阅读说明：本技术 可用于离子交换色谱和质谱分析的方法、组合物和试剂盒 (Methods, compositions and kits useful for ion exchange chromatography and mass spectrometry ) 是由 王�琦 M·A·劳伯 S·伊波利蒂 俞映清 于 2020-03-06 设计创作，主要内容包括：本公开涉及可用于多种分析物的增强pH梯度阳离子交换色谱的方法、组合物和试剂盒。在各个方面,本公开涉及色谱洗脱试剂盒,该色谱洗脱试剂盒包括(a)具有第一pH并包含第一浓度的第一有机酸盐的第一水性缓冲溶液和(b)具有第二pH并包含第二浓度的第一有机酸盐的第二水性缓冲溶液,其中第一有机酸盐包括第一有机酸铵盐,其中第二pH大于第一pH,并且其中第二浓度大于第一浓度。在各个方面,本公开涉及在色谱分离中使用此类水性缓冲溶液的方法。(The present disclosure relates to methods, compositions, and kits useful for enhanced pH gradient cation exchange chromatography of a variety of analytes. In various aspects, the present disclosure relates to a chromatography elution kit comprising (a) a first aqueous buffer solution having a first pH and comprising a first concentration of a first organic acid salt and (b) a second aqueous buffer solution having a second pH and comprising a second concentration of the first organic acid salt, wherein the first organic acid salt comprises a first organic acid ammonium salt, wherein the second pH is greater than the first pH, and wherein the second concentration is greater than the first concentration. In various aspects, the disclosure relates to methods of using such aqueous buffer solutions in chromatographic separations.)

1. A chromatography elution kit comprising (a) a first aqueous buffer solution having a first pH and comprising a first concentration of a first organic acid salt and (b) a second aqueous buffer solution having a second pH and comprising a second concentration of the first organic acid salt, wherein the first organic acid salt comprises a first organic acid ammonium salt, wherein the second pH is greater than the first pH, and wherein the second concentration is greater than the first concentration.

2. The chromatography elution kit of claim 1, wherein each of the first aqueous buffer solution and the second aqueous buffer solution comprises up to 20% of a second organic acid ammonium salt different from the first organic acid ammonium salt.

3. The chromatography elution kit of claim 1, wherein the first organic acid salt consists essentially of the first organic acid ammonium salt.

4. The chromatography elution kit of claim 1, wherein the first ammonium salt of an organic acid is the only ammonium salt of an organic acid in each of the first aqueous buffer solution and the second aqueous buffer solution.

5. The chromatography elution kit of any one of claims 1 to 4, wherein the first aqueous buffer solution and the second aqueous buffer solution each have a concentration of sodium and potassium of less than 100 ppb.

6. The chromatography elution kit according to any one of claims 1 to 5, wherein the first aqueous buffer solution has a pH between 4 and 6 and a concentration of the first ammonium salt of an organic acid between 20mM and 120mM, wherein the second aqueous buffer solution has a pH between 7.5 and 9.0 and a concentration of the first ammonium salt of an organic acid between 100mM and 300 mM.

7. The chromatography elution kit according to any one of claims 1 to 5, wherein the first aqueous buffer solution has a pH between 4 and 6 and a concentration of the first ammonium salt of an organic acid between 30mM to 100mM, and wherein the second aqueous buffer solution has a pH between 7.5 and 9.0 and a concentration of the first ammonium salt of an organic acid between 100mM to 200 mM.

8. The chromatography elution kit of any one of claims 1 to 7, wherein the first aqueous buffer solution has a conductivity in the range of 0.1 milliSiemens (mS) to 10mS, and wherein the second aqueous buffer solution has a conductivity in the range of 3mS to 100 mS.

9. The chromatography elution kit of any one of claims 1 to 8, wherein a plot of pH versus volume percentage of the first aqueous buffer solution relative to the total volume of the binary mixture of the first aqueous buffer solution and the second aqueous buffer solution is linear.

10. The chromatography elution kit of any one of claims 1 to 9, wherein a plot of conductivity versus volume percentage of the first aqueous buffer solution relative to the total volume of the binary mixture of the first aqueous buffer solution and the second aqueous buffer solution is linear.

11. The chromatography elution kit of any one of claims 1 to 10, wherein a plot of conductivity versus volume percentage of the first aqueous buffer solution relative to the total volume of a binary mixture of the first aqueous buffer solution and the second aqueous buffer solution does not exhibit a negative slope.

12. The chromatography elution kit of any one of claims 1 to 5, further comprising instructions for diluting each of the first and second aqueous buffer solutions, resulting, when followed by the instructions, in a diluted first aqueous buffer solution having a pH between 4 and 6 and a concentration of the first organic acid ammonium salt between 20mM and 120mM and a diluted second aqueous buffer solution having a pH between 7.5 and 9.0 and a concentration of the first organic acid ammonium salt between 100mM and 300 mM.

13. The chromatography elution kit of claim 12, wherein when the instructions for diluting each of the first and second aqueous buffer solutions are followed, a diluted first aqueous buffer solution having a pH between 4.5 and 5.5 and a concentration of the first organic acid ammonium salt between 40mM and 100mM and a diluted second aqueous buffer solution having a pH between 8.0 and 8.5 and a concentration of the first organic acid ammonium salt between 120mM and 200mM result.

14. The chromatography elution kit according to any one of claims 12 to 13, wherein the diluted first aqueous buffer solution has a conductivity in the range of 0.1mS to 10mS, and wherein the diluted second aqueous buffer solution has a conductivity in the range of 3mS to 100 mS.

15. The chromatography elution kit of any one of claims 12 to 14, wherein a plot of pH versus volume percentage of the diluted first aqueous buffer solution relative to the total volume of the binary mixture of the diluted first aqueous buffer solution and the diluted second aqueous buffer solution is linear.

