A formulation capable of collecting low pI protein charge variant components and its application

Abstract

The present invention belongs to the field of biology, and particularly relates to a formulation capable of collecting low pI protein charge variant components and its application. The composition of the collection solution is: Tween 80 with a mass-volume ratio of 0.03-0.07%, perfluorooctanoic acid sodium with a mass-volume ratio of not less than 0.2%, and the rest is an acetic acid aqueous solution with a pH of 2-3. Through the collection solution developed by the present invention and based on the MauriceFlex system, the chemical migration and complete collection of low pI protein charge variant components can be achieved, while avoiding the hydrolysis and destruction of proteins by low pH solutions. The collected components have a high purity and can be used for further in-depth characterization research of each component peak.

Description

Technical Field

[0001] The present invention belongs to the biological field, and particularly relates to a formulation capable of collecting low pI protein charge variant components and its application. Background Art

[0002] Charge variants formed by various modifications such as deamidation, N-terminal modification, isomerization, sialylation, and C-terminal cleavage in biologic macromolecule protein drugs (multiple types of samples such as monoclonal antibodies, bispecific antibodies, antibody-drug conjugates, recombinant proteins, and fusion proteins) may affect the stability, safety, and efficacy of the products. Whole-column imaging capillary isoelectric focusing (icIEF) can be used as a powerful tool for characterizing protein charge heterogeneity by utilizing the molecular pI (isoelectric point) characteristics. Its principle is to apply a DC voltage across the two ends of the capillary. The carrier ampholytes in the tube can form a pH gradient within a certain range. Proteins move towards the anode or cathode according to the charges they carry until they stop at a certain pH value (i.e., the isoelectric point pI) where the net charge is 0. Eventually, the proteins are focused into very narrow segments, thus achieving the purpose of separation.

[0003] MauriceFlex, as an icIEF-based component collection instrument, can separate and collect charge variant components within one day and is flexibly used for further characterization and analysis, such as post-translational modification analysis, glycan analysis, activity analysis, etc. Its collection principle is that after the separation and focusing of each charge variant of the sample, the original cathode solution sodium hydroxide is automatically replaced with ammonium acetate, which changes the pH gradient within the capillary, so that the focused charge variant proteins can be chemically migrated back towards the cathode end. Finally, the protein variant components are chemically migrated towards the collection module end (96-well plate) in order of decreasing pI, first the high pI Marker, followed by the basic variant components, the protein main peak component, the acidic variant components, and finally the low pI Marker. The cathode solution in the chemical migration should ensure that the low pI Marker component completes the chemical migration and collection, so as to determine the distribution area of the collected components in the 96-well plate based on the fluorescence tracing of the high and low pI Markers for the next step of icIEF component confirmation.

[0004] Low pI proteins refer to proteins with a relatively low isoelectric point (pI), which usually carry a positive charge under acidic conditions. Due to their unique physical and chemical properties, they are often used in the development of therapeutic drugs, vaccines, and diagnostic reagents. Common low pI proteins include certain subtypes of antibodies (IgG4), antibody fragments (Fab or scFv), PEGylated antibodies, cytokines, fusion proteins, vaccine-related proteins, etc. Since the pH value of ammonium acetate solution is close to neutral, and the pH of the charge variant components of low pI proteins after isoelectric focusing is lower than 7, it is difficult for the separated components to completely migrate into the collection wells, and the purity cannot meet the requirements of subsequent analysis, which is a difficult problem in the collection and characterization of charge variant components of low pI proteins. Summary of the Invention

[0005] In the MauriceFlex system, ammonium acetate solution is usually used as the solution at the cathode end. The ammonium acetate solution plays a role in changing the pH gradient inside the capillary during the experiment, enabling the originally focused proteins to move and achieve component collection, and can effectively separate and collect proteins with a pI greater than 7; in the MauriceFlex system, the selection of the solution at the cathode end is restricted by the device design and experimental requirements. According to the general principles of isoelectric focusing technology, the solution at the cathode end needs to be able to maintain a stable pH gradient and support the focusing and separation of proteins.

