A buffer for size exclusion chromatography

Abstract

The application discloses a buffer for volume exclusion chromatography, which is composed of the following components with the following concentrations: 50mM-1000mM sodium iodide, 1mM-100mM buffer salt, water as a solvent, and pH value of 6.0-8.0. The buffer can accurately characterize the physical stability of monoclonal antibodies and antibody conjugated drugs, reduce the non-specific interaction between the protein and the chromatographic column filler, reduce the tailing, improve the column efficiency, is not prone to long bacteria, and improves the work efficiency.

Description

Technical Field

[0001] This invention relates to buffer solutions that can be used in size exclusion chromatography (SEC) for a variety of analytes. Background Technology

[0002] Monoclonal antibodies (mAbs) and related products, such as antibody-drug conjugates (ADCs), are widely used to treat autoimmune diseases, cancer, and as therapeutic agents for transplant rejection. However, due to the inherent structural instability of mAbs, they are susceptible to physical or chemical changes during production, storage, and transportation, including exposure to high temperatures, stirring, shaking, and surface adsorption. These changes can alter the mAb structure, leading to aggregate formation and altering the biological activity of the drug protein. In some cases, this can even result in immunogenicity, endangering patient lives. Therefore, assessing and monitoring aggregates in mAbs is crucial in the biopharmaceutical field.

[0003] Size exclusion chromatography (SEC), also known as gel permeation chromatography, is one of the most commonly used methods for characterizing monoclonal antibody polymers. SEC separates proteins based on differences in their hydrodynamic radii in solution. By selecting appropriate mobile phase combinations for different analytes, the interaction between proteins and the SEC column packing material can be reduced, resulting in well-separated and highly efficient separation profiles, enabling the separation of mAb polymers, monomers, and fragments.

[0004] Column efficiency refers to the ability of a chromatographic column to separate two substances in a chromatogram. Higher column efficiency results in better separation and peak shape. Column efficiency can be characterized by four chromatographic parameters: plate number (N), resolution (R), full width at half maximum (FWHM), and tailing factor (T). N reflects the sharpness of a peak; generally, a larger N indicates a sharper peak in a specific region, resulting in a better signal-to-noise ratio. R measures the separation between two adjacent chromatographic peaks; the Chinese Pharmacopoeia stipulates that an R value > 1.5. FWHM reflects column efficiency; generally, a smaller FWHM and a larger N indicate better separation. T reflects the degree of peak tailing or leading edge. The Chinese Pharmacopoeia stipulates that when using peak area as a quantitative parameter, general peak tailing or leading edge does not affect the peak area integral, but severe tailing can affect the baseline, peak start / end determination, and the accuracy of peak area integration.

[0005] During SEC analysis, proteins readily interact with the SEC column packing material through electrostatic and hydrophobic interactions. These non-specific interactions can lead to protein adsorption, causing peak tailing, retention time shifts, and reduced resolution, resulting in decreased column efficiency. A common method to reduce electrostatic interactions in SEC is to increase the ionic strength or salt concentration of the mobile phase, which can improve peak symmetry. ArgHCl or NaCl is commonly used as a mobile phase component to reduce electrostatic interactions. However, mobile phases with added ArgHCl are prone to bacterial growth, and bacterial-laden mobile phases can shorten column life after flowing through the column. Conversely, mobile phases with added NaCl, if the NaCl concentration is too high, can increase hydrophobic interactions, leading to protein adsorption, especially for hydrophobic proteins, causing peak tailing or reduced resolution. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a buffer solution for use as a mobile phase in size exclusion chromatography, suitable for SEC analysis of monoclonal antibodies and antibody-drug conjugates. Conventional SEC buffer solutions consist of a combination of a non-buffered salt and a buffered salt; the non-buffered salt in the buffer solution of this invention is selected from an iodide, specifically sodium iodide.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a buffer solution for a mobile phase in size exclusion chromatography, the buffer solution being composed of the following components at the following concentrations: 50 mM to 1000 mM sodium iodide, 1 mM to 100 mM buffer salt, water as the solvent, and a pH value of 6.0 to 8.0.

[0009] The concentration of sodium iodide is 50mM to 1000mM, which can effectively reduce non-specific interactions. More preferably, it is 50mM to 500mM, and most preferably, it is 200mM.

[0010] The pH of the buffer solution used in the size exclusion chromatography mobile phase is adjusted with an aqueous sodium hydroxide solution (usually 0.5-5M).

