A method for quality control of roxadustat capsules

Abstract

This invention relates to the field of pharmaceutical analysis technology, and more particularly to a quality control method for roxadustat capsules. The method involves preparing a biomimetic dissolution medium containing polyvalent metal ions, comprising at least three buffer solutions with pH values ​​ranging from 1.2 to 6.8. Dissolution tests are conducted on the roxadustat capsule sample and a reference formulation using the biomimetic dissolution medium to determine the dissolution curve similarity factor, thereby evaluating the dissolution consistency of roxadustat capsules in the presence of polyvalent metal ions. The contents of the roxadustat capsules with consistent dissolution are accurately weighed, dissolved in an acidic diluent, and diluted to a predetermined concentration to obtain the test solution. High-performance liquid chromatography (HPLC) is used to analyze the test solution using a gradient elution method. The chromatogram of the test solution is recorded, and the content of each impurity is calculated using the main component self-comparison method, while the roxadustat content is calculated using the external standard method. This invention achieves integrated quality control from dissolution behavior evaluation to chemical quality inspection.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a quality control method for roxadustat capsules. Background Technology

[0002] Roxadustat is a novel oral hypoxia-inducible factor prolyl hydroxylase inhibitor used to treat anemia caused by chronic kidney disease (CKD). However, since CKD patients often need to take multiple medications at the same time, roxadustat faces complex drug interaction issues in clinical use.

[0003] Studies have shown that roxadustat poses a significant risk of interaction with drugs containing polyvalent cations. Specifically, concomitant use of roxadustat with sevelamer carbonate or calcium acetate can lead to a 67% and 46% decrease in the area under the plasma curve (AUC) of roxadustat, respectively, and a 66% and 52% decrease in the maximum plasma concentration, respectively. Therefore, clinical guidelines recommend that roxadustat be taken at least 1 hour before or after taking phosphate binders, oral iron, magnesium / aluminum antacids, or other drugs containing polyvalent cations.

[0004] Currently, quality control research on roxadustat capsules mainly focuses on routine dissolution evaluation and content determination. Existing dissolution tests typically use standard dissolution media specified in the Chinese Pharmacopoeia, such as hydrochloric acid solution with pH 1.2, acetate buffer with pH 4.5, and phosphate buffer with pH 6.8, etc., and use similarity factors as indicators to examine the dissolution consistency between the self-made formulation and the reference formulation.

[0005] Chinese Patent Publication No. CN113855638A discloses a roxadustat drug formulation comprising a cyclodextrin derivative and polacrine potassium, wherein the dissolution rate is improved by controlling the particle size of the active pharmaceutical ingredient.

[0006] Therefore, it is evident that existing quality control methods have the following shortcomings:

[0007] 1. Conventional dissolution media do not take into account the actual environment of polyvalent metal ions coexisting in the gastrointestinal tract when CKD patients take iron supplements and calcium phosphate binders in combination, and cannot effectively assess the changes in the dissolution behavior of roxadustat capsules in this special medication scenario;

[0008] 2. Roxadustat contains β-keto acid groups in its molecular structure, which are prone to oxidative degradation under alkaline conditions. The diluents used in the conventional preparation of test solutions are neutral or weakly alkaline, which causes the test solution to undergo partial degradation before chromatographic analysis, resulting in the content and impurity determination results deviating from the actual values.

[0009] 3. Existing methods separate dissolution evaluation from content and impurity determination into independent processes, lacking an integrated quality control approach from biomimetic dissolution evaluation to chromatographic analysis;

[0010] 4. There is a lack of further verification and remedial solutions for cases where the similarity factor determination of dissolution curves fails.

[0011] To address the aforementioned issues, this invention provides a quality control method for roxadustat capsules. By preparing a biomimetic dissolution medium containing polyvalent metal ions, the gastrointestinal metal ion conditions in the combined medication environment of CKD patients are simulated. The dissolution curve similarity between the test sample and the reference preparation is evaluated, and the content and impurities are determined in an integrated manner using high-performance liquid chromatography to more comprehensively reflect the quality attributes of roxadustat capsules in actual clinical use scenarios. Summary of the Invention

[0012] Therefore, the present invention provides a quality control method for roxadustat capsules to solve the problems in the prior art where conventional dissolution media cannot simulate the gastrointestinal environment of polyvalent metal ions coexisting in patients with chronic kidney disease taking iron supplements and calcium phosphate binders in combination, resulting in a disconnect between dissolution evaluation and actual clinical medication scenarios, and the problem that dissolution evaluation and content and impurity determination are separated and cannot form an integrated quality control.

[0013] To achieve the above objectives, the present invention provides a quality control method for roxadustat capsules, comprising:

[0014] A biomimetic dissolution medium containing polyvalent metal ions was prepared, wherein the biomimetic dissolution medium is at least three buffer solutions with a pH of 1.2-6.8, to simulate the gastrointestinal metal ion environment when patients with chronic kidney disease take iron supplements and calcium phosphate binders in combination.

[0015] Dissolution tests were conducted on the roxadustat capsule test sample and the reference preparation using the aforementioned biomimetic dissolution medium to determine the dissolution curve similarity factor, so as to evaluate the dissolution consistency of roxadustat capsule in the presence of multivalent metal ions.

[0016] Accurately weigh the contents of roxadustat capsules that have dissolved uniformly, dissolve and dilute them with an acidic diluent to the predetermined concentration to obtain the test solution.

[0017] The test solution was analyzed by high performance liquid chromatography with gradient elution. The chromatogram of the test solution was recorded. The content of each impurity was calculated by the main component self-comparison method, and the content of roxadustat was calculated by the external standard method.

[0018] Furthermore, the multivalent metal ions include Fe 3+ and Ca 2+ ;

[0019] The Fe 3+ The concentration of Ca is 0.05-0.20 mmol / L. 2+The concentration is 1.0-3.0 mmol / L.

[0020] Furthermore, the at least three buffer solutions include:

[0021] Hydrochloric acid solution with pH 1.2±0.05, acetate-sodium acetate buffer solution with pH 4.5±0.05, and phosphate buffer solution with pH 6.8±0.05.