[0006] The pI value of the charge variants of low pI proteins is usually less than 7. In the MauriceFlex system, aiming at the problem that ammonium acetate solution cannot effectively achieve the migration and collection of the charge variants of low pI proteins, the present invention develops a formulated solution suitable for the MauriceFlex system, which is used as the solution at the cathode end during the collection of the MauriceFlex system, and completes the chemical migration of the charge variant components of low pI proteins, thereby achieving the complete collection and high-purity preservation of the charge variants.

[0007] For the above purpose, the present invention provides the following technical solutions:

[0008] The first aspect of the present invention discloses a collection solution for charge variants of low pI proteins suitable for MauriceFlex. The composition of the collection solution is: Tween 80 with a mass-volume ratio of 0.03 - 0.07%, perfluorooctanoic acid sodium with a mass-volume ratio of not less than 0.2%, and the rest is acetic acid aqueous solution with a pH of 2 - 3.

[0009] Preferably, the composition of the collection solution is: Tween 80 with a mass-volume ratio of 0.05%, perfluorooctanoic acid sodium with a mass-volume ratio of 0.2%, and the rest is acetic acid aqueous solution with a pH of 2.5.

[0010] The preparation method of the collection solution is as follows: Weigh Tween 80 and sodium perfluorooctanoate according to the mass-to-volume ratio and place them in a beaker. Then dissolve them with an acetic acid aqueous solution with a pH of 2.5, stir and perform ultrasonic treatment, and mix well to obtain the collection solution.

[0011] The second aspect of the present invention discloses the application of the above collection solution in collecting low pI protein charge variants in the MauriceFlex system.

[0012] The pI value of the low pI protein charge variant is less than 7.

[0013] The present invention discloses a new collection solution formula: an acidic collection solution containing a protein stabilizer (suitable for the migration and collection of low pI protein charge variants), and the proportion of each component is as follows: Tween 80 with a mass-to-volume ratio of 0.05%, sodium perfluorooctanoate C7F15COONa with a mass-to-volume ratio of 0.2%, and an acetic acid aqueous solution with a pH of 2.5.

[0014] Tween 80 can help maintain the pH stability of the solution and reduce the impact of pH fluctuations on low pI proteins. At the same time, it can bind to the hydrophobic regions of proteins, reduce the hydrophobic interactions between protein molecules, and help maintain their native conformation, thereby inhibiting the aggregation and precipitation of low pI proteins.

[0015] Sodium perfluorooctanoate C7F 15 COONa is a fluorinated anionic surfactant, which replaces all hydrogen atoms on the straight-chain carbon with fluorine atoms. Its main structure is a non-polar fluorocarbon chain and a polar group carboxylic acid. Due to the very strong bond energy of the carbon-fluorine bond, it has high surface activity, high thermodynamic and chemical stability, and can be used in systems such as high temperature, strong acid, strong base, and strong oxidation media; it has excellent compatibility and can be widely used in various pH value ranges, and is often applied in fields such as detergents, latexes, coatings, pigment additives, and emulsifiers. Since the electronegativity of fluorine atoms in the fluoroalkyl chain is very high and the electron-withdrawing ability is strong, the present invention finds that using sodium perfluorooctanoate can synergistically act with the acidic solution, which is beneficial for the smooth migration of low pH proteins to the collection holes.

[0016] Compared with the prior art, the present invention has the following technical effects:

[0017] Through the collection solution developed by the present invention and based on the MauriceFlex system, the chemical migration and complete collection of low pI protein charge variant components can be achieved, while avoiding the hydrolysis and destruction of proteins by low pH solutions. The collected components have a relatively high purity and can be used for further in-depth characterization and research of each component peak. Detailed implementation mode

[0018] The present invention will be further illustrated by way of examples below, but the present invention is not limited to the scope of the described examples. The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0019] Example 1 (0.05% Tween 80, 0.2% sodium perfluorooctanoate, acetic acid aqueous solution with pH 2.5):

[0020] The collection solution formula is 0.05% Tween 80 in mass-volume ratio, 0.2% sodium perfluorooctanoate in mass-volume ratio, and acetic acid aqueous solution with pH 2.5.

