Formula capable of collecting low pI protein charge variant component and application thereof

Abstract

The invention belongs to the field of biology, and particularly relates to a formula capable of collecting low pI protein charge variant components and application of the formula. The collection liquid comprises 0.03-0.07% by mass volume of Tween 80, not less than 0.2% by mass volume of sodium perfluorooctanoate, and the balance of an acetic acid aqueous solution with the pH value of 2-3. According to the collection liquid developed by the invention, based on a MauriceFlex system, chemical migration and complete collection of low-pI protein charge variant components can be completed, meanwhile, hydrolysis damage of a low-pH-value solution to proteins is avoided, and the collected components are relatively high in purity and can be used for further performing deep characterization research on peaks of the components.

Description

Technical Field

[0001] The invention belongs to the biological field, and in particular relates to a formula capable of collecting low pI protein charge variant components and an application thereof. Background Art

[0002] Biomacromolecule protein drugs (monoclonal antibodies, bispecific antibodies, antibody-drug conjugates, recombinant proteins, fusion proteins and other types of samples) may affect the stability, safety and effectiveness of their products due to charge variants formed by various modifications such as deamidation, N-terminal modification, isomerization, sialylation and C-terminal shearing. Full-column imaging capillary isoelectric focusing (icIEF) can be used as a powerful tool to characterize protein charge heterogeneity using the molecular pI (i.e., isoelectric point) characteristics. The principle is to apply a DC voltage at both ends of the capillary, and the carrier amphoteric electrolyte in the tube can form a pH gradient within a certain range. The protein moves to the anode or cathode according to its charge until it stops at a certain pH value (i.e., isoelectric point pI) with a net charge of 0. Finally, the protein is focused into a very narrow segment, thereby achieving the purpose of separation.

[0003] As a component collection instrument based on icIEF, MauriceFlex can separate and collect charge variant components within one day, and can be flexibly used for further characterization analysis, such as post-translational modification analysis, glycoform analysis, activity analysis and other applications. The collection principle is that after the charge variants of the sample are separated and focused, the original cathode end solution sodium hydroxide is automatically replaced by ammonium acetate, so that the pH gradient in the capillary changes, so that the focused charge variant proteins can be moved back to the cathode end through chemical migration. Finally, the protein variant components chemically migrate to the collection module end (96-well plate) in order from high to low pI, first the high pI Marker, followed by the alkaline variant component, the main peak component of the protein, the acidic variant component, and finally the low pI Marker. The cathode end solution in the chemical migration should ensure that the low pI Marker component completes chemical migration and collection, so as to determine the distribution area of ​​the collected components in the 96-well plate according to the fluorescent tracing of the high and low pI Markers, so as to carry out the next step of icIEF component confirmation.

[0004] Low pI proteins refer to proteins with a low isoelectric point (pI), which are usually positively charged 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, the pH of low pI protein charge variant components after isoelectric focusing is lower than 7, so it is difficult for the separated components to completely migrate to the collection wells, and the purity does not meet the requirements of subsequent analysis, which is a difficult problem in the collection and characterization of low pI protein charge variant components. Summary of the invention

[0005] In the MauriceFlex system, the cathode end solution usually uses ammonium acetate solution. During the experiment, the ammonium acetate solution plays a role in changing the pH gradient in the capillary, allowing 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 choice of cathode end solution is limited by equipment design and experimental requirements. According to the general principles of isoelectric focusing technology, the cathode end solution needs to be able to maintain a stable pH gradient and support the focusing and separation of proteins.

[0006] The pI value of low pI protein charge variants is usually less than 7. In the MauriceFlex system, in view of the problem that ammonium acetate solution cannot effectively migrate and collect low pI protein charge variants, the present invention develops a formula solution suitable for the MauriceFlex system. The formula solution is used as the cathode end solution during collection in the MauriceFlex system to complete the chemical migration of low pI protein charge variant components, thereby achieving complete collection of charge variants and high-purity preservation.

[0007] In order to achieve the above-mentioned purpose, the present invention provides the following technical solutions: The first aspect of the present invention discloses a low pI protein charge variant collection solution suitable for MauriceFlex, the collection solution comprising: 0.03-0.07% Tween 80 by mass volume, not less than 0.2% sodium perfluorooctanoate by mass volume, and the rest being an acetic acid aqueous solution with a pH of 2-3.

