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Douthwaite J.A.,Protein Discovery
Methods in Molecular Biology | Year: 2012

Ribosome display is a powerful in vitro technology for the selection and directed evolution of proteins. Cell-free translation is central to the ribosome display process and is performed in such a way that the ribosome provides the link between genotype and phenotype that allows genes encoding proteins with desired properties to be identified by selection. Prokaryotic cell-free translation reagents, based initially on E. coli cell extracts and more recently containing purified and recombinant factors, have dominated the ribosome display literature. Eukaryotic cell extracts are also suitable for ribosome display; however, protocols for prokaryotic ribosome display are not directly transferable to the use of eukaryotic cell extracts. This chapter describes an optimised methodology for the use of rabbit reticulocyte lysate for ribosome display selections. © 2012 Springer Science+Business Media, LLC.

Buchanan A.,Protein Discovery
Methods in Molecular Biology | Year: 2012

The opportunity to enhance protein stability has a number of potential benefits for biological therapeutics -for example extending in vivo half-life, enabling a longer shelf life, reducing the propensity to aggregate, or enabling soluble expression. Engineering protein stability has been attempted empirically, rationally, and using directed evolution based on phage display. Ribosome display is a powerful in vitro technology for the selection and directed evolution of proteins. Ribosome display is typically used for the generation of high-affinity proteins and peptides. This method extends the utility of ribosome display to selecting for stability, defined as the propensity of a molecule to exist in its folded and active state. © 2012 Springer Science+Business Media, LLC.

Ravn P.,Protein Discovery
Methods in Molecular Biology | Year: 2012

A large antibody fragment library (>10 12) has been generated in ribosome display format. The library was constructed in a two-step process. First, variable (V) genes were isolated from human B cells from a panel of 14 donors and cloned into designated ribosome display vectors to create a gene bank. Second, RD-VH and RD-VL genes from individual immunoglobulin families were combined in vitro resulting in 112 scFv ribosome display sub-libraries. These were subsequently pooled to form a master library. This library was used to isolate a panel of antibodies to the IL4 receptor by three rounds of selections on a soluble target. © 2012 Springer Science+Business Media, LLC.

Elvin J.G.,Protein Discovery | Couston R.G.,University of Strathclyde | Van Der Walle C.F.,University of Strathclyde
International Journal of Pharmaceutics | Year: 2013

Antibodies are well established in mainstream clinical practice and present an exciting area for collaborative research and development in industry and academia alike. In this review, we will provide an overview of the current market and an outlook to 2015, focussing on whole antibody molecules while acknowledging the next generation scaffolds containing variable fragments. The market will be discussed in the context of disease targets, particularly in the areas of oncology and immune disorders which generate the greatest revenue by a wide margin. Emerging targets include central nervous system disorders which will also stimulate new delivery strategies. It is becoming increasingly apparent that a better understanding of bioprocessing is required in order to optimize the steps involved in the preparation of a protein prior to formulation. The latter is outside the scope of this review and nor is it our intention to discuss protein delivery and pharmacokinetics. The challenges that lie ahead include the discovery of new disease targets and the development of robust bioprocessing operations. © 2011 Published by Elsevier B.V. All rights reserved.

Smith A.J.,Protein Discovery
Journal of Biomolecular Screening | Year: 2015

Antibody drugs have become an increasingly significant component of the therapeutic landscape. Their success has been driven by some of their unique properties, in particular their very high specificity and selectivity, in contrast to the off-target liabilities of small molecules (SMs). Antibodies can bring additional functionality to the table with their ability to interact with the immune system, and this can be further manipulated with advances in antibody engineering. This review summarizes what antibody therapeutics have achieved to date and what opportunities and challenges lie ahead. The target landscape for large molecules (LMs) versus SMs and some of the challenges for antibody drug development are discussed. Effective penetration of membrane barriers and intracellular targeting is one challenge, particularly across the highly resistant blood-brain barrier. The expanding pipeline of antibody-drug conjugates offers the potential to combine SM and LM modalities in a variety of creative ways, and antibodies also offer exciting potential to build bi- and multispecific molecules. The ability to pursue more challenging targets can also be further exploited but highlights the need for earlier screening in functional cell-based assays. I discuss how this might be addressed given the practical constraints imposed by high-throughput screening sample type and process differences in antibody primary screening. © 2014 Society for Laboratory Automation and Screening.

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