Structural characterization of antibody drug conjugate by a combination of intact, middle-up and bottom-up techniques using sheathless capillary electrophoresis – Tandem mass spectrometry as nanoESI infusion platform and separation method

Antibody-drug conjugates (ADCs) represent a fast growing class of biotherapeutic products. Their production leads to a distribution of species exhibiting different number of conjugated drugs overlaying the inherent com-plexity resulting from the monoclonal antibody format, such as glycoforms. ADCs require an additional level of characterization compared to first generation of biotherapeutics obtained through multiple analytical tech-niques for complete structure assessment. We report the development of complementary approaches imple-menting sheathless capillary electrophoresis-mass spectrometry (sheathless CE-MS) to characterize the differ-ent aspects defining the structure of brentuximab vedotin. Native MS using sheathless CE-MS instrument as a nanoESI infusion platform enabled accurate mass measurements and estimation of the average drug to anti-body ratio alongside to drug load distribution. Middle-up analysis performed after limited IdeS proteolysis allowed to study independently the light chain, Fab and F(ab’)2 subunits incorporating 1, 0 to 4 and 0 to 8 pay-loads respectively. Finally, a CZE-ESI-MS/MS methodology was developed in order to be compatible with hy-drophobic drug composing ADCs. From a single injection, complete sequence coverage could be achieved. Using the same dataset, glycosylation and drug-loaded peptides could be simultaneously identified revealing robust information regarding their respective localization and abundance. Drug-loaded peptide fragmentation mass spectra study demonstrated drug specific fragments reinforcing identification confidence, undescribed so far. Results reveal the method ability to characterize ADCs primary structure in a comprehensive manner while reducing tremendously the number of experiments required. Data generated showed that sheathless CZE-ESI-MS/MS characteristics position the methodology developed as a relevant alternative for comprehensive multi-level characterization of these complex biomolecule

Novel sheathless CE-MS interface as an original and powerful infusion platform for nanoESI study: from intact proteins to high molecular mass noncovalent complexes.:

Development of nano-electrospray (nanoESI) sources allowed to increase significantly the sensitivity which is often lacking when studying biological noncovalent assemblies. However, the flow rate used to infuse the sample into the mass spectrometer cannot be precisely controlled with nanoESI and the robustness of the system could represent an issue. In this study, we have used a sheathless capillary electrophoresis-mass spectrometry (CESI) prototype as a nanoESI infusion device. The hydrodynamic mobilization of the capillary content was characterized and the ability of the system to generate a stable electrospray under controlled flow rate conditions ranging from 4 up to 900 nL/min was demonstrated. The effect of the infusing flow rate on the detection of an intact model protein analyzed under native conditions was investigated. Results demonstrated a significant increase in sensitivity of 46-fold and a signal-to-noise ratio improvement of nearly 5-fold when using an infusing flow rate from 456.9 down to 13.7 nL/min. The CESI prototype was further used to detect successfully the β ring homodimer in its native conformation. Obtained results were compared with those achieved with conventional ESI. Intensity signals were increased by a factor of 5, while sample consumption decreased 80 times. β ring complexed with the P14 peptide was also studied. Finally, the CESI interface was used to observe the quaternary structure of native hemocyanins from Carcinus maenas crabs; this high molecular complex coexisting under various degrees of complexation and resulting in masses ranging from 445 kDa to 1.34 MD

Monoclonal antibodies biosimilarity assessment using transient isotachophoresis capillary zone electrophoresis-tandem mass spectrometry

