboxypiperazinyl ] methyl) -phenol sulfophthalic acid, 3' -bis (4-morpholinomethyl) -5', 5' -dichlorophenol sulfophthalein, 3' -bis (4-morpholinomethyl) -o-cresol sulfophthalein, 2, 4-bis- (4-morpholinomethyl) -6-nitrophenol, 2, 4-bis [ (4-methyl-1-piperazinyl) methyl ] -6-nitrophenol and 2-nitro-4, 6-bis (1-piperidinylmethyl) phenol, wherein each isoelectric point substance corresponds to different isoelectric points and is respectively 3.0, 4.0, 4.7, 5.3, 6.4, 7.5, 8.5 and 10.1, the isoelectric point substances have colors which can be directly observed by naked eyes, and the isoelectric points of the isoelectric point substances are between 3.8 and 10.1.
The effect of the isoelectric point standard substance is verified by adopting a pH5-8 separation system, and the specific system parameters of the pH5-8 separation system are shown in the third table:
watch III
The verification results are as follows: FIG. 5 is a 420nm light absorption diagram (isoelectric point substances in isoelectric point standard have light absorption at 420nm, and can be detected by directly detecting light absorption at 420nm with an instrument), and the standard substances with different isoelectric points are uniformly focused in different shunt holes; in fig. 6, it can be seen that the standard substances with different colors are uniformly dispersed in different shunting holes. In the process of separating and preparing protein isomers, the isomers can be enriched and prepared according to the position positioning of different standards.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.
Claims (4)
1. An isoelectric point standard for free-flow electrophoresis comprising: an isoelectric substance characterized by: the number of the isoelectric point substances is 8, each isoelectric point substance corresponds to different isoelectric points, the isoelectric point substances have colors which can be directly observed by naked eyes, the isoelectric point of the isoelectric point substances is between 3.8 and 10.1, the isoelectric point substances are composed of small molecular substances diluted by ultrapure water, and the molecular weights of the isoelectric point substances are between 200 and 600KDa and are benzene rings and heterocyclic substances.
2. The isoelectric point standard for free-flow electrophoresis according to claim 1, wherein: the isoelectric point substances are respectively 4, 6-diaminoazobenzene-2, 2-dicarboxylic acid, 3' -bis [ N- (4-carboxypiperazinyl) methyl ] -o-cresol sulfophthalein, 3', 5', 3' 5' -tetra (N- [ 4-carboxypiperazinyl ] methyl) -phenol sulfophthalic acid, 3' -bis (4-morpholinylmethyl) -5', 5' -dichlorophenol sulfophthalein, 3' -bis (4-morpholinylmethyl) -o-cresol sulfophthalein, 2, 4-bis- (4-morpholinylmethyl) -6-nitrophenol, 2, 4-bis [ (4-methyl-1-piperazinyl) methyl ] -6-nitrophenol and 2-nitro-4, 6-bis (1-piperidinylmethyl) phenol.
3. The isoelectric point standard for free-flow electrophoresis according to claim 2, wherein: the isoelectric point of the isoelectric point substance is 3.0, 4.0, 4.7, 5.3, 6.4, 7.5, 8.5 and 10.1 respectively.
4. The isoelectric point standard for free-flow electrophoresis according to any one of claims 1 or 3 wherein: the isoelectric point substances have light absorption at 420nm, and can be detected by directly detecting the light absorption at 420nm by an instrument.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003172725A (en) * | 2001-12-05 | 2003-06-20 | Olympus Optical Co Ltd | Free flow electrophoresis |
| WO2009133153A1 (en) * | 2008-04-29 | 2009-11-05 | Becton, Dickinson And Company | Methods and systems for the separation and analysis of analytes using ffe |
| CN106814122A (en) * | 2015-11-30 | 2017-06-09 | 成都金凯生物技术有限公司 | The improved method that a kind of capillary isoelectric focusing determines recombinant protein isoelectric point |
-
2021
- 2021-06-23 CN CN202110695878.1A patent/CN113588361A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003172725A (en) * | 2001-12-05 | 2003-06-20 | Olympus Optical Co Ltd | Free flow electrophoresis |
| WO2009133153A1 (en) * | 2008-04-29 | 2009-11-05 | Becton, Dickinson And Company | Methods and systems for the separation and analysis of analytes using ffe |
| CN106814122A (en) * | 2015-11-30 | 2017-06-09 | 成都金凯生物技术有限公司 | The improved method that a kind of capillary isoelectric focusing determines recombinant protein isoelectric point |
Non-Patent Citations (2)
| Title |
|---|
| HERZOG, C ET.AL: "Rapid Isoelectric Point Determination in a Miniaturized Preparative Separation Using Jet-Dispensed Optical pH Sensors and Micro Free-Flow Electrophoresis", ANALYTICAL CHEMISTRY, vol. 86, no. 19, 12 November 2014 (2014-11-12) * |
| KOHLHEYER, D ET.AL: "Microfluidic high-resolution free-flow isoelectric focusing By", ANALYTICAL CHEMISTRY, vol. 79, no. 21, 1 November 2007 (2007-11-01) * |
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