INTRODUCTION:

An autoimmune disorder known as rheumatoid arthritis is a condition where inflammation in joints is observed and also inflammation is seen in some parts of body in rare cases. The major cause is disability which impact on the daily activities of the person effected by this disease^1-2. Sanofi and Regeneron Pharmaceuticals, Inc. collaborated to develop Sarilumab, it was received the United States Food and Drug Administration approval in May 2017 and the approval of European Union in June 2017. With combination of methotrexate, the sarilumab is used to treat rheumatoid arthritis disease, which is the reason for approval of drug sarilumab³. The patients who are not tolerable to DMARDS (Disease modifying antirheumatic drugs) are prescribed the drug sarilumab for the treatment of the disease⁴.

Sarilumab is an IgG1 mAb antibody that targets the IL-6 receptor and inhibits cis and trans inflammatory signalling pathways of IL-6 by binding to both types of IL-6 receptor on the membranes and soluble IL-6 receptor variants⁵.

Brand name of Sarilumab is Kevzara⁶. The chemical formula and molecular weight of sarilumab is C₆₃₈₈ H₉₉₁₈ N₁₇₁₈ O₁₉₉₈ S₄₄ and 150kDa⁷. IL-6 is a protein that plays a major role in the immune response, triggering the release of biomarkers such as CRP, serum amyloid A, and fibrinogen, which are symptomatic of RA activity. It is also present in joint fluid, where it leads to both inflammation and damage. Sarilumab act by inhibiting the IL-6 receptor which plays a major role in inflammation of joints and damage of some parts which effect by rheumatoid arthritis disease. By inhibiting IL-6 signaling in both the joints and throughout the body, Sarilumab helps to decrease inflammation and effectively manage RA symptoms. Additionally, in critically ill COVID-19 patients receiving organ support in intensive care units, treatment with IL-6 receptor antagonists such as tocilizumab and sarilumab has been illustrated to enhance output, including survival^8-11. Using LC-MS/MS. the peramivir and its compounds were separated in which the sarilumab was used as an internal standard¹². There are currently no documented techniques for using ultra-performance liquid chromatography (UPLC) to quantify sarilumab in a pharmaceutical formulation. In addition to conducting forced degradation studies to examine the stability of sarilumab under various stress settings, this research attempts to design and validate a UPLC method for separating sarilumab. The ICH Q2R1 guidelines, which specify the requirements for analytical methods and associated validation parameters, will be followed in the validation of the method. Furthermore, stability testing will adhere to the ICH Q1A(R2) guidelines to identify any degradation products and study the stability of Sarilumab in different solvents. A stability-indicating method helps to separate the drug from its breakdown products, providing a clear and reliable analysis.¹³ Sarilumab structure is represented in fig 1.

Figure 1: Structure of Sarilumab

MATERIALS AND METHODS:

Standard Drug and Reagents:

Sarilumab API samples with purity of 98-100% was obtained from Pharma Life Research facility in Hyderabad, India and other chemicals were of AR-grade from Ranken Chemicals, India.

Methodology:

This method intends to improve the ultra-high-resolution liquid chromatography process for estimating Sarilumab. Important factors would include buffer pH, mobile phase ratio, and the construction and evaluation of the UPLC and gain all the concepts to maximize the performance of the technique.

UPLC instrumentation:

The present study was accomplished using an Acuity UPLC CSH C18 Column with Empower 2 software, UV detector, auto-injector, Shimadzu UV-Visible spectrophotometer, Ohaus Electronic balance, Utech pH Meter and Phoenix 4.5 L digital ultrasonic cleaner.

Chromatographic conditions:

Flow rate	0.5ml/min
Column	Acquity UPLC CSH C18 Column, 130Å, 1.7µm, 2.1mm X 100mm
Detector wave length	212.0 nm
Column temperature	43°C
Injection volume	2.0mL
Run time	2.0 minutes

Standard Preparation:

Following precise measurements, we transferred 10mg of Sarilumab from the prepared syringe into 25ml of neat dry volumetric flask, then 10ml of diluent was added and the solution was sonicated for 10minutes then adjusted the final volume with diluent to get 400µg/ml Sarilumab.

