CN115286686A

CN115286686A - Free-flow isoelectric focusing electrophoresis buffer system

Info

Publication number: CN115286686A
Application number: CN202210914660.5A
Authority: CN
Inventors: 师要辉; 徐琼琼; 马校卫
Original assignee: Shanghai Lanxie Biotechnology Co ltd
Current assignee: Shanghai Lanxie Biotechnology Co ltd
Priority date: 2022-08-01
Filing date: 2022-08-01
Publication date: 2022-11-04

Abstract

The invention provides a free current isoelectric focusing electrophoresis buffer system, which comprises: the device comprises an anolyte, an anodic space occupying buffer solution, a separation buffer solution, a cathodic stabilization buffer solution and a catholyte, wherein the anolyte is 100mM phosphoric acid and 200mM pyridine ethanol, and the catholyte is 100mM NaOH and 300mM ACA. During electrophoresis, the system forms a stable pH gradient between pH8.5-10.0 under the action of an electric field, the optimal resolution can reach 0.02pH, and the system is used for separating and preparing the charge isomers of the antibody drugs.

Description

Free-flow isoelectric focusing electrophoresis buffer system

Technical Field

The invention relates to the technical field of free flow electrophoresis, in particular to a free flow isoelectric focusing electrophoresis buffer system.

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, reaches the isoelectric point, does not transversely move any more, is forcibly focused at a certain position, is not influenced by diffusion and convection, and only moves in the flowing direction of the solution. Benign substances such as proteins or peptides having different isoelectric points can be separated by IEF-FFE.

As the charge isomer is a monitoring condition of biological therapeutic protein, the activity, chemical modification and molecular weight of each isomer are different, analysis of each component is favorable for improvement of subsequent medicines, and for antibody medicines, the isoelectric point is basically concentrated between 8.5 and 10.0, so that the sensitivity in the pH range is improved, the single-component isomer can be obtained better, and the activity, molecular weight and modification of the isomer can be better analyzed.

Disclosure of Invention

The technical problem to be solved by the present invention is to provide a free-flow isoelectric focusing electrophoresis buffer system to solve the problems mentioned in the background. In order to achieve the purpose, the invention provides the following technical scheme: a free-flow isoelectric focusing electrophoresis buffer system comprising: the device comprises an anolyte, an anode occupying buffer solution, a separation buffer solution, a cathode stabilizing buffer solution and a catholyte, wherein the anolyte is 100mM phosphoric acid and 200mM pyridine ethanol, and the catholyte is 100mM NaOH and 300mM EACA.

Preferably, the anode-occupying buffer is 80mM phosphoric acid, 200mM pyridylethanol, 80mM oxalic acid.

Preferably, the separation buffer is 1.5% pharmalytete 8-10.5, 0.1% pharmalyte 5-8.

Preferably, the cathodic stabilization buffer is 150mM KOH, 300mM EACA.

Preferably, the optimal resolution of the buffer system is at 0.02pH.

Compared with the prior art, the invention has the following beneficial effects: during electrophoresis, the system forms a stable pH gradient between pH8.5-10.0 under the action of an electric field, has the optimal resolution of 0.02pH, and is used for separating and preparing charge isomers of antibody drugs.

Drawings

In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to these drawings without inventive effort.

FIG. 1 is a graph plotting the absorption at 420nm for standards of different isoelectric points in the examples;

FIG. 2 shows that the standard substances with different isoelectric points are uniformly dispersed in different flow-dividing holes in the examples;

FIG. 3 is a graph showing the results of separation of charge isomers by capillary isoelectric focusing in the examples;

FIG. 4 is a graph plotting the absorption of 420nm light for the charge isomer obtained in the present system in the examples.

Detailed Description

In order to make the technical means, the creation features, the work flow and the using method of the present invention easily understand and understand the purpose and the efficacy, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any inventive step, and other conclusions that can be drawn without inventive extensions, are intended to fall within the scope of the present invention.

Examples

A free-flow isoelectric focusing electrophoresis buffer system comprising: an anolyte, a separation buffer and a catholyte stabilization buffer, a catholyte, the anolyte being 100mM phosphoric acid, 200mM pyridylethanol, the catholyte being 100mM NaOH, 300mM EACA, the anolyte being 80mM phosphoric acid, 200mM pyridylethanol, 80mM oxalic acid, the separation buffer being 1.5% pharmalyte8-10.5, 0.1% pharmalyte 5-8, the catholyte stabilization buffer being 150mM KOH, 300mM EACA, wherein the anolyte pH is 5.7, the anolyte pH is 6.2, the separation buffer pH 8.91 and the catholyte stabilization buffer pH 11.4, the catholyte pH 10.82. The results of using the buffer system to form a linear pH gradient in the direction of the electric field in the separation chamber and adding standards with different isoelectric points are shown in FIGS. 1 and 2. It can be seen from fig. 2 that the standards with different colors are uniformly dispersed in different diversion holes. It can be seen from fig. 1 that the pH of the last two markers was 8.5 and 10.1, 65 wells were dispersed at 1.6 pH units, the pH between wells differed by 0.0246 pH units, thus the sensitivity was within 0.025 pH;

the original sample was subjected to capillary isoelectric focusing to separate the charge isomers, and as a result, it was found that there were four charge isomers in the original sample as shown in fig. 3. The original sample is separated by using the buffer system, and the 420nm light absorption of the separation result is plotted as shown in FIG. 4, wherein the absorption peaks on the graph and the four isomers of the original sample can be in one-to-one correspondence.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A free-flow isoelectric focusing electrophoresis buffer system is characterized in that: the method comprises the following steps: the device comprises an anolyte, an anode occupying buffer solution, a separation buffer solution, a cathode stabilizing buffer solution and a catholyte, wherein the anolyte is 100mM phosphoric acid and 200mM pyridine ethanol, and the catholyte is 100mM NaOH and 300mM EACA.

2. The buffer system of claim 1, wherein: the anode occupying buffer solution is 80mM phosphoric acid, 200mM pyridine ethanol and 80mM oxalic acid.

3. The buffer system of claim 1, wherein: the separation buffer was 1.5% pharmalyte8-10.5, 0.1% pharmalyte 5-8.

4. The buffer system for free-flow isoelectric focusing electrophoresis according to claim 1, characterized in that: the cathode stabilization buffer is 150mM KOH, 300mM EACA.

5. The buffer system for free-flow isoelectric focusing electrophoresis according to claim 1, characterized in that: the optimal resolution of the buffer system is at 0.02pH.