Development and validation of a mass spectrometric method to determine the identity of rituximab based on its microheterogeneity profile

Highlights

• First reported validation design of an identity MS-UPLC method for biotherapeutic analysis.

• Validation was designed according to ICH Q2 (R1).

• The proposed validation could be applied to other similar analytical methods for mAbs analysis.

Abstract

Analytical methods have been considered the “eyes” for development, characterization and batch releasing of biotherapeutics over the past 40 years. One of the most powerful analytical platform for biotherapeutic analysis is mass spectrometry coupled to liquid chromatography (LC-MS). Due to its wide flexibility and instrumental configurations, LC-MS can determine different physicochemical attributes of proteins, e.g. molecular mass, primary sequence, and posttranslational modifications. Intact molecular mass analysis of therapeutic proteins is essential to confirm their identity. Analytical methods must be validated to support drug quality information during its approval process. Although there are international guidelines that provide general information on validation of analytical methods, practical examples about the design, selection of validation attributes and acceptance criteria of identity LC-MS methods are scarce. Here, according to the recommendations of Q2R1 ICH guideline, we showcase the validation of an LC-MS-TOF method to identity rituximab by determining its intact and deglycosylated molecular mass profiles. The proposed method specifically identified the m/z profile and deconvoluted mass profile of rituximab from deglycosylated rituximab and from excipient blank (specificity) with a maximum error of 76.63 ppm (accuracy) and a maximum Relative Standard Deviation (RSD) of 0.00315% (precision). Besides, the system suitability test, which was based on the expected mass value of the mass calibrator, confirmed the reliability of the analytical results. In summary, validation showed that the proposed method is suitable for identifying rituximab based on its glycosylated (intact) and deglycosylated mass profile.

Introduction

Batch-to-batch consistency of biotherapeutics is demanded by regulatory agencies during the approval process of any biotherapeutic, given that it is crucial to guarantee product quality and to ensure the expected efficacy and safety [1], [2], [3], [4]. Batch reproducibility is a valuable piece of information of the Quality Module of the Common Technical Document (CTD), the document that follows the ICH guidelines used to submit drug product information to regulatory agencies for approval purposes [5]. Also, Quality Module contains the evidence of the characterization assays that describe the physicochemical and functional properties of the biotherapeutic, which are taken into account to identify critical quality attributes (CQAs) and quality specifications of the drug product [6]. Notwithstanding, some CQAs must be supported by cutting-edge analytical technology due to the complexity of biotherapeutics.

It is well-known that most of the therapeutic recombinant proteins are subjected to posttranslational modifications during biosynthesis and to chemical modifications during purification, formulation and storage [7], [8], [9]; both processes confer the characteristic microheterogeneity profile to each biotherapeutic. Microheterogeneity of monoclonal antibodies (mAbs) is mainly related to a conserved N-glycosylations at Asp 297 on both heavy chains, where three different oligosaccharide structure could be attached: high mannose, complex and hybrid, with or without a core of fucosylation (F) and/or sialic acid (S) [10], [11]. In consequence, glycosylated mAbs exhibit different pairs of glycan structures, namely G0(F), G1(F), G2(F), G2S1(F) and G2S2(F) as described elsewhere [12]. On the other hand, analysis of deglycosylated mAbs can easily detect isoforms related to one or two additional C-Lys residues at C-terminal of heavy chains, or spontaneous glutamic acid cyclization (pyroglutamic acid) of Gln residues at N-terminal of light or heavy chain (pQ isoform) [13]. Oxidation, deamination, isomerization and proteolytic cleavage of some amino acid residues could be also detected [14].

The combination of posttranslational and chemical modifications gives the characteristic molecular mass profile of biotherapeutics, which regulatory agencies strongly recommend to characterize considering its high impact on safety (immunogenicity) and efficacy (pharmacodynamics and pharmacokinetics) [15], [16]. In this sense, it is well known that mAbs with a higher content of fucosylated isoforms exhibit less Antibody-dependent Cell-mediated Cytotoxicity (ADCC), which might compromise efficacy [17]. In contrast, extra C-terminal lysine residues induce basic variants and increase isoelectric point values, but it does not alter in vivo activity given that extra C-terminal lysines are removed by endogenous carboxypeptidases [18], [19], [20].

Several orthogonal analytical methods based on cutting-edge technologies have been rapidly adopted by pharmaceutical industries for characterization and batch releasing of biotherapeutics. Among them, LC-MS has been evolving to become a confident high-throughput technology routinely employed for characterization and quality control of therapeutic proteins [21], [22], [23]. For example, intact mass analysis of monoclonal antibodies by LC-MS allows determining the microheterogeneity profile based on their posttranslational and chemical modifications [24], [25], [26], [27]. Notwithstanding, despite MS determines an absolute characteristic of the analyte (Molecular Mass), validation of LC-MS methods is mandatory when used for pharmaceutical purposes. Nowadays, regulatory agencies have published guidelines on validation of LC-MS methods for determining the identity of therapeutic proteins, but a practical validation strategy has not been published yet. Here, we describe the development and validation of an LC-MS for determining the identity of a monoclonal antibody based on its intact (glycosylated) mass according to Q2R1 ICH guideline using rituximab as model.