[0022] Furthermore, the process of determining the dissolution curve similarity factor includes:

[0023] The test sample and the reference preparation were subjected to dissolution tests in the biomimetic dissolution medium. The dissolution solutions of the test sample and the reference preparation were collected at multiple preset time points to determine the average cumulative dissolution of the test sample and the reference preparation at each time point.

[0024] The average cumulative dissolution rate of the reference formulation at each time point is used as the reference curve, and the average cumulative dissolution rate of the test sample at each time point is used as the test curve. The average value of the sum of squares of the differences between the reference curve and the test curve at each time point is calculated to determine the dissolution curve similarity factor.

[0025] Furthermore, when the dissolution curve similarity factor is greater than or equal to 50, it is determined that the dissolution of roxadustat capsules is consistent in the environment of coexistence of multivalent metal ions;

[0026] When the dissolution curve similarity factor is less than 50, it is determined that the dissolution of roxadustat capsules is inconsistent in the presence of multiple metal ions, and then an additional product containing Al is added. 3+ The Al was subjected to a verification test using a dissolution medium. 3+ The concentration is 0.1-0.5 mmol / L.

[0027] Furthermore, the acidic diluent is a 0.05% aqueous solution of phosphoric acid with a pH of 2.0 ± 0.2, to inhibit the oxidative degradation of roxadustat via β-keto acid groups under alkaline conditions;

[0028] The predetermined concentration is 0.10-0.50 mg / mL for roxadustat.

[0029] Furthermore, the chromatographic conditions for the high performance liquid chromatography are as follows: using octadecylsilane-bonded silica gel as the packing material, the column temperature is 35-40℃, the detection wavelength is 260nm, and the flow rate is 1.0mL / min.

[0030] Furthermore, the gradient elution uses 0.02 mol / L potassium dihydrogen phosphate solution as mobile phase A and acetonitrile as mobile phase B;

[0031] The mobile phase A was adjusted to a pH of 3.0 ± 0.1 with phosphoric acid;

[0032] The gradient elution procedure is as follows:

[0033] 0-5 min: Mobile phase A 85% → 85%, Mobile phase B 15% → 15%;

[0034] 5-20 min: Mobile phase A 85% → 60%, Mobile phase B 15% → 40%;

[0035] 20-30 min: Mobile phase A 60% → 30%, Mobile phase B 40% → 70%;

[0036] 30-35 min: Mobile phase A 30% → 30%, Mobile phase B 70% → 70%;

[0037] 35-40 min: Mobile phase A 30% → 85%, Mobile phase B 70% → 15%;

[0038] 40-48 min: Mobile phase A 85% → 85%, Mobile phase B 15% → 15%.

[0039] Furthermore, the limits for roxadustat content are 95.0%-105.0% of the labeled amount, the limit for a single known impurity is ≤0.2%, the limit for a single unknown impurity is ≤0.1%, and the limit for total impurities is ≤1.0%.

[0040] Furthermore, it also includes system suitability testing:

[0041] Take roxadustat reference standard and at least one known impurity reference standard, dissolve and dilute them with the acidic diluent to prepare a system suitability solution, and inject it into the liquid chromatograph;

[0042] Among them, the separation degree between the main peak of roxadustat and the adjacent impurity peak is ≥1.5, the tailing factor is 0.8-1.5, and the theoretical plate number is ≥3000.

[0043] Compared with the prior art, the beneficial effect of the present invention is that by adding Fe to the conventional leaching medium... 3+ and Ca 2 + A biomimetic dissolution medium was constructed to simulate the gastrointestinal environment of polyvalent metal ions in patients with chronic kidney disease taking iron supplements and calcium phosphate binders in combination. The ortho- and hydroxyl groups and carbonyl groups in the roxadustat molecule have the ability to chelate polyvalent metal ions. When Fe is present in the dissolution medium... 3+ and Ca 2+At this time, some roxadustat molecules can form soluble or insoluble complexes with these ions, thereby affecting their dissolution rate and extent. Conventional dissolution media, lacking these ions, cannot reflect the effect of this chelation on dissolution. In conventional dissolution media, the similarity factors of the dissolution curves of the test sample and the reference preparation are 78.6, 75.3, and 71.2, respectively, while in the Fe-containing medium of this invention… 3+ / Ca 2+ In the biomimetic dissolution medium, the similarity factor changed to 72.3, 68.5, and 65.1, respectively, but all were greater than 50, indicating consistent dissolution. It should be noted that the direction and magnitude of the change in the similarity factor in the biomimetic medium are related to the formulation composition and process parameters, and different formulations show different trends. This result shows that by introducing clinically relevant polyvalent metal ions, the biomimetic dissolution medium can reveal the potential impact of ion chelation / complexation effects on dissolution behavior that cannot be reflected by conventional media, making the in vitro dissolution consistency evaluation closer to the actual clinical drug use scenario and improving the clinical relevance and discriminative power of the evaluation.

[0044] Furthermore, roxadustat contains β-keto acid groups in its molecular structure. These groups are prone to oxidative degradation under alkaline conditions but are relatively stable in acidic environments. If neutral or alkaline diluents are used, the test solution may degrade during preparation and storage before chromatographic analysis, leading to lower results for the main component content and higher results for degradation impurities, thus failing to accurately reflect the quality attributes of the sample. This invention uses a 0.05% phosphoric acid aqueous solution with a pH of 2.0 as an acidic diluent for the test solution. By providing a protonated environment, the oxidation reaction of the β-keto acid groups is inhibited, thereby ensuring the chemical stability of the test solution throughout the chromatographic analysis process. As shown in Comparative Example 2, when using the acidic diluent of this invention, the peak area of ​​roxadustat in the test solution decreased by only 0.9% within 24 hours; while the degradation rate reached 5.0% when using the neutral diluent and as high as 15.0% when using the alkaline diluent. This indicates that the acidic diluent can effectively inhibit the degradation of roxadustat during the preparation and storage of the test solution, ensuring that the content determination results are not lowered due to degradation in the sample preparation process and that the impurity determination results are not higher due to artificial degradation, so that the quality control data truly reflects the inherent quality attributes of the product.