[0021] Preparation process: Weigh 0.01 g of Tween 80 and 0.04 g of sodium perfluorooctanoate and place them in a beaker. After dissolving with 20 mL of acetic acid aqueous solution with pH 2.5, stir with a glass rod and perform ultrasonic treatment. After mixing, it can be used. Take 30 μL of the collection solution and add it to wells 1B1 to 1H12 of a 96-well plate (numbered 1) in sequence.

[0022] Prepare another 20 mL of acetic acid aqueous solution with pH 2.5. Take 30 μL of the acetic acid aqueous solution (pH 2.5) and add it to wells 1B1 to 1H12 of a 96-well plate (numbered 2) in sequence.

[0023] Sample pretreatment: If the sample buffer contains salt, the sample needs to be ultrafiltered to desalt, then use a micro-spectrophotometer to perform ultraviolet scanning to detect the concentration, and use ultrapure water to dilute the desalted sample to a concentration of 2.0 mg / mL.

[0024] Preparation of the sample for the instrument: Take 10 μL of the sample, 10 μL of 200 mM IDA, 36 μL of ultrapure water, 35 μL of 1% MC, 1 μL of amphoteric electrolyte 3 - 10, 3 μL of amphoteric electrolyte 5 - 8, 2 μL of pI Marker 3.21, and 3 μL of pI Marker 7.05 into a 1.5 mL centrifuge tube, with a total volume of 100 μL. Prepare 1 parallel sample for the instrument. After centrifuging the 2 samples for the instrument at 12000×g for 5 min, take 90 μL and add it to the 96-well plate (numbered 1) at position 1A1; take 90 μL and add it to the 96-well plate (numbered 2) at position 1A1.

[0025] Preparation of the Maurice Flex cartridge: Place the cartridge flat with the electrode chamber facing up. Add 2 mL of the anolyte (0.08 M H3PO4, 0.1% methyl cellulose) to the anode chamber, and cover it with a red rubber stopper. Place the cartridge into the instrument.

[0026] Maurice Flex instrument method settings: sample chamber temperature 10°C; focusing Period 1, 500V, 10 min; focusing Period 2, 1000V, 10 min; focusing Period 3, 1500V, 25 min; migration module, 1000V, 25 min; collection module, 1000V, 60 min, and set to move in a serpentine pattern from wells 1B1 to 1H12 on a 96-well plate when collecting icIEF components. Conduct icIEF component collection experiments using 96-well plates (number 1) and 96-well plates (number 2) to run the Maurice Flex sequence respectively.

[0027] Confirmation of icIEF collected component purity: Based on the wells where the protein main peak components are located as predicted and analyzed by the software, re-conduct icIEF experiments on the main peak components of the 96-well plate (number 1) and the 96-well plate (number 2) respectively to confirm the purity.

[0028] Experimental results:

[0029] Table 1 icIEF purity results of Example 1

[0030] Collection liquid formulation Purity of the main protein peak component (Area%) 0.05% Tween 80, 0.2% sodium perfluorooctanoate, acetic acid aqueous solution with pH 2.5 90% Acetic acid aqueous solution with pH 2.5 40%

[0031] Discussion: Using an aqueous acetic acid solution with pH 2.5 can achieve the collection of low pI proteins. And by using 0.05% Tween 80 and 0.2% sodium perfluorooctanoate simultaneously, the aqueous acetic acid solution with pH 2.5 can not only completely collect the charge variant components of low pI proteins, but also this formulation protects the protein variants under low pH conditions, and the icIEF purity is 50% higher than that of the formulation of aqueous acetic acid solution with pH 2.5 (without protective agents).

