How carfilzomib is measured

Abstract

Provided herein is an improved method for measuring the concentration of carfilzomib in a sample, the method comprising: diluting the sample with a diluent comprising water and less than 50% by volume of acetonitrile to form an analytical sample; and subjecting the analytical sample to high performance liquid chromatography (HPLC) to determine the carfilzomib concentration of the sample.

Description

[Background technology]

[0001] Accurately assessing the concentration of an active pharmaceutical ingredient (API) in solution prior to formulation is important to ensure the correct amount of API is dispensed in the resulting formulation. Carfilzomib is an API approved for the treatment of multiple myeloma and is sold under the name KYPROLIS®. Accurate measurement of carfilzomib during formulation is required to ensure the desired amount of carfilzomib is contained in the resulting formulation. Therefore, a method is needed that provides an accurate measurement of carfilzomib in a sample. Summary of the Invention [Means for solving the problem]

[0002] Provided herein are methods for measuring the concentration of carfilzomib in a sample in a formulation of carfilzomib. The method for measuring the concentration of carfilzomib in a sample includes diluting the sample with a diluent comprising water and less than 50% acetonitrile by volume to form an analytical sample; and subjecting the analytical sample to high performance liquid chromatography (HPLC) to obtain the carfilzomib concentration of the sample. In various embodiments, the sample is from a formulation of carfilzomib. In some embodiments, the sample is from a bulk lyophilized solution of carfilzomib. In some embodiments, the sample is a solubilized sample. In some embodiments, the sample is a post-dilution sample. In various cases, the diluent is water and 0% to 45% acetonitrile by volume. In some cases, the diluent is water and 25% to 40% acetonitrile by volume. In some cases, the diluent is water and 30% acetonitrile by volume. In some cases, the diluent is water and 40% acetonitrile by volume. In various embodiments, the method provides a relative standard deviation (RSD) value of less than 2% determined from multiple analytical samples of the sample. In some cases, the RSD% is less than 1%. In some cases, the RSD% is less than 0.5%. In various cases, the concentration of carfilzomib in the sample is between 3 mg / mL and 8 mg / mL. In some cases, the concentration of carfilzomib in the sample is between 5 mg / mL and 6 mg / mL. In various embodiments, the analytical sample is at a temperature of 2-8°C prior to HPLC analysis. In various embodiments, the analytical sample is at room temperature prior to HPLC analysis. In various embodiments, the methods disclosed herein further include preparing a lyophilizate from the bulk lyophilization solution. In some cases, the lyophilizate contains 10 mg of carfilzomib. In some cases, the lyophilizate contains 30 mg of carfilzomib. In some cases, the lyophilizate contains 60 mg of carfilzomib. [Brief explanation of the drawings]

[0003] [Figure 1]FIG. 1 shows the results of carfilzomib concentration for samples analyzed using a series of diluents with varying volume percentages of acetonitrile in water. [Figure 2] FIG. 1 shows the concentration of analytical sample solutions prepared from samples having a concentration of approximately 7.8 mg / mL using a diluent of 50% v / v ACN when the analytical samples were at 5° C. prior to HPLC analysis, analyzed on days 1 and 2. [Figure 3] Figure 1 shows the concentrations of sample solutions ranging from 3.7 mg / mL to 7.8 mg / mL, corresponding to a nominal concentration range of the method of 60% to 130%, when analytical samples were prepared using diluents of 30% and 40% v / v ACN and the analytical samples were at 5 °C prior to HPLC analysis. DETAILED DESCRIPTION OF THE INVENTION

[0004] Carfilzomib (KYPROLIS®) is approved for the treatment of multiple myeloma and is prepared as a solution in single-dose vials, such as vials containing 10 mg, 30 mg, or 60 mg of carfilzomib, and as a lyophilizate for injection.

[0005] It is important to determine the concentration of carfilzomib before formulating it into a lyophilizate so that the lyophilizate contains an appropriate amount of carfilzomib. Disclosed herein are improved methods for measuring the concentration of carfilzomib in a sample, which allow for a more accurate assessment of carfilzomib in the sample. In some cases, the carfilzomib concentration of the sample is between 3 mg / mL and 8 mg / mL. In some cases, the carfilzomib concentration is between 5 mg / mL and 6 mg / mL.