[0008] Preferably, the collection liquid is composed of: 0.05% by weight volume of Tween 80, 0.2% by weight volume of sodium perfluorooctanoate, and the rest is an acetic acid aqueous solution with a pH of 2.5.

[0009] The preparation method of the collection liquid is as follows: Tween 80 and sodium perfluorooctanoate are weighed in a mass-to-volume ratio and placed in a beaker, then dissolved with an acetic acid aqueous solution with a pH of 2.5, stirred and ultrasonically treated, and then mixed to obtain the collection liquid.

[0010] The second aspect of the present invention discloses the use of the above-mentioned collection solution in collecting low pI protein charge variants in the MauriceFlex system.

[0011] The low pI protein charge variant has a pI value of less than 7.

[0012] The present invention discloses a novel collection liquid formula: an acidic collection liquid containing a protein stabilizer (suitable for the migration and collection of low pI protein charge variants), wherein the proportions of the components are: 0.05% Tween 80 by mass volume, 0.2% sodium perfluorooctanoate C7F15COONa by mass volume, and an acetic acid aqueous solution with a pH of 2.5.

[0013] Tween 80 can help maintain the pH stability of the solution and reduce the impact of pH fluctuations on low pI proteins. It can also bind to the hydrophobic region of the protein, reduce the hydrophobic interactions between protein molecules, and help maintain its natural conformation, thereby inhibiting the aggregation and precipitation of low pI proteins.

[0014] Sodium perfluorooctanoate C 7 F 15 COONa is a fluorinated anionic surfactant, which is a fluorinated carbon chain that replaces all hydrogen atoms with fluorine atoms. Its main structure is a non-polar fluorinated carbon chain and a polar group carboxylic acid. Since the bond energy of the carbon-fluorine bond is very strong, it has high surface activity, high thermodynamics and chemical stability, and can be used in systems such as high temperature, strong acid, strong base, and strong oxidizing medium; it has excellent compatibility and can be widely used in various pH ranges, and is often used in detergents, latex, coatings, pigment additives, emulsifiers and other fields. Since the electronegativity of the fluorine atom in the fluoroalkyl chain is very high and has a strong electron-withdrawing ability, the present invention finds that the use of sodium perfluorooctanoate can cooperate with the effect of the acidic solution, which is conducive to the smooth migration of low pH proteins to the collection hole.

[0015] Compared with the prior art, the present invention has the following technical effects: Through the collection liquid developed by the present invention and based on the MauriceFlex system, the low pI protein charge variant components can complete chemical migration and complete collection, while avoiding the hydrolysis damage of the protein by the low pH solution. The collected components have high purity and can be used for further in-depth characterization research on each component peak. DETAILED DESCRIPTION

[0016] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0017] Example 1 (0.05% Tween 80, 0.2% sodium perfluorooctanoate, pH 2.5 acetic acid aqueous solution): The collection liquid formula is 0.05% Tween 80 by mass volume, 0.2% sodium perfluorooctanoate by mass volume, and acetic acid aqueous solution with a pH of 2.5.

[0018] Preparation process: Weigh 0.01 g Tween 80 and 0.04 g sodium perfluorooctanoate respectively and place them in a beaker. Dissolve them in 20 mL of pH 2.5 acetic acid aqueous solution, stir with a glass cup and ultrasonicate. Mix well and use. Take 30 μL of the collected solution and add them to wells 1B1 to 1H12 of a 96-well plate (number 1) in sequence.

[0019] Separately, prepare 20 mL of pH 2.5 acetic acid aqueous solution, and add 30 μL of the acetic acid aqueous solution (pH 2.5) to wells 1B1 to 1H12 of a 96-well plate (No. 2) in sequence.

[0020] Sample pretreatment: If the sample buffer contains salt, the sample needs to be desalted by ultrafiltration, and then the concentration is detected by ultraviolet scanning using a micro-spectrophotometer. The desalted sample is diluted with ultrapure water to a concentration of 2.0 mg / mL.