16. The chromatography elution kit of any one of claims 12 to 15, wherein a plot of conductivity versus volume percentage of the diluted first aqueous buffer solution relative to the total volume of the binary mixture of the diluted first aqueous buffer solution and the diluted second aqueous buffer solution is linear.

17. The chromatography elution kit of any one of claims 12 to 16, wherein a plot of conductivity versus volume percentage of the diluted first aqueous buffer solution relative to the total volume of a binary mixture of the diluted first aqueous buffer solution and the diluted second aqueous buffer solution does not exhibit a negative slope.

18. The chromatography elution kit of any one of claims 1-17, wherein the first organic acid ammonium salt is formed from an organic acid anion selected from formate, acetate, difluoroacetate, trifluoroacetate, propionate, butyrate, carbonate, bicarbonate, oxalate, malonate, succinate, maleate, glutarate, glycolate, lactate, malate, citrate, or gluconate, and an ammonium cation selected from ammonium, monoalkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium.

19. The chromatography elution kit of any one of claims 1-17, wherein the first organic acid ammonium salt is selected from ammonium formate, ammonium acetate, tetramethylammonium formate, tetramethylammonium acetate, triethylammonium acetate, or triethylammonium formate.

20. The chromatography elution kit of any one of claims 1-19, wherein each of the first aqueous buffer solution and the second aqueous buffer solution is contained in a polymer container.

21. The chromatography elution kit of any one of claims 1-20, wherein each of the first aqueous buffer solution and the second aqueous buffer solution further comprises a miscible organic co-solvent at a concentration ranging from 1% to 50%.

22. The chromatographic elution kit of claim 21, wherein the miscible organic cosolvent is selected from the group consisting of acetonitrile, methanol, ethanol, n-propanol, isopropanol.

23. The chromatography elution kit of any one of claims 1-23, further comprising ion exchange chromatography material.

24. The chromatography elution kit of claim 23, comprising a separation device comprising a housing comprising an inlet and an outlet and configured to accept and retain the ion exchange chromatography material.

25. The chromatography elution kit according to any one of claims 23 to 24, wherein the ion exchange chromatography material is a cation exchange chromatography material.

26. The chromatography elution kit of claim 25, wherein the cation exchange chromatography material comprises carboxylate groups, sulfonate groups, or both.

27. A method for analyzing a sample comprising a plurality of analytes, the method comprising:

loading the sample onto an ion exchange chromatography material, thereby binding the plurality of analytes to the ion exchange chromatography material, and eluting the plurality of analytes from the ion exchange chromatography material with a mobile phase comprising varying amounts of: (a) a first aqueous buffer solution having a pH between 4.0 and 6.0 and comprising a first organic acid salt at a concentration between 20mM and 120mM, and (b) a second aqueous buffer solution having a pH between 7.5 and 9.0 and comprising the first organic acid salt at a concentration between 100mM and 300mM to separate at least some of the plurality of analytes in a concentration gradient separation manner, and wherein the first organic acid salt comprises a first organic acid ammonium salt.

28. The method of claim 27, wherein each of the first aqueous buffer solution and the second aqueous buffer solution comprises up to 20% of a second organic acid ammonium salt different from the first organic acid ammonium salt.

29. The method of claim 27, wherein the first organic acid salt consists essentially of the first organic acid ammonium salt.

30. The method of claim 27, wherein the first ammonium salt of an organic acid is the only ammonium salt of an organic acid in each of the first aqueous buffer solution and the second aqueous buffer solution.

31. The method of any one of claims 27 to 30, wherein the volume percentage of the first aqueous buffer solution decreases during the elution, and wherein the volume percentage of the second aqueous buffer solution increases during the elution.

32. The method according to any one of claims 27 to 30, wherein the volume percentage of the first aqueous buffer solution is reduced from 100% to 0% during the elution, and wherein the volume percentage of the second aqueous buffer solution is increased from 0% to 100% during the elution.

33. The method of any one of claims 27 to 32, wherein there is a linear increase in the volume percentage of the second aqueous buffer solution during the elution.

34. The method of any one of claims 27 to 33, wherein the first aqueous buffer solution and the second aqueous buffer solution are mixed to form the mobile phase using an automated system.

35. The method of any one of claims 27 to 31, wherein the mobile phase further comprises different amounts of the first aqueous buffer solution, the second aqueous buffer solution, and water.

36. The method of claim 35, wherein the first aqueous buffer solution, the second aqueous buffer solution, and the water are mixed using an automated system.

37. The method of any one of claims 27 to 36, further comprising detecting the plurality of analytes.

38. The method of claim 37, wherein the plurality of analytes are detected using electrospray ionization mass spectrometry.

39. The method of any one of claims 27-38, wherein the plurality of analytes comprises a plurality of biomolecules.

40. The method of any one of claims 27-38, wherein the plurality of analytes comprises a plurality of proteins.

41. The method of any one of claims 27 to 38, wherein the plurality of analytes comprises a plurality of mAb species.

42. The method according to claim 41, wherein the ion exchange chromatography material is a cation exchange chromatography material, and wherein the plurality of mAb species have pI values in the range of 6 to 10.

Technical Field

The present disclosure relates to methods, compositions, and kits useful for enhanced gradient ion exchange chromatography of multiple analytes.

Background

Ion exchange chromatography (IEX) has been widely used for the separation and analysis of proteins. In IEX, proteins are separated based on their ionic interaction with oppositely charged moieties present on a stationary phase. Under conditions where the pH is below the isoelectric point (pI), the protein is positively charged. As the mobile phase pH increases, the protein gradually loses positive charge and becomes neutral, then becomes negatively charged. In cation exchange chromatography, positively charged proteins are adsorbed into a negatively charged stationary phase. These proteins can be eluted by a salt or pH gradient mechanism. In salt gradient separation, proteins with more charge require higher concentrations of salt, whereas in pH gradient techniques, proteins with different pI can be separated by changes in the pH of the mobile phase.