[0006] The in-process control (IPC) method for the bulk (lyophilized) solution of carfilzomib for injection is a high-performance liquid chromatography (HPLC) assay used to determine the concentration of carfilzomib in IPC samples during formulation, both after solubilization and post-dilution of the bulk solution. The in-process bulk solution is diluted, e.g., 10-fold, using a diluent of acetonitrile (ACN) and water. In some embodiments, the carfilzomib sample is diluted at least 2-fold using the diluent, and in some cases, 8-15-fold, 8-12-fold, or 9-11-fold using the diluent. As used herein, an "analytical sample" is a sample that has been diluted using a diluent and can be analyzed using HPLC. In this context, the concentration of a sample refers to the concentration of carfilzomib in the sample as assessed using an analytical sample prepared from that sample and a diluent.

[0007] Previous methods for measuring carfilzomib concentration used a diluent with a 50 / 50 volume-to-volume (v / v) ratio of water and ACN. It has been determined herein that excess ACN (>50% by volume) in the diluent can impair sample solubility, which can affect the results of the IPC assay. Furthermore, as described in detail herein, previous diluents with a 50 / 50 (v / v) ratio of water and ACN are believed to be sensitive to slight perturbations that can affect the amount of carfilzomib in each phase, thereby affecting the accuracy of carfilzomib concentration measurements.

[0008] To assess the sensitivity of carfilzomib concentration measurements to the amount of ACN in the diluent, analytical samples of solubilized and post-diluted bulk solution samples from an in-process bulk (lyophilized) solution of carfilzomib for injection were prepared in a series of diluents ranging from 0% to 60% ACN by volume and analyzed by HPLC using the assay described in the Examples section. In some cases, the samples were from bulk solution from an in-process bulk (lyophilized) solution of carfilzomib for injection. In some cases, the samples were solubilized samples. In some cases, the samples were post-diluted samples. In some cases, the analytical samples were refrigerated (e.g., having a temperature of 2-8°C) during or before HPLC analysis. The results for different ACN concentration conditions for carfilzomib samples are shown in Figure 1.

[0009] It is contemplated that samples can be collected at various stages of carfilzomib manufacture and formulation (e.g., during compounding of carfilzomib), and that the methods described herein can be used to measure carfilzomib concentration in these samples from various stages. For example, manufacture may involve solubilizing carfilzomib in a formulated bulk solution, followed by diluting the formulated bulk solution (e.g., by adding water for injection) to obtain a drug product containing a target concentration of carfilzomib for filling into vials. Measurements of carfilzomib concentration can be performed on the formulated bulk solution (e.g., to determine the amount of diluent needed to achieve the target concentration in the drug product) and again on post-dilution of the formulated bulk product (e.g., to confirm that the drug product contains the target concentration or carfilzomib). As used herein, the term "solubilized sample" refers to a sample containing solubilized carfilzomib that has not yet been diluted to the target concentration of the drug product.

[0010] As used herein, the term "post-dilution bulk solution sample" refers to a sample comprising carfilzomib bulk solution and a diluent. The carfilzomib may be at a target concentration or may be a candidate target concentration. The post-dilution bulk solution may include a drug product used to fill vials, e.g., single-dose vials.

[0011] As seen in Figure 1, the measured carfilzomib concentration decreases as the percentage of ACN in the diluent exceeds 50%. These data highlight the risk that excessive ACN in the diluent can lead to inaccurate and low IPC assay results. Accordingly, the methods disclosed herein include diluting a carfilzomib sample with a diluent comprising water and less than 50% ACN by volume to form an analytical sample. In some embodiments, the diluent comprises 0.1% to 49.9% ACN by volume. In some embodiments, the diluent comprises 0% to 45% ACN by volume. In some embodiments, the diluent is 25 to 40% ACN by volume. In some cases, the diluent is 30% ACN by volume. In some cases, the diluent is 40% ACN by volume. In some cases, the diluent is less than 50%, 49%, 45%, 40%, or 25% ACN by volume. In some cases, the diluent comprises ACN and is less than 50%, 49%, 45%, 40%, or 25% ACN by volume.