[0021] Preparation of samples for the machine: 10 μL sample, 10 μL 200 mM IDA, 36 μL ultrapure water, 35 μL 1% MC, 1 μL ampholyte 3-10, 3 μL ampholyte 5-8, 2 μL pI Marker 3.21, 3 μL pI Marker 7.05 were placed in a 1.5 mL centrifuge tube, with a total volume of 100 μL. Prepare 1 sample for the machine in parallel. Centrifuge the 2 samples at 12000×g for 5 min, take 90 μL and add to the 96-well plate (number 1), position 1A1; take 90 μL and add to the 96-well plate (number 2), position 1A1.

[0022] Maurice Flex cartridge preparation: Lay the cartridge flat with the electrode slot facing up and add 2 mL of anode solution (0.08 MH 3 PO 4 , 0.1% methylcellulose), cover with a red rubber stopper. Place the cassette into the instrument.

[0023] MauriceFlex instrument method settings: sample chamber temperature 10°C; focus period 1, 500V, 10 min; focus period 2, 1000V, 10 min; focus period 3, 1500V, 25 min; migration module, 1000V, 25 min; collection module, 1000V, 60 min, and set to move in a serpentine pattern from 1B1 to 1H12 on the 96-well plate when collecting icIEF components. Maurice Flex sequences were run using 96-well plates (No. 1) and 96-well plates (No. 2) for icIEF component collection experiments.

[0024] Confirmation of purity of icIEF collected components: Based on the wells where the main peak components of the protein are located based on the software prediction analysis, icIEF experiments are performed again on the main peak components of the 96-well plate (No. 1) and the 96-well plate (No. 2) to confirm the purity.

[0025] Experimental results: Table 1 Example 1 icIEF purity results Collection fluid formula Purity of main protein peak component (Area%) 0.05% Tween 80, 0.2% sodium perfluorooctanoate, pH 2.5 acetic acid solution 90% pH 2.5 acetic acid aqueous solution 40% Discussion: The use of pH 2.5 acetic acid aqueous solution can achieve the collection of low pI proteins, and the use of 0.05% Tween 80 and 0.2% sodium perfluorooctanoate, pH 2.5 acetic acid aqueous solution can not only completely collect the low pI protein charge variant components, but also protect the protein variants under low pH conditions. The icIEF purity is 50% higher than that of the pH 2.5 acetic acid aqueous solution (without protective agent).

[0026] Example 2 (0.05% Tween 80, 0.5% sodium perfluorooctanoate, pH 2.5 acetic acid aqueous solution): The collection solution formula is 0.05% Tween 80 by mass volume, 0.5% sodium perfluorooctanoate by mass volume, and acetic acid aqueous solution with a pH of 2.5.

[0027] Preparation process: Weigh 0.01 g Tween 80 and 0.1 g sodium perfluorooctanoate respectively in a beaker, dissolve in 20 mL of pH 2.5 acetic acid aqueous solution, stir with a glass cup and ultrasonicate, mix well and then use. Take 30 μL of the collected solution and add it to wells 1B1 to 1H12 of a 96-well plate (number 3) in sequence.

[0028] Sample pretreatment: If the sample buffer contains salt, the sample needs to be desalted by ultrafiltration, and the concentration is detected by ultraviolet scanning using a micro-spectrophotometer. The desalted sample is diluted with ultrapure water to a concentration of 2.0 mg / mL.

[0029] Sample preparation for the machine: 10 μL sample, 10 μL 200 mM IDA, 36 μL ultrapure water, 35 μL 1% MC, 1 μL ampholyte 3-10, 3 μL ampholyte 5-8, 2 μL pI Marker 3.21, 3 μL pI Marker 7.05 were placed in a 1.5 mL centrifuge tube, with a total volume of 100 μL. Centrifuge the sample at 12000 × g for 5 min, and take 90 μL and add it to the 96-well plate (No. 3), position 1A1.

[0030] Maurice Flex cartridge preparation: Lay the cartridge flat with the electrode slot facing up and add 2 mL of anode solution (0.08 MH 3 PO 4 , 0.1% methylcellulose), cover with a red rubber stopper. Place the cassette into the instrument.

