Analytical Method Development and Validation of Some Biosimilar Drugs by High Performance Thin Layer Chromatography


A simple and rapid HPTLC analytical method has been developed and validated for the determination of Etanercept and Filgrastim in pure form and in marketed formulation. Both the drugs were chromatographed on silica gel 60 F254s HPTLC plates, as stationary phase. The mobile phase optimized for Filgrastim and Etanercept was Water: n-butanol (7.5:2.5 v/v) and Isopropyl alcohol: water (6.5:4.5 v/v), respectively. The chromatogram obtained was scanned at 225 nm and 222 nm for filgrastim and etanercept which resulted in a retention factor of 0.45 ± 0.07 and 0.32 ± 0.03, respectively. The method was validated for parameters like linearity, accuracy, precision, specificity and robustness. Recovery studies were performed at three concentration levels, to demonstrate suitability, accuracy and precision of proposed method. Statistical analysis proved that the proposed method is accurate and reproducible with linearity in the range of 500 to 3000 ng/band for filgrastim and 200 to 1200 ng/band for etanercept. The limit of detection and limit of quantification for filgrastim was found to be 63.418 ng/band and 192.177 ng/band. For etanercept, LOD and LOQ were found to be 33.381 ng/band and 101.153 ng/band, respectively. The proposed method can be employed for the routine analysis of selected biosimilars.

Share and Cite:

Qureshi, H. and Veeresham, C. (2023) Analytical Method Development and Validation of Some Biosimilar Drugs by High Performance Thin Layer Chromatography. American Journal of Analytical Chemistry, 14, 121-133. doi: 10.4236/ajac.2023.143007.

1. Introduction

Etanercept is a dimeric fusion protein drug produced by recombinant DNA technology from Chinese Hamster Ovaries expression system [1] [2] . It acts as a Tumour Necrosis Factor (TNF) inhibitor. Etanercept blocks the interaction of TNF with receptors on the cell surface, henceforth preventing TNF-mediated immune and inflammatory responses. It belongs to the class of biological disease modifying anti-rheumatic drugs (bDMARDS).

Filgrastim or Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is one in the family of hematopoietic growth factors which regulates the proliferation and differentiation of cells of neutrophil lineage. It is produced by recombinant DNA technology in genetically engineered Escherichia coli cells. It is used for reducing the risk of infection in cancer patients by improving the neutrophil count and in patients receiving chemotherapy or a bone marrow transplant [3] [4] .

High Performance Thin Layer Chromatography (HPTLC) is an analytical technique that includes a widely standardized and systematized methodology. It is used for qualitative and quantitative analysis and also for validation of not only plant extracts but also a number of pharmaceutical formulations [5] [6] . HPTLC meets all quality requirements as par with any analytical techniques. HPTLC can reproduce data with the best resolution and with more accurate quantitative measurements [6] [7] . HPTLC has the advantage of being reliable in quantitation [8] , simple in procedure [9] , and sensitive analysis not only in micrograms but also in nanograms levels above all being cost effective [6] . The volume of solvent required is very less in comparison to HPLC. This minimizes the time and expenditure of analysis. It also diminishes the possibilities of pollution of the environment. Concurrent assay of many components in a composite formulation or extracts is also possible [8] [10] [11] . Hence, the present research aimed to develop and validate a simple, rapid and specific HPTLC method for the determination of Etanercept and Filgrastim in accordance with International Conference on Harmonization (ICH) guidelines.

2. Materials and Method

2.1. Reagents Used

Etanercept and Filgrastim were obtained from European Medicine agency, Strasbourg, France. Etacept® and Grafeel®, marketed formulation was obtained as gift sample from Cipla Limited, Mumbai and Dr. Reddy’s Laboratories, Hyderabad, India, respectively. Merck Millipore Direct Q UV water system was used to obtain double distilled water for analysis. Isopropyl alcohol and n-Butanol was purchased from Merck.

