VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF AXITINIB AND AVELUMAB BY USING ANALYTICAL QUALITY BY DESIGN (AQbD) METHOD

VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF AXITINIB AND AVELUMAB BY USING ANALYTICAL QUALITY BY DESIGN (AQbD) METHOD

K. Nithiyananthan * and K. V. S. Prasada Rao

Acharya Nagarjuna University, Guntur, Andhra Pradesh, India.

ABSTRACT: The quantitative measurement of Axitinib and Avelumab has been created using a simple, quick, precise, sensitive, and reproducible reverse-phasehigh-performance liquid chromatography (RP-HPLC) method. It is more difficult to analyse varying amounts of pharmaceutical active medicinal ingredients in dose forms without any interferences. Therefore, the objective of the current work is to estimate Axitinib and Avelumab simultaneously by adopting an Analytical Quality by Design (AQbD) arotatable central composite-based technique using RP-HPLC-based method development and validation. Axitinib and Avelumab were separated by chromatography using a Hyperclone 5µ BDS C18 130A (150 x 4.6 mm, 5μ) column and a mobile phase made up of Acetonitrile: 0.1% TEA pH-2.5/OPA in a ratio of 45:55 v/v. The flow rate was 1.2 ml/min, and a Photodiode Array Detector operating at room temperature was used to detect absorption at 219 nm. ICH criteria have been used to validate the offered techniques' linearity, accuracy, precision, and other attributes. The degradation study's findings showed that the medications deteriorated in high-stress situations. The chemical and pharmaceutical sectors might easily implement this unique AQbD-based analytical technique for routine analysis without any regulatory constraints.

Keywords: RP-HPLC, Analytical quality by design (AQbD), Central Composite Design, Axitinib and Avelumab

INTRODUCTION: Clear-cell renal-cell carcinoma, the most common kind of renal cancer, is characterised by genetic defects that cause excessive production of vascular endothelial growth factor (VEGF), a crucial regulator of angiogenesis. Even though sunitinib is a first-line treatment option that is considered to be the standard of care for patients with advanced renal cell carcinoma, many of these patients have an innate resistance to antiangiogenic medications or have a progressing disease.

Avelumab, an anti-programmed death ligand 1 (PD-L1) antibody, is one type of immune checkpoint inhibitor. As first- and second-line treatments for patients with a variety of tumour types, including advanced renal-cell carcinoma, these medications have been found to have acceptable safety and long-lasting anticancer activity ¹.

VEGF receptor (VEGFR) inhibitors have immunomodulatory effects in addition to antiangiogenic effects. These effects include increased immune cell infiltration of tumours and decreased immunosuppressive activity of myeloid-derived suppressor cells. We predicted that the complimentary modes of action of an immune checkpoint inhibitor and a VEGF-targeted antiangiogenic treatment may increase the therapeutic benefits. Advanced renal cell carcinoma is a condition for which axitinib, a highly selective VEGFR inhibitor, is approved for treatment ². Since, the drug combination was being tested in clinical studies for the treatment of renal cancer, relatively few analytically fundamental approaches were reported. However, up until this point, no reports have been made on the estimation of axitinib and avelumab stability using the quality-by-design method. This research's initial report will include a thorough analysis of techniques variables, a complete profile of the targeted medicine, and risk assessment considerations. The QbD methodology, which provides the precise interaction of several variables at a time on method response, completely overcame the drawbacks of the classic one-factor altering method. To estimate Axitinib and Avelumab via the QbD technique, a simple, sensitive, precise, robust selective stability indicating RP-HPLC method was devised.

MATERIALS AND METHODS:

Drugs, Chemicals, Solvents, Instruments and Software: Axitinib and Avelumabare generously gifted from the Shree Icons laboratories, in Vijayawada, India. HPLC grade Tri Ethyl Amine (TEA) was purchased from Thermo Fisher Scientific (Maharashtra, India). HPLC grade acetonitrile and orthophosphoric acid (OPA) from Rankem fine chemicals limited (NewDelhi, India).