In practice, 1EX is used to analyze many different types of biomolecules and many different types of proteins. A great deal of information can be gleaned from these separations, particularly when they are applied to the analysis of protein therapeutics. As one type of protein therapeutic, monoclonal antibodies (mabs) have been used to treat a number of diseases. As an inherent result of production, post-translational modification (PTM) of protein therapeutics needs to be carefully characterized, as minor structural differences can have a significant impact on drug stability, efficacy, and efficacy. Some modifications, such as deamidation, sialylation, C-terminal lysine variation, etc., cause changes in the net charge of the protein. IEX is a valuable means of detecting and monitoring the formation of these unique protein variants.

However, due to the widespread use of high ionic strength non-volatile buffers in both salt and pH gradient methods, most examples of detailed analysis rely on time consuming off-line fraction collection or cumbersome multidimensional LC Mass Spectrometry (MS). More ideally, it would be desirable to achieve an optimized, robust IEX separation based on a volatile mobile phase composition that facilitates direct coupling with mass spectrometry. To date, two exemplary IEX-MS analyses have been published. Leblanc and colleagues developed a double salt/pH gradient method for charge variant characterization of mabs using the middle-up approach. Leblanc, y.; ramon, c.; bihreau, n.; chevreux, g., "characterization of charge variants of monoclonal antibodies by ion exchange chromatography coupled in-line with natural mass spectrometry: case studies after long-term storage at +5 ℃ (Charge variants catalysis of a monomeric antibody by and exchange chromatography coupled on-line to negative mass spectrometry: Case study after a long-term storage at +5 ℃ C) ", Journal of chromatography. B, Analytical technologies in the biological and life sciences 2017, 1048, 130. sup. th. f. in FIGS. In this work, the IdeS digestion subunits of mabs were directly analyzed with IEX-MS under native conditions using volatile ammonium formate and ammonium acetate at pH range 3.9 to 7.4. In practice, the chromatographic resolution and quality of the MS obtained with this technique has proven to be undesirable, and the mobile phase composition is unnecessarily complicated by having more components than necessary, as it continues with both ammonium formate and ammonium acetate. Finally, the abundance of the salt adduct impairs the interpretability of the mass spectrum from the method and it depends on high salt concentrations. Recently, Fassl and coworkers developed a pH gradient process based on ammonium bicarbonate, acetic acid and ammonium hydroxide. Fussl, f.; cook, k.; scheffller, k.; farrell, a.; mittermayr, s.; bones, J., "Charge variant analysis of Monoclonal antibodies by High Resolution Native Mass Spectrometry Using Direct Coupled pH Gradient Cation Exchange Chromatography (Charge variable analysis of Monoclonal antibodies Using Direct Coupled pH Gradient Chromatography pH Gradient Exchange Change Chromatography to High-Resolution Natural Mass Spectrometry.)" Analytical Chemistry 2018, 90(7), 4669-. The mobile phase system provides relatively constant conductivity over a pH range of 5.3 to 10.18 to analyze intact mabs with different pI. However, the gradient needs to be fine-tuned for each analyte and since the mobile phase contains ammonium bicarbonate, carbon dioxide adducts are easily observed in the resulting MS spectra.

Disclosure of Invention

The present disclosure provides novel methods, mobile phase compositions, and kits that facilitate gradient ion exchange chromatography of analytes. The methods, mobile phase compositions, and kits of the present disclosure are advantageous because they are compatible with MS detection of analytes.

In various aspects, the present disclosure relates to a chromatography elution kit comprising (a) a first aqueous buffer solution having a first pH and comprising a first concentration of a first organic acid salt and (b) a second aqueous buffer solution having a second pH and comprising a second concentration of the first organic acid salt, wherein the first organic acid salt comprises a first organic acid ammonium salt, wherein the second pH is greater than the first pH, and wherein the second concentration is greater than the first concentration.

In some embodiments that may be used in combination with any of the above aspects, each of the first aqueous buffer solution and the second aqueous buffer solution comprises less than 20%, less than 10%, less than 5%, or less than 1% of an ammonium salt of a second organic acid that is different from the ammonium salt of the first organic acid.

In some embodiments that may be used in combination with any of the above aspects, the first organic acid salt consists essentially of the first organic acid ammonium salt.

In some embodiments that may be used in combination with any of the above aspects, the first organic acid ammonium salt is the only organic acid ammonium salt in each of the first aqueous buffer solution and the second aqueous buffer solution.

In some embodiments that may be used in combination with any of the above aspects and embodiments, each of the first aqueous buffer solution and the second aqueous buffer solution does not comprise ammonium bicarbonate.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the first aqueous buffer solution and the second aqueous buffer solution each have a sodium and potassium concentration of less than 100ppb, advantageously less than 20 ppb.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the first aqueous buffer solution has a pH of between 4 and 6, more advantageously between 4.5 and 5.5, and a concentration of between 20mM and 120mM, more advantageously between 40mM and 100 mM.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the second aqueous buffered solution has a pH of between 7.5 and 9.0, more advantageously between 8 and 8.5, and a concentration of between 100mM and 300mM, more advantageously between 120mM and 200 mM.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the first aqueous buffer solution has a conductivity in a range of 0.1 millisiemens (mS) to 10mS, and the second aqueous buffer solution has a conductivity in a range of 3mS to 100 mS.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the plot of pH versus volume percent of the first aqueous buffer solution relative to the total volume of the binary mixture of the first aqueous buffer solution and the second aqueous buffer solution is linear.