Standard Working Preparation:

Fill a 10ml volumetric flask with 1ml of the stock solution mentioned above, then top it off with 10ml. (40µg/ml Sarilumab).

Sample Preparation:

The study sample of the sarilumab synthesis process sample was prepared and the injection formulation equivalent to the label claim of 200mg was taken at the 1.14ml filled volume. It was transferred into a 100ml volumetric flask. After adding ¾th of the diluent, the solution was sonicated for 30 minutes and then adjusted to the final volume with diluent, and filtered with 0.25um nylon filter (2000µg/ml Sarilumab).

Sample Working Preparation:

To make 10ml of the aforesaid stock solution (40µg/ml Sarilumab), 0.5ml was transferred into a 25ml volumetric flask.

Buffer solution:

Potassium dihydrogen phosphate

Preparation of buffer:

Sterilize the solution by passing it through a 0.22-micron filter after adding 1.36 grams of potassium dihydrogen phosphate to a 1000 milliliter solution of distilled water in an appropriate vessel. The solution should be kept at ambient temperature or at 4°C in firmly sealed containers. Acetic acid was added in 1 milliliter (0.1%) to bring the pH down to 5.0.

Mobile Phase: Buffer and water were taken in the ratio 70:30.

Method development:

For method development of a drug the crucial conditions for an optimizing chromatogram are column chemistry, mobile phase composition, column temperature, and flow rate these are necessary for an evaluation of chromatographic parameters. The process helps to obtain a symmetrical peak shape with enhanced resolution of drug compounds. The optimized chromatogram is presented in Figure 3, showcasing the enhanced performance of the method.

Method validation:

Validation is defined as documented evidence to evaluate whether the analytical system is within the specified limits and meet the required specifications. Method validation is a process or the steps necessary to assess the performance of the analytical system present as per the standard guidelines. In liquid chromatography the crucial parameters evaluated are specificity, precision, accuracy, linearity, robustness, recovery, detection and quantification limits, and ruggedness. Validation requires a clear and comprehensive protocol method to ensure concentration ranges, and specific test materials are accurate and reliable. It improves the overall precision of the entire analytical system, rather than just the individual method. The validation process is perfectly planned to estimate both the analytical system and the procedural steps which are involved^14-18.

Linearity, Precision, Accuracy, Robustness studies: ^19-21

In accordance to ICH guidelines, linearity is defined as the ability of the method to produce results that are directly proportional to the concentration of the substance being tested across a set range, which reflects the distance between the lowest and highest concentrations in which the method can determinately and reliably identify the substance with accuracy and precision. These measures are important to guarantee that the method promises what it intends to do. Precision is the ability of a method to yield the same result for the same sample multiple times. Accuracy is the ability of a method to return the true value of the substance being identified. Robustness is the ability of the method withstands slight deviations in experimental conditions such as minute variations in temperature or flow rate. The absence of interfering peaks in the chromatograms of the blank, placebo, and sample solutions during the retention time of Sarilumab during analysis confirmed the specificity of the suggested UPLC approach. This demonstrates that Sarilumab will be uniquely identified by the suggested method, with other samples having no effect on the outcomes. The blank and placebo chromatograms are presented in figures 4 & 5. In Table 1 and Figure 3, the optimized chromatogram and the associated data are displayed.

Assay and Forced degradation studies^22-25

UPLC analysis was performed by injecting 10µL of standard and sample solution separately. Using peak areas obtained from the chromatograms, the quantification of the drug in the solution was done, which was accurate for measuring the concentration of drug in the sample. To examine the intrinsic stability of the drug substance, stress testing was conducted based on the recommendations provided by the International Council for Harmonization (ICH). Suitably, stress testing was performed against the standard solution with several environmental stress conditions.

· Acid degradation: The sampled drug was exposed to 2 N HCl. The sample was refluxed at 60 °C for 30 minutes.

· Alkali degradation: The sampled drug was exposed to 2 N NaOH. The sample was refluxed with distilled water at 60 °C for 30 minutes.

· Peroxide degradation: The sampled drug was exposed to 20% hydrogen peroxide. The sample was heated at 60 °C for 30 minutes.

· Thermal degradation: The sampled drug was placed in a hot air oven at 105 °C for 6 hours.