[0045] Furthermore, in the determination of dissolution curve similarity factor, this invention designs a method where a dissolution similarity factor greater than or equal to 50 directly indicates consistent dissolution, and when the dissolution curve similarity factor is less than 50, additional Al is added. 3+ The logic for determining the verification hierarchy; the test sample and the reference preparation in the presence of Fe 3+ / Ca 2+When the similarity factor of the dissolution curves in the biomimetic medium is less than 50, the differences may stem from two different reasons: first, differences in the formulation or process of the preparation itself leading to differences in intrinsic quality; second, differences in the degree of response of the test sample and the reference preparation to this specific ionic environment, i.e., the differences exhibit ion selectivity; Al 3+ with Fe 3+ Although both are trivalent cations, their coordination chemistry differs. This can be addressed by adding Al... 3+ Verification can distinguish between the two reasons mentioned above. If in AI... 3+ If the similarity factor of the dissolution curve in the medium is still less than 50, then the difference originates from the formulation itself; if in Al 3+ If the similarity factor of the dissolution curves in the medium is greater than or equal to 50, then the difference has ion selectivity; this hierarchical judgment logic provides a more comprehensive and diagnostically valuable quality evaluation dimension for dissolution consistency evaluation.

[0046] Furthermore, this invention establishes a more clinically relevant in vitro dissolution evaluation method by adding polyvalent metal ions to the dissolution medium, overcoming the limitation of conventional dissolution media in reflecting the influence of metal ion chelation effects on formulation release behavior; in a pH 6.8 medium, the solubility of roxadustat meets the sink conditions, effectively evaluating Fe 3+ / Ca 2+ Dissolution consistency under ion coexistence environment; for media with pH < 4.5, although a complete dissolution curve cannot be obtained due to the extremely low drug solubility, it can still help reflect the early release trend of the formulation in the acidic ion environment of the stomach, and provide supplementary information for the evaluation of ion interference in different pH ranges. Attached Figure Description

[0047] Figure 1 This is a flowchart of the quality control method for roxadustat capsules as described in an embodiment of the present invention;

[0048] Figure 2 This is a dissolution curve of the test sample and the reference preparation in Comparative Example 1 of the present invention in conventional phosphate buffer at pH 6.8;

[0049] Figure 3 In this embodiment of the invention, the test sample and the reference preparation are prepared at pH 6.8 containing Fe. 3+ / Ca 2+ Dissolution curves in biomimetic dissolution media;

[0050] Figure 4 The figure shows the stability test results of the roxadustat test solution under different pH diluent conditions in Comparative Example 2 of this invention. Detailed Implementation

[0051] To make the objectives and advantages of the present invention clearer, the present invention will be further described below with reference to embodiments; it should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0052] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0053] Please see Figure 1 As shown, Figure 1 This is a flowchart illustrating the quality control method for roxadustat capsules according to an embodiment of the present invention. The quality control method for roxadustat capsules according to an embodiment of the present invention includes:

[0054] Step S1: Prepare a biomimetic dissolution medium containing polyvalent metal ions. The biomimetic dissolution medium is at least three buffer solutions with a pH of 1.2-6.8 to simulate the gastrointestinal metal ion environment when patients with chronic kidney disease take iron supplements and calcium phosphate binders in combination.

[0055] Step S2: Dissolution tests were conducted on the roxadustat capsule test sample and the reference preparation respectively using the biomimetic dissolution medium to determine the dissolution curve similarity factor, so as to evaluate the dissolution consistency of roxadustat capsule in the environment of coexistence of multivalent metal ions.

[0056] Step S3: Take the contents of the roxadustat capsules that have dissolved consistently, weigh them accurately, dissolve and dilute them with an acidic diluent to the predetermined concentration to obtain the test solution.

[0057] Step S4: The test solution is analyzed by high performance liquid chromatography with gradient elution. The chromatogram of the test solution is recorded. The content of each impurity is calculated by the main component self-comparison method, and the content of roxadustat is calculated by the external standard method.

[0058] In the embodiments of this invention, all raw materials and reagents used are commercially available. Experimental methods without specific conditions are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0059] In this embodiment of the invention, roxadustat, chemically named N-[(4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carbonyl)-glycine], contains a β-keto acid group in its molecular structure and is prone to oxidative degradation under alkaline conditions. Previous studies have shown that specific impurities of roxadustat, such as RM-R152407, undergo slow degradation after 24 hours in alkaline solution, with a peak area relative standard deviation of 3.89%, while remaining relatively stable in acidic and neutral solutions. Therefore, this embodiment of the invention uses an acidic diluent to prepare the test solution to ensure the stability of the sample solution.

[0060] It is understood that the biomimetic dissolution medium described in this embodiment of the invention is positioned as a quality consistency evaluation tool for specific clinical drug use scenarios (CKD patients taking iron supplements and calcium phosphate binders in combination), in order to provide an evaluation method with greater clinical discriminative power than conventional dissolution media, rather than replacing the conventional batch release dissolution test method in the legal standards.

[0061] Example 1

[0062] 1. Preparation of biomimetic dissolution media

[0063] In this embodiment of the invention, the multivalent metal ion includes Fe. 3+ and Ca 2+ ;

[0064] The Fe 3+ The concentration of Ca is 0.05-0.20 mmol / L. 2+ The concentration is 1.0-3.0 mmol / L.

[0065] Specifically, the preparation process of the biomimetic dissolution medium includes:

[0066] To prepare a hydrochloric acid solution with a pH of 1.2 ± 0.05, take approximately 7.65 mL of hydrochloric acid, dilute it to 1000 mL with water, shake well, and adjust the pH to 1.2 ± 0.05 with hydrochloric acid or water to obtain a basic hydrochloric acid solution with a pH of 1.2. Accurately weigh 27.03 mg of ferric chloride hexahydrate and 294.02 mg of calcium chloride dihydrate, place them in a 1000 mL volumetric flask, add an appropriate amount of the above basic hydrochloric acid solution with a pH of 1.2, sonicate or shake to dissolve, cool to room temperature, and dilute to the mark with the above basic hydrochloric acid solution with a pH of 1.2, shake well. After preparation, measure the pH of the solution. If necessary, adjust the pH to 1.2 ± 0.05 with hydrochloric acid or water to ensure Fe... 3+ The concentration was 0.10 mmol / L, Ca 2+ The concentration is 2.0 mmol / L.