[0011] Preferably, the pH of the buffer solution used as the mobile phase for size exclusion chromatography is 7.4 ± 0.1.

[0012] Furthermore, the buffer salt is one or a mixture of two of NaH2PO4 and L-histidine hydrochloride (HisHCl), with NaH2PO4 being particularly preferred.

[0013] Preferably, the concentration of the buffer salt is 1 mM to 50 mM.

[0014] The present invention particularly recommends that the buffer solution for the mobile phase used in size exclusion chromatography be composed of the following components at the following concentrations: 200 mM sodium iodide, 20 mM NaH2PO4, with water as the solvent and a pH of 7.4 ± 0.1.

[0015] Secondly, the present invention provides an application of the above-mentioned buffer solution for the mobile phase of size exclusion chromatography in the separation of biomacromolecules.

[0016] In one embodiment of the present invention, the application is on TSKgel G3000SW XL The operation was carried out in a chromatographic column.

[0017] Furthermore, the biomacromolecule is a protein, and in one embodiment of the present invention, it is HS630, HS022, HS626, HS628 or HS627.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention can accurately characterize the physical stability of monoclonal antibodies and antibody-drug conjugates, while reducing non-specific interactions between proteins and chromatographic column packing, reducing tailing, improving column efficiency, reducing bacterial growth, and improving work efficiency. Attached Figure Description

[0020] Figure 1 This is a comparison chart of the SEC separation column efficiency in Example 1;

[0021] Figure 2 The SEC separation results for different concentrations of NaI in Example 2;

[0022] Figure 3 The results of SEC separation in buffer solutions with different pH values ​​in Example 3;

[0023] Figure 4 The results of SEC separation of different concentrations of NaH2PO4 buffer salt in Example 4;

[0024] Figure 5 The SEC separation results of HisHCl in Example 5 are shown. Detailed Implementation

[0025] The present invention will be further illustrated below through specific embodiments. It must be noted that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0026] This invention can accurately characterize the physical stability of monoclonal antibodies and antibody-drug conjugates, while reducing non-specific interactions between proteins and hydrophilic silica packing material in SEC chromatography columns, reducing tailing, improving column efficiency, and increasing work efficiency.

[0027] Example 1: Comparison of column efficiency after SEC injection using conventional methods and the method of this invention.

[0028] The purpose of this embodiment is to compare the column efficiency differences between the traditional method and the method of the present invention, using NaI as a buffer component.

[0029] Methods: Prepare the corresponding buffer solution as the mobile phase, and inject the prepared sample for SEC analysis.

[0030] SEC mobile phase 1 (conventional method 1): 20 mM NaH2PO4 and 200 mM NaCl, pH = 7.4

[0031] SEC mobile phase 2 (conventional method 2): 20 mM NaH2PO4 and 200 mM ArgHCl, pH = 7.4

[0032] SEC mobile phase 3 (method of this invention): 20 mM NaH2PO4 and 200 mM NaI, pH = 7.4

[0033] Preparation method of SEC mobile phase 1: Weigh out NaH2PO4 with a final concentration of 20mM and NaCl with a final concentration of 200mM and pour them into the same beaker. Add a certain amount of pure water to dissolve them. Adjust the pH to 7.4 with 5M NaOH and then make up the volume to 500mL. Filter the solution using a 0.22μm filter membrane, sonicate it in an ultrasonic instrument, and then remove it for use.

[0034] Preparation method of SEC mobile phase 2: Weigh 20 mM NaH2PO4 and 200 mM ArgHCl into the same beaker, add a certain amount of pure water to dissolve, adjust the pH to 7.4 with 5 M NaOH, and then make up to 500 mL. Filter with a 0.22 μm filter membrane, sonicate in an ultrasonic instrument, and then take it out for use.

[0035] Preparation method of SEC mobile phase 3: Weigh 20 mM NaH2PO4 and 200 mM NaI into the same beaker, add a certain amount of pure water to dissolve, adjust the pH to 7.4 with 5 M NaOH, and make up to 500 mL. Filter with a 0.22 μm filter membrane, sonicate in an ultrasonic instrument, and then remove for use.

[0036] The samples to be tested consisted of one ADC drug (HS630) and four monoclonal antibodies (HS022, HS626, HS628, and HS627), all provided by Zhejiang Hisun Pharmaceutical Co., Ltd., hereinafter the same.

