Development and validation of a mass spectrometric method to determine the identity of rituximab based on its microheterogeneity 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.
Section snippets
Chemicals and reagents
Solvents and the pH modifier used to prepare chromatographic solutions were MS grade: formic acid (FA) and acetonitrile (AcN) were purchased from Thermo Fisher Scientific (MA, USA), and water from Honeywell® (NJ, USA). The spectrometric mass corrector [Glu1]-fibrinopeptide B (Glufib) was obtained from Waters® (MA, USA). Deglycosylated rituximab samples were prepared using sequencing grade PNGase F, which was acquired from New England Biolabs (MA, USA). Deglycosylation procedure also involved
Method development
The first step to develop an identity MS method comprises (i) tuning the mass spectrometer acquisition parameters, (ii) the ion source parameters and (iii) determining the concentration of the analyte samples to obtain suitable m/z signals, whose signal/noise ratio should be higher than 10 [31], [32]. This value is highly recommended to reduce the variability of method accuracy and precision. In this sense, the optimal m/z signal of rituximab samples was obtained at 2 mg/mL employing the MS
Conclusion
International guidelines, such as ICH Q2 (R1), provide valuable suggestions to perform the validation of pharmaceutical analytical methods. Notwithstanding, the design should be adapted depending on the know-how of the analytical method developer and the intended purpose of the analytical method. Here, we describe the design and performance of an MS-UPLC method for determining the identity of rituximab based on its microheterogeneity (glycosylated and deglycosylated variants). The obtained
Declaration of Competing Interest
Authors declare no conflict of interest.
Acknowledgements
This work was carried out with equipment of “Laboratorio Nacional para Servicios Especializados de Investigación, Desarrollo e Innovación (I+D+i) para Farmoquímicos y Biotecnológicos”, LANSEIDI-FarBiotec-CONACyT, which is part of Unidad de Desarrollo e Investigación en Bioprocesos (UDIBI)-IPN”.
References (37)
N-glycosylation heterogeneity and the influence on structure, function and pharmacokinetics of monoclonal antibodies and Fc fusion proteins
Eur. J. Pharm. Biopharm.
(2016)Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins
J. Pharm. Sci.
(2015)Heterogeneity of monoclonal antibodies
J. Pharm. Sci.
(2008)- et al.
Glycan analysis for protein therapeutics
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
(2019) Protocols for the analytical characterization of therapeutic monoclonal antibodies. II - Enzymatic and chemical sample preparation
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
(2017)- et al.
High performance liquid chromatography (HPLC) based direct and simultaneous estimation of excipients in biopharmaceutical products
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
(2019) Evaluation of recent very efficient wide-pore stationary phases for the reversed-phase separation of proteins
J. Chromatogr. A
(2012)- et al.
Development of an analytical reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry method for characterization of recombinant antibodies
J. Chromatogr. A
(2004) - et al.
Importance of manufacturing consistency of the glycosylated monoclonal antibody adalimumab (Humira (R)) and potential impact on the clinical use of biosimilars
Gabi J.-Generics Biosimilars Initiative J.
(2016) Physicochemical characteristics of transferon batches
Biomed. Res. Int.
(2016)
Process development in the QbD paradigm: Role of process integration in process optimization for production of biotherapeutics
Biotechnol. Prog.
ICH guideline Q11 on development and manufacture of drug substances (chemical entities and biotechnological/ biological entities)
ICH guideline M4 on the organisation of the common technical document for the registration of pharmaceuticals for human use
ICH guideline Q6B on specifications: test procedures and acceptance criteria for biotechnological/biological products
Glycosylation control technologies for recombinant therapeutic proteins
Appl. Microbiol. Biotechnol.
Prediction of methionine oxidation risk in monoclonal antibodies using a machine learning method
MAbs
Impact of mammalian cell culture conditions on monoclonal antibody charge heterogeneity: an accessory monitoring tool for process development
J. Ind. Microbiol. Biotechnol.
Fc sialylation prolongs serum half-life of therapeutic antibodies
J. Immunol.
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Both authors contributed equally to this work.