2.2. Instrumentation

CAMAG® Linomat 5 Automatic TLC Sampler 4 (ATS 4) with TLC scanner 3, Switzerland. Silica gel 60 RP-18 F254s HPTLC plates were obtained from Merck.

3. Methodology

3.1. Preparation of Standard Stock Solution

Standard stock solution of Etanercept was prepared by dissolving 10 mg of drug in 10 ml of water to get concentration of 1000 μg/ml. From the standard stock solution, working standard solution was prepared containing 100 μg/ml of Etanercept.

Working stock solution containing 250 μg/ml of Filgrastim was prepared from standard filgrastim solution. The stock solution was used for detection of wavelength for maximum absorbance.

3.2. Selection of Detection Wavelength

Working stock solution was scanned over the range of 200 - 400 nm and the spectrum was obtained. It was observed that Etanercept showed considerable absorbance at 222 nm (Figure 1) and filgrastim showed at 225 nm. Representative UV spectrum of Filgrastim is shown in Figure 2.

3.3. HPTLC Analysis

Solution of Etanercept (100 µg/ml) and Filgrastim (200 µg/ml) was prepared. 4 µl (400 ng/band for etanercept) and (1000 ng/band of filgratim) of solution was applied on pre-activated HPTLC plate with the help of Hamilton syringe (100 μl), using Linomat 5 sample applicator. After spotting the plate, it was placed in the saturated chamber and developed up to 80 mm distance. The plate was dried and was scanned over 90 mm distance at 222 nm for etanercept and 225 nm for

Figure 1. UV spectrum of Etanercept.

Figure 2. UV spectrum of Filgrastim.

filgrastim. The retention factor was found to be 0.32 ± 0.03 for Etanercept and 0.45 ± 0.07 for Filgrastim.

3.4. Validation

The HPTLC method was validated according to the International Conference on Harmonization (ICH) Q2 (R1) guideline [12] for linearity, precision, accuracy and recovery, limit of detection, limit of quantification, specificity and robustness.

3.4.1. Specificity

The specificity of the method was ascertained by analyzing standard compound and by peak purity profile studies

3.4.2. Linearity and Range

To evaluate linearity range, the calibration curve was plotted based on the peak area obtained against concentration of etanercept and Filgrastim. Scanning over the concentration range 200 - 1200 ng/ml for etanercept and 500 - 3000 ng/ml for Filgrastim was carried out. The correlation coefficient (r) values were calculated.

3.4.3. Precision

% RSD is used to estimate the Intraday and Interday precision of the method. Precision was calculated on the same day (Intraday) and on three consecutive days (Interday). The analysis was done in triplicate for each sample for both the drugs.

3.4.4. Accuracy

The standard addition method was used to determine the accuracy of the method. Known amounts of Etanercept and filgrastim was added at three different levels (50%, 100% and 150%) and analysis was carried out for both the drugs in triplicate.

3.4.5. Robustness

Robustness is a measure of the ability of a developed method to remain stable and unaffected by minor and deliberate changes in the experimental conditions. It indicates reliability of the method.

3.4.6. Limit of Detection (LOD) and Limit of Quantitation

They are calculated from the following formula,

LOD = 3.3 σ/S

LOQ = 10 σ/S

where, σ is the standard deviation of Y intercept and S is slope of the calibration curve.

3.4.7. Assay (Marketed Formulation)

Etacept® and Grafeel® pre filled injections were taken and a concentration equivalent to 400 ng/band for Etanercept and 1000 ng/ml for Filgrastim were analyzed from formulations, respectively. Concentration and % recovery were determined. The sample was analyzed six times.

4. Results

4.1. HPTLC Analysis (Table 1)

The retention factor was found to be 0.32 ± 0.03 for ETA and 0.45 ± 0.07 for FILG.

4.2. Validation of Analytical Method

The proposed method was validated as per International Council for Harmonisation (ICH Q2 (R1)) guidelines.