[0037] Sample preparation method: Dilute the samples with ultrapure water to 1 mg / mL, briefly vortex and blow until well mixed, centrifuge and collect the supernatant, and add them to different liquid chromatography injection bottles for injection.

[0038] Liquid chromatography injection correlation settings:

[0039] Flow rate: 0.5 mL / min; Wavelength: 280 nm; Column temperature: 25 °C; Injection pan temperature: 5–8 °C; Injection volume: 50 μL; Isocratic elution: 30 min

[0040] Column type: TSKgel G3000SW XL

[0041] Liquid Chromatography Instrument Model: Agilent 1260

[0042] The injection sequence is as follows: SEC mobile phase 1 (containing NaCl) is used to analyze the above-mentioned 1 ADC drug and 4 monoclonal antibodies according to the above liquid phase injection settings. After the analysis is completed, SEC mobile phase 2 (containing ArgHCl) is used to analyze the above-mentioned 1 ADC drug and 4 monoclonal antibodies according to the same liquid phase injection settings. After the analysis is completed, SEC mobile phase 3 (containing NaI) is used to analyze the above-mentioned 1 ADC drug and 4 monoclonal antibodies according to the same liquid phase injection settings.

[0043] The results are as follows Figure 1 As shown, from the perspective of column efficiency characterization parameters, the mobile phase using NaI as the buffer component has better resolution R (the higher the better to a certain extent), plate number N (the higher the better to a certain extent), and full width at half maximum (FWHM) than the mobile phase using NaCl. At the same time, it can reduce the effect of tailing. For HS630, HS022, HS628, and HS627, it is better than the mobile phase using ArgHCl.

[0044] Example 2: Effect of different NaI concentrations in the buffer solution on SEC analysis results

[0045] The purpose of this embodiment is to explore the suitable concentration range of NaI.

[0046] Methods: Prepare the corresponding buffer solution as the mobile phase, and inject the prepared sample for SEC analysis.

[0047] SEC mobile phase: 20 mM NaH₂PO₄ and different concentrations of NaI, pH = 7.4

[0048] The final concentrations of NaI were 0 mM, 1 mM, 5 mM, 10 mM, 50 mM, 100 mM, 200 mM, 500 mM, and 1000 mM.

[0049] Table 1. Composition of mobile phases with different NaI concentrations

[0050]

[0051] Take 9 beakers, numbered ① to ⑨. Weigh out NaH2PO4 with a final concentration of 20mM and NaI with different final concentrations, and pour them into the corresponding numbered beakers. Add a certain amount of pure water to dissolve them. Adjust the pH to 7.4 with 5M NaOH, and then bring the volume to 500mL. Filter each beaker using a 0.22μm filter membrane, and then sonicate them in an ultrasonic instrument before removing them for later use.

[0052] The analytes were an ADC drug (HS630) and a monoclonal antibody (HS022).

[0053] Sample preparation method: Dilute the samples with ultrapure water to 1 mg / mL, briefly vortex and blow until well mixed, centrifuge and collect the supernatant, and add them to different liquid chromatography injection bottles for injection.

[0054] Liquid chromatography injection correlation settings:

[0055] Flow rate: 0.5 mL / min; Wavelength: 280 nm; Column temperature: 25 °C; Injection pan temperature: 5–8 °C; Injection volume: 50 μL; Isocratic elution: 30 min

[0056] Column type: TSKgel G3000SW XL

[0057] Liquid Chromatography Instrument Model: Agilent 1260

[0058] The injection sequence is as follows: Use the SEC mobile phase numbered ① to analyze the above-mentioned ADC drug and monoclonal antibody according to the above liquid phase injection settings. After the analysis is completed, switch to the SEC mobile phase numbered ② and analyze the above-mentioned ADC drug and monoclonal antibody according to the same liquid phase injection settings. Continue in this manner until all 9 mobile phases have been analyzed.

[0059] The results are as follows Figure 2 As shown, the results indicate that different concentrations of NaI have different effects on column efficiency in the analytical chromatogram. When using NaI concentrations of 0 mM to 10 mM for injection analysis, the column efficiency is not ideal, and the chromatogram cannot accurately display the peak shape. When using NaI concentrations of 50 mM to 1000 mM, the peak shape can be accurately displayed. The preferred NaI concentration is 50 mM to 1000 mM. Since 1000 mM is too high, 50 mM to 500 mM is more preferred.