As used herein, a plot of one variable versus another is "linear" when a linear least squares regression analysis yields a determination coefficient (R2) value of at least 0.90, more typically at least 0.95.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the plot of conductivity versus volume percent of the first aqueous buffer solution relative to the total volume of the binary mixture of the first aqueous buffer solution and the second aqueous buffer solution is linear.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the plot of conductivity versus volume percentage of the first aqueous buffer solution relative to the total volume of the binary mixture of the first aqueous buffer solution and the second aqueous buffer solution does not exhibit a negative slope.

In some embodiments that may be used in conjunction with any of the above aspects and embodiments, the chromatographic elution kit further comprises instructions for diluting each of the first and second aqueous buffer solutions, resulting when followed by said instructions in a diluted first aqueous buffer solution having a pH of between 4 and 6, more advantageously between 4.5 and 5.5, and a concentration of the first organic acid ammonium salt of between 20mM and 120mM, more advantageously between 40mM and 100mM, and a diluted second aqueous buffer solution having a pH of between 7.5mM and 9.0mM, more advantageously between 8mM and 8.5mM, and a concentration of the first organic acid ammonium salt of between 100mM and 300mM, more advantageously between 120mM and 200 mM.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the diluted first aqueous buffer solution has a conductivity in a range of 0.1mS to 10mS, and the diluted second aqueous buffer solution has a conductivity in a range of 3mS to 100 mS.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the plot of pH versus volume percent of the diluted first aqueous buffer solution relative to the total volume of the binary mixture of the diluted first aqueous buffer solution and the diluted second aqueous buffer solution is linear.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the plot of conductivity versus volume percent of the diluted first aqueous buffer solution relative to the total volume of the binary mixture of the diluted first aqueous buffer solution and the diluted second aqueous buffer solution is linear.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the plot of conductivity versus volume percent of the diluted first aqueous buffer solution relative to the total volume of the binary mixture of the diluted first aqueous buffer solution and the diluted second aqueous buffer solution does not exhibit a negative slope.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the first organic acid ammonium salt is formed from: (a) an organic acid anion selected from formate, acetate, difluoroacetate, trifluoroacetate, propionate, butyrate, carbonate, bicarbonate, oxalate, malonate, succinate, maleate, glutarate, glycolate, lactate, malate, citrate or gluconate, and (b) an ammonium cation selected from ammonium, monoalkylammonium, dialkylammonium, trialkylammonium or tetraalkylammonium.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the first organic acid ammonium salt is selected from ammonium formate, ammonium acetate, tetramethylammonium formate, tetramethylammonium acetate, triethylammonium acetate, or triethylammonium formate.

In some embodiments that may be used in conjunction with any of the above aspects and embodiments, each of the first aqueous buffer solution and the second aqueous buffer solution is contained in a glass-free container, e.g., a polymer container, such as those formed from polyolefins, such as polyethylene (e.g., HDPE), or fluoropolymers, such as Polytetrafluoroethylene (PTFE).

In some embodiments that may be used in combination with any of the above aspects and embodiments, each of the first aqueous buffer solution and the second aqueous buffer solution further comprises a miscible organic co-solvent at a concentration ranging from 1% to 50%. For example, the miscible organic co-solvent is selected from acetonitrile, methanol, ethanol, n-propanol or isopropanol, among other possibilities.

In some embodiments that may be used in conjunction with any of the above aspects and embodiments, the first aqueous buffer solution and the second aqueous buffer solution may be formulated with trace amounts of a biocide (including, but not limited to, about 100ppm to 400ppm chloroform) for extended shelf life.

In some embodiments that may be used in conjunction with any of the above aspects and embodiments, the first aqueous buffer solution and the second aqueous buffer solution may be packaged with an oxygen absorbing material for the purpose of extending shelf life.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the chromatography elution kit further comprises an ion exchange chromatography material.

In some embodiments that may be used in conjunction with any of the above aspects and embodiments, the chromatography elution kit further comprises an ion exchange chromatography material and a separation device (e.g., a column, a sample preparation device, a centrifugation/spin column, or a microextraction plate) comprising a housing having an inlet and an outlet, the housing configured to receive and retain the ion exchange chromatography material.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the chromatography elution kit further comprises a cation exchange chromatography material. The cation exchange chromatography material can comprise, for example, carboxylate groups, sulfonate groups, or both.

In other aspects, the present disclosure relates to methods for analyzing a sample comprising a plurality of analytes, the method comprising: loading a sample onto the ion exchange chromatography material according to any of the above aspects and embodiments, thereby binding the plurality of analytes to the ion exchange chromatography material; and eluting the plurality of analytes from the ion exchange chromatography material with a mobile phase comprising different amounts of (a) the first aqueous buffer solution according to any of the above aspects and embodiments and (b) the second aqueous buffer solution according to any of the above aspects and embodiments, thereby separating at least some of the plurality of analytes.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the volume percentage of the first aqueous buffer solution decreases during elution and the volume percentage of the second aqueous buffer solution increases during elution.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the volume percentage of the first aqueous buffer solution is reduced from 100% to 0% during elution, and the volume percentage of the second aqueous buffer solution is increased from 0% to 100% during elution.

In some embodiments that may be used in combination with any of the above aspects and embodiments, there is a linear increase in the volume percentage of the second aqueous buffer solution during elution.

In some embodiments that may be used in conjunction with any of the above aspects and embodiments, an automated system is used to mix the first aqueous buffer solution and the second aqueous buffer solution to form the mobile phase.

In some embodiments that may be used in combination with any of the above aspects and embodiments, the method includes varying the amounts of the first aqueous buffer solution, the second aqueous buffer solution, and the water. In some of these embodiments, an automated system may be used to mix the first aqueous buffer solution, the second aqueous buffer solution, and water.

In some embodiments that may be used in conjunction with any of the above aspects and embodiments, the method may further comprise detecting a plurality of analytes. In some of these embodiments, the plurality of analytes can be detected using mass spectrometry techniques, such as electrospray ionization mass spectrometry.

In some embodiments that may be used in conjunction with any of the above aspects and embodiments, the plurality of analytes comprises a plurality of biomolecules.

