The growing role of structural mass spectrometry in the discovery and development of therapeutic antibodies

The Analyst ◽  
2018 ◽  
Vol 143 (11) ◽  
pp. 2459-2468 ◽  
Author(s):  
Yuwei Tian ◽  
Brandon T. Ruotolo
Keyword(s):  
Mass Spectrometry ◽  
State Of The Art ◽  
Therapeutic Antibodies ◽  
Critical Importance ◽  
Current State ◽  
Measurement Science ◽  
Structural Mass Spectrometry ◽  
Structural Mass

The comprehensive structural characterization of therapeutic antibodies is of critical importance for the successful discovery and development of such biopharmaceuticals, yet poses many challenges to modern measurement science. Here, we review the current state-of-the-art mass spectrometry technologies focusing on the characterization of antibody-based therapeutics.

scholarly journals Perspectives on the future of multi-dimensional platforms

2019 ◽  
Vol 218 ◽  
pp. 72-100 ◽  
Author(s):  
Gino Groeneveld ◽  
Bob W. J. Pirok ◽  
Peter J. Schoenmakers
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
High Resolution ◽  
High Resolution Mass Spectrometry ◽  
State Of The Art ◽  
Two Dimensional ◽  
Current State ◽  
Recent Developments ◽  
Resolution Mass

A practical example, the characterization of polysorbates by high-resolution comprehensive two-dimensional liquid chromatography in combination with high-resolution mass spectrometry, is described as a culmination of recent developments in 2D-LC and as an illustration of the current state of the art.

scholarly journals State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

2021 ◽  
Vol 14 (6) ◽  
pp. 498
Author(s):  
Evolène Deslignière ◽  
Anthony Ehkirch ◽  
Bastiaan L. Duivelshof ◽  
Hanna Toftevall ◽  
Jonathan Sjögren ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Ion Mobility ◽  
State Of The Art ◽  
Native Mass Spectrometry ◽  
Site Specific ◽  
Drug Conjugates ◽  
Conjugation Process ◽  
Antibody Drug

Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.

scholarly journals Nucleolin Controls Ribosome Biogenesis through Its RNA-Binding Properties

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5056-5056 ◽  
Author(s):  
Julia Fremerey ◽  
Pavel Morozov ◽  
Cindy Meyer ◽  
Aitor Garzia ◽  
Marianna Teplova ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Sites ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Binding Properties ◽  
Expression Levels ◽  
Rna Targets ◽  
Polymerase I

Abstract Introduction Nucleolin (NCL) is a multifunctional, proliferation-associated factor that is overexpressed in many cancers and has already been demonstrated to play a profound role in leukemogenesis (Abdelmohsen and Gorospe, 2012; Shen et al., 2014). This can be linked to an increased synthesis of ribosomal RNA (rRNA). Thus, in leukemic cells, high expression levels of NCL contribute to malignant transformation through the increase of rRNA synthesis, which is required to sustain high levels of protein synthesis. Physiologically, NCL is a highly abundant, nucleolar RNA-binding protein that is implicated in the regulation of polymerase I transcription, post-transcriptional gene regulation, and plays a central role in ribosome biogenesis (Srivastava and Pollard, 1999). To further elucidate the exact role of NCL, this study focused on the characterization of the RNA-binding properties and protein-interactions of NCL in the context of ribosome biogenesis. Methods In order to identify transcriptome-wide binding sites and the cellular RNA targets of NCL, PAR-CLIP (photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation) and RIP-Seq (RNA immunoprecipitation sequencing) analyses were carried out in HEK 293 cells. PAR-CLIP is characterized by the incorporation of 4-thiouridine into newly transcribed RNA that causes a T to C conversion in the corresponding cDNA of crosslinked RNA (Hafner et al., 2010). The RNA-binding properties and the interaction of NCL with its identified RNA targets were elucidated by electrophoretic mobility shift assays, isothermal titration calorimetry and size-exclusion chromatography. To further define the role of NCL in ribosome biogenesis and the effect on precursor rRNA levels, siRNA mediated knockdown of NCL was employed followed by RNA sequencing. Furthermore, to characterize the interaction network of NCL on a proteome-wide level, mass-spectrometry was performed. Results This study focuses on the characterization of the RNA-binding properties of NCL and provides the first PAR-CLIP data set of NCL and identifies small nucleolar RNAs (snoRNA) and precursor rRNA as main targets of NCL, both of which were further confirmed by RIP-Seq analysis. Binding sites of NCL were identified in the 5'ETS (external transcribed spacer), after the first cleavage site, in ITS1 and ITS2 (internal transcribed spacer) within the precursor rRNA, indicating that NCL might play a role in the early processing steps of ribosome biogenesis within the nucleolus. Biochemical and structural binding analyses reveal that NCL interacts along the complete precursor region and shows high binding affinity to G/C/U-rich repeat sequences, which is in agreement with the nucleotide composition of the primary rRNA transcript. Moreover, we propose that siRNA mediated knockdown of NCL inhibits polymerase I transcription, which is shown by decreased expression levels of the precursor rRNA transcript. On the proteome-wide level, mass-spectrometry analysis of NCL identified several interaction partners including block of proliferation 1 (BOP1), DEAD-box RNA helicase 18 (DDX18), and 5'-3' exoribonuclease 2 (XRN2) and numerous ribosomal proteins of the small and the large ribosomal subunits including RPS24, RPL11, RPL35A, and RPL36. Conclusion This study provides evidence that NCL is highly associated with the process of ribosome biogenesis on the proteome- and transcriptome-wide level. Therefore, NCL might serve as a promising biochemical target in the context of increased ribosome biogenesis in cancer. Disclosures No relevant conflicts of interest to declare.

scholarly journals Geomorphometry: Today and Tomorrow

2018 ◽  
Author(s):  
John P Wilson
Keyword(s):  
Content Knowledge ◽  
State Of The Art ◽  
High Fidelity ◽  
Terrain Modeling ◽  
Actionable Knowledge ◽  
Digital Terrain ◽  
Current State ◽  
Digital Terrain Modeling ◽  
Key Innovations

This paper summarizes the current state-of-the-art in geomorphometry and describes the innovations that are close at hand and will be required to push digital terrain modeling forward in the future. These innovations will draw on concepts and methods from computer science and the spatial sciences and require greater collaboration to produce “actionable” knowledge and outcomes. The key innovations include rediscovering and using what we already know, developing new digital terrain modeling methods, clarifying and strengthening the role of theory, developing high-fidelity DEMs, developing and embracing new visualization methods, adopting new computational approaches, and making better use of provenance, credibility, and application-content knowledge.

The interaction between the natural metalloendopeptidase inhibitor BJ46a and its target toxin jararhagin analyzed by structural mass spectrometry and molecular modeling

2020 ◽  
Vol 221 ◽  
pp. 103761
Author(s):  
Viviane A. Bastos ◽  
Francisco Gomes-Neto ◽  
Surza Lucia G. Rocha ◽  
André Teixeira-Ferreira ◽  
Jonas Perales ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Modeling ◽  
Structural Mass Spectrometry ◽  
Structural Mass
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