The High-performance liquid chromatographic (HPLC) system utilized for the whole analysis was Waters e 2695HPLC (Wilmslow, England), united with a double solvent manager with a photodiode array detector (PDA) along with an auto sampler. Unichrome ultrasonic baths have been used to solubilize and degas the sample and solvents. The pH of the mobile phase was adjusted with an Eutech Digital pH Meter (Maharashtra, India). Waters HPLC system unified with Empower 2.0 software for data management. AQbD was developed using Design-Expert® trail version 13 (Stat-Ease Inc., Minneapolis-USA).

Solutions Preparations:

Preparation of Standard: Accurately weigh and transfer 5 mg of Axitinib, and 20 mg of Avelumab working standard into a 10 ml clean dry volumetric flask add Diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent (Stock solution). Further pipette 1 ml of the above stock solutions into a 10 ml volumetric flask and dilute up to the mark with diluent. (50ppm of Axitinib, 200ppm of Avelumab)

Preparation of Sample: Accurately weigh and transfer 83.4 mg of Axitinib tablet powder and 1ml of Avelumab sample into a 10mL clean dry volumetric flask add diluent and sonicate it up to 30 mins to dissolve, and centrifuge for 30min. to dissolve it completely and make volume up to the mark with the same solvent. Then it is filtered through a 0.45-micron Injection filter (Stock solution). Further pipette 1 ml of the above stock solutions into a 10 ml volumetric flask and dilute up to the mark with diluents. (50ppm of Axitinib, 200ppm a of Avelumab).

Final Optimized Chromatographic Conditions in RP-HPLC using AQbD ^{3, 4}: The separation and determination of two drugs have been achieved with the help of rotatable central composite-based AQbD using Hyperclone 5µ BDS C18 130A (150x4.6 mm, 5 µ) column. The mobile phase comprises HPLC grade acetonitrile: 0.1% TEA (pH adjusted to 2.5 using orthophosphoric acid) in a ratio of 45: 55 v/v. The optimized chromatographic conditions were validated according to the ICH Q2R1 guidelines for specificity, linearity, accuracy, precision, precision, LOD & LOQ and ICH Q2B for degradation studies.

System Suitability ⁵: The tailing factor for the peaks due to Axitinib and Avelumab in the Standard solution should not be more than 2.0. Theoretical plates for the Axitinib and Avelumab peaks in the Standard solution should not be less than 2000. Resolution for the Axitinib and Avelumab peaks in standard solution should not be less than 2.

Assay: Inject 10 µL of the standard, sample into the chromatographic system and measure the areas for Axitinib and Avelumab peaks and calculate the percentage.

Specificity: The specificity of an analytical method is the ability to measure specifically the analyte of interest without interference from blank and known impurities. For this purpose, blank chromatogram, standard chromatogram and sample chromatogram were recorded. The chromatogram of blank shows no response at the retention times of drugs which confirms the reaction of drugs was specific ⁶.

Linearity:

Preparation of Stock Solution: Accurately weigh and transfer 5mg of Axitinib, and 20mg of Avelumab working standard into a 10ml clean dry volumetric flask add Diluent and sonicate to dissolve it entirely and make volume up to the mark with the same solvent (Stock solution). Prepare 6 levels of samples with varying concentrations viz., 12.5, 25, 37.5, 50, 62.5, and 75 ppm of Axitinib and 50, 100, 150, 200, 250, 300 ppm of Avelumab respectively.

Procedure: Inject each level into the chromatographic system and measure the peak area. Plot a graph of peak area versus concentration (on the X-axis concentration and Y-axis Peak area) and calculate the correlation coefficient ⁷.

Accuracy ⁸: Concerning target Assay concentration were prepared i.e., 50, 100 & 150 % solutions of Axitinib and Avelumab and determined.

Procedure: Inject the standard solution, Accuracy -50%, Accuracy -100% and Accuracy -150% keys.

Acceptance Criteria: The percentage recovery for each level should be between 98.0 to 102.0%.

Precision ⁹: Precision is the degree of repeatability of an analytical method under normal operation conditions. Precision is of 3 types

1. System precision
2. Method precision (Repeatability)
3. Intermediate precision (a. Intraday precision, b. Inter day precision)

System precision is checked by using standard chemical substances to ensure that the analytical system is working properly. In this peak area and % of drug of six determinations is measured and % RSD should be calculated. In method precision, a homogenous sample of a single batch should be analyzed 6 times. This indicates whether a method is giving constant results for a single batch. In this analyze the sample six times and calculate the % RSD. The precision of the instrument was checked by repeatedly injecting (n=6) solutions of 50ppm of Axitinib and 200ppm of Avelumab).