In various embodiments that can be used in conjunction with any of the above aspects and embodiments, the plurality of analytes can include a plurality of peptides or a plurality of proteins, including a plurality of mAb proteins, a plurality of non-mAb proteins, a plurality of fusion proteins, a plurality of Antibody Drug Conjugates (ADCs), and the like. In certain embodiments, the plurality of analytes may comprise a plurality of proteins having pI values in the range of 6 to 10, among other possible values.

Drawings

FIGS. 1A-1D show the normalized MS response from SEC-MS, observed as a function of mobile phase pH and ionic strength. Showing that from Size Exclusion Chromatography (SEC) -MSNormalized MS signal response for IdeS digested (shown in figure 1A) and intact NIST mAb (shown in figure 1B) with 50mM ammonium acetate mobile phase at different pH values. Also shown are normalized MS signal responses on SEC-MS with different ionic strengths of pH 5 ammonium acetate mobile phase, IdeS digested (shown in figure 1C) and intact NIST mAb (shown in figure 1D). For figures 1A-1D, normalized MS signal response of IdeS digested NIST mAb was calculated as the percentage ratio of the total peak area of m/z 4245.8 ± 1.5 and 3376.7 ± 1.5 in the extracted ion chromatogram to the total peak area in the IdeS NISTmAb elution window in the UV chromatogram. Normalized MS signal response of intact NIST mAb was calculated as the percentage ratio of the peak area of m/z 5295.1 ± 1.5 in the extracted ion chromatogram to the total peak area in the elution window of intact NIST mAb in the UV chromatogram. MS Signal response at 4.6mm × 150mm ACQUITY UPLC protein BEH SEC column ((R))1.7 μm) using ACQUITY coupled to a Xevo G2-S QTOF mass spectrometerI-Class System measurement. The separation conditions can be found in example 1. Figures 1A-1D show that an increase in mobile phase pH has a greater effect on the Mass Spectral (MS) signal of the intact and IdeS digested subunits of the monoclonal antibody (mAb) than an increase in ionic strength.

Figures 2A-2C show the effect of pH and ionic strength of the mobile phase on the on-line IEX-MS analysis of mabs. The UV chromatograms of the obtained NIST mAb (shown in figure 2A) along with pH (shown in figure 2B) and conductivity trace (shown in figure 2C) are shown, with the mobile phase consisting of 50mM ammonium formate (pH 3.9) as buffer a and 150mM or 300mM ammonium acetate (pH 8.0 or 9.0) as buffer B. In FIGS. 2A-2C, the acid is shown withChromatograms were obtained on a 2.1X50mm BioResolve SCX mAb column from H-Class Bio System, and pH and conductivity traces were obtained with GE medical monitor pH/C-900. The separation conditions can be found in example 2.

Figures 3A-3C show mobile phases of ammonium formate/acetate versus ammonium acetate alone for on-line IEX-MS analysis of mabs. UV chromatograms (shown in figure 3A) and normalized MS signal responses (shown in figure 3B) of intact NIST mabs, as well as pH and conductivity traces (shown in figure 3C) obtained using a mobile phase system consisting of 40mM ammonium formate, 50mM ammonium acetate (pH 5.0) or 90mM ammonium acetate (pH 5.0) as buffer a, 200mM ammonium acetate (pH 8.2) as buffer B, with a linear gradient of 0% to 100% B, from 1.7 to 20.0 minutes, are shown. In FIGS. 3A-3C, the acid is shown withChromatograms were collected on a 2.1X50mm BioResolve SCX mAb column of H-Class Bio System and pH and conductivity traces were obtained with GE medical monitor pH/C-900, and normalized MS signal responses were measured as the ratio in thousandths of the total peak area in the basal peak chromatogram and UV chromatogram on a 2.1X50mm BioResolve SCX mAb column with ACQUITY coupled to a Xevo G2-S QTOF mass spectrometerI-Class System. The separation conditions can be found in example 2 and example 3.

Figures 4A-4C show additional mobile phase ionic strength and gradient optimization to improve the resolution of intact and IdeS digested mabs for online IEX-MS analysis. A UV chromatogram obtained with a mobile phase consisting of: 90mM ammonium acetate pH 5.0 as buffer A and 200mM ammonium acetate pH 8.4 as buffer B (FIG. 4A), 45mM ammonium acetate pH 5.0 as buffer A and 150mM ammonium acetate pH 8.4 as buffer B (FIG. 4B), or 20mM ammonium acetate pH 5.0 as buffer A and 120mM ammonium acetate pH 8.4 as buffer B (FIG. 4C). In FIGS. 4A-4C, the liquid crystal display panel is shown with ACQUITYChromatograms were obtained on 2.1X50mm BioResolve SCX mAb columns from I-Class System. The separation conditions can be found in example 3.

FIGS. 5A-5C illustrate a method for improving the score for an online IEX-MSMobile phase optimization of the resolution of the intact and IdeS digested mabs. Acquiring a UV chromatogram of intact infliximab and IdeS digested trastuzumab with a mobile phase, wherein the mobile phase consists of the following substances: 25mM ammonium bicarbonate 30mM acetic acid pH 5.3 as buffer A and 10mM ammonium hydroxide in 2mM acetic acid pH 10.18 as buffer B (as shown in FIG. 5A), 50mM ammonium formate pH 3.9 as buffer A and 500mM ammonium acetate pH 7.4 as buffer B (as shown in FIG. 5B), or 45mM ammonium acetate pH 5.0 as buffer A and 150mM ammonium acetate pH 8.4 as buffer B (as shown in FIG. 5C). In FIGS. 5A-5C, the liquid crystal display panel is shown with ACQUITYChromatograms were obtained on 2.1X50mm BioResolve SCX mAb columns from I-Class System. The separation conditions can be found in example 3.

