Method Validation for Determination of Charge Variants in Adalimumab by Ion-Exchange Chromatography

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Kawalin Raksasorn
Sudatip Sunsin
Saiwarul Jadoonkittinan


Background: Adalimumab, fully human recombinant immunoglobulin G1, is a highly specific monoclonal antibody that binds to Tumor necrosis factor-alpha (TNF-α) and prevents its interaction with receptors. Currently, all versions of adalimumab both originator and biosimilar, are widely used to treat rheumatoid arthritis and other inflammatory diseases. During its commercial manufacturing, adalimumab is produced by biotechnological techniques that may change the charge variants on biomolecules. These modifications may impact on biological activity, immunogenicity, and stability. Consequently, the quality control of these drugs is significant in order to ensure that patients receive quality drugs according to the international standard. Characterization of protein charge variants using ion-exchange chromatography, including ionic strength condition or pH gradient mode, is useful for qualitative and quantitative analysis.

Objectives: This study aimed to validate a method for the determination of charge variants in adalimumab by ion-exchange chromatography according to the International Conference on Harmonization (ICH) guidelines and to be used as a standard protocol in laboratory quality control of marketed adalimumab prior to release for supply in the country.

Methods: Validation for the determination of charge isoforms in adalimumab was performed by a pH-gradient cation-exchange chromatography with testing parameters including specificity, precision, accuracy, linearity, limit of quantitation, limit of detection, and robustness.

Results: The results indicated that the method was specific for adalimumab determination with linearity over the concentration range of 1.00 to 10.00 mg/ml. The percentage of relative standard deviations of repeatability and intermediate precision were less than 5%. The percentage of recovery in the accuracy range was 90% to 110%, while the limits of quantitation and detection were 2.50 and 0.70 mg/ml, respectively. The method demonstrates robustness when changing the shelf life of the mobile phase.

Conclusions: The results of the study suggested that this method had specificity, precision, accuracy, linearity, and robustness. Hence, it is suitable for a standard method in quality control of adalimumab products for registration purposes in Thailand.

Article Details

How to Cite
Raksasorn K, Sunsin S, Jadoonkittinan S. Method Validation for Determination of Charge Variants in Adalimumab by Ion-Exchange Chromatography. TFDJ [Internet]. 2023 Aug. 3 [cited 2024 Apr. 20];30(2):49-67. Available from:
Research Article
Author Biographies

Kawalin Raksasorn, Institute of Biological Products, Department of Medical Sciences

Institute of Biological Products, Department of Medical Sciences, Tiwanon Road, Mueang District, Nonthaburi, 11000, Thailand

Sudatip Sunsin, Institute of Biological Products, Department of Medical Sciences

Institute of Biological Products, Department of Medical Sciences, Tiwanon Road, Mueang District, Nonthaburi, 11000, Thailand

Saiwarul Jadoonkittinan, Institute of Biological Products, Department of Medical Sciences

Institute of Biological Products, Department of Medical Sciences, Tiwanon Road, Mueang District, Nonthaburi, 11000, Thailand


Vena GA, Cassano N. Drug focus: adalimumab in the treatment of moderate to severe psoriasis. Biologics 2007;1(2):93-103.

Lu X, Hu R, Peng L, Liu M, Sun Z. Efficacy and safety of adalimumab biosimilars: Current Critical Clinical Data in Rheumatoid Arthritis. Frontiers in immunology 2021;12.

Ellis CR, Azmat CE. Adalimumab. In: StatPearls [Internet]. 2022 [cite 2022 Jul 31]. Available from: [Internet]. 2022 [cited 2022 Jul 31]. Available from:

Exemptia Adalimumab EMPOWERING YOU. Exemptia - World’s First Adalimumab Biosimilar [Internet].2022 [cited 2022 Aug 14]. Available from:

U.S. Food and Drug Administration. Biosimilar Drug Information [Internet]. 2022 [cited 2022 Aug 14]. Available from:

Kaur R, Borgayari D, Rathore A. Impact of media components on CQAs of monoclonal antibodies. BioPharm International 2017;30(9)40-6.