Acceptance Criteria: The % RSD for the absorbance of six replicate injection results should not be more than 2%.

Degradation Studies:

Preparation of Stock: Accurately weigh and transfer 83.4 mg of Axitinib and 1 ml of Avelumab sample into a 10 ml clean dry volumetric flask add Diluent and sonicate to dissolve it entirely and make volume up to the mark with the same solvent. (Stock solution)

Acid Degradation: Pipette 1 ml of the aforementioned solution was added to a 10 ml vacuum flask, followed by 1 ml of 1N HCl. The vacuum flask was then maintained at 60°C for 1 hour before being neutralised with 1 N NaOH and diluted to 10ml with diluent. Filter the solution using 0.22-micron syringe filters and transfer it to bottles.

Alkali Degradation: Pipette 1 ml of the above solution into a 10 ml volumetric flask and add 1ml of 1N NaOH was added. Then, the volumetric flask was kept at 60ºC for 1 hour and then neutralized with 1N HCl and made up to 10ml with diluent. Filter the solution with 0.22-micron syringe filters and place it in vials.

Thermal Degradation: Axitinib and Avelumab sample was taken in a Petri dish and kept in a Hot air oven at 105°C for 3 hours. Then the sample was taken and diluted with diluents injected into HPLC and analysed.

Peroxide Degradation: Pipette 1 ml above stock solution was added to a 10 ml vacuum flask, 1 ml of 3 per cent w/v hydrogen peroxide was added to the flask and the volume was built up to the mark using diluent. The vacuum flask was then maintained at 60^oC for 1 hour. After that, the vacuum flask was left at room temperature for 15 minutes. Filter the solution using 0.22-micron syringe filters and transfer it to bottles.

Reduction Degradation: Pipette 1ml of the above-stock solution was added to a 10ml vacuum flask, 1ml of 10% Sodium bisulphate was added to a flask and the volume was built up to the required volume with diluent. The vacuum flask was then maintained at 60^oC for 1 hour. After that, the vacuum flask was left at room temperature for 15 minutes. Filter the solution using 0.22-micron syringe filters and transfer it to bottles.

Photolytic Degradation: Axitinib and Avelumab sample was placed in a Photo stability chamber for 3 hours. Then the sample was taken and diluted with diluents injected into HPLC and analysed.

Hydrolysis Degradation: Pipette 1ml of above-stock solution was added to a 10ml vacuum flask, 1ml of HPLC grade water was added to a flask and the volume was built up to the required volume with diluent. The vacuum flask was then maintained at 60°C for 1 hour. After that, the vacuum flask was left at room temperature for 15 minutes. Filter the solution using 0.22-micron syringe filters and transfer it to bottles.

RESULTS AND DISCUSSION:

Method Development: The mobile phase used for chromatographic separation was acetonitrile: 0.1% TEA (pH adjusted to 2.5 using orthophosphoric acid) in a ratio of 45: 55 v/v at a flow rate of 1 mL/min. The column temperature was kept at 25°C, and 219 nm detection was used. Axitinib and Avelumab’sretention times were 2.141 minutes and 3.832 minutes, respectively Fig. 1. The optimized chromatographic conditions were shown in Table 1.

FIG. 1: OPTIMIZED CHROMATOGRAM OF AXITINIB AND AVELUMAB

TABLE 1: OPTIMIZED CHROMATOGRAPHIC CONDITIONS

Design of Experiment using AQbD: Mobile phase ratio, mobile phase pH, and column temperature were shown to be the analytical characteristics that have an impact on the performance of the method based on the chosen chromatographic settings. The method responses selected were the number of theoretical plates and resolution between degradant peak 1 and degradant peak 2 which likely co-elute and lead to method failure often. 2-factor 3-level factorial designs were preferred in the response surface method. The selected method responses and their levels are given in Table 1. Table 2 lists the summary of solutions used and selected. Thirteen chromatogram runs were carried out using the central composite design in Design Expert software Table 3.

