Methods of measuring carfilzomib

Abstract

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

Description

Background Technology

[0001] Accurately assessing the concentration of the active pharmaceutical ingredient (API) in the solution before preparation is important to ensure that the correct amount of API is dispensed into the resulting formulation. Carfilzomib is an approved API for the treatment of multiple myeloma and is marketed under the name Carfilzomib. Sales. Accurate measurement of carfilzomib during formulation is required to ensure that the resulting formulation contains the desired amount of carfilzomib. Therefore, there is a need for a method for accurately measuring carfilzomib in samples. Summary of the Invention

[0002] This document provides a method for measuring the concentration of carfilzomib in a sample during the preparation 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 performing high-performance liquid chromatography (HPLC) on the analytical sample to obtain the carfilzomib concentration of the sample. In various embodiments, the sample is derived from a carfilzomib complex. In some embodiments, the sample is derived from a bulk lyophilized solution of carfilzomib. In some embodiments, the sample is a dissolved sample. In some embodiments, the sample is a diluted 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, these methods provide 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 from 3 mg / mL to 8 mg / mL. In some cases, the concentration of carfilzomib in the sample is from 5 mg / mL to 6 mg / mL. In various embodiments, the sample is analyzed at a temperature of 2°C to 8°C prior to HPLC analysis. In various embodiments, the sample is analyzed at room temperature prior to HPLC analysis. In various embodiments, the method disclosed herein further comprises preparing a lyophilized product from a bulk lyophilized solution. In some cases, the lyophilized product contains 10 mg of carfilzomib. In some cases, the lyophilized product contains 30 mg of carfilzomib. In some cases, the lyophilized product contains 60 mg of carfilzomib. Attached Figure Description

[0003] Figure 1 The results of carfilzomib concentration analysis of samples using a series of diluents containing different percentages of acetonitrile by volume in water are shown.

[0004] Figure 2The concentrations of analytical sample solutions prepared from a sample with a concentration of approximately 7.8 mg / mL using 50% v / v ACN diluent are shown, with the analytical samples being analyzed on days 1 and 2 at 5°C prior to HPLC analysis.

[0005] Figure 3 The concentrations of sample solutions ranging from 3.7 mg / mL to 7.8 mg / mL are shown, corresponding to 60% to 130% of the method nominal concentration, wherein the analytical samples were prepared with 30% and 40% v / v ACN diluent and the analytical samples were at 5°C prior to HPLC analysis. Detailed Implementation

[0006] Carfilzomi Approved for the treatment of multiple myeloma and prepared as a lyophilized product for reconstitution and injection in single-dose vials, including vials containing 10 mg, 30 mg, or 60 mg carfilzomib.

[0007] Determining the concentration of carfilzomib before formulating it into a lyophilized product is important to ensure that the lyophilized product contains an appropriate amount of carfilzomib. This article discloses an improved method for measuring the concentration of carfilzomib in a sample, providing a more accurate assessment of the carfilzomib concentration in the sample. In some cases, the carfilzomib concentration in the sample is from 3 mg / mL to 8 mg / mL. In other cases, the carfilzomib concentration is from 5 mg / mL to 6 mg / mL.

[0008] The process control (IPC) method for carfilzomib in bulk (lyophilized) solutions for injection is determined by high-performance liquid chromatography (HPLC) to determine the concentration of carfilzomib in the IPC sample after dissolution and dilution of the bulk solution during the compounding process. The bulk solution is diluted using a diluent of acetonitrile (ACN) and water, for example, by a factor of ten. In some embodiments, the carfilzomib sample is diluted at least twice with the diluent, and in some cases, by a factor of eight to fifteen, eight to twelve, or nine to eleven. As used herein, “analytical sample” is a sample diluted with the diluent and capable of being analyzed by HPLC. In this context, references to sample concentration refer to the concentration of carfilzomib in a sample evaluated as if using an analytical sample prepared from that sample and the diluent.