Fig. 6A-6C show the best practice for mobile phase preparation for online IEX-MS analysis of mabs. Mass spectra of IdeS digested infliximab and intact NIST mAb were acquired with a mobile phase consisting of: 90mM ammonium acetate pH 5.0 as buffer A and 200mM ammonium acetate pH 8.4 as buffer B, prepared in glass vials and glass labware with LC/MS grade water (shown in FIG. 6A), or with 0.2 μ M filtered 18.2M Ω water and plastic labware, with pH measurements using a glass electrode filled with 3M potassium chloride (shown in FIG. 6B) and without a glass electrode filled with 3M potassium chloride (shown in FIG. 6C). In FIGS. 6A-6C, ACQUITY coupled to a Xevo G2-S QTOF mass spectrometer was usedI-Class System Mass spectra were acquired on a 2.1X50mm BioResolve SCX mAb column. The separation conditions can be found in example 3.

Detailed Description

Described herein are mobile phases and methods of use thereof that provide for robust and high resolution IEX separation of proteins that can be directly coupled to electrospray ionization mass spectrometry. As can be seen from the following detailed description of certain advantageous embodiments, ammonium salt solutions have been developed.

In particular, the following describes a volatile mobile phase system based on ammonium salt solutions with certain beneficial pH, concentration and/or purity, taking into account the electrospray ionization effect of proteins not associated with ion exchange chromatography. The effects of mobile phase pH and ionic strength were also studied by Size Exclusion Chromatography (SEC) -MS to obtain an orthogonal view of protein ionization efficiency and potential method considerations. The effect of the mobile phase pH was investigated by increasing the pH of the 50mM ammonium acetate mobile phase from 5 to 7 to 9. Intact and IdeS digested NIST mAb (reference material 8671) was studied. Using buffers of different pH, no change in the charge state distribution of IdeS digested or intact NIST mAb was observed. The MS signal response of the IdeS digested NIST mAb was normalized to the total peak areas of m/z 4245.8 + -1.5 and 3376.7 + -1.5 in the extracted ion chromatogram (corresponding to F (ab')₂And (Fc/2)₂The most abundant charge state of the subunit) to the total peak area in the IdeS NIST mAb elution window in the UV chromatogram. The MS signal response of the intact NIST mAb was normalized to the ratio of the peak area of m/z 5295.1 ± 1.5 in the extracted ion chromatogram (which corresponds to the most abundant charge state of the intact NIST mAb) to the total peak area in the elution window of the intact NIST mAb in the UV chromatogram. A major decrease in MS signaling response was observed on both IdeS digested and intact NIST mAb when buffer pH was increased from 5 to 9 (fig. 1A and 1B). Meanwhile, the effect of the ionic strength of the mobile phase was investigated by increasing the concentration of the ammonium acetate mobile phase from 50mM to 300mM while maintaining the pH at 5. No change in charge state distribution was observed on IdeS digested or intact NIST mAb using different concentrations of buffer. Only a slight decrease in signal intensity was observed when the ammonium acetate concentration was increased from 50mM to 300mM (fig. 1C and 1D). These observations support the use of the dual pH/salt gradient method of the present disclosure, as applied to 1EX-MS analysis of IdeS digested and intact mabs. That is, it is believed that the pH gradient only method will exhibit lower MS sensitivity, and the salt gradient only method will require pH adjustment to adapt it to different analytes.

To assess the resolving power of volatile mobile phase systems with ion exchange chromatography, intact and IdeS digested NIST mabs (referenceCoop material 8671), infliximab, and trastuzumab. Peak-to-trough ratio (p/v) values for the primary lysine variant of NIST mAb were calculated from UV chromatograms. Method optimization was performed to determine the pH and ionic strength of the elution buffer solution in the mobile phase system. High pI mAb, NIST mAb (pI 9.2) was monitored using a linear gradient between 50mM ammonium formate pH 3.9 as initial buffer (buffer A) and 150mM or 300mM ammonium acetate pH 8.0 or 9.0 as elution buffer (buffer B)³) Retention and resolution of. The strongest retention and best resolution was observed with the eluent comprising 150mM ammonium acetate at pH 8 as confirmed in the UV chromatogram taken on the 2.1x50mM strong cation exchange stationary phase (fig. 2A). Increasing the pH of 150mM ammonium acetate from 8 to 9 resulted in co-elution of the main peak and the first lysine variant. A linear pH trace was observed using 150mM ammonium acetate pH 8 as eluent, while a sudden pH increase was observed at the retention window of the NIST mAb after titrating the pH of the eluent to 9 (fig. 2B), which is believed to be the most likely cause of poor resolution. Similarly, resolution of NIST mAb was compromised after increasing the ionic strength of ammonium acetate from 150mM to 300 mM. After titrating the pH of 300mM ammonium acetate buffer to 9, a sudden pH increase and a shortened pH linear range were observed. The linear conductivity trace was observed using 150mM or 300mM ammonium acetate pH 8 solution as eluent (fig. 2C), while a slight deviation from linearity was observed in the conductivity trace after titration of the eluent pH to 9. Thus, an advantageous composition of the eluent in the mobile phase system is an ammonium acetate solution having a pH between 7.5 and 9.0, more advantageously between 8 and 8.5, and a concentration between 100mM and 300mM, more advantageously between 120mM and 200 mM.