[0067] To prepare an acetate-sodium acetate buffer solution with a pH of 4.5 ± 0.05, take 2.99 g of sodium acetate, add 14.0 mL of 2 mol / L acetic acid solution, dissolve and dilute with water to 1000 mL, shake well, and adjust the pH to 4.5 ± 0.05 with acetic acid or sodium acetate solution. Accurately weigh 27.03 mg of ferric chloride hexahydrate and 294.02 mg of calcium chloride dihydrate, place them in a 1000 mL volumetric flask, add an appropriate amount of the above-mentioned acetate-sodium acetate buffer solution with a pH of 4.5 to dissolve, cool to room temperature, dilute to the mark with the above-mentioned buffer solution, and shake well. After preparation, measure the pH of the solution. If necessary, adjust the pH to 4.5 ± 0.05 with acetic acid or sodium acetate solution to ensure Fe... 3+The concentration was 0.10 mmol / L, Ca 2+ The concentration is 2.0 mmol / L.

[0068] To prepare a phosphate buffer solution with a pH of 6.8 ± 0.05, take 250 mL of 0.2 mol / L potassium dihydrogen phosphate solution and 118 mL of 0.2 mol / L sodium hydroxide solution, dilute with water to 1000 mL, shake well, and adjust the pH to 6.8 ± 0.05 with phosphoric acid or sodium hydroxide solution to obtain the base solution of the phosphate buffer solution with a pH of 6.8; accurately weigh 27.03 mg of ferric chloride hexahydrate and 294.02 mg of calcium chloride dihydrate, and place them in a 1000 mL volumetric flask. Add an appropriate amount of the above-mentioned phosphate buffer solution with pH 6.8, slowly adding it while stirring. After complete dissolution, cool to room temperature and dilute to the mark with the above-mentioned phosphate buffer solution with pH 6.8, then shake well. Measure the pH of the prepared solution. If necessary, adjust the pH to 6.8 ± 0.05 with phosphoric acid or sodium hydroxide solution. Check the clarity of the solution. If slight turbidity occurs, filter it through a 0.45 μm filter membrane before use. This medium should be prepared and used immediately. Complete the dissolution test within 4 hours after preparation.

[0069] Contains Al 3+ For the dissolution medium, take an appropriate amount of the above pH 6.8 phosphate buffer base solution, accurately weigh approximately 72.4 mg of aluminum chloride hexahydrate, add it to the buffer solution, and shake well to dissolve, so that Al 3+ The concentration is 0.30 mmol / L. Dilute to 1000 mL with phosphate buffer (pH 6.8), shake well, and the solution is ready. Check the clarity of the solution after preparation; if turbidity occurs, filter before use.

[0070] In this embodiment of the invention, the above-mentioned leaching media are used only after degassing treatment, and the degassing treatment includes vacuum filtration, heating and boiling, or ultrasonic degassing.

[0071] In this embodiment of the invention, Al 3+ Both are trivalent cations, and with Fe 3+ It has similar charge density and coordination chemistry, but differs from roxadustat in its chelation constant, with the addition of Al. 3+ The verification was conducted to determine whether the inconsistency in the dissolution profiles was due to ion selectivity or to differences in the formulation itself.

[0072] In this embodiment of the invention, roxadustat has the highest solubility in a pH 6.8 medium. After the drug is fully dissolved, the ortho-phenolic hydroxyl and carbonyl groups in its molecular structure exist in a deprotonated form, which has the strongest chelation coordination with trivalent cations. The interference effect of metal ions on dissolution behavior is most significant under this pH condition. Therefore, pH 6.8 is the most sensitive discrimination window for this verification test, and the diagnostic conclusions obtained under this condition are the most representative.

[0073] In this embodiment of the invention, a biomimetic dissolution medium containing polyvalent metal ions is formulated to simulate the real multi-ion coexistence environment in the gastrointestinal tract of patients with chronic kidney disease when taking oral iron supplements and calcium phosphate binders in combination during clinical treatment. Fe 3+ and Ca 2+ The concentrations were referenced to the physiological concentration ranges of soluble ions in the gastrointestinal tract for oral iron and calcium preparations, respectively. This introduced clinically relevant interfering factors into the in vitro dissolution evaluation, so that the comparison of the dissolution behavior of the test sample and the reference preparation was no longer limited to the conventional buffer system specified in the pharmacopoeia, but could reflect the actual release differences of the preparation when faced with the chelation or complexation of multivalent metal ions.

[0074] 2. Dissolution test of roxadustat capsules (test sample and reference preparation)

[0075] 2.1 Test sample and reference preparation

[0076] In this embodiment of the invention, the test sample is a self-made roxadustat capsule with a specification of 50 mg / capsule, prepared according to the following formula and process:

[0077] Prescription composition: Roxadustat 50mg, lactose 289.8mg, microcrystalline cellulose 9.36mg, croscarmellose sodium 1.8mg (added internally), povidone 5.4mg (added internally), magnesium stearate 3.6mg (added externally).

[0078] The preparation process includes:

[0079] A high-shear wet granulation process was adopted. Roxadustat active pharmaceutical ingredient with a D90 of 20-26 μm, lactose, microcrystalline cellulose, and croscarmellose sodium were added to a wet mixing granulator and premixed for 5 minutes at a stirring speed of 100 rpm and a cutter speed of 1000 rpm. An 8% povidone aqueous solution was added for wet granulation at a stirring speed of 150 rpm and a cutter speed of 2000 rpm. The granules were then dried in a fluidized bed at 70±5℃ until the LOD ≤ 2%. After granulation, magnesium stearate was added and mixed in, and then the mixture was filled into capsules.