[0060] Example 3: Effect of different pH buffer solutions on SEC analysis results

[0061] The purpose of this embodiment is to explore a suitable pH value.

[0062] Methods: Prepare the corresponding buffer solution as the mobile phase, and inject the prepared sample for SEC analysis.

[0063] SEC mobile phase: 20 mM NaH2PO4 and 200 mM NaI, under different pH conditions, where the pH is 6.0, 6.7, 7.4 and 8.0.

[0064] Table 2. Composition of mobile phase at different pH values

[0065]

[0066] Take four beakers, numbered ① to ④. Weigh out NaH2PO4 with a final concentration of 20 mM and NaI with a final concentration of 200 mM respectively, and pour them into the corresponding numbered beakers. Add a certain amount of pure water to dissolve them. Adjust the pH to 6.0, 6.7, 7.4 and 8.0 respectively using 5 M NaOH. Then, make up the volume to 500 mL. Filter each beaker using a 0.22 μm filter membrane, and sonicate them in an ultrasonic instrument before removing them for use.

[0067] The analytes were an ADC drug (HS630) and a monoclonal antibody (HS022).

[0068] Sample preparation method: Dilute the samples with ultrapure water to 1 mg / mL, briefly vortex and blow until well mixed, centrifuge and collect the supernatant, and add them to different liquid chromatography injection bottles for injection.

[0069] Liquid chromatography injection correlation settings:

[0070] Flow rate: 0.5 mL / min; Wavelength: 280 nm; Column temperature: 25 °C; Injection pan temperature: 5–8 °C; Injection volume: 50 μL; Isocratic elution: 30 min

[0071] Column type: TSKgel G3000SW XL

[0072] Liquid Chromatography Instrument Model: Agilent 1260

[0073] The injection sequence is as follows: Use the SEC mobile phase numbered ① to analyze the above-mentioned ADC drug and monoclonal antibody according to the above liquid phase injection settings. After the analysis is completed, switch to the SEC mobile phase numbered ② and analyze the above-mentioned ADC drug and monoclonal antibody according to the same liquid phase injection settings. Continue in this manner until all four mobile phases have been analyzed.

[0074] The results are as follows Figure 3As shown, the results indicate that when using mobile phases containing 20 mM NaH2PO4 and 200 mM NaI at different pH values, the liquid chromatography chromatograms obtained in the pH range of 6.0–8.0 show good column efficiency. Therefore, the preferred pH range is 6.0–8.0.

[0075] Example 4: Effect of different concentrations of NaH2PO4 buffer salt in the buffer solution on SEC analysis results

[0076] The purpose of this embodiment is to explore the suitable concentration range of NaH2PO4 buffer salt.

[0077] Methods: Prepare the corresponding buffer solution as the mobile phase, and inject the prepared sample for SEC analysis.

[0078] SEC mobile phase: different concentrations of NaH₂PO₄ and 200 mM NaI, pH = 7.4

[0079] The final concentrations of NaH2PO4 were 0 mM, 1 mM, 5 mM, 10 mM, 20 mM, 50 mM, and 100 mM, respectively.

[0080] Table 3. Composition of mobile phases with different concentrations of NaH2PO4

[0081]

[0082] Take 7 beakers, numbered ① to ⑦. Weigh out NaH2PO4 of different final concentrations and NaI of 200mM final concentration and pour them into the corresponding numbered beakers. Add a certain amount of pure water to dissolve them. Except for ①, which uses 50mM NaOH to adjust the pH to 7.4, the other mobile phases are adjusted to pH 7.4 using 5M NaOH. Make up to 500mL for each mobile phase. Filter each mobile phase using a 0.22μm filter membrane. After sonication in an ultrasonic instrument, remove the mobile phases for later use.

[0083] The analytes were an ADC drug (HS630) and a monoclonal antibody (HS022).

[0084] Sample preparation method: Dilute the samples with ultrapure water to 1 mg / mL, briefly vortex and blow until well mixed, centrifuge and collect the supernatant, and add them to different liquid chromatography injection bottles for injection.