Further process optimization was performed to determine the most effective pH and ionic strength of the initial buffer solution applied in the mobile phase system. The retention and resolution of NIST mAb was monitored using a mobile phase system based on 40mM ammonium formate 50mM ammonium acetate pH 5.0 (buffer a1) or 90mM ammonium acetate pH 5.0 (buffer a2) and 200mM ammonium acetate pH 8.15 as elution buffer (buffer B). Similar resolution was observed at the same linear gradient from 0% to 100% B from 1.7 min to 20 min (fig. 3A), while retention was stronger with buffer a2 than a 1. The normalized MS signal response was measured as the ratio of the total peak area in the base peak chromatogram taken on a QTOF mass spectrometer to the UV chromatogram measured at 280nm on a 2.1x50mm strong cation exchange column (fig. 3B). Slightly higher MS signal response was observed with buffer a 2. Linear pH traces were observed using buffer a1 or a2 as the initial buffer solution and 200mM ammonium acetate pH 8.2 as the elution buffer solution (fig. 3C), but a broader linear range was observed with buffer a 2. Buffer a1 showed a conductivity slightly higher than a2 as measured with an off-line conductivity meter (9.19 mS at 21.6 ℃ for buffer a1, 8.58mS at 22.9 ℃ for buffer a2), despite the fact that they showed similar on-line conductivity traces. Although comparable LC resolution and MS sensitivity were observed with buffers consisting of a mixture of ammonium formate and ammonium acetate and buffers consisting of ammonium acetate alone, buffer systems consisting of a single ammonium salt offer the advantage of simple and more stringent control of reagent purity. Thus, an advantageous initial buffer solution for the mobile phase system of the present disclosure is ammonium acetate having a pH between 4 and 6, more advantageously between 4.5 and 5.5, and a concentration between 20mM and 120mM, more advantageously between 40mM and 100 mM.

Additional optimization of the mobile phase ionic strength and gradient obtained further improved the resolution of intact and IdeS digested mabs. Although the pH of the initial buffer solution was kept at 5.0 and the pH of the salting-out of the elution buffer was kept at 8.4, it was found that a decrease in the ionic strength of ammonium acetate in the total mobile phase system can lead to an improved chromatographic separation. It has proved particularly effective to reduce the concentration of the initial buffer solution and the elution buffer solution from 90mM and 200mM to 45mM and 150mM, respectively. For example, acidic variants of the major peak of the intact NIST mAb were improved by these changes (fig. 4A and 4B). Similar observations were made for the first major peak of intact infliximab. However, further reduction of the buffer ionic strength to 20mM and 120mM (initial solution compared to eluate) did not show much benefit relative to the resolution of the separation of IdeS digested trastuzumab, intact NIST mAb or intact infliximab (fig. 4C). In summary, well-defined boundaries have been established to construct a mobile phase system for implementing a robust IEX-MS method. Finally, this effort resulted in a simple volatile buffer system that produced linear pH and conductivity traces and provided elution of mabs and mAb subunits with different pI values and retention behavior.

The chromatographic capabilities of the buffer systems and methods described herein are exemplary and this is readily demonstrated by comparison to alternatives. To this end, a study was conducted to compare the buffer system of the present disclosure with a pH gradient buffer based on ammonium bicarbonate, acetic acid and ammonium hydroxide prepared by F ü ssi et al (supra) and the double salt/pH gradient method of Leblanc et al (supra). A direct comparison of intact and IdeS digested mAb charge variant separation using three methods was performed using a 3 μm non-porous sulfonated cation exchange stationary phase. The implementation of the buffer system described by fussl et al failed to resolve intact infliximab (its three main peaks co-eluted) (fig. 5A). This method also failed to provide an accurate distribution of IdeS digested trastuzumab using a universal gradient of 40% to 100% elution solution. Observed from the peak at an unreserved retention time of 0.8 to 1.0 minute corresponds to (Fc/2)₂And only F (ab')₂Weak signal of subunit. The disadvantage of this method is that it requires very careful method optimization for different analytes and it is not designed for analysis of IdeS digested subunits of mabs. In contrast, the Leblanc method was designed to serve as a platform method for analysis of IdeS digested subunits of mabs (fig. 5B). By comparison with the LeBlanc method, it is clearly shown that the best resolution of intact infliximab and IdeS digested trastuzumab is achieved with the composition of the invention (fig. 5C). For the separation of intact infliximab and trastuzumab subunits, a significant increase in resolution was observed.

Finally, the procedure for mobile phase preparation was optimized to improve mass spectral quality. The levels of sodium and potassium adducts in the mass spectra of IdeS digested infliximab and intact NIST mAb were monitored on a strong cation exchange column coupled to a QTOF mass spectrometer using a mobile phase consisting of 90mM ammonium acetate (pH 5.0) as the initial buffer solution (buffer a) and 200mM ammonium acetate (pH 8.4) as the elution buffer solution (buffer B). Significant amounts of sodium adduct were observed with the mobile phase prepared with LC/MS grade water in glass bottles and glass laboratory dishes (fig. 6A). In another case, high levels of potassium adduct were observed in mass spectra collected using mobile phase prepared from 0.2 μ M filtered 18.2M Ω water, plastic lab dishes, and pH measurements were performed using glass electrodes filled with 3M potassium chloride (fig. 6B). Minimal levels of sodium or potassium adducts were achieved with 0.2 μ M filtered 18.2M Ω water, plastic labware and mobile phase prepared without pH measurements (fig. 6C). Advantageously, the application of the glass-free process to the preparation, storage and application of mobile phase concentrates and/or ready-to-use mobile phases minimizes the sodium and potassium adducts and allows easy interpretable mass spectra to be obtained.

Thus, described herein is a buffer system that has been found to provide an attractive means for IEX-MS analysis of proteins, including intact and IdeS digested mabs. A mobile phase solution of the method may consist of ammonium acetate having a pH between 4 and 6, more advantageously between 4.5 and 5.5, and a concentration between 20mM and 120mM, more advantageously between 40mM and 100 mM. Another mobile phase solution may consist of ammonium acetate having a pH between 7.5 and 9.0, more advantageously between 8 and 8.5, and a concentration between 100mM and 300mM, more advantageously between 120mM and 200 mM. In alternative embodiments, the mobile phase solution can be formed from ammonium formate, tetramethylammonium acetate, triethylammonium formate, or the like. In order to achieve high quality mass spectra of proteins with minimal salt adducts, it is also advantageous that the sodium and potassium content of the ammonium acetate salt is less than 100ppb, more advantageously less than 20 ppb. Also, in a preferred embodiment, the mobile phase solution may be prepared in a glass-free process and provided in a glass-free container in the form of a buffer concentrate and/or a ready-to-use mobile phase.

