Method for treating polysorbate-containing protein formulations

Abstract

Provided herein are methods for precipitating proteins in compositions containing proteins and polysorbates. These methods allow for accurate determination of the concentration or amount of polysorbate in the composition.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 63 / 393,575, filed July 29, 2022, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION Disclosed herein are methods for precipitating proteins from polysorbate-containing protein formulations. [Background technology]

[0003] Nonionic surfactants, such as polysorbate 20 / 80 (PS20 / PS80), commercially known as Tween 20 / 80, are commonly used in protein drug formulations to increase protein stability from surface and shear stress. Polysorbate degradation has attracted attention throughout the industry because it can reduce product stability and potentially form invisible and / or visible particles in drug products during their shelf life, raising patient safety concerns. Therefore, monitoring polysorbate concentrations in drug product formulations is crucial. However, for samples containing large amounts of protein, existing methods for analyzing polysorbate concentrations can be adversely affected by several factors, including high sample viscosity, gel formation, column clogging, interfering peaks, and inaccurate results. Summary of the Invention

[0004] Disclosed herein is a method for precipitating a protein from a polysorbate-containing composition, the method comprising adding to a polysorbate-containing composition comprising a protein and a polysorbate an amount of a chelating agent and an amount of a C1-C6 alcohol, the amount of the chelating agent and the amount of a C1-C6 alcohol being combined and effective to precipitate the protein. [Brief explanation of the drawings]

[0005] The Summary of the Invention, as well as the Detailed Description that follows, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the disclosed method, there are shown in the drawings exemplary embodiments of the method. However, the method is not limited to the particular embodiments disclosed therein. In the drawings: [Figure 1] Figure 1 shows a schematic diagram of an exemplary EDTA / EtOH precipitation scheme (black squares) prior to polysorbate analysis by mixed-mode anion-exchange-hydrophobic (MAX) high-performance liquid chromatography (HPLC) using an evaporative light scattering detector (ELSD). Volumes can be scaled proportionally. [Figure 2A] MAX-ELSD chromatogram of PS80 standard (400 μg / mL) is shown. [Figure 2B] MAX-ELSD chromatogram of Antibody 4 drug product (containing 400 μg / mL of PS80) is shown. [Figure 2C] Shown is the MAX-ELSD chromatogram of EDTA / EtOH precipitated antibody 4 drug product (containing 400 μg / mL PS80). DETAILED DESCRIPTION OF THE INVENTION

[0006] The methods of the present disclosure may be understood more readily by reference to the following detailed description taken in conjunction with the accompanying drawings, which form a part of this disclosure: It is to be understood that the methods of the present disclosure are not limited to the specific methods described and / or illustrated herein, and further, the terminology used herein is for the purpose of describing particular embodiments, by way of example only, and is not intended to limit the methods claimed.

[0007] Unless otherwise stated, any description of possible mechanisms or modes of operation or reasons for improvement is intended to be illustrative only, and the methods of the present disclosure are not limited by the merits or demerits of the proposed mechanisms or modes of operation or reasons for improvement.

[0008] When a range of numerical values is recited or established herein, this range includes its endpoints, and all individual integers and fractions within that range, and also includes each of the narrower ranges formed by all the various possible combinations of these endpoints and internal integers and fractions, each of which forms a subgroup of the larger group of values within the recited range, as if each of those narrower ranges were explicitly recited. When a range of numerical values is described herein that exceeds the recited value, the range is nevertheless finite, with its upper limit defined by a value operable within the context disclosed herein. When a range of numerical values is described herein that is less than the recited value, the range is nevertheless defined by a lower limit defined by a non-zero value. It is not intended that the scope of the present method be limited to the specific values recited when defining the range. All ranges are inclusive and combinable.

[0009] When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. Reference to a particular numerical value is intended to include at least that particular value unless the context dictates otherwise.

[0010] It should be understood that certain features of the methods of the present disclosure, which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods of the present disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0011] As used herein, the singular forms "a," "an," and "the" are intended to include plurals.

[0012] Various terms relating to aspects of the present specification are used throughout the specification and claims. Unless otherwise indicated, such terms shall be given their ordinary meaning in the art. Other specifically defined terms shall be construed in a manner consistent with the definition provided herein.

[0013] Similarly, the term "comprising" is intended to include examples encompassed by the terms "consisting essentially of" and "consisting of." Similarly, the term "consisting essentially of" is intended to include examples encompassed by the term "consisting of."

[0014] Disclosed herein is a method for precipitating a protein from a polysorbate-containing composition, the method comprising adding to a polysorbate-containing composition comprising a protein and a polysorbate an amount of a chelating agent and an amount of a C1-C6 alcohol, the amount of the chelating agent and the amount of a C1-C6 alcohol being effective for precipitating the protein when combined. The method can include providing a composition comprising a protein and a polysorbate, and adding to the composition an amount of a chelating agent and an amount of a C1-C6 alcohol that are effective for precipitating the protein.