4.2.1. Specificity

The specificity of the developed method was established by peak purity profiling studies. The peak purity values were found to be more than 0.996 for Etanercept and 0.998 for Filgrastim, indicating the non-interference of any other peak of degradation product or impurity.

4.2.2. Linearity

From the 100 µg/ml solution of Etanercept, five replicates per concentration were spotted. The linearity was determined by analyzing six concentrations over the concentration range of 200 - 1200 ng/band. Against the corresponding concentrations the peak areas were plotted to obtain the densitogram as shown in Figure 3. The result was found to be linear with regression equation of y = 9.0888x + 5321.3 (Figure 4) and r2 value of 0.9905.

Working standard solution containing 250 μg/ml of Filgrastim was further used for preparing dilutions and spotting. Six replicates per concentration were spotted. The linearity (relationship between peak area and concentration) was

Table 1. HPTLC Analysis.

Figure 3. Densitogram of Etanercept.

Figure 4. Calibration Graph of Etanercept.

determined by analyzing six concentrations over the concentration range 500 - 3000 ng/band to obtain calibration curve. The results were found to be linear with regression equation of y = 5.5173x + 2453.8 and r2 value was found to be 0.9931. The densitogram and calibration curve are shown in Figure 5 and Figure 6, respectively.

4.2.3. Precision

For both the drugs, the intra-day studies was analyzed using 3 replicates of 3

Figure 5. Densitogram of Filgrastim reference standard.

Figure 6. Calibration Graph of Filgrastim.

concentrations on the same day and percentage RSD were calculated. For the inter day variation studies, 3 replicates of 3 concentrations were analyzed on 3 consecutive days were calculated along with %RSD. For intraday precision and inter-day precision results obtained are shown in Table 2 and Table 3, respectively.

4.2.4. Accuracy

To check accuracy of the method, recovery studies were carried by spiking the standard drug to the sample solution, at three different levels 50%, 100% and

Table 2. Intraday and Interday Precision of Etanercept.

Table 3. Intraday and Interday Precision of Filgrastim.

150%. Basic concentration of sample chosen was 400 ng/band for etanercept and 1000 ng/ml for filgrastim. % Recovery was determined from linearity equation. The results obtained are represented in Table 4 and Table 5.

4.2.5. Limit of Detection (LOD) and Limit of Quantification (LOQ)

The LOD and LOQ of Etanercept was found to be 33.381 ng/band and 101.153 ng/band, respectively whereas for filgrastim it was found to be 63.418 ng/band and 192.177 ng/band, respectively.

4.2.6. Robustness

Robustness of the method was determined by carrying out the analysis under conditions during which scanning wavelength was altered. Time was also changed from spotting to development and development to scanning and the effect on the

Table 4. Recovery studies of Etanercept.

Table 5. Recovery studies of Filgrastim.

area were noted. It was found that method is robust. Table 6 and Table 7 represent the results obtained.

4.2.7. Assay

Etacept® 25 mg injection which is a lyophilized powder for reconstitution, contained 25 mg of Etanercept. Grafeel® pre filled syringes contained 300 µg/ml of Filgrastim. Analysis for both marketed formulations was carried out. Procedure was repeated for six times. Sample was spotted, scanned and area was recorded. Basic concentration of sample chosen was 400 ng/band for Etanercept and 1000 ng/ml for Filgrastim. Concentration and % recovery were determined from linear equation. Assay results obtained are shown in Table 8 and Table 9.

Table 6. Robustness data of Etanercept.

Table 7. Robustness data of Filgrastim.

Table 8. Assay of marketed formulation (Etacept®).

5. Discussion

For analyzing biosimilar drugs requires state of the art technologies. Literature revealed research work showcasing the use of RP-HPLC, Peptide Mapping, circular dichroism (CD) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy,

Table 9. Assay of marketed formulation (Grafeel®).