Out of all categories, monoclonal antibody (mAb) therapeutics attract the most interest due to their strong therapeutic potency and specificity. Six of the ten top-selling drugs are antibody-based therapeutics that will lose patent protection soon. The European Medicines Agency has pioneered the regulatory framework for approval of biosimilar products and approved the first biosimilar antibodies by the end of 2013. As highly complex glycoproteins with a wide range of micro-variants, mAbs require extensive characterization through multiple analytical methods for structure assessment rendering manufacturing control and biosimilarity studies particularly product and time-consuming. Here, capillary zone electrophoresis coupled to mass spectrometry by a sheathless interface (CESI-MS) was used to characterize marketed reference mAbs and their respective biosimilar candidate simultaneously over different facets of their primary structure. CESI-MS/MS data were compared between approved mAbs and their biosimilar candidates to prove/disconfirm biosimilarity regarding recent regulation directives. Using only a single sample injection of 200 fmol, CESI-MS/MS data enabled 100% amino acids (AA) sequence characterization, which allows a difference of even one AA between two samples to be distinguished precisely. Simultaneously glycoforms were characterized regarding their structures and position through fragmentation spectra and glycoforms semiquantitative analysis was established, showing the capacity of the developed methodology to detect up to 16 different glycans. Other posttranslational modifications hotspots were characterized while their relative occurrence levels were estimated and compared to biosimilars. These results proved the value of using CESI-MS because the separation selectivity and ionization efficiency provided by the system allowed substantial improvement in the characterization workflow robustness and accuracy. Biosimilarity assessment could be performed routinely with a single injection of each candidate enabling improvements in the biosimilar development pipeline

Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry

Monoclonal antibodies (mAbs) are highly complex proteins that display a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product - and time - consuming. This work presents the characterization of trastuzumab sequence using sheathless capillary electrophoresis (referred as CESI) – tandem mass spectrometry (CESIMS/MS). Using this bottom-up proteomic-like approach, CESI-MS/MS provided 100% sequence coverage for both heavy and light chain via peptide fragment fingerprinting (PFF) identification. The result was accomplished in a single shot, corresponding to the analysis of 100 fmoles of digest. The same analysis also enabled precise characterization of the post-translational hot spots of trastuzumab, used as a representative widely marketed therapeutic mAb, including the structural confirmation of the five major N-glycoforms

Development of high sensitivity capillary electrophoresis - mass spectrometry coupling : application to multi-level characterization of proteins

Les interfaces permettant le couplage entre l'électrophorèse capillaire (CE) et la spectrométrie de masse (MS) à source ESI souffrent actuellement d'un manque de robustesse ou de sensibilité. Les travaux présentés décrivent la mise en oeuvre d'un nouveau type d'interface CE-ESl-MS â haute sensibilité CESl-MS. La caractérisation du spray généré par CESl-MS a montré la production d'un nanoESI induisant une augmentation importante de la sensibilité par rapport au régime ESI classique. Le système CESl-MS a pu ainsi être mis en oeuvre comme plateforme d'infusion nanoESI et utilisé pour l'étude de complexes non-covalents de haut poids moléculaires en MS native. Une méthodologie par CESl-MS/MS a également été développée pour la caractérisation complète de la structure primaire d'anticorps monoclonaux (mAbs). Les résultats montrent la possibilité en une unique injection de caractériser l'ensemble de la séquence d'acides aminés, un nombre significatif de glycosylations et l'ensemble des modifications d'intérêts. Cette méthodologie a pu être appliquée pour déterminer la similarité entre des mAbs commerciaux et leur candidat biosimilaire respectif.Interfacings allowing the hyphenation of capillary electrophoresis (CE) to ESI mass spectrometry(MS) currently suffer from lack of robustness and sensitivity. This work describes the application of a new design of CE-ESl-MS coupling referred as the CESl-MS. Characterization of the ESI generated through the CESl-MS system showed the production of a nanoESI allowing to increase drastically the sensitivity compared to conventional ESI. The CESl-MS was used as a nanoESI infusion platform allowing to study high molecular masses noncovalent complexes in native MS. A CESl-MS/MS method was developed enabling the complete primary structure characterization o fmonoclonal antibodies (mAbs). Results showed the ability of the methodology in a single injection to simultaneously characterize the entire amino acid sequence, a significant number of glycosylation and all the posttranslational modifications of interest. Finally the methodotogy was applied to assess the similarity between marketed mAbs and their respective biosimilar candidate

Development of high sensitivity capillary electrophoresis - mass spectrometry coupling : application to multi-level characterization of proteins

