Improved drug-like properties of therapeutic proteins by directed evolution

Buchanan, Andrew; Ferraro, Franco; Rust, Steve; Sridharan, Sridha; Franks, Ruth; Dean, Greg; McCourt, Matthew; Jermutus, Lutz; Minter, Ralph

doi:10.1093/protein/gzs054

Cited by 20 publications

(20 citation statements)

References 41 publications

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“…3B). The behavior of β 2 m contrasts starkly with that of the C3 variant of G-CSF (37). This 175-residue, 4-helical, all α-protein was found to be extremely sensitive to the effects of extensional flow.…”

Section: Design and Computational Characterization Of Extensional Flowmentioning

confidence: 86%

Inducing protein aggregation by extensional flow

Dobson

Kumar

Willis

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

129

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Relative to other extrinsic factors, the effects of hydrodynamic flow fields on protein stability and conformation remain poorly understood. Flow-induced protein remodeling and/or aggregation is observed both in Nature and during the large-scale industrial manufacture of proteins. Despite its ubiquity, the relationships between the type and magnitude of hydrodynamic flow, a protein's structure and stability, and the resultant aggregation propensity are unclear. Here, we assess the effects of a defined and quantified flow field dominated by extensional flow on the aggregation of BSA, β 2 -microglobulin (β 2 m), granulocyte colony stimulating factor (G-CSF), and three monoclonal antibodies (mAbs). We show that the device induces protein aggregation after exposure to an extensional flow field for 0.36-1.8 ms, at concentrations as low as 0.5 mg mL −1 . In addition, we reveal that the extent of aggregation depends on the applied strain rate and the concentration, structural scaffold, and sequence of the protein. Finally we demonstrate the in situ labeling of a buried cysteine residue in BSA during extensional stress. Together, these data indicate that an extensional flow readily unfolds thermodynamically and kinetically stable proteins, exposing previously sequestered sequences whose aggregation propensity determines the probability and extent of aggregation.extensional flow | aggregation | unfolding | bioprocessing | antibody P roteins are dynamic and metastable and consequently have conformations that are highly sensitive to the environment (1). Over the last 50 y the effect of changes in temperature, pH, and the concentration of kosmatropic/chaotropic agents on the conformational energy landscape of proteins has become well understood (1). This, in turn, has allowed a link to be established between the partial or full unfolding of proteins and their propensity to aggregate (2). The force applied onto a protein as a consequence of hydrodynamic flow has also been observed to trigger protein aggregation and has fundamental (3), medical (4), and industrial relevance, especially in the manufacture of biopharmaceuticals (5-8). Although a wealth of studies have been performed (7,(9)(10)(11)(12)(13), no consensus has emerged on the ability of hydrodynamic flow to induce protein aggregation (7,14,15). This is due to the wide variety of proteins used (ranging from lysozyme, BSA, and alcohol dehydrogenase to IgGs), differences in the type of flow field generated (e.g., shear, extensional, or mixtures of these), and to the presence or absence of an interface (16). A shearing flow field (Fig. 1A, Top) is caused by a gradient in velocity perpendicular to the direction of travel and is characterized by the shear rate (s −1 ). This results in a weak rotating motion of a protein alongside translation in the direction of the flow. An extensional flow field (Fig. 1A, Bottom) is generated by a gradient in velocity in the direction of travel and is characterized by the strain rate (s −1 ). A protein in this type of flow would experien...

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Section: Design and Computational Characterization Of Extensional Flowmentioning

confidence: 86%

Inducing protein aggregation by extensional flow

Dobson

Kumar

Willis

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

129

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“…[7][8][9][10] Nevertheless, engineered scaffolds can complement antibodies, particularly in applications requiring small multispecific agents or where Fc-mediated functions are not required. These limitations of antibodies are being addressed by engineering for improved stability and developments in eukaryotic, microbial, and cell free translation systems.…”

Section: Engineered Scaffoldsmentioning

confidence: 99%

Novel Therapeutic Proteins and Peptides

Buchanan

Revell

2015

Novel Approaches and Strategies for Biologics, Vaccines and Cancer Therapies

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“…In addition to generation of antibodies, ribosome display has been explored to select different proteins and peptides, including scaffolds, tags, enzymes, DNA‐binding proteins, receptors, membrane proteins and engineered vaccines . It has also been used to identify novel DNA sequence for promoting protein expression/solubility and identify molecular interactions . Combining with next‐generation sequencing (NGS) technology, ribosome display has been used to rapidly produce novel molecules from very large libraries .…”

Section: Introductionmentioning

confidence: 99%

“…It has also been used to identify novel DNA sequence for promoting protein expression/solubility and identify molecular interactions [3][4][5]. Combining with next-generation sequencing (NGS) technology, ribosome display has been used to rapidly produce novel molecules from very large libraries [5,6].…”

Section: Introductionmentioning

confidence: 99%

Cell‐free ribosome display and selection of antibodies on arrayed antigens

Cong¹,

Yu²,

He³

et al. 2016

Proteomics

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In vitro display technology is a powerful tool for discovery and optimisation of novel antibodies. With increasing demands on various binding molecules in proteomics studies, techniques for a large-scale generation of antibodies or antibody fragments are needed. Here, we describe a novel method for parallel generation of different antibody fragments (scFv) by integrating cell-free ribosome display with array technology. We have demonstrated the procedure by successfully isolating scFv antibodies specific to 16 different cancer biomarkers via a single process. Our results provide proof of principle for multiple production of various scFv antibodies simultaneously.

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Improved drug-like properties of therapeutic proteins by directed evolution

Cited by 20 publications

References 41 publications

Inducing protein aggregation by extensional flow

Inducing protein aggregation by extensional flow

Novel Therapeutic Proteins and Peptides

Cell‐free ribosome display and selection of antibodies on arrayed antigens

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