(MALDI-TOF) mass spectrometry, liquid chromatography electrospray ionization (LC-ESI) mass spectrometry, fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance size-exclusion chromatography, dynamic light scattering (DLS), light obscuration, extrinsic fluorescence (Bis-ANS), far-UV circular dichroism (CD) spectroscopy, second derivative UV spectroscopy (UV), and enzyme-linked immunosorbent assay (ELISA) [13] - [16] for comparing innovator biopharmaceutical with marketed biosmilars. Batlovska-Borozanova et al., 2010; Dalmora et al., 2006; Qureshi et al., 2021 reported research work of analysis using RP-HPLC and UV for filgrastim. The LOD and LOQ values ranged from 0.1813 µg/ml to 24.35 µg/ml [17] [18] [19] . The LOD and LOQ values of the proposed method for Filgrastim were found to be 63.418 ng/band and 192.177 ng/band, for Etanercept 33.381 ng/band and 101.153 ng/band, respectively accentuating method’s sensitivity. The selected biosimilars, filgrastim (18.8 KDa) and etanercept (150 KDa) contains 175 [3] and 934 amino acids [1] , respectively and are protein in nature. Biller, 2015 reported for separation of proteins by HPTLC, in which different proteins with molecular weights from 5777.5 Da (Insulin) to 66,432.9 Da (Bovine serum albumin) [20] were analyzed. In collation to sophisticated and complex techniques, the present research work offers the use of simple, easy and economical technique for analysis of biosimilar drugs.

6. Conclusion

Biosimilars structural characterization and comparison with that of innovator biopharmaceuticals requires the most advanced techniques. These techniques are neither cheaper nor less time consuming. The authors endeavored in developing the first simple, rapid and less expensive analytical technique using HPTLC for selected biosimilars, which is the need of the hour. The developed method can be used for comparing different biosimilars with the marketed formulations. It can also be used for the determination of presence of any post-translational modifications, degradation products, etc. as complex biopharmaceuticals are prone to these changes and HPTLC offers easy detection of degradation products [21] .

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.