[0080] In this embodiment of the invention, the reference formulation is the original formulation, with a specification of 50 mg / capsule.

[0081] 2.2 Dissolution test conditions

[0082] In this embodiment of the invention, the dissolution media were hydrochloric acid solution with a pH of 1.2 ± 0.05, acetate-sodium acetate buffer solution with a pH of 4.5 ± 0.05, and phosphate buffer solution with a pH of 6.8 ± 0.05, prepared according to the examples. The media volume was 900 mL, the temperature was 37 ± 0.5 °C, and the paddle speed was 75 rpm. Samples were taken at 5, 10, 15, 20, 30, 45, and 60 minutes, with each sample volume not less than 5 mL. Immediately after sampling, the sample was filtered through a microporous membrane with a pore size not exceeding 0.45 μm, and the filtrate was used as the test sample dissolution solution. At the same time, the reference preparation was processed in the same way to obtain the reference preparation dissolution solution.

[0083] 2.3 Dissolution test

[0084] In this embodiment of the invention, high-performance liquid chromatography (HPLC) was used to determine the concentration of roxadustat in the dissolution solutions of the test sample and the reference preparation at each time point. The chromatographic conditions were as follows: octadecylsilane-bonded silica gel (YMCTriart C18 type, 4.6 mm × 150 mm, 3 μm particle size) was used as the stationary phase; 0.02 mol / L potassium dihydrogen phosphate solution was used as mobile phase A, with the pH adjusted to 3.0 ± 0.1 by phosphoric acid; acetonitrile was used as mobile phase B; and elution was performed according to the gradient program shown in Table 2. The flow rate was 1.0 mL / min, the column temperature was 35-40℃, the detection wavelength was 260 nm, and the injection volume was 20 μL. The cumulative dissolution rate at each time point was calculated using the external standard method based on the peak area. The average cumulative dissolution rate of the test sample and the reference preparation at each time point was calculated separately. Six samples were measured at each time point.

[0085] 2.4 Determination of similarity factor for dissolution curves

[0086] Using the average cumulative dissolution rate of the reference formulation at each time point as the reference curve, and the average cumulative dissolution rate of the test sample at each time point as the test curve, the dissolution similarity factor is calculated as follows: Calculate the sum of squares of the differences between the two curves at each corresponding time point, and obtain the similarity factor through mathematical transformation. When the dissolution similarity factor is greater than or equal to 50, it is determined that the dissolution of the test sample and the reference formulation in the biomimetic dissolution medium is consistent; when the dissolution similarity factor is less than 50, it is determined that the dissolution of the test sample and the reference formulation in the biomimetic dissolution medium is inconsistent.

[0087] If the average cumulative dissolution rate of the test sample and the reference preparation is not less than 85% at 15 minutes, the two dissolution curves are directly determined to be similar, and there is no need to calculate the dissolution curve similarity factor.

[0088] 2.5 Additional Verification Tests

[0089] When the dissolution curve similarity factor between the test sample and the reference preparation is less than 50, an additional sample containing Al is added. 3+The dissolution medium was subjected to a verification test. The dissolution test method described above was repeated, the similarity factor was calculated, and the results were recorded. Specifically, the Al... 3+ The concentration is 0.1-0.5 mmol / L.

[0090] 2.6 Dissolution evaluation results of three batches of samples

[0091] Three batches of roxadustat capsules, batch numbers 20250101, 20250102, and 20250103, were taken and dissolution tests were performed according to the above method. The results are shown in Table 1.

[0092] Table 1. Similarity factor of dissolution curves of three batches of roxadustat capsules in biomimetic dissolution medium.

[0093]

[0094] The results showed that the dissolution curves of the three batches of samples had similarity factors greater than 50 with the reference formulation in the three biomimetic dissolution media, indicating that their dissolution behavior was consistent.

[0095] In this embodiment of the invention, the evaluation of the biomimetic dissolution medium aims to simulate the multivalent metal ion environment in the gastrointestinal tract of CKD patients taking iron supplements and calcium phosphate binders in combination. Given that roxadustat has extremely low solubility at pH < 4.5, dissolution tests in pH 1.2 and pH 4.5 media mainly examine the differences in early release behavior of the formulation and the initial performance of ion chelation effects under acidic ionic environments. The core evaluation criterion for formulation dissolution consistency is the pH 6.8 biomimetic medium, under which the drug solubility meets the leakage conditions and can fully reflect the release characteristics of the formulation.

[0096] 3. Preparation and chromatographic determination of the test solution

[0097] 3.1 Preparation of the test solution

[0098] Take an appropriate amount of the contents of the roxadustat capsules that have dissolved consistently, approximately equivalent to 50 mg of roxadustat, accurately weigh it, place it in a 100 mL brown volumetric flask, add approximately 80 mL of acidic diluent (0.05% phosphoric acid aqueous solution), adjust the pH to 2.0 ± 0.2 with phosphoric acid, sonicate for approximately 15 minutes to dissolve, cool to room temperature, dilute to the mark with the acidic diluent, shake well, filter, and take the filtrate as the test solution.

[0099] In this embodiment of the invention, roxadustat contains a β-keto acid group in its molecular structure, which is prone to oxidative degradation under alkaline conditions. Previous studies have shown that certain impurities in roxadustat undergo slow degradation after 24 hours in alkaline solutions, while remaining relatively stable in acidic and neutral solutions. This embodiment of the invention uses a 0.05% phosphoric acid aqueous solution with a pH of 2.0 as a diluent to inhibit the degradation of roxadustat during the preparation of the test solution and chromatographic analysis, ensuring that the content and impurity determination results reflect the true quality attributes.

[0100] 3.2 Preparation of reference solution

[0101] Accurately weigh approximately 50 mg of roxadustat reference standard and place it in a 100 mL amber volumetric flask. Dissolve and dilute to the mark with acidic diluent, and shake well to prepare a stock solution of the reference standard. Accurately measure an appropriate amount of the stock solution of the reference standard and dilute it with acidic diluent to prepare a series of reference standard solutions with concentrations of 0.10 mg / mL, 0.25 mg / mL, and 0.50 mg / mL.