TABLE 2: SUMMARY OF SOLUTIONS USED AND SELECTED

TABLE 3: CENTRAL COMPOSITE DESIGN FOR SCREENING OF METHOD PARAMETERS

Statistical Analysis of Method Response 1 – Theoretical Plates: ANOVA for method response 1: The Design Expert programme provided the analysis of variance (ANOVA) regression parameters for the projected response surface quadratic model for the number of theoretical plates Table 4. The model is important given its Model F-value of 175.22. A "Model F-value" this large could only occur owing to noise with a 0.0001% chance. When "Prob > F" is less than 0.0500, model terms are considered significant. A, B, and A2 are important model terms in this instance. Model terms are not significant if the value is higher than 0.1000. The "Lack of Fit F-value" of 6.24 indicates that there is a 5.46 per cent possibility that noise might be the cause of a "Lack of Fit F-value" this significant. As one may typically anticipate, the "Pred R-squared" of 0.9514 is close to the "Adj R-squared" of 0.9864. The signal-to-noise ratio is measured using "Adeq Precision." A ratio of at least 4 is preferred. An acceptable signal is indicated by the model ratio of 40.7549. The design space can be explored using this model.

TABLE 4: ANOVA FOR QUADRATIC MODEL - RESPONSE 1: PEAK ONE THEORETICAL PLATES

3D and Contour Graph for the Number of Theoretical Plates: From the ANOVA report on the number of theoretical plates, it is clearly stated that the interaction effect of flow rate and mobile phase has a p-value higher than 0.05 so the interaction effect of these terms does not have any significant effect on the number of theoretical plates. By keepinga constant temperature at 25°C, the interaction effect of pH of the organic phase and flow rate were studied from contour and 3D graphs. From the graph, a lower flow rate and lower level of the mobile phase can give a lesser number of theoretical plates Fig. 2 & 3.

FIG. 2: CONTOUR PLOTS FOR THEORETICAL PLATES AS A FUNCTION OF MOBILE PHASE AND FLOW RATE (CONSTANT TEMPERATURE 30^OC)

FIG. 3: RESPONSE SURFACE FOR THEORETICAL PLATES AS A FUNCTION OF MOBILE PHASE AND FLOW RATE (CONSTANT TEMPERATURE 30^OC)

Statistical analysis of Method Response 2 – Resolution: ANOVA for method response 2: The Design Expert programme provided the analysis of variance (ANOVA) regression parameters for the projected response surface quadratic model for the number of theoretical plates Table 5.

The model is important given its Model F-value of 7.86. A "Model F-value" this large could only occur owing to noise with a 0.0001% chance. When "Prob > F" is less than 0.0500, model terms are considered significant. Model terms are not significant if the value is higher than 0.1000. The "Lack of Fit F-value" of 4.77 indicates that there is a 5.26 per cent possibility that noise might be the cause of a "Lack of Fit F-value" this significant. As one may typically anticipate, the "Pred R-squared" of 0.1082 is close to the "Adj R-squared" of 0.7409. The signal-to-noise ratio is measured using "Adeq Precision." A ratio of at least 4 is preferred. An acceptable signal is indicated by the model ratio of 7.9619.

TABLE 5: ANOVA FOR QUADRATIC MODEL - RESPONSE 2: RESOLUTION

3D and Contour Graph for Resolution: From the ANOVA report on resolution, it is clearly stated that the interaction effect of organic phase and flow rate has a p-value higher than 0.05 so the interaction effect of these terms does not have any significant effect on resolution. By keeping a constant temperature at 25°C, the interaction effect of flow rate and organic phase were studied from contour and 3D graphs Fig. 4 & 5.

FIG. 4: CONTOUR PLOTS FOR RESOLUTION AS A FUNCTION OF MOBILE PHASE AND FLOW RATE (CONSTANT TEMPERATURE 30^OC)

FIG. 5: RESPONSE SURFACE FOR RESOLUTION AS A FUNCTION OF MOBILE PHASE AND FLOW RATE (CONSTANT TEMPERATURE 30^OC)

System Suitability: All the system suitability parameters were within the range and satisfactory as per ICH guidelines. According to ICH guidelines, the plate count should be more than 2000, the tailing factor should be less than 2 and the resolution must be more than 2. All the system-suitable parameters were passed and were within the limits Table 6.