[0009] Previous methods for measuring carfilzomib concentration used a diluent with a 50 / 50 volume-to-volume (v / v) ratio of water and ACN. This paper has determined that excess ACN (>50% by volume) in the diluent can impair sample solubility, potentially affecting IPC determination results. Furthermore, as detailed herein, conventional diluents that account for a 50 / 50 (v / v) ratio of water and ACN are sensitive to slight perturbations, which can affect the amount of carfilzomib in each phase and thus potentially the accuracy of carfilzomib concentration measurements.

[0010] To assess the sensitivity of carfilzomib concentration measurements to the amount of ACN in the diluent, analytical samples from dissolved and diluted bulk solutions of carfilzomib for injection were prepared in a range of diluents (ranging from 0% to 60% ACN by volume) and analyzed by HPLC using the assays described in the Examples section. In some cases, the samples were from bulk solutions of carfilzomib for injection. In some cases, the samples were dissolved samples. In some cases, the samples were diluted samples. In some cases, the analytical samples were refrigerated (e.g., at temperatures from 2°C to 8°C) during or prior to HPLC analysis. Results for different ACN concentration conditions of carfilzomib samples were presented in... Figure 1 As shown in the image.

[0011] Samples can be collected at various stages of the manufacture and formulation of carfilzomib (e.g., during carfilzomib compounding), and the methods described herein can be used to measure the concentration of carfilzomib in these samples from each stage. For example, manufacturing may include dissolving carfilzomib in a formulated bulk solution, and subsequently may include diluting the formulated bulk solution (e.g., by adding water for injection) to obtain a pharmaceutical product containing a target concentration of carfilzomib for filling vials. Measurement of carfilzomib concentration can be performed in the formulated bulk solution (e.g., to determine how much diluent is needed to achieve the target concentration in the pharmaceutical product), and furthermore, it can be performed after dilution of the formulated bulk product (e.g., to verify that the pharmaceutical product contains the target concentration or carfilzomib). As used herein, the term "dissolved sample" refers to a sample containing dissolved carfilzomib that has not yet been diluted to the target concentration of the pharmaceutical product.

[0012] As used herein, the term "diluted bulk solution sample" refers to a sample containing a bulk carfilzomib solution and a diluent. Carfilzomib may be at a target concentration or may be a candidate at a target concentration. The diluted bulk solution may include pharmaceutical products for filling vials (e.g., single-dose vials).

[0013] like Figure 1As can be seen, when the percentage of ACN in the diluent exceeds 50%, the measured carfilzomib concentration decreases. These data highlight the risk that excessive ACN in the diluent may lead to inaccurate or low IPC results. Therefore, the method disclosed herein involves diluting a carfilzomib sample with a diluent containing water and less than 50% ACN by volume to form an analytical sample. In some embodiments, the diluent contains 0.1% to 49.9% ACN by volume. In some embodiments, the diluent contains 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 contains ACN and is less than 50%, 49%, 45%, 40%, or 25% ACN by volume.

[0014] A series of experiments were conducted to identify alternative diluents for the IPC method used in bulk (lyophilized) carfilzomib solution for injection, in order to reduce the risk of inaccurate and low IPC results. Figure 1 The data shown indicate that diluents with lower % ACN result in less accurate IPC assays, with a lower risk of inaccurate results. Two alternative diluents, 30% ACN 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, corresponding to 60% to 130% of the method nominal concentration. The obtained data are summarized in Table 1. In some cases, the carfilzomib concentration in the samples was 3 mg / mL to 8 mg / mL. In other cases, the carfilzomib concentration was 5 mg / mL to 6 mg / mL.

[0015] Table 1

[0016]

[0017] 1 Relative to the method nominal concentration

[0018] 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 methods disclosed in the Examples section below. Results showed good accuracy, as evidenced by low % RSD values. In some embodiments, the methods disclosed herein provide RSD% less than 2%. In various cases, RSD% is less than 1%. In various cases, RSD% is less than 0.5%.

[0019] A confirmatory study was subsequently conducted in which two dissolved and two diluted bulk IPC samples were analyzed using a 40% ACN diluent by volume. All analyzed samples showed good accuracy, as evidenced by low %RSD values, and no adverse trends were observed in the results. These data are summarized in Table 2.