[0012] To reduce the risk of inaccurate and low IPC assay results, a series of experiments was conducted to identify alternative diluents for the IPC method for a bulk (lyophilized) solution of carfilzomib for injection. The data, shown in Figure 1, demonstrate that diluents with a lower % ACN content are associated with a lower risk of inaccurate and low IPC assay results. Two alternative diluents, 30% ACN by volume and 40% ACN by volume, were evaluated. Each diluent was analyzed at three different representative IPC assay sample concentrations ranging from approximately 3.7 mg / mL to 7.8 mg / mL. These correspond to a range of 60% to 130% of the method's nominal concentration. The resulting data are summarized in Table 1. In some cases, the carfilzomib concentrations of the samples ranged from 3 mg / mL to 8 mg / mL. In some cases, the carfilzomib concentrations ranged from 5 mg / mL to 6 mg / mL.

[0013] [Table 1]

[0014] For each condition evaluated, all analytical sample solutions were prepared using a diluent containing 30% or 40% ACN by volume, or analyzed according to the analytical method disclosed in the Examples section below. The results demonstrated good precision, as evidenced by the low %RSD values. In some embodiments, the methods disclosed herein provide an RSD% of less than 2%. In various cases, the RSD% is less than 1%. In various cases, the RSD% is less than 0.5%.

[0015] A confirmatory study was then conducted in which two solubilized and two post-diluted bulk solution IPC samples were analyzed using a diluent of 40% ACN by volume. All analytical samples analyzed showed good precision as evidenced by low %RSD values, and no adverse trends were observed in the results. These data are summarized in Table 2.

[0016] [Table 2]

[0017] A series of comparative analyses was performed in which the same samples were analyzed by IPC for two different lots, A and B, using diluents containing 50% and 40% ACN by volume, respectively. Analytical samples from both solubilized and post-diluted bulk solution samples were analyzed. Carfilzomib concentrations determined by IPC were comparable for both diluents. These data are shown in Table 3.

[0018] [Table 3]

[0019] The carfilzomib concentration in the solubilized or post-diluted bulk solution sample can be used to appropriately dispense the desired amount of carfilzomib for the resulting formulation, e.g., lyophilizate in a single-dose vial. In some cases, the single-dose vial of lyophilizate contains 10 mg of carfilzomib. In some cases, the single-dose vial of lyophilizate contains 30 mg of carfilzomib. In some cases, the single-dose vial of lyophilizate contains 60 mg of carfilzomib. [Example]

[0020] Carfilzomib Concentration Assay: Analytical samples containing carfilzomib are evaluated using a high-performance liquid chromatography (HPLC) assay. Analytical samples are prepared in triplicate. 5.0 mL of sample solution is transferred to a 50 mL volumetric flask, diluted with sample diluent containing the appropriate ratio (v / v) of ACN and water, and mixed thoroughly. Samples are analyzed on a Phenomenex Gemini™ C18 column (50 x 4.6 mm, 3 μm particle size) with a column temperature of 30°C ± 2°C, an autosampler temperature of 5°C ± 3°C, a detection wavelength of 220 nm, a flow rate of 1.5 mL / min, an injection volume of 10 μL, and a total run time of 4 minutes.

[0021] Two different IPC samples of carfilzomib were evaluated for concentration using various amounts of water and ACN as diluents. The results are shown in Table 4 below. It was observed that the measured carfilzomib concentration decreased significantly when the amount of ACN in the diluent exceeded 50%.

[0022] [Table 4]

[0023] An additional set of carfilzomib samples was evaluated for concentration using diluents containing varying amounts of ACN in water. As shown in Table 5, the measured concentrations decreased above 50% ACN.

[0024] [Table 5]

[0025] We evaluated whether the temperature of the analytical sample prior to HPLC analysis affected the measured carfilzomib concentration. Evidence of phase separation was observed in the sample solution prepared with a 50% v / v ACN diluent at a carfilzomib concentration of approximately 7.8 mg / mL (Figure 2). In this case, the lowest carfilzomib concentration was detected at a needle offset of 0 mm (i.e., the bottom of the HPLC vial), and the highest carfilzomib concentration was detected at a needle offset of 15 mm (i.e., the top of the HPLC vial). This carfilzomib concentration gradient remained comparable even when the vial was reinjected the day after dispensing (Figure 2). Importantly, this concentration gradient was not observed in either the sample solutions prepared with a 30% v / v or 40% v / v ACN diluent, nor was it detected in any sample solutions analyzed in an HPLC sample tray held at room temperature.