[1] Goffe, B. and Cather, J.C. (2003) Etanercept: An Overview. Journal of the American Academy of Dermatology, 49, 105-111.
[2] Strand, V., Girolomoni, G., Schiestl, M., Mayer, R.E., Friccius-Quecke, H. and McCamish, M. (2017) The Totality-of-the-Evidence Approach to the Development and Assessment of GP2015, a Proposed Etanercept Biosimilar. Current Medical Research and Opinion, 33, 993-1003.
[3] Dalmora, S.L., D’Avila, F.B., da Silva, L.M., Bergamo, A.C. and Zimmermann, E.S. (2009) Development and Validation of a Capillary Zone Electrophoresis Method for Assessment of Recombinant Human Granulocyte Colony-Stimulating Factor in Pharmaceutical Formulations and Its Correlation with Liquid Chromatography Methods and Bioassay. Journal of Chromatography B, 877, 2471-2476.
[4] Sörgel, F., Schwebig, A., Holzmann, J., Prasch, S., Singh, P. and Kinzig, M. (2015) Comparability of Biosimilar Filgrastim with Originator Filgrastim: Protein Characterization, Pharmacodynamics, and Pharmacokinetics. BioDrugs, 29, 123-131.
[5] Sonia, K., Bhavyashree, B. and Lakshmi, K.S. (2017) HPTLC Method Development and Validation: An Overview. Journal of Pharmaceutical Sciences and Research, 9, 652-657.
[6] Srivastava, M.M. (2010) An Overview of HPTLC: A Modern Analytical Technique with Excellent Potential for Automation, Optimization, Hyphenation, and Multidimensional Applications. In: Srivastava, M.M., Ed., High Performance Thin Layer Chromatography (HPTLC), Springer, New York, 3-24.
[7] Weins, C. and Hauck, H.E. (1996) Advances and Developments in Thin Layer Chromatography. LC-GC International, 14, 455-471.
[8] Chandel, S., Barhate, C.R., Srivastava, A.R., Kulkarni, S.R. and Kapadia, C.J. (2012) Development and Validation of HPTLC Method for Estimation of Tenoxicam and Its Formulations. Indian Journal of Pharmaceutical Sciences, 74, 36-40.
[9] Monireh, H., Masoumeh, G., Solmaz, G., Hossein, A. and Ghanbari, M.M. (2016) Development and Validation of a HPTLC Method for Analysis of Sunitinib Malate. Brazilian Journal of Pharmaceutical Sciences, 52, 595-601.
[10] Tayade, N.G. and Nagarsenker, M.S. (2007) Validated HPTLC Method of Analysis for Artemether and Its Formulations. Journal of Pharmaceutical and Biomedical Analysis, 43, 839-844.
[11] Dixit, R.P., Barhate, C.R. and Nagarsenkar, M.S. (2008) Stability Indicating HPTLC Method for Simultaneous Estimation of Ezetimibe and Simvatsatin. Chromatographia, 67, 101-107.
[12] ICH Harmonized Guideline Q2R1 (2005) Validation of Analytical Procedures: Text and Methodology (ICH Q2 (R1)).
[13] Halim, L.A., Márquez, M., Maas-Bakker, R.F., Castañeda-Hernández, G., Jiskoot, W. and Schellekens, H. (2018) Quality Comparison of Biosimilar and Copy Filgrastim Products with the Innovator Product. Pharmaceutical Research, 35, Article No. 226.
[14] Magalhaes, V., Mantovani, M., Caruso, C., Facchini, F., Pascon, R. and Cagnacci, P. (2016) Physicochemical and Biological Comparison of the First Brazilian Biosimilar Filgrastim with Its Reference Product. Biosimilars, 6, 45-60.
[15] Cho, I.H., Lee, N., Song, D., Jung, S.Y., Bou-Assaf, G., Sosic, Z., Zhang, W. and Lyubarskaya, Y. (2016) Evaluation of the Structural, Physicochemical, and Biological Characteristics of SB4, a Biosimilar of Etanercept. mAbs, 8, 1136-1155.
[16] Maarschalkerweerd, A., Wolbink, G.J., Stapel, S.O., Jiskoot, W. and Hawe, A. (2011) Comparison of Analytical Methods to Detect Instability of Etanercept during Thermal Stress Testing. European Journal of Pharmaceutics and Biopharmaceutics, 78, 213-221.
[17] Batkovska-Borozanova, I., Tonic-Ribarska, J. and Trajkovic-Jolevska, S. (2010) New High Performance Liquid Chromatography Method for Analysis of Filgrastim in Pharmaceutical Formulations. Webmedcentral Pharmaceutical Sciences, 1, WMC001117.
[18] Luiz Dalmora, S., Maria Krug Masiero, S., Renato de Oliveira, P., da Silva Sangoi, M. and Brum Junior, L. (2006) Validation of an RP-LC Method and Assessment of rhG-CSF in Pharmaceutical Formulations by Liquid Chromatography and Biological Assay. Journal of Liquid Chromatography & Related Technologies, 29, 1753-1767.
[19] Qureshi, H.K., Veeresham, C. and Srinivas, C. (2021) Analytical Method Development and Validation of Filgrastim by UV and RP-UFLC Methods. American Journal of Analytical Chemistry, 12, 333-346.
[20] Biller, J., Morschheuser, L., Riedner, M. and Rohn, S. (2015) Development of Optimized Mobile Phases for Protein Separation by High Performance Thin Layer Chromatography. Journal of Chromatography A, 1415, 146-154.
[21] Abo-Gharam, A.H. and El-Kafrawy, D.S. (2022) Eco-Friendly Stability-Indicating HPTLC Micro-Determination of the First FDA Approved SARS-CoV-2 Antiviral Prodrug Remdesivir: Study of Degradation Kinetics and Structural Elucidation of the Degradants Using HPTLC-MS. Sustainable Chemistry and Pharmacy, 29, Article ID: 100744.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.