Bioanalytical Workflow for Novel Scaffold Protein–Drug Conjugates: Quantitation of Total Centyrin Protein, Conjugated Centyrin and Free Payload for Centyrin–Drug Conjugate in Plasma and Tissue Samples Using Liquid Chromatography–Tandem Mass Spectrometry

Shi, Chuan; Goldberg, Shalom D.; Lin, Tricia; Dudkin, Vadim Y.; Widdison, Wayne C.; Harris, Luke; Wilhelm, Sharon; Jmeian, Yazen; Davis, Darryl; O’Neil, Karyn T.; Weng, Naidong; Jian, Wenying

doi:10.4155/bio-2018-0201

Cited by 14 publications

(7 citation statements)

References 47 publications

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“…Small non-antibody formats also offer advantages such as fine-tuning of affinity to low nanomolar levels, enabling them to pass through the outer layers of tumour cells and avoid a 'barrier effect' [59]. The absence of cysteines in FN3 domains allows for the introduction of a single conjugation-compatible cysteine for site-specific loading with drugs, thus avoiding potential disulphide-mediated aggregation [60,61]. This feature is critical as cytotoxic drugs can be disruptive to protein structure due to their hydrophobic nature, and small protein scaffolds require well-controlled conjugation to ensure resistance to aggregation [35,57].…”

Section: Delivery Agentsmentioning

confidence: 99%

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

Chandler

Buckle

2020

Cells

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As a non-antibody scaffold, monobodies based on the fibronectin type III (FN3) domain overcome antibody size and complexity while maintaining analogous binding loops. However, antibodies and their derivatives remain the gold standard for the design of new therapeutics. In response, clinical-stage therapeutic proteins based on the FN3 domain are beginning to use native fibronectin function as a point of differentiation. The small and simple structure of monomeric monobodies confers increased tissue distribution and reduced half-life, whilst the absence of disulphide bonds improves stability in cytosolic environments. Where multi-specificity is challenging with an antibody format that is prone to mis-pairing between chains, multiple FN3 domains in the fibronectin assembly already interact with a large number of molecules. As such, multiple monobodies engineered for interaction with therapeutic targets are being combined in a similar beads-on-a-string assembly which improves both efficacy and pharmacokinetics. Furthermore, full length fibronectin is able to fold into multiple conformations as part of its natural function and a greater understanding of how mechanical forces allow for the transition between states will lead to advanced applications that truly differentiate the FN3 domain as a therapeutic scaffold.

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Section: Delivery Agentsmentioning

confidence: 99%

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

Chandler

Buckle

2020

Cells

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“…Small non-antibody formats also offer advantages such as fine-tuning of affinity to low nanomolar levels, enabling them to pass through the outer layers of tumour cells and avoid a 'barrier effect' [58]. The absence of cysteines in FN3 domains allows for the introduction of a single conjugation-compatible cysteine for site-specific loading with drugs, thus avoiding potential disulphide-mediated aggregation [59,60]. This feature is critical as cytotoxic drugs can be disruptive to protein structure due to their hydrophobic nature and a well-controlled conjugation in a small protein scaffold is required to ensure resistance to aggregation [35,56].…”

Section: Delivery Agentsmentioning

confidence: 99%

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

Chandler

Buckle

2020

Preprint

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As a non-antibody scaffold, monobodies based on the fibronectin type III (FN3) domain overcome antibody size and complexity while maintaining analogous binding loops. However, antibodies and their derivatives remain the gold standard for design of new therapeutics. In response, clinical therapeutic proteins based on the FN3 domain are beginning to use native fibronectin function as a point of differentiation. The small and simple structure of monomeric monobodies confers increased tissue distribution and reduced half-life, whilst the absence of disulphide bonds improves stability in cytosolic environments. Where multi-specificity is challenging with an antibody format that is prone to mis-pairing of chains, FN3 domains in the fibronectin assembly already interact with a large number of molecules. As such, multiple monobodies engineered for interaction with therapeutic targets are being combined in a similar beads-on-a-string assembly which improves both efficacy and pharmacokinetics. Furthermore, full length fibronectin is able to fold into multiple conformations as part of its natural function and a greater understanding of how mechanical forces allow for the transition between states will lead to advanced applications that truly differentiate the FN3 domain as a therapeutic scaffold.

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“…A similar approach was followed to study the in vivo fate of a protein-drug conjugate (PDC): a surrogate peptide from the unconjugated part of the protein was measured next to a peptide containing a small-molecule drug attached via a linker. After enrichment using immobilized metal affinity chromatography, the sample was digested and the two peptides were quantified as a measure for the total and conjugated forms of the PDC, respectively [77]. Biotransformation has also been investigated at the intact protein level by affinity capture and quantification of a fusion protein and two catabolites by LC-HRMS with deconvolution.…”

Section: Biotransformationmentioning

confidence: 99%

Protein Quantification by LC–MS: A Decade of Progress Through the Pages of Bioanalysis

Merbel

2019

Bioanalysis

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Over the past 10 years, there has been a remarkable increase in the use of LC–MS for the quantitative determination of proteins, and this technique can now be considered an established bioanalytical platform for the quantification of macromolecular drugs and biomarkers, next to the traditional ligand-binding assays. Many researchers have contributed to the field and helped improve both the technical possibilities of LC–MS-based workflows and our understanding of the meaning of the results that are obtained. As a tribute to Bioanalysis, which has published many important contributions, this report gives a high-level overview of the most important trends in the field of protein LC–MS, as published in this journal since its inauguration a decade ago. It describes the major technical developments with regard to sample handling, separation and MS detection of both digested and intact protein analysis. In addition, the relevance of the complex structure and in vivo behavior of proteins is discussed and the effect of protein–protein interactions, biotransformation and the occurrence of isoforms on the analytical result is addressed.

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Bioanalytical Workflow for Novel Scaffold Protein–Drug Conjugates: Quantitation of Total Centyrin Protein, Conjugated Centyrin and Free Payload for Centyrin–Drug Conjugate in Plasma and Tissue Samples Using Liquid Chromatography–Tandem Mass Spectrometry

Cited by 14 publications

References 47 publications

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

Protein Quantification by LC–MS: A Decade of Progress Through the Pages of Bioanalysis

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