[0020] Table 2

[0021]

[0022]

[0023] A series of comparative analyses were performed, in which identical samples were analyzed using the IPC method for two different batches A and B, respectively, with diluents containing 50% and 40% ACN by volume. Analytical samples from each dissolved sample and diluted bulk solution sample were analyzed. Carfilzomib concentrations determined by the IPC method were comparable between the two diluents. These data are shown in Table 3.

[0024] Table 3

[0025]

[0026] The concentration of carfilzomib in the dissolved or diluted bulk solution sample can be used to appropriately dispense the desired amount of carfilzomib into the resulting formulation, such as the lyophilized product in a single-dose vial. In some cases, the lyophilized product in a single-dose vial contains 10 mg of carfilzomib. In some cases, the lyophilized product in a single-dose vial contains 30 mg of carfilzomib. In some cases, the lyophilized product in a single-dose vial contains 60 mg of carfilzomib.

[0027] Example

[0028] Carfilzomib Concentration Determination: Analytical samples containing carfilzomib were evaluated using high-performance liquid chromatography (HPLC). Three aliquots of the analytical sample were prepared. 5.0 mL of the sample solution was transferred to a 50 mL volumetric flask and diluted with a sample diluent containing ACN and water at an appropriate (v / v) ratio, and thoroughly mixed. The sample was run on a Phenomenex Gemini™ C18 column (50 × 4.6 mm 3 μm particle size) at 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.

[0029] The concentrations of two different carfilzomib IPC samples were evaluated using varying amounts of water and ACN as diluents, and the results are shown in Table 4 below. A significant decrease in the measured carfilzomib concentration was observed when the amount of ACN in the diluent exceeded 50%.

[0030] Table 4

[0031] Diluent composition Sample 1 Sample 2 40% ACN 6.00 5.04 50% ACN 6.03 5.04 51% ACN 5.72 5.01 52.5% ACN 5.10 4.94 55% ACN 4.29 4.14 60% ACN 2.75 2.82

[0032] The concentration of another group of carfilzomib samples was evaluated using different amounts of ACN diluent in water, and the concentrations measured at and above 50% ACN decreased, as shown in Table 5.

[0033] Table 5

[0034]

[0035]

[0036] When the HPLC sample tray cools to 5°C, use the sample solution diluted with 50% v / v ACN.

[0037] The effect of sample temperature on the measured carfilzomib concentration was evaluated prior to HPLC analysis. Evidence of phase separation was observed in sample solutions prepared with 50% v / v ACN diluent at a carfilzomib concentration of approximately 7.8 mg / mL. Figure 2 In this scenario, 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 when the vials were re-injected the day after aliquoting. Figure 2 Importantly, this concentration gradient was not observed in any sample solutions prepared with 30% v / v or 40% v / v ACN diluent, nor was it detected in any sample solutions analyzed using an HPLC sample tray held at room temperature.

[0038] This difference is hypothesized to be caused by phase separation between the diluent's ACN and the aqueous component, as well as the varying solubility of carfilzomib in these components. Due to its poor water solubility, a higher carfilzomib concentration is detected in the upper (lower density) ACN layer. Without being theoretically constrained, the phenomenon that sugars in carfilzomib pharmaceutical products (such as cyclodextrin, which can be used in some formulations) contribute to "glycan precipitation," where sugars above a certain concentration threshold lead to an increase in the concentration of ACN in the upper organic phase, is considered.

[0039] When analyzed using an HPLC sample tray at 5°C, all analytical sample solutions prepared with 30% v / v ACN and 40% v / v ACN diluents produced results with good accuracy, as evidenced by low %RSD values. However, for analytical sample solutions prepared with 50% v / v ACN diluent, particularly samples at 60% and 7.8 mg / mL carfilzomib concentrations (relative to 130% of the method nominal concentration), considerable variability was observed, with most of these conditions having %RSD > 2.0% (Table 6).