· Photolytic degradation: The sampled drug was placed in a ultraviolet (UV) chamber for seven days.

· Hydrolysis: The sampled drug was subjected to hydrolytic conditions to obtain the degradation in a moisture environment.

As noted above, the stressed samples peak areas were measured. Each of the stressed drug samples peak area was compared to the standard sample peak area, allowing for long term use and storage.

RESULTS AND DISCUSSION:

A new stability- indicating UPLC method has been devised and validated for the quantitative determination of Sarilumab. It was developed utilizing an Acquit UPLC CSH C18 column with a eluent of Potassium dihydrogen phosphate buffer and water (70:30 v/v). This mobile phase settled for effective separation and quantification of Sarilumab. A wavelength of 212 nm was chosen for the analysis, and optimization for increased sensitivity and specificity meant the linearity and analytical methods produced satisfactory results. The full analysis time was 2 minutes, making the method highly useful for day-to-day laboratory testing. This fast analysis time is very useful to have at hand to improve efficiencies and improve a facilities throughput within the pharmaceutical testing industry, all while maintaining the integrity of the analysis. The UV spectrum of Sarilumab can be seen in figure 2 which displays the characteristic absorption by Sarilumab at 212 nm. The retention time of Sarilumab is shown in figure 3 and determined to be 0.819 minutes, allowing for an effective separation of Sarilumab in a timely manner. The UPLC method designed for Sarilumab is precise, dependable, and effective for the regular quantification of pharmaceutical products and research and commercial endeavours.

Figure 2: UV spectrum of Sarilumab

Figure 3: Optimized chromatogram

Table 1: Optimized Chromatogram data

S. No	Peak name	Retention Time	Area	USP Tailing	USP Plate Count
1	Sarilumab	0.819	580998	1.1	8743.2

Specificity:

By ensuring that the chromatograms of the empty, the control group, and the test did not contain any interfering peaks during the Sarilumab retention time, the specificity of the suggested UPLC method was crucially established. This guarantees that no other compounds or formulation excipients will interfere with the suggested method's ability to accurately assess sarilumab. Chromatograms for blank and placebo samples are shown in figures 4 & 5 respectively.

Both figures show that there are no other peaks occurring during the retention time of Sarilumab, verifying that the proposed method can specifically quantify Sarilumab without interference from other components. Since there was no interference from the other components, the method should be able to differentiate Sarilumab from the other ingredients allowing for accurate and reproducible quantifying and validating during the research and pharmaceutical quality control process.

Figure 4: Blank

Figure 5: Placebo

Linearity, Precision, Accuracy, Robustness, LOD, LOQ studies.

The UPLC method developed in this work revealed excellent linearity of Sarilumab within the concentration range of 10-60µg/mL. The corresponding regression equation (y=14130x + 3582.7) indicates that there is a strong relationship between concentration and response (Table 2), which confirms that the UPLC method is capable of producing an accurate and exact measurement. The calibration curve depicting this relationship is depicted in Figure 6. To evaluate the robustness of the method it was evaluated with accuracy, precision and robustness. All %RSD values for these parameters (Tables 3, 4 and 5) were below 2.0% which is satisfactory for ensuring that the method produces reliable and consistent data which can be used quantitatively for Sarilumab. In regard to sensitivity; the method had a LOD of 0.57μg/mL (Figure 7) and a LOQ of 1.72μg/mL (Figure 8), therefore low concentrations of Sarilumab can be detected and quantitated. More supporting data are included in Tables 6 and 7. Finally, the validated UPLC method described here is concise, accurate, reliable, and sensitive which can be conveniently applied to the analysis of Sarilumab in bulk as well as pharmaceutical formulations. Therefore, this method can be considered as a reliable and reproducible tool for quality control and regulatory purposes in pharmaceutical research.

