Innovative analytical methodologies for biotherapeutics analysis in biological samples – MethAmAbs

In this work, we have developed a new analytical strategy based on capillary electrophoresis coupled with tandem mass spectrometry (CE-MS / MS) for the quantification of infliximab (Remicade®) in human serum. The CESI800 system coupled to a 5600+ Q-TOF mass spectrometer was implemented in this study. A purification step was optimized in order to obtain sample conditions perfectly compatible with MS analysis. A buffer exchange on a 10 Da cutoff Amicon is carried out to remove excess salts. Then, the infliximab peptides obtained from the tryptic digestion were separated and analyzed by CE-MS / MS in a protemic bottom-up strategy. In order to guarantee optimal specificity, the absolute quantification was developed using peptides strictly specific to infliximab and an internal standard. The processing of the raw data making it possible to obtain the calibration curves was obtained using Skyline and Peakview processing software.

The CE-MS/MS method enabled to successfully perform the quantification of infliximab in spicked serum over a concentration range of 1-25 mg/L which represents the levels commonly found in treated patients. The characteristic of CE-MS/MS analysis enabled to use up to 7 different proteolytic peptides of Infliximab for the quantification. The structural assessment of this monoclonal antibody was also performed using the same dataset as the one used for quantification. Some mAbs post-translational modifications (PTMs) of interest, such as oxidations and deamidations were identified. The occurrence of PTMs can be identified from the mass shift between the intact peptide and its modified counterpart. In the case of MS/MS analysis, the peptide backbone fragmentation generated from CID allows to attribute in a precise manner the residue concern by the PTM. N-glycosylations of Infliximab could also be characterized in a detailed manner. The preliminary results obtained allowed to successfully determine the structures of the major glycan of the protein and to establish a detailed glyco-profiling, in agreement with the reference material. Different sample preparation protocols were investigated to enhance the signal intensity. To be compatible with CE-MS, the viscosity and salt concentration must be limited. Thus, the buffer exchange step was studied and improved to be adapted with the analysis.

Over the next 6 months, the project provides for the finalization of the optimization of the analytical conditions used for the analysis of serum samples by CE-MS/MS as well as the performance of a complete method validation in order to demonstrate the performance, accuracy, repeatability and intermediate reliability of the analytical strategy developed. The work will then be valued from the fundamental point of view concerning the analysis of mAbs in biological samples by CE-MS/MS, as well as on the sample preparation aspects compatible with the analysis of biological samples by CE-MS/ MS. Beyond 6 months, once the CE-MS/MS method has been validated, the project will continue with the performance of CE-MS / MS analyzes of samples from patients treated in gastroenterology.

Book Chapter

1. CE-MS methods for the characterization of monoclonal antibodies
T. Reinert, E. Leize-Wagner, P. Houzé, R. Gahoual, YN. Francois
Ramautar, Chen (Eds.): Capillary Electrophoresis- Mass Spectrometry (CE-MS) for Proteomics and Metabolomics Wiley-VCH (soumis)

Communication in France
1. Absolute quantification and concomitant structural characterization of infliximab in biological samples using sheathless capillary electrophoresis - mass spectrometry
T. Reinert
SEP21, Paris, 5-7 octobre 2021

Popularization conference
1. Quantification absolue et caractérisation structurale de l’Infliximab dans des échantillons biologiques par CE-MS
T. Reinert
Réunion UMR 7140, 5 mai 2021
2. Caractérisation et compréhension des aspects pharmacocinétiques et dynamiques de l’infliximab dans des sérums de patients atteints de la maladie de Crohn par CE-MS
T. Reinert
Séminaire UTCBS (UMR8258, 19 Janvier 2021)

Among therapeutic proteins, monoclonal antibodies (mAbs) are meeting a major interest which undoubtedly contributed to the advent of the biopharmaceutical industry with more than 80 products approved. Their domain of therapeutic application is currently driven by oncology or the treatment of immune disorder. Due to their recent introduction as therapeutic agent, the evolution of mAbs consequently to the injection is not yet fully understood. Thus, a significant portion of the patients are reacting differently to the treatment, with for instance the expression of antibodies targeting the therapeutic mAbs injected potentially cancelling the effect of the product. In addition, while the dose used is normalized, previous studies show that the clearance of mAbs is significantly different from patients to patients which suggests the dosage is not necessarily optimal in particular to guarantee a constant exposure to the treatment. The quantification of therapeutic mAbs in biological samples is commonly performed using ELISA immunoassays which are available only for a few mAbs. The constant introduction of novel mAbs requires the complete development of a dedicated assay which uses different materials. In addition, the assays are potentially sensitive to interferences originating from the matrix. In order to address the current limitations of ELISA immunoassay and provide a deeper insight regarding the evolution of therapeutic mAbs after injection to the patient, this research project is aiming for the implementation of an innovative analytical strategy for the simultaneous quantification and structural assessment of mAbs in complex biological matrices. This methodology has the particularity to employ high sensitivity capillary electrophoresis hyphenated to tandem mass spectrometry (CE-MS/MS). This approach relies on an innovative instrumentation in order to provide an optimal specificity as well as sensitivity. Additionally, the CE-MS/MS analysis will allow to investigate the structural integrity of the protein over time consequently to the administration to the patient in order to determine the occurrence of post-translational modifications (PTMs) which may impact the pharmacological properties of the treatment or induce immune reactions for example. Especially, the characteristics provided by the CE separation will enable to study the evolution of the different glycoforms independently to unravel the complex mechanistic influencing therapeutic mAbs based treatments. The strategy developed for this research project will focus primarily on the analysis of the mAbs targeting the tumor necrosis factor (TNF-a) in the serum of patients. Consequently, to the statistical validation of the methodology, the CE-MS/MS approach will be applied to study serum samples originating from patients treated for chronic inflammatory bowel disease such as Crohn disease. The quantitative information and structural characterization obtained from the CE-MS/MS analysis will be correlated with the pathophysiological profile of the patients in order to monitor the relevance of the dosage over an extended period of time. In parallel, the structural information will be used to investigate the occurrence of PTMs with the specific reactions of the patient to the treatment. The information delivered by the CE-MS/MS analysis could reveal crucial aspects regarding the evolution of mAbs after their administration to the patient. The specific monitoring provided will help the practitioner on the dosage used depending on the profile of the patients in order to rationalize the cost of these high-value treatments which represents a significant cost for the national healthcare system. Finally, the transposition of the CE-MS/MS methodology to the analysis of other types of therapeutic mAbs will be implemented in order to demonstrate the transferability of this innovative analytical strategy to other types of mAbs with minimal additional developments.