[0085] Liquid chromatography injection correlation settings:

[0086] Flow rate: 0.5 mL / min; Wavelength: 280 nm; Column temperature: 25 °C; Injection pan temperature: 5–8 °C; Injection volume: 50 μL; Isocratic elution: 30 min

[0087] Column type: TSKgel G3000SWXL

[0088] Liquid Chromatography Instrument Model: Agilent 1260

[0089] The injection sequence is as follows: Use the SEC mobile phase numbered ① to analyze the above-mentioned ADC drug and monoclonal antibody according to the above liquid phase injection settings. After the analysis is completed, switch to the SEC mobile phase numbered ② and analyze the above-mentioned ADC drug and monoclonal antibody according to the same liquid phase injection settings. Continue in this manner until all 7 mobile phases have been analyzed.

[0090] The results are as follows Figure 4 As shown, the results indicate that using a mobile phase containing different concentrations of NaH2PO4 and 200 mM NaI at pH 7.4 for analysis, the liquid chromatography chromatograms obtained with NaH2PO4 concentrations ranging from 0 mM to 100 mM showed good column efficiency. Since it is difficult to adjust the pH when NaH2PO4 is at 0 mM, a concentration of 1 mM to 100 mM is preferred. Since adjusting the pH when NaH2PO4 is at 100 mM requires a higher concentration of alkali, a concentration range of 1 mM to 50 mM of NaH2PO4 is preferred.

[0091] Example 5: The effect of different types of buffer salts in the buffer solution on SEC analysis results

[0092] The purpose of this embodiment is to investigate whether normal SEC analysis can be performed and normal spectra can be generated after replacing NaH2PO4 with HisHCl, thereby broadening the range of buffer salts.

[0093] Methods: Prepare the corresponding buffer solution as the mobile phase, and inject the prepared sample for SEC analysis.

[0094] SEC mobile phase: 20 mM HisHCl and 200 mM NaI, pH = 7.4.

[0095] Preparation method: Weigh HisHCl with a final concentration of 20mM and NaI with a final concentration of 200mM into the same beaker, add a certain amount of pure water to dissolve, adjust the pH to 7.4 with 5M NaOH, and make up to 500mL. Filter with a 0.22μm filter membrane, sonicate in an ultrasonic instrument, and then remove for use.

[0096] The samples to be tested consisted of one ADC drug (HS630) and one monoclonal antibody (HS022).

[0097] Sample preparation method: Dilute the samples with ultrapure water to 1 mg / mL, briefly vortex and blow until well mixed, centrifuge and collect the supernatant, and add them to different liquid chromatography injection bottles for injection.

[0098] Liquid chromatography injection correlation settings:

[0099] Flow rate: 0.5 mL / min; Wavelength: 280 nm; Column temperature: 25 °C; Injection pan temperature: 5–8 °C; Injection volume: 50 μL; Isocratic elution: 30 min

[0100] Column type: TSKgel G3000SW XL

[0101] Liquid Chromatography Instrument Model: Agilent 1260

[0102] The injection sequence is as follows: using SEC mobile phase, analyze one ADC drug and one monoclonal antibody according to the above liquid phase injection settings.

[0103] The results are as follows Figure 5 As shown, the results indicate that the liquid chromatography chromatograms obtained using a mobile phase containing HisHCl and 200 mM NaI at pH 7.4 exhibit good column efficiency.

Claims

1. The application of a buffer solution for the mobile phase in size exclusion chromatography in the separation of biological macromolecules, characterized in that... The buffer solution for the mobile phase used in size exclusion chromatography consists of the following components at the following concentrations: 50 mM to 1000 mM sodium iodide, 1 mM to 100 mM buffer salt, water as the solvent, and a pH of 6.0 to 8.

0. The buffer salt is one or a mixture of two of NaH2PO4 and L-histidine hydrochloride.

2. The application as described in claim 1, characterized in that: The concentration of sodium iodide is 50 mM to 500 mM.

3. The application as described in claim 2, characterized in that: The concentration of sodium iodide is 200 mM.

4. The application as described in claim 1, characterized in that: The buffer solution used as the mobile phase in size exclusion chromatography has a pH of 7.4 ± 0.

1.

5. The application as described in claim 1, characterized in that: The concentration of the buffer salt is 1 mM to 50 mM.

6. The application as described in claim 1, characterized in that: The buffer solution for the mobile phase used in size exclusion chromatography consists of the following components at the following concentrations: 200 mM sodium iodide, 20 mM NaH2PO4, with water as the solvent and a pH of 7.4 ± 0.

1.

7. The application as described in claim 1, characterized in that: The application was performed on a TSKgel G3000SWXL column.

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