[0032] Example 2 (0.05% Tween 80, 0.5% sodium perfluorooctanoate, aqueous acetic acid solution with pH 2.5):

[0033] The collection solution formulation is 0.05% Tween 80 by mass-volume ratio, 0.5% sodium perfluorooctanoate by mass-volume ratio, and an aqueous acetic acid solution with pH 2.5.

[0034] Preparation process: Weigh 0.01 g of Tween 80 and 0.1 g of sodium perfluorooctanoate respectively and place them in a beaker. Dissolve them with 20 mL of aqueous acetic acid solution with pH 2.5, then stir with a glass rod and perform ultrasonic treatment. After mixing, it can be used. Take 30 μL of the collection solution respectively and add them into wells 1B1 to 1H12 of the 96-well plate (number 3) in sequence.

[0035] Sample pretreatment: If the sample buffer contains salt, the sample needs to be ultrafiltered to desalt, then use a micro-spectrophotometer to perform ultraviolet scanning to detect the concentration, and dilute the desalted sample to a concentration of 2.0 mg / mL with ultrapure water.

[0036] Preparation of sample for machine injection: Take 10 μL of sample, 10 μL of 200 mM IDA, 36 μL of ultrapure water, 35 μL of 1% MC, 1 μL of ampholyte 3 - 10, 3 μL of ampholyte 5 - 8, 2 μL of pI Marker 3.21, and 3 μL of pI Marker 7.05 and put them into a 1.5 mL centrifuge tube. The total volume is 100 μL. After centrifuging the sample for machine injection at 12000×g for 5 min, take 90 μL and add it to a 96 - well plate (number 3) at position 1A1.

[0037] Preparation of Maurice Flex cartridge: Place the cartridge flat with the electrode chamber facing up. Add 2 mL of anolyte (0.08M H3PO4, 0.1% methyl cellulose) to the anodic chamber and cover it with a red rubber stopper. Put the cartridge into the instrument.

[0038] MauriceFlex instrument method settings: Sample chamber temperature 10°C; Focusing Period 1, 500V, 10 min; Focusing Period 2, 1000V, 10 min; Focusing Period 3, 1500V, 25 min; Migration module, 1000V, 25 min; Collection module, 1000V, 60 min, and set the icIEF component collection to move in a serpentine pattern from well 1B1 to well 1H12 on the 96 - well plate. Run the Maurice Flex sequence for the icIEF component collection experiment.

[0039] Confirmation of purity of icIEF collected components: According to the well predicted by software analysis where the main protein peak component is located, perform the icIEF experiment again on the main peak component of the 96 - well plate (number 3) to confirm the purity.

[0040] Experimental results:

[0041] Table 2 icIEF purity results of Example 2

[0042] Collection liquid formulation Purity of the main protein peak component (Area%) 0.05% Tween 80, 0.5% sodium perfluorooctanoate, acetic acid aqueous solution with pH 2.5 90%

[0043] Discussion: Further use 0.5% sodium perfluorooctanoate in the collection liquid formula. The electron - withdrawing ability is enhanced, which can cooperate with the function of the acidic collection liquid, facilitating the migration of each charge variant component to the cathode end and into the collection wells, making the collection of low - pI protein charge variant components more complete. At the same time, it protects the protein variants under low - pH conditions, and the icIEF purity is 30% higher than the formula without sodium perfluorooctanoate.

[0044] Example 3 (0.05% Tween 80, acetic acid aqueous solution with pH 2.5):

[0045] The collection solution formulation is Tween 80 with a mass-volume ratio of 0.05% and an acetic acid aqueous solution with a pH of 2.5.

