Development and Validation of Salt Gradient CEX Chromatography Method for Charge Variants Separation and Quantitative Analysis of the IgG mAb-Cetuximab
- 53 Downloads
Ion exchange chromatography is widely used for charge variant analysis of proteins, including monoclonal antibodies. In this study, a simple and robust salt gradient cation exchange chromatography was developed and validated for quantitative determination of cetuximab in biopharmaceutical formulations. For this purpose, we investigated the effect of various parameters including buffer composition, column temperature, pH, gradient volume and flow rate on chromatographic separation of charge variants to achieve the acceptable peak separation, and the optimum condition was selected. Validation of the method was done in accordance with the International Conference on Harmonization (ICH) guidelines. The developed method was found to provide a linear regression over the concentration range of 0.06–2.00 mg mL−1 yielding a correlation coefficient of 0.9972. The limits of detection and quantification for the developed method were 0.02 and 0.06 mg mL−1, respectively. The intra-day and inter-day precision had relative standard deviation values ≤ 2.7%. The robustness of the method was assessed by changes in the applied pH range of buffer, temperature, mobile phase composition, and flow rate. Specificity of the method was confirmed by evaluation of baseline resolution of the mAb variants from product excipients, which showed no interference between excipients and cetuximab. The stability indicating capability of this method was determined using photodegraded, and mechanically and thermally stressed samples. The proposed method could be applied as a simple, precise, and robust quantitative technique which can be reproduced in any labs for the high-throughput quality control and stability assessment of in-process and final product samples.
KeywordsCation exchange chromatography Charge heterogeneity Method validation Monoclonal antibody Cetuximab
This work is a part of A. farjami thesis, submitted for Ph.D. degree (no. 117) and supported by Research Council, Tabriz University of Medical Sciences and we would like to thank the CinnaGen Medical Biotechnology Center for kindly providing all of the cetuximab medicinal samples.
Compliance with Ethical Standards
Conflict of interest
The authors state no conflict of interest.
Research involving human participants and/or animals
This article does not contain any studies with human participants or animals performed by any of the authors.
- 12.Talebi M, Nordborg A, Gaspar A, Lacher NA, Wang Q, He XZ, Haddad PR, Hilder EF (2013) Charge heterogeneity profiling of monoclonal antibodies using low ionic strength ion-exchange chromatography and well-controlled pH gradients on monolithic columns. J Chromatogr A 1317:148–154CrossRefGoogle Scholar
- 23.Ayoub D, Jabs W, Resemann A, Evers W, Evans C, Main L, Baessmann C, Wagner-Rousset E, Suckau D, Beck A (2013) Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques. MAbs 5(5):699–710CrossRefGoogle Scholar
- 28.Guideline IHT (2005) Validation of analytical procedures: text and methodology Q2 (R1). In: International conference on harmonization, Geneva, Switzerland, pp 11–12Google Scholar
- 30.Guidance for industry: Analytical procedures and methods validation for drugs and biologics, US Department of Health and Human Services (2014). https://www.fda.gov/downloads/drugs/guidances/ucm386366.pdf. Accessed July 2018
- 31.ICH: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Topic Q1B: Stability testing: photostability testing of new drug substances and products (1996). https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q1B/Step4/Q1B_Guideline.pdf. Accessed July 2018
- 33.ICH: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Topic Q5C: Stability testing of biotechnological/biological products (1995). http://www.ich.org/fleadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q5C/Step4/Q5C_Guideline.pdf. Accessed July 2018
- 35.Wei Z, Feng J, Lin H-Y, Mullapudi S, Bishop E, Tous GI, Casas-Finet J, Hakki F, Strouse R, Schenerman MA (2007) Identification of a single tryptophan residue as critical for binding activity in a humanized monoclonal antibody against respiratory syncytial virus. Anal Chem 79(7):2797–2805CrossRefGoogle Scholar