[0015] The chelating agent can be added to the composition before the alcohol is added to the composition, or the chelating agent and alcohol can be added to the composition simultaneously.

[0016] Suitable chelating agents include one or more of ethylenediaminetetraacetic acid (EDTA), triethylamine (TEA), (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) (EGTA), n-(2-hydroxyethyl)ethylenediamine-N,N',N' (HEDTA), nitrilotriacetic acid (NTA), and 2-hydroxybenzoic acid (salicylic acid; SA). In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA). In some embodiments, the chelating agent is triethylamine (TEA). In some embodiments, the chelating agent is (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) (EGTA). In some embodiments, the chelating agent is n-(2-hydroxyethyl)ethylenediamine-N,N',N' (HEDTA). In some embodiments, the chelating agent is nitrilotriacetic acid (NTA). In some embodiments, the chelating agent is 2-hydroxybenzoic acid (salicylic acid; SA). In some embodiments, the chelating agent is any combination of ethylenediaminetetraacetic acid (EDTA), triethylamine (TEA), (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) (EGTA), n-(2-hydroxyethyl)ethylenediamine-N,N',N (HEDTA), nitrilotriacetic acid (NTA), and 2-hydroxybenzoic acid (salicylic acid; SA).

[0017] The concentration of the chelating agent can be about 5 mM to about 50 mM. Suitable concentrations of the chelating agent can be, for example, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the concentration of the chelating agent is 15 mM.

[0018] Suitable C1-C6 alcohols include methanol, ethanol, propanol, butanol, pentanol, and hexanol. In some embodiments, the C1-C6 alcohol is methanol. In some embodiments, the C1-C6 alcohol is ethanol. In some embodiments, the C1-C6 alcohol is propanol. The propanol may be isopropanol. In some embodiments, the C1-C6 alcohol is butanol. In some embodiments, the C1-C6 alcohol is pentanol. In some embodiments, the C1-C6 alcohol is hexanol. In some embodiments, the C1-C6 alcohol is any combination of methanol, ethanol, propanol, butanol, pentanol, and hexanol.

[0019] The alcohol concentration can be about 45% to about 55%. Suitable alcohol concentrations can be, for example, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, or about 55%. In some embodiments, the alcohol concentration is about 50% by volume. The alcohol concentration added to the composition can be any concentration that is effective for precipitating proteins in the composition.

[0020] The disclosed methods can include a C1-C6 alcohol, which is ethanol, together with a chelating agent selected from EDTA, TEA, EGTA, HEDTA, NTA, and salicylic acid. The disclosed methods can include adding to a composition comprising a protein and a polysorbate amounts of both of the following: ethanol and EDTA; ethanol and TEA; ethanol and EGTA; ethanol and HEDTA; ethanol and NTA; or ethanol and salicylic acid, which, when combined, are effective to precipitate the protein. In some embodiments, the C1-C6 alcohol is ethanol and the chelating agent is EDTA. The method can include adding to a composition comprising a protein and a polysorbate an amount of EDTA and an amount of ethanol, which, when combined, are effective to precipitate the protein.

[0021] The protein can be an antibody. In some embodiments, the antibody is a monoclonal antibody.

[0022] The method can further include removing the protein from the composition. Removing the protein from the composition can include precipitating the protein, centrifuging the composition to pellet the precipitated protein, and separating the supernatant from the precipitated protein, thereby removing the protein from the composition. The method can further include determining the concentration or amount of polysorbate in the composition. Before determining the concentration or amount of polysorbate, the precipitated protein can be separated from the non-precipitated portion, thereby forming a supernatant. In some embodiments, the chelating agent, alcohol, and composition comprising the protein and polysorbate are mixed before separating the precipitated protein from the non-precipitated portion. In some embodiments, the precipitated protein is separated from the non-precipitated portion via centrifugation. Centrifugation can be performed until the supernatant is essentially free of precipitated protein. The supernatant essentially free of precipitated protein can have a residual protein concentration that is about 1 / 100th of the initial protein concentration before precipitation and centrifugation.

[0023] The disclosed methods can be performed on compositions containing a protein greater than about 10 mg / mL. Protein concentrations can range from about 10 mg / mL to about 250 mg / mL. Suitable protein concentrations can be, for example, about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, about 210 mg / mL, about 220 mg / mL, about 230 mg / mL, about 240 mg / mL, or about 250 mg / mL. In some embodiments, the protein concentration is about 20 mg / mL of protein.