Harris R, Shire S, Winter C. Commercial manufacturing scale formulation and analytical characterization of therapeutic recombinant antibodies. Drug Development Research 2004;61(3):137-54.

Huang L, Lu J, Wroblewski V, Beals J, Riggin R. In Vivo Deamidation Characterization of Monoclonal Antibody by LC/MS/MS. Analytical Chemistry 2005;77(5):1432-439.

Khawli L, Goswami S, Hutchinson R, Kwong Z, Yang J, Wang X et al. Charge variants in IgG1. mAbs 2010;2(6):613-24.

Singh S, Narula G, Rathore A. Should charge variants of monoclonal antibody therapeutics be considered critical quality attributes?. Electrophoresis 2016;37(17-18):2338-46.

Hermeling S, Crommelin D, Schellekens H, Jiskoot W. Structure-immunogenicity relationships of therapeutic proteins. Pharmaceutical Research 2004;21(6):897-903.

Monoclonal antibodies for human use. In: European pharmacopoeia 10.0thed. Strasbourg: EDQM Council of Europe; 2020. P.878-880

Santora L, Krull I, Grant K. Characterization of recombinant human monoclonal tissue necrosis factor-α antibody using cation - exchange HPLC and capillary isoelectric focusing. Analytical Biochemistry 1999;275(1):98-108.

Ahrer K, Jungbauer A. Chromatographic and electrophoretic characterization of protein variants. Journal of Chromatography B 2006;841(1-2):110-22.

Du Y, Walsh A, Ehrick R, Xu W, May K, Liu H. Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies. mAbs 2012;4(5):578-85.

Fekete S, Beck A, Fekete J, Guillarme D. Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part II: pH gradient approach. Journal of Pharmaceutical and Biomedical Analysis 2015;102:282-9.

Farnan D, Moreno G. Multiproduct High-Resolution Monoclonal Antibody Charge Variant Separations by pH Gradient Ion-Exchange Chromatography. Analytical Chemistry 2009;81(21):8846-857.

Talebi M, Nordborg A, Gaspar A, Lacher N, Wang Q, He X et al. Charge heterogeneity profiling of monoclonal antibodies using low ionic strength ion-exchange chromatography and well-controlled pH gradients on monolithic columns. Journal of Chromatography A 2013;1317:148-54.

Ahamed T, Nfor B, Verhaert P, van Dedem G, van der Wielen L, Eppink M et al. pH-gradient ion-exchange chromatography: An analytical tool for design and optimization of protein separations. Journal of Chromatography A 2007;1164(1-2):181-88.

ICH Harmonised Tripartite Guideline. Validation of analytical procedures: text and methodology (Q2(R1)). London: European Medicines Agency; 2005.

Center for Drug Evaluation and Research (CDER). Reviewer guidance, validation of Chromatographic methods [Internet]. U.S. Food and Drug Administration. FDA; [cited 2023 Feb 22]. Available from:

สำนักยา สำนักงานคณะกรรมการอาหารและยา. คู่มือและหลักเกณฑ์การขึ้นทะเบียนตำรับยาชีววัตถุคล้ายคลึง (Biosimilars). กรุงเทพฯ: สำนักพิมพ์อักษรกราฟฟิคแอนด์ดีไซน์; 2561.

Bandyopadhyay S, Mahajan M, Mehta T, Singh AK, Gupta AK, Parikh A, et al. Physicochemical and functional characterization of a biosimilar adalimumab ZRC-3197. Biosimilars 2015;5:1-18.

Rea JC, Moreno GT, Lou Y, Farnan D. Validation of a pH gradient-based ion-exchange chromatography method for high-resolution monoclonal antibody charge variant separations. Journal of Pharmaceutical and Biomedical Analysis 2011;54(2):317–23.

Mohan C. A guide for the preparation and use of buffers in biological s ystems [Internet]. [cited 2023 Feb 26]. Available from:

Fekete S, Beck A, Veuthey J, Guillarme D. Ion-exchange chromatography for the characterization of biopharmaceuticals. Journal of Pharmaceutical and Biomedical Analysis 2015;113:43-55.