Development and Validation of an Intact Mass Spectrometry Method for the Characterisation of Rituximab

Abstract

Background: Rituximab, a monoclonal antibody (mAb), is of significant medical importance, particularly in the treatment of rheumatoid arthritis, non-Hodgkin's lymphoma, and chronic lymphocytic leukemia. Since rituximab is produced using recombinant DNA technology, it exhibits structural heterogeneity and various post-translational modifications (PTMs), such as glycosylation variants. These variations can directly impact the product's safety, efficacy, and quality. Therefore, high-accuracy analytical techniques are essential for the quality control of both originator rituximab and the biosimilars available on the market. The use of UHPLC-Q/TOF MS is a standard approach, relying on the direct analysis of the molecule's mass-to-charge ratio (m/z) to confirm the primary structure (based on overall molecular weight) and to characterise PTMs. This method is considered a standard guideline for quality assessment.

Objectives: This study aimed to develop an intact mass spectrometry method for rituximab using UHPLC-Q/TOF in accordance with ICH guidelines Q2(R2), in order to establish a standard method for identity testing and quality control of rituximab at the Institute of Biological Products.

Methods: This experimental research involved the method development through literature review and optimisation of conditions. High-purity samples of the originator drug and biosimilars were prepared by desalting and adjusting the protein concentration to 1.0 mg/ml to enhance ionisation efficiency. Optimal analysis conditions included a reversed-phase column and gradient elution using 0.1% v/v formic acid in water (mobile phase A) and 0.1% v/v formic acid in acetonitrile (mobile phase B). Method validation was performed for key parameters: specificity, precision, and robustness to confirm reliability. Two other monoclonal antibodies, trastuzumab and bevacizumab, were used to test the method's effectiveness in verifying the primary structure and PTM data. The study was conducted from 1 October 2022 to 30 September 2023.

Results: The System Suitability Test using the NIST monoclonal antibody standard showed that the mass accuracy of the major glycoforms was within acceptable limits (±20 ppm), confirming the reliability of the high-accuracy instrument. Validation results demonstrated: (1) specificity: analysis of the blank did not show any signal corresponding to rituximab's major glycoforms. The major glycoforms of the biosimilar were consistent with the originator, with an overall molecular weight of 147,243 kDa and a deviation from the target mass of only 3 Da, which is significantly less than the specified limit of ±10 Da. Furthermore, analysis of other monoclonal antibodies (trastuzumab and bevacizumab) did not show major glycoforms consistent with rituximab, confirming the method's high specificity for rituximab; (2) Precision: testing for intra-day repeatability and inter-analyst/inter-day accuracy showed very low Relative Standard Deviation (%RSD) values. The % RSD for intra-day repeatability was 0.0004, and the % RSD for inter-day/inter-analyst precision was 0, all of which were lower than the specified acceptance criteria.; robustness: testing by changing the sample storage time (stored at -30 °C for 7 days) showed that the mass values of the major glycoforms were not statistically significantly different (p>0.05) when compared to samples analysed immediately, confirming the method's robustness against changes in sample storage conditions.

Conclusions: The developed UHPLC-Q/TOF MS intact mass analysis method for rituximab demonstrates high specificity, precision, and robustness. It is suitable for use as a standard laboratory method for confirming the identity and controlling the quality and efficacy of rituximab before commercial release. This method is also a crucial tool for assessing the structural similarity of the primary structure between biosimilars and the originator drug. However, to enhance the completeness of the analysis, future considerations should include the use of deglycosylase enzyme to remove sugar moieties, which would simplify the analysis and provide aglycosylated mass data. Comparative studies with analytical columns from other manufacturers should also be added to increase operational options and mitigate the risk of equipment shortage.