[0024] The method can further include determining the concentration of polysorbate in the non-precipitated portion of the composition. In some embodiments, the method further includes determining the amount of the polysorbate in the non-precipitated portion of the composition. For example, the method can include precipitating protein, centrifuging the composition to pellet the precipitated protein, and determining the concentration / amount of polysorbate in the supernatant. Polysorbate can be measured by any method known in the art. In some embodiments, the concentration or amount of polysorbate is determined by mixed-mode anion exchange-hydrophobic high-performance liquid chromatography using an evaporative light scattering detector (MAX-ELSD). During MAX-ELSD, an acidified mobile phase elutes positively charged proteins in the void volume, while neutral polysorbate molecules are retained and eluted separately from the proteins. In some embodiments, the concentration or amount of polysorbate is determined by mixed-mode anion exchange-hydrophobic high-performance liquid chromatography using a charged aerosol detector (MAX-CAD). [Example]

[0025] Polysorbates are common stabilizers used in protein drug formulations. However, degradation of polysorbates can result in less stable products and the formation of sub- and / or visible particles that may be unsuitable for patient administration. To evaluate whether the amount of polysorbate in antibody-containing formulations can be accurately measured using chromatography, various antibody-containing formulations were subjected to initial chromatographic conditions by exposing the formulations to an aqueous solution containing 10% isopropanol and 2% formic acid. Table 1 lists 11 antibodies, their respective protein concentrations, and whether they formed gels when mixed with acidified isopropanol, which mimics the chromatographic conditions experienced by samples during analysis. Seven of the 11 antibody-containing formulations, each containing a protein concentration greater than 100 mg / mL, formed gels under these conditions. These results demonstrate the risk of protein gel formation during high-performance liquid chromatography (HPLC).

[0026] [Table 1]

[0027] Organic solvents such as ethanol (EtOH) and acetone are widely used to precipitate proteins. However, organic solvents can result in gel formation. To determine whether antibodies can be precipitated using organic solvents prior to chromatography, antibody-containing formulations were incubated with EtOH. As shown in Table 2 below, much of the antibody did not precipitate, and gel formation was observed (Table 2). The limited success of using organic solvents such as ethanol to precipitate proteins limits the applicability of this protein precipitation method as a universal strategy.

[0028] [Table 2] N / A: No precipitate was formed so PS80 was not analyzed in the sample.

[0029] To determine whether EDTA can aid in organic solvent-induced precipitation of antibodies, antibody-containing formulations were first mixed with 30 mM EDTA in a 1:1 volume ratio, followed by EtOH in a 1:1 volume ratio. Sample volumes could be, for example, 100 μL of sample, 100 μL of 30 mM EDTA, and 200 μL of EtOH. The volumes of sample, chelating agent, and alcohol could be scaled up proportionally. Precipitation occurred without extended incubation. An exemplary EDTA / EtOH precipitation and removal procedure is shown in Figure 1. The combination of EDTA and EtOH resulted in efficient precipitation and acceptable polysorbate recovery (with analytical targets of 80–120%) for each antibody-containing formulation tested (Table 3).

[0030] [Table 3] Recovery % being greater than 100% due to tolerances.

[0031] Although only three antibody-containing formulations precipitated upon the addition of EtOH alone (Table 2), the addition of EDTA to the samples prior to EtOH precipitation (Table 3) resulted in protein precipitation for all antibody-containing formulations tested (Table 3). Thus, EDTA improved the EtOH precipitation process. EDTA / EtOH precipitation was a highly effective strategy for analyzing polysorbates in samples with high protein concentrations that could not be analyzed by existing MAX-ELSD methods due to viscosity, gel formation, column clogging, or peak interference.

[0032] Four polysorbate-containing antibody samples were analyzed using MAX-ELSD. Figure 2A shows the MAX-ELSD chromatogram of the PS80 standard, showing a single peak. Analysis of the four polysorbate-containing antibody samples showed the presence of peaks eluting between 2.5 and 7.5 minutes, with one peak eluting at 4.2 minutes, potentially interfering with the PS80 peak (Figure 2B). Removing protein from the samples by EDTA / EtOH precipitation prior to PS80 analysis resulted in no interfering peaks and improved PS80 recovery (Figure 2C). Protein precipitation by EDTA / EtOH prior to MAX-ELSD resulted in a chromatogram similar to the PS80 standard (Figure 2A).

[0033] Efficient protein removal by EDTA / EtOH precipitation was further demonstrated by measuring the protein concentration in the supernatant after precipitation using a variable pathlength spectrophotometer, SoloVPE®. Protein concentrations in all antibody-containing formulations tested showed at least a 100-fold reduction in the amount of antibody in solution (Table 4). As a result, the performance of the PS80 method improved when analyzing samples with high protein concentrations (Figure 2C). Good recovery of PS80 using the EDTA / EtOH precipitation method was demonstrated by recoveries within the desired range of 80%–120% of spiked PS80 compared to antibody-containing formulations (Table 3).