TABLE 6: SYSTEM SUITABILITY PARAMETERS FOR AXITINIB & AVELUMAB

Assay:

TABLE 7: ASSAY OFAXITINIB & AVELUMAB

FIG. 6: CHROMATOGRAM OF ASSAY-1

FIG. 7: CHROMATOGRAM OF ASSAY-2

Method Validation: Axitinib and Avelumab had retention durations of 2.141 minutes and 3.832 minutes, respectively. At the retention time of the medications in this approach, we did not detect any interfering peaks in the placebo or blank samples. This approach was therefore said to be specific Fig. 8. The devised technique was linear over the concentration ranges of 12.50-75 g/ml for axitinib and 50-300 g/ml for avelumab, with correlation coefficients of 0.9998 and 0.9997, respectively Table 8 & Fig. 9. The per cent recovery of the drug was determined to be within 99-100.4 per cent for the accuracy studies at 50, 100, and 150 per cent levels Table 10. System, method, and intermediate precision were used, and the results showed that the per cent RSD values were less than 1% Table 11-13.

Specificity:

FIG. 8: OPTIMIZED CHROMATOGRAM FOR SPECIFICITY

TABLE 8: RESULTS OF LINEARITY FOR AXITINIB & AVELUMAB

FIG. 9: CALIBRATION CURVE FOR AXITINIB AND AVELUMAB

Accuracy:

TABLE 9: ACCURACY RESULTS OF AXITINIB AND AVELUMAB BY RP-HPLC METHOD

Precision:

System Precision:

TABLE 10: SYSTEM PRECISION TABLE OF AXITINIB & AVELUMAB

Method Precision:

TABLE 11: METHOD PRECISION FOR AXITINIB & AVELUMAB BY RP-HPLC METHOD

Intermediate Precision:

TABLE 12: INTERMEDIATE PRECISION (DAY VARIATION) FOR AXITINIB AND AVELUMAB BY RP-HPLC METHOD

Degradation Studies: Forced degradation studies of Axitinib and Avelumab were observed in various conditions such as acidic, basic, peroxide, reduction, thermal, photolytic, and hydrolytic conditions. The Axitinib and Avelumab were stable under, reduction, thermal, photolytic and hydrolytic conditions. The drug showed significant degradation in acidic, basic and peroxide conditions represented in Fig. 11-13. The results of forced degradation studies are presented in Table 14.

TABLE 13: FORCED DEGRADATION RESULTS FOR AXITINIB AND AVELUMAB

FIG. 10: CHROMATOGRAM OF ACID DEGRADATION

FIG. 11: CHROMATOGRAM OF ALKALI DEGRADATION

FIG. 12: CHROMATOGRAM OF PEROXIDE DEGRADATION

CONCLUSION: Using an analytical quality-by-design methodology, a straightforward, reliable, and robust RP-HPLC method was created for the quantification of Axitinib and Avelumab. Flow rate and the percentage of organic material in the mobile phase were chosen as the key method parameters (CMPs). Retention time and theoretical plates are the essential quality characteristics. A rotating central composite design was used to methodically tune the CMPs (CCD). Mobile phase acetonitrile: 0.1% TEA (pH adjusted to 2.5 using orthophosphoric acid) in a ratio of 45:55 v/v pumped at a flow rate of 1.2 ml/min constitute the optimal chromatographic conditions. For the drugs Axitinib and Avelumab, the retention times were determined to be 2.141 and 3.832 minutes, respectively. Asymmetry and theoretical plates were discovered to be within the bounds. The created method was accepted by ICH Q2 (R1) recommendations. Studies on drug degradation under different stress conditions revealed that the medication deteriorated more quickly in acidic, basic, and peroxide environments.

ACKNOWLEDGEMENT: We like to thank Shree ICON Pharmaceutical laboratories, Vijayawada for providing analytical support.

CONFLICTS OF INTEREST: The authors declares that there is no conflicts of interest involved.

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   How to cite this article:

   Nithiyananthan K and Rao KVSP: Validation of stability indicating RP-HPLC method for simultaneous estimation of Axitinib and Avelumab by using analytical quality by design (AQbD) method. Int J Pharm Sci & Res 2024; 15(3): 944-55. doi: 10.13040/IJPSR.0975-8232.15(3).944-55.

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