[0102] 3.3 Preparation of System Suitability Solution

[0103] Take appropriate amounts of roxadustat reference standard and known impurity reference standards, dissolve and dilute them with acidic diluent to prepare a system suitability solution containing approximately 0.4 mg of roxadustat and approximately 0.6 μg of each impurity per 1 mL.

[0104] The known impurity reference standards include: impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity SM1, impurity IN1, impurity IN2, impurity IN3, and impurity IN4; wherein, impurity A is 4-hydroxy-N,N-dimethyl-7-phenoxyisoquinoline-3-carboxamide; impurity B is 4-hydroxy-1-(hydroxymethyl)-7-phenoxyisoquinoline-3-yl ketone; impurity C is 1-(hydroxymethyl)-4-(acetoxy)-7-phenoxyisoquinoline-3-yl ketone; impurity D is... 4-Hydroxy-1-methyl-7-phenoxyisoquinoline-3-carboxylic acid; impurity E is methyl 4-hydroxy-1-(hydroxymethyl)-7-phenoxyisoquinoline-3-carboxylic acid; impurity F is methyl 2-(4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carboxamido)-acetate; impurities G, SM1, IN1, IN2, IN3, and IN4 are byproducts or degradation products that may exist in the synthesis process of roxadustat, and their chemical structures and CAS numbers can be obtained with reference to the known techniques in this field.

[0105] 3.4 Chromatographic conditions

[0106] Chromatographic column: Octadecylsilane-bonded silica gel, YMCTriart C18 type, with dimensions of 4.6 mm × 150 mm and a particle size of 3 μm, was used as the stationary phase A. 0.02 mol / L potassium dihydrogen phosphate solution was used as the mobile phase A, and the pH of the solution was adjusted to 3.0 ± 0.1 with phosphoric acid. Acetonitrile was used as the mobile phase B. Elution was performed according to the gradient program shown in Table 2. The flow rate was 1.0 mL / min, the column temperature was 35-40℃, the detection wavelength was 260 nm, and the injection volume was 20 μL.

[0107] Table 2 Gradient elution program

[0108]

[0109] In this embodiment of the invention, the detection wavelength of 260 nm is the characteristic wavelength of roxadustat in the ultraviolet region, which can take into account the detection sensitivity of both the main component and impurities. The gradient elution program is designed to gradually increase the mobile phase B from 15% to 70%, achieving effective separation of roxadustat and multiple impurity components with large polarity differences. At the same time, the retention time reproducibility of the next analysis is ensured by isocratic rebalancing for 40-48 minutes.

[0110] 3.5 System Suitability Test

[0111] Accurately inject 20 μL of the system suitability solution into the liquid chromatograph and record the chromatogram. System suitability requirements: the resolution between the roxadustat main peak and the adjacent impurity peak should be no less than 1.5; the tailing factor should be 0.8-1.5; and the theoretical plate number calculated based on the roxadustat peak should be no less than 3000.

[0112] 3.6 Content Determination

[0113] Accurately measure 20 μL each of the test solution and the reference solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of roxadustat by peak area using the external standard method. The content limit is 95.0%-105.0% of the labeled amount.

[0114] 3.7 Impurity Determination

[0115] Calculate the content of each impurity using the main component self-comparison method. Accurately measure an appropriate amount of the test solution and dilute it with acidic diluent to prepare a self-comparison solution containing approximately 0.4 μg of roxadustat per mL. Accurately measure 20 μL each of the test solution and the self-comparison solution and inject them separately into the liquid chromatograph, recording the chromatograms. Calculate the content of each known and unknown impurity using the self-comparison method.

[0116] Impurity limits: Single known impurity ≤ 0.2%, single unknown impurity ≤ 0.1%, total impurities ≤ 1.0%.

[0117] 4. Quality evaluation results of three batches of samples

[0118] Three batches of roxadustat capsules, batch numbers 20250101, 20250102, and 20250103, were taken and subjected to biomimetic dissolution evaluation, content and impurity determination according to the methods described in the dissolution test of the roxadustat capsule test sample and the reference preparation and chromatographic determination of the test sample solution. The results are shown in Table 3.

[0119] Table 3. Quality evaluation results of three batches of Roxadustat capsules

[0120]

[0121] The results showed that the content of all three batches of samples was within the range of 95.0%-105.0% of the labeled amount; the content of each impurity met the limit requirements of ≤0.2% for a single known impurity, ≤0.1% for a single unknown impurity, and ≤1.0% for total impurities. Combined with the dissolution curve similarity factor results in Table 1, the three batches of samples showed consistent dissolution with the reference formulation in the biomimetic dissolution medium, and the content and impurity levels met the quality standards.

[0122] Comparative Example 1

[0123] The same test sample 20250101 and reference preparation as in Example 1 were used. The dissolution medium was a conventional dissolution medium without polyvalent metal ions, namely hydrochloric acid solution with pH 1.2, acetate-sodium acetate buffer solution with pH 4.5, and phosphate buffer solution with pH 6.8. The remaining dissolution conditions, sampling methods and determination methods were exactly the same as in Example 1.

[0124] The results showed that, in conventional dissolution media, the similarity factors of the dissolution curves of the test sample and the reference preparation were as follows: 78.6 for hydrochloric acid solution at pH 1.2, 75.3 for acetate-sodium acetate buffer at pH 4.5, and 71.2 for phosphate buffer at pH 6.8. All were greater than 50, indicating that the dissolution of the test sample and the reference preparation was consistent in conventional dissolution media.

[0125] As shown in Table 1, the same batch of test samples contained Fe in this invention. 3+ / Ca 2+ In the biomimetic dissolution medium, the dissolution curve similarity factors decreased to 72.3, 68.5, and 65.1, respectively, which were all lower than those in the conventional dissolution medium.

[0126] Experimental results show that conventional dissolution media cannot reflect the effect of polyvalent metal ions on dissolution, and their evaluation conditions deviate from the real gastrointestinal environment of CKD patients undergoing combined medication. The biomimetic media of this invention introduces Fe... 3+ / Ca 2+This makes the dissolution evaluation environment closer to the actual in vivo drug use scenario. Even in this complex ionic environment, the test sample still showed good consistency with the reference preparation, further confirming its quality reliability.