[0046] Preparation process: Weigh 0.01 g of Tween 80 and place it in a beaker. After dissolving it with 20 mL of acetic acid aqueous solution with a pH of 2.5, stir with a glass rod and perform ultrasonic treatment. After mixing, it can be used. Take 30 μL of the collection solution each and add them sequentially to wells 1B1 to 1H12 of a 96-well plate (numbered 4).

[0047] Sample pretreatment: If the buffer solution of the sample contains salt, the sample needs to be ultrafiltered to desalt, and then the concentration is detected by ultraviolet scanning using a micro-spectrophotometer. The desalted sample is diluted to a concentration of 2.0 mg / mL with ultrapure water.

[0048] Preparation of the sample for the instrument: Take 10 μL of the sample, 10 μL of 200 mM IDA, 36 μL of ultrapure water, 35 μL of 1% MC, 1 μL of ampholyte 3-10, 3 μL of ampholyte 5-8, 2 μL of pI Marker 3.21, and 3 μL of pI Marker 7.05 and place them in a 1.5 mL centrifuge tube. The total volume is 100 μL. Centrifuge the sample for the instrument at 12000×g for 5 min, and then take 90 μL and add it to the 96-well plate (numbered 4) at position 1A1.

[0049] Preparation of the Maurice Flex cartridge: Place the cartridge flat with the electrode chamber facing up. Add 2 mL of the anode solution (0.08 M H3PO4, 0.1% methyl cellulose) to the anode chamber and cover it with a red rubber stopper. Place the cartridge into the instrument.

[0050] Method settings for the MauriceFlex instrument: The sample chamber temperature is 10°C; Focusing Period 1, 500 V, 10 min; Focusing Period 2, 1000 V, 10 min; Focusing Period 3, 1500 V, 25 min; Migration module, 1000 V, 25 min; Collection module, 1000 V, 60 min, and set the icIEF component collection to move in a snake-like pattern from wells 1B1 to 1H12 on the 96-well plate. Run the Maurice Flex sequence to perform the icIEF component collection experiment.

[0051] Confirmation of the purity of the icIEF collected components: According to the prediction and analysis by the software, the well where the main protein peak component is located is determined, and the icIEF experiment is performed again on the main peak component of the 96-well plate (numbered 4) to confirm the purity.

[0052] Experimental results:

[0053] Table 3 icIEF purity results of Example 3

[0054] Collection liquid formulation Purity of the main protein peak component (Area%) 0.05% Tween 80, acetic acid aqueous solution with pH 2.5 60%

[0055] Discussion: Using 0.05% Tween 80 in an aqueous acetic acid solution at pH 2.5 can help maintain the native conformation of the protein, prevent pH-induced denaturation, and contribute to improving the stability of the charge variant components of low pI proteins in acidic collection solutions. The icIEF purity is 20% higher than that of the formulation in Example 1 (aqueous acetic acid solution at pH 2.5).

[0056] Example 4 (0.2% sodium perfluorooctanoate, aqueous acetic acid solution at pH 2.5):

[0057] The collection solution formulation is an aqueous acetic acid solution containing 0.2% sodium perfluorooctanoate by mass / volume ratio and having a pH of 2.5.

[0058] Preparation process of the collection solution: Weigh 0.04 g of sodium perfluorooctanoate and place it in a beaker. Dissolve it with 20 mL of aqueous acetic acid solution at pH 2.5, stir with a glass rod and sonicate, and it can be used after mixing. Take 30 μL of the collection solution respectively and add them into wells 1B1 to 1H12 of a 96-well plate (number 5) in sequence.

[0059] Sample pretreatment: If the sample buffer contains salt, the sample needs to be ultrafiltered to remove salt, then the concentration is detected by ultraviolet scanning using a micro-spectrophotometer, and the desalted sample is diluted to a concentration of 2.0 mg / mL with ultrapure water.