[0040] Table 6

[0041]

[0042] a The average values ​​and %RSD calculations included results from repeated injections from the same HPLC vial at different HPLC needle positions (0 mm, 7.5 mm, 15 mm needle offset).

[0043] b Relative to the method nominal concentration

[0044] *%RSD>2.0%

[0045] When the HPLC sample tray cools to 5°C, use the sample solution diluted with 40% v / v ACN.

[0046] When analyzed using an HPLC sample tray at 5°C, analytical sample solutions prepared with 50% v / v ACN diluent showed a significant gradient in results, while all analytical sample solutions prepared with 30% and 40% v / v ACN diluents showed ≤2.0% RSD when analyzed under the same conditions. This observation was also consistent with HPLC sample vials re-injected the day after preparation. These data suggest that reducing the percentage of ACN in the diluent by at least 10% within the concentration range of 70% to 130% (relative to the method nominal concentration) yields more accurate results, and this accuracy remains even when vials are stored for extended periods of 24 hours on a cooled HPLC sample tray. Figure 3 The average carfilzomib concentration results and corresponding %RSD values ​​for each sample concentration prepared with 30% and 40% v / v ACN are shown.

[0047] When the percentage of ACN in the diluent exceeded 50%, the detected carfilzomib concentration decreased, but consistent carfilzomib concentrations were achieved at %ACN levels ranging from 50% to 0% (comparable to values ​​obtained with 50% v / v ACN diluent). Sample solutions prepared using 40% v / v ACN diluent and cooled to 5°C in an HPLC sample tray showed the best results in all four tests. In IPC samples, the results remained accurate and precise. %RSD ≤ 2.0% was achieved under all conditions (Table 7).

[0048] Table 7

[0049]

[0050] a The average values ​​and %RSD calculations included results from repeated injections from the same HPLC vial at different HPLC needle positions (0 mm, 7.5 mm, 15 mm needle offset).

[0051] b The mean and %RSD calculations included results from all repeated injections (0 mm, 7.5 mm, 15 mm offset) and repeated sample preparations (flasks 1, 2, and 3).

[0052] When the HPLC sample tray is at room temperature, use sample solutions diluted with 30% / 40% / 50% v / v ACN.

[0053] When the HPLC sample tray was kept at room temperature on day 1, all conditions met the %RSD sample acceptance criteria.

Claims

1. A method for measuring the concentration of carfilzomib in a sample containing carfilzomib, the method comprising the following steps: The sample was diluted with a diluent containing water and 25% to 40% acetonitrile by volume to form the analytical sample; and The concentration of carfilzomib in the sample was determined by high performance liquid chromatography (HPLC).

2. The method as described in claim 1, wherein, The sample was obtained from a carfilzomib combination drug.

3. The method as described in claim 1, wherein, The sample was obtained from a bulk lyophilized solution of carfilzomib.

4. The method of claim 1, wherein, This sample is a dissolved sample.

5. The method of claim 1, wherein, This sample is a diluted sample.

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

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

8. The method according to any one of claims 1 to 5, wherein, This method includes a relative standard deviation (RSD) value of less than 2%.

9. The method of claim 8, wherein, The RSD is less than 1%.

10. The method of claim 8, wherein, The RSD is less than 0.5%.

11. The method according to any one of claims 1 to 5, wherein, The concentration of this carfilzomib is 3 mg / mL to 8 mg / mL.

12. The method of claim 11, wherein, The concentration of this carfilzomib is 5 mg / mL to 6 mg / mL.

13. The method according to any one of claims 1 to 5, wherein, The temperature of the sample to be analyzed was between 2°C and 8°C prior to HPLC analysis.

14. The method according to any one of claims 1 to 5, wherein, The sample was at room temperature prior to HPLC analysis.

15. The method of any one of claims 3 to 5, further comprising the step of preparing a lyophilized product from the sample.

16. The method of claim 15, wherein, This lyophilized product contains 10 mg of carfilzomib.

17. The method of claim 15, wherein, This lyophilized product contains 30 mg of carfilzomib.

18. The method of claim 15, wherein, This lyophilized product contains 60 mg of carfilzomib.

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