[0026] This difference is hypothesized to be caused by phase separation between the ACN and water components of the diluent and the differential solubilities of carfilzomib in these components. Due to its poor solubility in water, higher concentrations of carfilzomib would be detected in the upper (lower density) ACN layer. Without being bound by theory, it is believed that sugars in carfilzomib drug product (such as cyclodextrins, which may be used in some formulations) contribute to "sugarization," a phenomenon in which sugars above a certain concentration threshold cause an increase in the concentration of ACN in the upper organic phase.

[0027] All analytical sample solutions prepared using the 30% v / v and 40% v / v ACN diluents yielded good precision results, as evidenced by low %RSD values, when analyzed using an HPLC sample tray at 5° C. However, significant variability was observed for analytical sample solutions prepared using the 50% v / v ACN diluent, especially for samples with carfilzomib concentrations of 60% and 7.8 mg / mL (130% of the method nominal concentration), with %RSDs >2.0% for the majority of these conditions (Table 6).

[0028] [Table 6]

[0029] While analytical sample solutions prepared with a 50% v / v ACN diluent revealed a significant gradient in the results when analyzed using an HPLC sample tray at 5°C, all analytical sample solutions prepared with 30% v / v and 40% v / v ACN diluents showed RSDs of ≤2.0% when analyzed under the same conditions. This observation also held true for HPLC sample vials reinjected the day after preparation. The data suggest that reducing the percentage of ACN in the diluent by at least 10% yields more accurate assay results across a concentration range of 70% to 130% (compared to the method's nominal concentration), and that these results remain accurate even with long-term storage of vials on a refrigerated HPLC sample tray for up to 24 hours. The mean carfilzomib concentration results and the respective %RSD values ​​for each sample concentration prepared with 30% and 40% v / v ACN are shown in Figure 3.

[0030] Although the detected carfilzomib concentration decreased as the percentage of ACN in the diluent increased above 50%, consistent carfilzomib concentrations (comparable to those obtained with a diluent containing 50% v / v ACN) were achieved at %ACN levels ranging from 50% to 0%. Sample solutions prepared using a diluent containing 40% v / v ACN and an HPLC sample tray cooled to 5°C demonstrated that assay results remained precise and accurate for all four Kyprolis® IPC samples tested. %RSDs of ≤2.0% were achieved for all conditions (Table 7).

[0031] [Table 7]

[0032] When the HPLC sample tray was kept at room temperature on day 1, all conditions passed the %RSD sample acceptance criteria.

Claims

1. 1. A method for measuring the concentration of carfilzomib in a sample containing carfilzomib, comprising: diluting the sample with a diluent comprising water and 25% to 40% by volume of acetonitrile to form an analytical sample; and subjecting the analytical sample to high performance liquid chromatography (HPLC) to determine the carfilzomib concentration of the sample; A method comprising:

2. 10. The method of claim 1, wherein the sample is obtained from a formulation of carfilzomib.

3. 10. The method of claim 1, wherein the sample is obtained from a bulk lyophilized solution of carfilzomib.

4. The method of claim 1 , wherein the sample is a solubilized sample.

5. The method of claim 1 , wherein the sample is a post-dilution sample.

6. The method of any one of claims 1 to 5, wherein the diluent comprises water and 30% by volume of acetonitrile.

7. The method of any one of claims 1 to 5, wherein the diluent comprises water and 40% by volume of acetonitrile.

8. 8. The method of any one of claims 1 to 7, wherein the concentration of carfilzomib is 3 mg / mL to 8 mg / mL.

9. 9. The method of claim 8, wherein the concentration of carfilzomib is 5 mg / mL to 6 mg / mL.

10. 10. The method of any one of claims 1 to 9, wherein the temperature of the analytical sample is at a temperature of 2°C to 8°C prior to HPLC analysis.

11. The method of any one of claims 1 to 9, wherein the temperature of the analytical sample is at room temperature prior to HPLC analysis.

12. The method of any one of claims 3 to 11, further comprising the step of preparing a lyophilisate from the sample.

13. 13. The method of claim 12, wherein the lyophilizate contains 10 mg of carfilzomib.

14. 13. The method of claim 12, wherein the lyophilizate contains 30 mg of carfilzomib.

15. 13. The method of claim 12, wherein the lyophilizate contains 60 mg of carfilzomib.

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