[0060] Preparation of the sample for the instrument: Take 10 μL of the sample, 10 μL of 200 mM IDA, 36 μL of ultrapure water, 35 μL of 1% MC, 1 μL of ampholyte 3 - 10, 3 μL of ampholyte 5 - 8, 2 μL of pI Marker 3.21, and 3 μL of pI Marker 7.05 into a 1.5 mL centrifuge tube, with a total volume of 100 μL. After centrifuging the sample for the instrument at 12000×g for 5 min, take 90 μL and add it to the 96-well plate (number 5) at position 1A1.

[0061] Preparation of the Maurice Flex cartridge: Place the cartridge flat with the electrode chamber facing up, add 2 mL of anolyte (0.08M H3PO4, 0.1% methyl cellulose) to the anodic chamber, and cover it with a red rubber stopper. Put the cartridge into the instrument.

[0062] MauriceFlex instrument method settings: sample chamber temperature 10°C; focusing period 1, 500V, 10 min; focusing period 2, 1000V, 10 min; focusing period 3, 1500V, 25 min; migration module, 1000V, 25 min; collection module, 1000V, 60 min. The icIEF fraction collection was set to a serpentine pattern from wells 1B1 to 1H12 on a 96-well plate. The MauriceFlex sequence for the icIEF fraction collection experiment was run using a 96-well plate (number 5).

[0063] Confirmation of the purity of the collected components by icIEF: Based on the well where the main peak component of the protein is located, the main peak component of the 96-well plate (No. 5) is subjected to icIEF experiment again to confirm the purity.

[0064] Experimental results:

[0065] Table 4 icIEF purity results of Example 4

[0066] Collection liquid formulation Purity of the main protein peak component (Area%) 0.2% sodium perfluorooctanoate, acetic acid aqueous solution with pH 2.5 75%

[0067] Discussion: Combining the icIEF purity tests of Examples 1, 3, and 4, it can be demonstrated that a collection solution containing 0.2% sodium perfluorooctanoate and 0.05% Tween 80 by mass ratio has a better effect. Compared with the use of Tween 80 or sodium perfluorooctanoate alone, the combination of the two can significantly improve the separation and migration of proteins, and is more suitable for the collection and preservation of icIEF components of low-pI proteins.

[0068] Experimental conclusion: Example 1 is the optimal ratio of the collection solution suitable for the separation and migration of low pI protein icIEF components, which is: 0.05% Tween 80 by weight volume, 0.2% sodium perfluorooctanoate by weight volume, and acetic acid aqueous solution with a pH of 2.5.

[0069] As the cathode end solution during the MauriceFlex system collection, it can complete the chemical migration of low pI protein charge variant components, thereby achieving complete collection and high-purity preservation of charge variants.

[0070] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A low pI protein charge variant collection solution applicable to MauriceFlex, characterized in that, The composition of the collection liquid is: Tween 80 with a mass-volume ratio of 0.03 - 0.07%, perfluorooctanoic acid sodium with a mass-volume ratio of 0.2% - 0.5%, and the rest is an acetic acid aqueous solution with a pH of 2 - 3.

2. The low pI protein charge variant collection solution applicable to MauriceFlex according to claim 1, characterized in that, The composition of the collection liquid is: Tween 80 with a mass-volume ratio of 0.05%, perfluorooctanoic acid sodium with a mass-volume ratio of 0.2%, and the rest is an acetic acid aqueous solution with a pH of 2.

5.

3. A low pI protein charge variant collection solution applicable to MauriceFlex as described in any one of claims 1-2, characterized in that, The preparation method of the collection liquid is: Weigh Tween 80 and perfluorooctanoic acid sodium according to the mass-volume ratio and place them in a beaker, then dissolve them with an acetic acid aqueous solution with a pH of 2.5, stir and perform ultrasonic treatment, and then mix evenly to obtain the collection liquid.

4. Application of the collection liquid according to any one of claims 1 - 2 in collecting low pI protein charge variants in the MauriceFlex system.

5. The application according to claim 4, wherein The pI value of the low pI protein charge variant is less than 7.