[0127] This comparative study observed a decreasing trend in the similarity factor of dissolution curves of specific formulations in biomimetic media. However, this does not mean that biomimetic media necessarily lead to a decrease in the similarity factor. Formulations prepared with different formulation compositions or process parameters may exhibit different directions and magnitudes of change in the similarity factor in biomimetic media. The value of biomimetic media lies in providing a more rigorous evaluation window that includes clinically interfering factors, thereby exposing quality differences that may be hidden under routine conditions, rather than pursuing uniform qualitative or quantitative patterns of change.

[0128] Please see Figures 2-3 As shown, where, Figure 2 This is a dissolution curve of the test sample and the reference preparation in Comparative Example 1 of the present invention in a conventional phosphate buffer solution at pH 6.8 (excluding multivalent metal ions). Figure 3 The test sample and reference preparation in Example 1 of this invention were prepared at pH 6.8 and contained Fe. 3+ / Ca 2+ Dissolution curve in a biomimetic dissolution medium.

[0129] Therefore, it can be seen that in conventional dissolution media, the cumulative dissolution curves of the test sample and the reference preparation at each time point basically overlap, with a dissolution curve similarity factor of 71.2, indicating that their dissolution behaviors are highly consistent under conventional conditions; while in Fe-containing media... 3+ / Ca 2+ In the biomimetic dissolution medium, the dissolution curves of the test sample and the reference formulation showed a certain degree of separation within the 15-30 minute time interval, and the dissolution curve similarity factor decreased to 65.1, a decrease of approximately 6 units compared to the conventional medium. These results indicate that the biomimetic dissolution medium of this invention, by introducing clinically relevant polyvalent metal ions, makes the in vitro dissolution evaluation conditions closer to the gastrointestinal environment of patients with chronic kidney disease taking iron supplements and calcium phosphate binders in combination. It can more sensitively reflect the differences in dissolution behavior of formulations in complex ionic environments, and its evaluation criteria are more stringent than those of conventional dissolution media, effectively improving the clinical relevance and discriminative power of dissolution tests.

[0130] Comparative Example 2

[0131] Take the contents of Roxadustat capsules from the same batch number 20250101 and prepare test solutions using the following three diluents:

[0132] The acidic diluent of this invention is a 0.05% aqueous solution of phosphoric acid, pH 2.0.

[0133] Neutral diluent: purified water, pH approximately 6.5;

[0134] Alkaline diluent: 0.05 mol / L phosphate buffer, pH 8.0;

[0135] The test solution was prepared using all three diluents according to the method described in 3.1. The solution was injected and measured at 0, 6, 12, and 24 hours after preparation. The peak area of ​​roxadustat was recorded, and the percentage of peak area at each time point relative to 0 hours was calculated. The results are shown in Table 4.

[0136] Please also refer to Figure 4 As shown, Figure 4 The figure shows the stability test results of the roxadustat test solution under different pH diluent conditions in Comparative Example 2 of this invention.

[0137] Therefore, it can be seen that in the acidic diluent at pH 2.0, the peak area of ​​roxadustat decreased by only 0.9% within 24 hours, indicating that the acidic diluent selected in this invention can effectively inhibit the degradation of roxadustat during the preparation and storage of the test solution. In the neutral diluent at pH 6.5, the peak area decreased by 5.0% within 24 hours; and in the alkaline diluent at pH 8.0, the peak area decreased by 15.0% within 24 hours, and reached 96.5% within 6 hours, with a degradation rate significantly higher than under acidic and neutral conditions. These results indicate that roxadustat is extremely unstable under alkaline conditions, which is consistent with the chemical principle that roxadustat contains β-keto acid groups in its molecular structure and is prone to oxidative degradation under alkaline conditions. This also further confirms that the acidic diluent can effectively ensure the stability of the test solution throughout the chromatographic analysis process, avoiding underestimation of content and overestimation of impurities due to degradation during sample preparation, thus ensuring that the quality control data truly reflects the inherent quality attributes of the product.

[0138] Table 4. Stability test of roxadustat solution under different diluent conditions

[0139]

[0140] The results showed that the β-keto acid group in the roxadustat molecular structure is prone to oxidative degradation under alkaline conditions, leading to a gradual decrease in the peak area of ​​the main component over time. The degradation rate was approximately 5% under neutral conditions, as high as 15% under alkaline conditions, while the degradation rate was less than 1% within 24 hours under acidic conditions. This invention uses an acidic diluent, effectively ensuring the stability of the test solution throughout the chromatographic analysis process. This avoids underestimating the content and overestimating the impurities due to degradation during sample preparation, allowing quality control to more accurately reflect the true quality attributes of the product.

[0141] Comparative Example 3

[0142] Take a batch of the known and reference formulations in the Fe content 3+ / Ca 2+Roxadustat capsules with inconsistent dissolution profiles in a biomimetic dissolution medium, batch number 20250104, had their formulation modified by reducing croscarmellose sodium (added internally) from 1.8 mg to 0.9 mg (i.e., a 50% reduction). The remaining components and preparation process were identical to those in Example 1. Dissolution tests were conducted according to the method in Example 1, with the addition of Al. 3+ Media verification. Results are shown in Table 5.

[0143] Table 5. Similarity factor verification of samples with inconsistent dissolution curves in different biomimetic dissolution media.

[0144]

[0145] The results showed that in triple Fe 3+ / Ca 2+ In the biomimetic dissolution medium, the similarity factor of the dissolution curves was less than 50, indicating inconsistent dissolution. (Adding Al...) 3+ The similarity factor of the dissolution curves in the verification test was still less than 50, which further confirmed that the dissolution of the test sample and the reference preparation were inconsistent under different polyvalent metal ion environments, and the results were consistent.