In some embodiments, these mobile phase solutions may comprise an organic co-solvent, including but not limited to acetonitrile, methanol, ethanol, or isopropanol, at a concentration ranging from 1% to 50%, more advantageously from 2% to 30% w/v, in order to slow bacterial growth.

In some embodiments, the mobile phase solution may be used in a binary gradient, and in other embodiments in the form of a ternary gradient with water. Having a ternary gradient of water may allow fine-tuning the separation of conductivity changes for pH changes, which may be an effective optimization parameter for developing separations of specific protein analytes.

In one embodiment, the present disclosure represents a method that requires the use of the MS-compatible mobile phase buffer system for charge variant distribution of protein therapeutics (including but not limited to mAb-based therapeutics).

Furthermore, it has been found to be advantageous to apply a glass-free process to the preparation of the mobile phase concentrate and/or the ready-to-use mobile phase.

It is particularly advantageous to employ such mobile phase buffer systems with cation exchange columns based on carboxylated or sulfonated polymer resins. Thus, the mobile phase buffer system is beneficial in pairing with a cation exchange stationary phase prepared as described in U.S. patent application Ser. No. 16/287,364 entitled "Polymer Particles with a Gradient Composition and Methods of Production therof" (Polymer Particles with Gradient Composition and Methods of preparation thereof), which is incorporated herein by reference. In addition, the mobile phase buffer system can be advantageously paired with a variety of commercially available cation exchange columns including, but not limited to, Waters BioResolve^TMSCX mAb, Thermo Scientific MAb Pac SCX, Thermo Scientific Pro Pac WCX, Thermo Scientific Pro Pac Elite WCX, Phenomenex BioZen WCX, Agilent Bio SCX, Agilent Bio WCX, Sepax proteom SCX, Sepax proteom WCX, Sepax Antibodix WCX, Tosoh TSKgel SP-STAT, Tosoh TSKgel SP-NPR, and YMC BioPro SP-F.

In various embodiments, the present disclosure provides the above-described concentrates of the buffered mobile phase, which are prepared at 2-fold to 100-fold concentration volume, more advantageously at 5-fold to 20-fold concentration volume. Alternatively, the mobile phase system may be provided in a ready-to-use form.

In other embodiments, the present disclosure provides kits wherein the user follows the provided instructions to prepare the mobile phase from the above-described buffer concentrate.

In another embodiment, a kit can be provided that includes a ready-to-use or concentrated set of buffers and a cation exchange column. In some embodiments, the ready-to-use buffers and buffer concentrates described above can be prepared with buffer salts containing metals (including but not limited to sodium, potassium, and iron) at concentrations of less than 100 ppb.

In addition, to extend their shelf life, these ready-to-use buffers and buffer concentrates can be formulated with trace amounts of a biocide (including but not limited to 200ppm chloroform) and packaged in weekly oxygen bags.

Although optimized to achieve high resolution of mabs, the methods, compositions, and kits described herein can be used to isolate other analytes, including other types of biomolecules, specific examples of which include peptides, other proteins, including naturally occurring non-mAb proteins, fusion proteins, and Antibody Drug Conjugates (ADCs), and the like.

More details are given in the examples below.

Example 1: SEC-UV-MS

Fig. 1 shows a histogram obtained with the following conditions:

LC Condition：

MS Condition：

Gradiometer：

Example 2: IEX-UV

Figures 2, 3A and 3C present chromatograms obtained with the conditions listed below:

LC Condition：

Mobile phase of figure 2：

Mobile phase A50 mM ammonium formate pH 3.9

Mobile phase B150 or 300mM ammonium acetate, titrated to pH 8.0 or 9.0

Mobile phase of figure 3：

Mobile phase a 40mM ammonium formate 50mM ammonium acetate pH 5.0, or 90mM ammonium acetate pH 5.0

Mobile phase B200 mM ammonium acetate pH 8.2

Gradiometer of figures 2 and 3：

Example 3: IEX-UV-MS

Fig. 3B, fig. 4, fig. 5 and fig. 6 present chromatograms obtained with the following conditions:

LC Condition：

MS Condition：

Mobile phase of fig. 3B：

Mobile phase a 40mM ammonium formate 50mM ammonium acetate pH 5.0, or 90mM ammonium acetate pH 5.0

Mobile phase B200 mM ammonium acetate pH 8.2

Gradiometer of figure 3B：

Mobile phase of fig. 4A and 6：

Mobile phase A90 mM ammonium acetate pH 5.0

Mobile phase B200 mM ammonium acetate pH 8.4

Mobile phase of fig. 4B and 5C：

Mobile phase A45 mM ammonium acetate pH 5.0

Mobile phase B150 mM ammonium acetate pH 8.4

Mobile phase of fig. 4C：

Mobile phase A20 mM ammonium acetate pH 5.0

Mobile phase B120 mM ammonium acetate pH 8.4

Gradiometer of figures 4, 5C and 6：

Mobile phase of fig. 5A：

Mobile phase a 25mM ammonium bicarbonate and 30mM acetic acid pH 5.3

Mobile phase B2 mM ammonium hydroxide in acetic acid 10mM pH 10.18

FIG. 5A gradient chart of whole infliximab：

FIG. 5A gradient of IdeS digested trastuzumab：

Mobile phase of fig. 5B：

Mobile phase A50 mM ammonium formate pH 3.9

Mobile phase B500 mM ammonium acetate pH 7.4

Gradiometer of figure 5B：