[0031] MauriceFlex instrument method settings: sample chamber temperature 10°C; focus period 1, 500V, 10 min; focus period 2, 1000V, 10 min; focus period 3, 1500V, 25 min; migration module, 1000V, 25 min; collection module, 1000V, 60 min, and set icIEF component collection to move in a serpentine pattern from wells 1B1 to 1H12 on a 96-well plate. Run the Maurice Flex sequence for icIEF component collection experiments.

[0032] Confirmation of purity of icIEF collected components: Based on the well where the main peak component of the protein is located, the icIEF experiment is performed again on the main peak component of the 96-well plate (No. 3) to confirm the purity.

[0033] Experimental results: Table 2 Example 2 icIEF purity results Collection fluid formula Purity of main protein peak component (Area%) 0.05% Tween 80, 0.5% sodium perfluorooctanoate, pH 2.5 acetic acid solution 90% Discussion: Further use of 0.5% sodium perfluorooctanoate in the collection fluid formula enhances the electron-withdrawing ability, which can synergize with the effect of the acidic collection fluid, facilitate the migration of each charge variant component to the cathode end into the collection well, making the collection of low pI protein charge variant components more complete. At the same time, it protects protein variants under low pH conditions. The purity of icIEF is 30% higher than that of the formula without sodium perfluorooctanoate.

[0034] Example 3 (0.05% Tween 80, pH 2.5 acetic acid aqueous solution): The collection solution is a 0.05% Tween 80 solution by mass volume and an acetic acid solution with a pH of 2.5.

[0035] Preparation process: Weigh 0.01 g of Tween 80 into a beaker, dissolve it in 20 mL of pH 2.5 acetic acid aqueous solution, stir it with a glass cup and ultrasonicate it, mix it well and it is ready for use. Take 30 μL of the collected solution and add it to wells 1B1 to 1H12 of a 96-well plate (number 4) in sequence.

[0036] Sample pretreatment: If the sample buffer contains salt, the sample needs to be desalted by ultrafiltration, and the concentration is detected by ultraviolet scanning using a micro-spectrophotometer. The desalted sample is diluted with ultrapure water to a concentration of 2.0 mg / mL.

[0037] Sample preparation for the machine: 10 μL sample, 10 μL 200 mM IDA, 36 μL ultrapure water, 35 μL 1% MC, 1 μL ampholyte 3-10, 3 μL ampholyte 5-8, 2 μL pI Marker 3.21, 3 μL pI Marker 7.05 were placed in a 1.5 mL centrifuge tube, with a total volume of 100 μL. Centrifuge the sample at 12000 × g for 5 min, and take 90 μL and add it to the 96-well plate (No. 4), position 1A1.

[0038] Maurice Flex cartridge preparation: Lay the cartridge flat with the electrode slot facing up and add 2 mL of anode solution (0.08 MH 3 PO 4 , 0.1% methylcellulose), cover with a red rubber stopper. Place the cassette into the instrument.

[0039] MauriceFlex instrument method settings: sample chamber temperature 10°C; focus period 1, 500V, 10 min; focus period 2, 1000V, 10 min; focus period 3, 1500V, 25 min; migration module, 1000V, 25 min; collection module, 1000V, 60 min, and set icIEF component collection to move in a serpentine pattern from wells 1B1 to 1H12 on a 96-well plate. Run the Maurice Flex sequence for icIEF component collection experiments.

[0040] Confirmation of purity of icIEF collected components: Based on the well where the main peak component of the protein is located, the icIEF experiment is performed again on the main peak component of the 96-well plate (No. 4) to confirm the purity.

[0041] Experimental results: Table 3 Example 3 icIEF purity results Collection fluid formula Purity of main protein peak component (Area%) 0.05% Tween 80, pH 2.5 acetic acid solution 60% Discussion: The use of 0.05% Tween 80 in pH 2.5 acetic acid aqueous solution can help maintain the native conformation of the protein, prevent pH-induced denaturation, and help improve the stability of the charge variant components of low pI proteins in the acidic collection solution. The icIEF purity is 20% higher than the formulation in Example 1 (pH 2.5 acetic acid aqueous solution).

[0042] Example 4 (0.2% sodium perfluorooctanoate, pH 2.5 acetic acid aqueous solution): The collection liquid formula is 0.2% sodium perfluorooctanoate by mass volume and an acetic acid aqueous solution with a pH of 2.5.