[0034] [Table 4] * Target concentrations of polysorbates in drug products

[0035] The performance of the EDTA / EtOH precipitation method was then evaluated by performing a test method validation for PS80 in antibody 7-containing formulations (Table 5) according to ICH guidelines (www.ema.europa.eu / en / ich-q2r2-validation-analytical-procedures). This test method has also been successfully validated for antibody 8 PS20 and antibody 5 PS20 analysis.

[0036] [Table 5]

[0037] [Table 6]

[0038] [Table 7]

[0039] Additional chelating agents were tested for their ability to aid in the precipitation of antibody 7 from antibody-containing formulations. As shown below, triethylamine (TEA), (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) (EGTA), n-(2-hydroxyethyl)ethylenediamine-N,N',N (HEDTA), nitrilotriacetic acid (NTA), and 2-hydroxybenzoic acid (salicylic acid; SA) each precipitated the antibody as effectively as EDTA.

[0040] [Table 8]

[0041] The presence of high protein concentrations in biopharmaceutical products can result in chromatographic peak interference and affect the accuracy of polysorbate analysis by MAX-ELSD. A universal and easy-to-perform protein precipitation method was developed herein. EDTA-mediated EtOH precipitation (EDTA / EtOH precipitation) removes monoclonal antibody (mAb) proteins from samples prior to HPLC-ELSD analysis, enabling successful and accurate polysorbate quantification in biopharmaceuticals with high protein concentrations (≥100 mg / mL).

[0042] The introduction of an EDTA / EtOH precipitation step prior to MAX-ELSD resulted in improved and accurate determination of polysorbate content in a wider range of samples. Protein removal by EDTA / EtOH precipitation had broader applicability compared to precipitation with organic solvents alone due to the presence of EDTA, which aids in protein removal by promoting precipitation and preventing gel formation. Due to its simplicity and robustness, this sample preparation method was also suitable for application in quality control environments and provided improved test method specificity and accuracy. EDTA / EtOH precipitation was simple, fast, and easy to perform. It was completed in three simple steps within a short time. This method was suitable for quality control environments where polysorbate concentrations are monitored by testing formulations before their release to the market and during their storage period to ensure product safety and efficacy.

[0043] Those skilled in the art will recognize that numerous changes and modifications can be made to the preferred embodiments disclosed herein and that such changes and modifications can be made without departing from the spirit of the present invention. It is, therefore, intended by the appended claims to cover all such equivalent variations that fall within the true spirit and scope of the invention.

Claims

1. A method comprising adding a certain amount of a chelating agent and a certain amount of C1-C6 alcohol to a composition containing a protein and a polysorbate, the latter being effective in precipitating the protein when combined.

2. To provide a composition containing protein and polysorbate, The composition is to be Methods that include...

3. The method according to claim 1 or 2, wherein the chelating agent is added to the composition before the alcohol is added to the composition.

4. The method according to claim 1 or 2, wherein the chelating agent is one or more of ethylenediaminetetraacetic acid (EDTA), triethylamine (TEA), (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) (EGTA), n-(2-hydroxyethyl)ethylenediamine-N,N',N(HEDTA), nitrilotriacetic acid (NTA), and 2-hydroxybenzoic acid (salicylic acid; SA).

5. The method according to claim 1 or 2, wherein the concentration of the chelating agent is approximately 5 mM to approximately 50 mM.

6. The method according to claim 1 or 2, wherein the C1 to C6 alcohols are ethanol.

7. The method according to claim 1 or 2, wherein the alcohol concentration is approximately 45% by volume to approximately 55% by volume.

8. The method according to claim 1 or 2, wherein the protein is an antibody.

9. The method according to claim 8, wherein the antibody is a monoclonal antibody.

10. The method according to claim 1 or 2, further comprising determining the concentration or amount of the polysorbate in the non-precipitated portion of the composition.

11. The method according to claim 10, wherein the concentration or amount of polysorbate is determined by mixed-mode anion exchange-hydrophobic high-performance liquid chromatography using an evaporative light scattering detector (MAX-ELSD).

12. The method according to claim 10, wherein the method comprises separating the precipitated protein from the non-precipitated portion to form a supernatant before determining the concentration or amount of the polysorbate.

13. The method according to claim 12, wherein the composition comprising the chelating agent, alcohol, protein, and polysorbate is mixed before separating the precipitated protein from the non-precipitated portion.

14. The method according to claim 12, wherein separating the precipitated protein from the non-precipitated portion includes centrifugation.

15. The method according to claim 14, wherein the centrifugation is carried out until the supernatant essentially no longer contains the precipitated protein.

16. The method according to claim 1 or 2, wherein the C1 to C6 alcohols are ethanol, and the chelating agent is selected from EDTA, TEA, EGTA, HEDTA, NTA, and salicylic acid.

17. The method according to claim 1 or 2, wherein the C1 to C6 alcohols are ethanol and the chelating agent is EDTA.

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