Figure 6: Calibration curve of Sarilumab

Table 2 Linearity Study

Sample	Sarilumab
	Concentration(ug/ml)	Peak Area
1	10	144800
2	20	283384
3	30	430538
4	40	570546
5	50	70892
6	60	850536
Slope = 14130 Intercept = 3582.7 R²= 0.9999

Table 3 Precision study

Peak Name	Sarilumab
Peak Name	Repeatability (sample)	Method precision	Day to day precision
1	570236	586790	587916
2	579964	583294	581336
3	580998	587019	584943
4	578294	583999	582640
5	578687	585640	584527
6	579402	580595	588090
Mean	577930	584556	584909
SD	3889.0	2442.3	2728.5
%RSD	0.7	0.4	0.5

Table 4 Accuracy study

Level	Sarilumab
Level	Peak area	% Recovery (%RSD)
50%	849226 851307 853645	99.24(0.8)
100%	1129551 1129362 1137867	99.70(0.9)
150%	1427141 1423288 1408961	100.3(1.1)

DEGRADATION STUDIES:

The stability of Sarilumab was characterized through its chromatographic properties, fundamentally providing a thorough overview of the degradation of the drug under different conditions. The UPLC chromatograms, presented in a summary table, offer a clear demonstration of the ability to detects small, but meaningful differences in drug stability. The results showed Sarilumab was unstable in acidic, oxidative (peroxide), and elevated thermal conditions; for example, even at -2°C sarilumab was relatively unstable in acid. This is important information to know when properly formulating and storing the drug to maintain potency. Between the UPLC and all other methods, this analytical method is reliable and useful for stability profiling of Sarilumab in pharmaceutical research and quality. Overall, knowing how a new drug reacts to different stresses helps ensure its safety and efficacy to patients. The degradation patterns and related data are illustrated in Figure 9 & Table 9.

Table 9: Forced Degradation

Degradation Condition	Sarilumab
	% Drug degraded	%Drug undegraded
Acid degradation	7.21	92.79
Alkali degradation	1.86	98.14
Peroxide degradation	9.49	90.51
Thermal degradation	5.88	94.12
Photolytic degradation	1.23	98.77
Hydrolysis degradation	0.76	99.24

CONCLUSION:

The sarilumab is separated using UPLC method. An efficient ultra-performance Liquid Chromatography method developed for the analysis of sarilumab because of the simplicity, speed, cost-effectiveness, specificity, and reliability. By this method an accurate, precise and reliable results were obtained making it highly suitable for routine laboratory testing. Results showed that the retention time was found to be 0.819min showing a fast and efficient separation with 2min run time. The validation results were also within their specified limit. The % RSD of every parameter was found to be below 2 which shows that the sarilumab is effectively suitable for the pharmaceutical formulations. The method proved to be accurate, precise, linear, and robust. The detection limit is very sensitive that is 0.52μg/mL and the quantification limit is 1.72μg/mL. The assay results are also within the acceptable limit which shows the purity of the drug. To study the stability of the sarilumab in different solvents the forced degradation studies were also performed for the forced degradation studies we conclude that the degradation was observed in acid, peroxide, and thermal conditions. By this we can conclude that for the bulk formulations using sarilumab we can prefer the alkali, hydrolysis or photolytic conditions for the long-term stability of the drug. This validation has shown its capacity for quality control of drug substances and pharmaceutical dosage forms. The efficiency and reproducibility make this UPLC method the ideal analytical method for pharmaceutical research and industry, with a high level of confidence.

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Received on 30.04.2025 Revised on 12.09.2025

Accepted on 28.11.2025 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2082-2088.

DOI: 10.52711/0974-360X.2026.00299

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Sarilumab
Condition	Flow rate (-) 0.4ml/min	Flow rate (+) 0.6ml/min	MP (-) 65:35 (v/v)	MP (+) 75:25 (v/v)	Temp (-) 33⁰C	Temp (+)53⁰C
Peak area	557952	578029	578233	549137	541803	576279
	555695	579050	573644	544276	542448	578365
	562259	578292	574513	541067	547008	575832
	559135	577833	581781	550821	549866	572121
	560389	585475	576822	547389	547055	580872
Mean	559086	579736	576999	546538	545636	576694
SD	2477.5	3241.4	3234.9	3901.0	3414.8	3243.3
% RSD	0.4	0.6	0.6	0.7	0.6	0.6


Acid Degradation	Alkali Degradation

Hydrolysis Degradation	Peroxide Degradation

Thermal Degradation	Photolytic Degradation
Figure 9: Typical chromatograms obtained during the forced degradation studies