[0043] Preparation of collection solution: Weigh 0.04 g of sodium perfluorooctanoate into a beaker, dissolve it in 20 mL of pH 2.5 acetic acid aqueous solution, stir with a glass cup and ultrasonicate, mix well and then use. Take 30 μL of the collection solution and add it to wells 1B1 to 1H12 of a 96-well plate (No. 5) in sequence.

[0044] Sample pretreatment: If the sample buffer contains salt, the sample needs to be desalted by ultrafiltration, and the concentration is detected by ultraviolet scanning using a micro-spectrophotometer. The desalted sample is diluted with ultrapure water to a concentration of 2.0 mg / mL.

[0045] Sample preparation for the machine: 10 μL sample, 10 μL 200 mM IDA, 36 μL ultrapure water, 35 μL 1% MC, 1 μL ampholyte 3-10, 3 μL ampholyte 5-8, 2 μL pI Marker 3.21, 3 μL pI Marker 7.05 were placed in a 1.5 mL centrifuge tube, with a total volume of 100 μL. Centrifuge the sample at 12000 × g for 5 min, and take 90 μL and add it to the 96-well plate (No. 5), position 1A1.

[0046] Maurice Flex cartridge preparation: Lay the cartridge flat with the electrode slot facing up and add 2 mL of anode solution (0.08 MH 3 PO 4 , 0.1% methylcellulose), cover with a red rubber stopper. Place the cassette into the instrument.

[0047] MauriceFlex instrument method settings: sample chamber temperature 10°C; Focus Period 1, 500V, 10 min; Focus Period 2, 1000V, 10 min; Focus 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 for icIEF fraction collection. A Maurice Flex sequence was run using a 96-well plate (number 5) for icIEF fraction collection experiments.

[0048] Confirmation of purity of icIEF collected components: Based on the well where the main peak component of the protein is located, the icIEF experiment is performed again on the main peak component of the 96-well plate (No. 5) to confirm the purity.

[0049] Experimental results: Table 4 Example 4 icIEF purity results Collection fluid formula Purity of main protein peak component (Area%) 0.2% sodium perfluorooctanoate, pH 2.5 acetic acid solution 75% Discussion: Combining the icIEF purity detection of Example 1, Example 3 and Example 4, it can be proved that the collection liquid using a mass ratio of 0.2% sodium perfluorooctanoate to 0.05% Tween 80 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.

[0050] Experimental conclusion: Example 1 is the optimal ratio of the collection solution suitable for the separation and migration of low pI protein icIEF components, and its formula is: 0.05% Tween 80 by mass volume, 0.2% sodium perfluorooctanoate by mass volume, and acetic acid aqueous solution with a pH of 2.5.

[0051] As the cathode end solution during the collection of the MauriceFlex system, it can complete the chemical migration of low pI protein charge variant components, thereby achieving complete collection and high-purity preservation of charge variants.

[0052] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A low pI protein charge variant collection solution suitable for MauriceFlex, characterized in that: The composition of the collected liquid is: 0.03-0.07% Tween 80 by mass volume, not less than 0.2% sodium perfluorooctanoate by mass volume, and the rest is acetic acid aqueous solution with a pH of 2-3.

2. A low pI protein charge variant collection solution suitable for MauriceFlex as claimed in claim 1, characterized in that: The collection solution is composed of: 0.05% Tween 80 by weight volume, 0.2% sodium perfluorooctanoate by weight volume, and the rest is acetic acid aqueous solution with a pH of 2.

5.

3. A low pI protein charge variant collection solution suitable for MauriceFlex according to any one of claims 1 to 2, characterized in that: The preparation method of the collection liquid is as follows: Tween 80 and sodium perfluorooctanoate are weighed according to a mass-to-volume ratio and placed in a beaker, then dissolved with an acetic acid aqueous solution with a pH of 2.5, stirred and ultrasonically treated, and then mixed to obtain the collection liquid.

4. Use of the collection solution according to any one of claims 1 to 2 for collecting low pI protein charge variants in a MauriceFlex system.

5. The use according to claim 4, characterized in that The low pi protein charge variant has a pi value of less than 7.