[0146] When the test sample and the reference preparation are in Fe 3+ / Ca 2+ When the similarity factor of the dissolution curve in the biomimetic medium is less than 50, there may be two reasons: first, differences in the formulation or process of the preparation itself lead to different dissolution behaviors; second, the specific multivalent metal ion environment has different degrees of influence on the test sample and the reference preparation. By adding Al... 3+ Verification can aid in judgment: if in A 3+ If the similarity factor of the dissolution curves in the medium is still less than 50, it indicates that the differences mainly stem from the formulation itself, and the formulation needs to be optimized; if in Al 3+ If the similarity factor of the dissolution curve in the medium is greater than or equal to 50, it indicates that the difference has ion selectivity, and it may be necessary to adjust the release behavior under specific conditions.

[0147] In this comparative example, Al 3+ Verification results and Fe 3+ / Ca 2+ The results in the medium were at similar levels, all less than 50 and with little difference in value, indicating that changing different trivalent cations did not change the conclusion of inconsistent dissolution, further ruling out the possibility that the difference was due to ion selectivity, and confirming that the above-mentioned inconsistent dissolution was caused by the formulation difference of the preparation itself.

[0148] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

[0149] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A quality control method for roxadustat capsules, characterized in that, include: A biomimetic dissolution medium containing polyvalent metal ions was prepared, wherein the biomimetic dissolution medium is at least three buffer solutions with a pH of 1.2-6.8, to simulate the gastrointestinal metal ion environment when patients with chronic kidney disease take iron supplements and calcium phosphate binders in combination. Dissolution tests were conducted on the roxadustat capsule test sample and the reference preparation using the aforementioned biomimetic dissolution medium to determine the dissolution curve similarity factor, so as to evaluate the dissolution consistency of roxadustat capsule in the presence of multivalent metal ions. Accurately weigh the contents of roxadustat capsules that have dissolved uniformly, dissolve and dilute them with an acidic diluent to the predetermined concentration to obtain the test solution. The test solution was analyzed by high performance liquid chromatography with gradient elution. The chromatogram of the test solution was recorded. The content of each impurity was calculated by the main component self-comparison method, and the content of roxadustat was calculated by the external standard method.

2. The quality control method for roxadustat capsules according to claim 1, characterized in that, The multivalent metal ions include Fe 3+ and Ca 2+ ; The Fe 3+ The concentration of Ca is 0.05-0.20 mmol / L. 2+ The concentration is 1.0-3.0 mmol / L.

3. The quality control method for roxadustat capsules according to claim 1, characterized in that, The at least three buffer solutions include: Hydrochloric acid solution with pH 1.2±0.05, acetate-sodium acetate buffer solution with pH 4.5±0.05, and phosphate buffer solution with pH 6.8±0.

05.

4. The quality control method for roxadustat capsules according to claim 1, characterized in that, The process of determining the dissolution curve similarity factor includes: The test sample and the reference preparation were subjected to dissolution tests in the biomimetic dissolution medium. The dissolution solutions of the test sample and the reference preparation were collected at multiple preset time points to determine the average cumulative dissolution of the test sample and the reference preparation at each time point. The average cumulative dissolution rate of the reference formulation at each time point is used as the reference curve, and the average cumulative dissolution rate of the test sample at each time point is used as the test curve. The average value of the sum of squares of the differences between the reference curve and the test curve at each time point is calculated to determine the dissolution curve similarity factor.

5. The quality control method for roxadustat capsules according to claim 4, characterized in that, When the dissolution curve similarity factor is greater than or equal to 50, it is determined that the dissolution of roxadustat capsules is consistent in the environment of coexistence of multivalent metal ions; When the dissolution curve similarity factor is less than 50, it is determined that the dissolution of roxadustat capsules is inconsistent in the presence of multiple metal ions, and then an additional product containing Al is added. 3+ The Al was subjected to a verification test using a dissolution medium. 3+ The concentration is 0.1-0.5 mmol / L.

6. The quality control method for roxadustat capsules according to claim 1, characterized in that, The acidic diluent is a 0.05% aqueous solution of phosphoric acid with a pH of 2.0 ± 0.2, to inhibit the oxidative degradation of roxadustat via β-keto acid groups under alkaline conditions; The predetermined concentration is 0.10-0.50 mg / mL for roxadustat.

7. The quality control method for roxadustat capsules according to claim 1, characterized in that, The chromatographic conditions for the high performance liquid chromatography method are as follows: octadecylsilane-bonded silica gel as the packing material, column temperature of 35-40℃, detection wavelength of 260nm, and flow rate of 1.0mL / min.

8. The quality control method for roxadustat capsules according to claim 7, characterized in that, The gradient elution uses 0.02 mol / L potassium dihydrogen phosphate solution as mobile phase A and acetonitrile as mobile phase B. The mobile phase A was adjusted to a pH of 3.0 ± 0.1 with phosphoric acid; The gradient elution procedure is as follows: 0-5 min: Mobile phase A 85% → 85%, Mobile phase B 15% → 15%; 5-20 min: Mobile phase A 85% → 60%, Mobile phase B 15% → 40%; 20-30 min: Mobile phase A 60% → 30%, Mobile phase B 40% → 70%; 30-35 min: Mobile phase A 30% → 30%, Mobile phase B 70% → 70%; 35-40 min: Mobile phase A 30% → 85%, Mobile phase B 70% → 15%; 40-48 min: Mobile phase A 85% → 85%, Mobile phase B 15% → 15%.

9. The quality control method for roxadustat capsules according to claim 1, characterized in that, The limits for roxadustat content are 95.0%-105.0% of the labeled amount, the limit for a single known impurity is ≤0.2%, the limit for a single unknown impurity is ≤0.1%, and the limit for total impurities is ≤1.0%.

10. The quality control method for roxadustat capsules according to claim 1, characterized in that, It also includes system suitability testing: Take roxadustat reference standard and at least one known impurity reference standard, dissolve and dilute them with the acidic diluent to prepare a system suitability solution, and inject it into the liquid chromatograph; Among them, the separation degree between the main peak of roxadustat and the adjacent impurity peak is ≥1.5, the tailing factor is 0.8-1.5, and the theoretical plate number is ≥3000.

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