Les interfaces permettant le couplage entre l'électrophorèse capillaire (CE) et la spectrométrie de masse (MS) à source ESI souffrent actuellement d'un manque de robustesse ou de sensibilité. Les travaux présentés décrivent la mise en oeuvre d'un nouveau type d'interface CE-ESl-MS â haute sensibilité CESl-MS. La caractérisation du spray généré par CESl-MS a montré la production d'un nanoESI induisant une augmentation importante de la sensibilité par rapport au régime ESI classique. Le système CESl-MS a pu ainsi être mis en oeuvre comme plateforme d'infusion nanoESI et utilisé pour l'étude de complexes non-covalents de haut poids moléculaires en MS native. Une méthodologie par CESl-MS/MS a également été développée pour la caractérisation complète de la structure primaire d'anticorps monoclonaux (mAbs). Les résultats montrent la possibilité en une unique injection de caractériser l'ensemble de la séquence d'acides aminés, un nombre significatif de glycosylations et l'ensemble des modifications d'intérêts. Cette méthodologie a pu être appliquée pour déterminer la similarité entre des mAbs commerciaux et leur candidat biosimilaire respectif.Interfacings allowing the hyphenation of capillary electrophoresis (CE) to ESI mass spectrometry(MS) currently suffer from lack of robustness and sensitivity. This work describes the application of a new design of CE-ESl-MS coupling referred as the CESl-MS. Characterization of the ESI generated through the CESl-MS system showed the production of a nanoESI allowing to increase drastically the sensitivity compared to conventional ESI. The CESl-MS was used as a nanoESI infusion platform allowing to study high molecular masses noncovalent complexes in native MS. A CESl-MS/MS method was developed enabling the complete primary structure characterization o fmonoclonal antibodies (mAbs). Results showed the ability of the methodology in a single injection to simultaneously characterize the entire amino acid sequence, a significant number of glycosylation and all the posttranslational modifications of interest. Finally the methodotogy was applied to assess the similarity between marketed mAbs and their respective biosimilar candidate

Développement du couplage électrophorèse capillaire - spectrométrie de masse haute sensibilité : application à la caractérisation fine de protéines

Interfacings allowing the hyphenation of capillary electrophoresis (CE) to ESI mass spectrometry(MS) currently suffer from lack of robustness and sensitivity. This work describes the application of a new design of CE-ESl-MS coupling referred as the CESl-MS. Characterization of the ESI generated through the CESl-MS system showed the production of a nanoESI allowing to increase drastically the sensitivity compared to conventional ESI. The CESl-MS was used as a nanoESI infusion platform allowing to study high molecular masses noncovalent complexes in native MS. A CESl-MS/MS method was developed enabling the complete primary structure characterization o fmonoclonal antibodies (mAbs). Results showed the ability of the methodology in a single injection to simultaneously characterize the entire amino acid sequence, a significant number of glycosylation and all the posttranslational modifications of interest. Finally the methodotogy was applied to assess the similarity between marketed mAbs and their respective biosimilar candidate.Les interfaces permettant le couplage entre l'électrophorèse capillaire (CE) et la spectrométrie de masse (MS) à source ESI souffrent actuellement d'un manque de robustesse ou de sensibilité. Les travaux présentés décrivent la mise en oeuvre d'un nouveau type d'interface CE-ESl-MS â haute sensibilité CESl-MS. La caractérisation du spray généré par CESl-MS a montré la production d'un nanoESI induisant une augmentation importante de la sensibilité par rapport au régime ESI classique. Le système CESl-MS a pu ainsi être mis en oeuvre comme plateforme d'infusion nanoESI et utilisé pour l'étude de complexes non-covalents de haut poids moléculaires en MS native. Une méthodologie par CESl-MS/MS a également été développée pour la caractérisation complète de la structure primaire d'anticorps monoclonaux (mAbs). Les résultats montrent la possibilité en une unique injection de caractériser l'ensemble de la séquence d'acides aminés, un nombre significatif de glycosylations et l'ensemble des modifications d'intérêts. Cette méthodologie a pu être appliquée pour déterminer la similarité entre des mAbs commerciaux et leur candidat biosimilaire respectif

Characterization of the Primary Structure of Cysteine-Linked Antibody-Drug Conjugates Using Capillary Electrophoresis with Mass Spectrometry

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