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Construction and Selection of Affilin® Phage Display Libraries

Affilin® molecules represent a new class of so-called scaffold proteins. The concept of scaffold proteins is to use stable and versatile protein structures which can be endowed with de novo binding properties and specificities by introducing mutations in surface exposed amino acid residues. Complex variations and combinations are generated by genetic methods of randomization resulting in large cDNA libraries. The selection for candidates binding to a desired target can be executed by display methods, especially the very robust and flexible phage display. Here, we describe the construction of ubiquitin based Affilin® phage display libraries and their use in biopanning experiments for the identification of novel protein ligands.

© Springer Science+Business Media LLC 2018

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Affinity chromatography for vaccines manufacturing: Finally ready for prime time?

Affinity chromatography is among the most powerful separation techniques, achieving the finest separation with high yields even in the most challenging feed streams. Incorporating affinity chromatography in vaccine purification has long been attempted by researchers to improve unit yield and purity with the secondary goal of reducing the number of downstream process operations. Despite the success in laboratory-scale proof of concept, implementation of this technique in pilot or cGMP manufacturing has rarely been realised due to technical and economic challenges in design and manufacturing of ideal ligands as well as availability of high-productivity chromatography media. This paper reviews evolving technologies in engineered ligands and chromatography media that are encouraging companies to revisit the possible use of affinity chromatography in larger scale vaccine purification. It is postulated that commercial-scale implementation of high throughput single-use affinity chromatography can significantly simplify process architecture, improve productivity and flexibility, and reduce cost of goods.

© 2018 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license.

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Mabfilin and Fabfilin - New antibody-scaffold fusion formats for multispecific targeting concepts

Protein based binding molecules have a broad applicability from therapeutic to technical use. Monoclonal antibodies represent the major class of this type of agents complemented by innovative approaches using scaffold proteins with tailor-made properties. Various concepts for new formats combining antibody chains or antibody fragments and fusions with other entities have been developed recently. This strategy opens up options to design molecules with biophysical, biochemical and pharmacological characteristics in a broad range while simultaneously addressing several targets or epitopes. The demand for such compounds is still growing as reflected by the literature and further new ideas are expected. In this context we developed so called Mabfilin and Fabfilin molecules. The formats synergistically bring together the classical antibody or fragments thereof supplemented with additional binding moieties, the Affilin® molecules. These are based on the scaffold ubiquitin endowed with novel targeting properties by local randomization and selection from combinatorial libraries. Mab-/Fabfilin variants show advantageous biochemical properties and open a new route for the development of multispecific compounds for flexible applications.

© 2018 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY-NC-ND license.

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Ubiquitin is a versatile scaffold protein for the generation of molecules with de novo binding and advantageous drug-like properties

In the search for effective therapeutic strategies, protein-based biologicals are under intense development. While monoclonal antibodies represent the majority of these drugs, other innovative approaches are exploring the use of scaffold proteins for the creation of binding molecules with tailor-made properties. Ubiquitin is especially suited for this strategy due to several key characteristics. Ubiquitin is a natural serum protein, 100% conserved across the mammalian class and possesses high thermal, structural and proteolytic stability. Because of its small size and lack of posttranslational modifications, it can be easily produced in Escherichia coli. In this work we provide evidence that ubiquitin is safe as tested experimentally in vivo. In contrast to previously published results, we show that, in our hands, ubiquitin does not act as a functional ligand of the chemokine receptor CXCR4. Cellular assays based on different signaling pathways of the receptor were conducted with the natural agonist SDF-1 as a benchmark. In none of the assays could a response to ubiquitin treatment be elicited. Furthermore, intravenous application to mice at high concentrations did not induce any detectable effect on cytokine levels or hematological parameters.

© 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license.

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Affinity Membrane Chromatography For Robust And Cost-Efficient Industrial Vaccine Purification

Due to the outdated manufacturing technologies used for majority of legacy products, many current vaccines processes suffer from a lack of control and robustness; hence, the highest levels of quality are maintained only at the expense of extensive QC release testing and high lot rejection rates. While manufacturers are engaged in life cycle management projects, the use of process chromatography is gaining its popularity but the implementation is still limited mostly due to the low throughput and vastly capital expense associated with resin columns. This work presents an innovative process architecture strategy taking advantage of the purification power of affinity chromatography and the high throughput of hydrogel membranes, while leveraging all the proven benefits of disposable technologies. Proof of concept studies combining Scil Affilin ligands and Natrix hydrogel membranes technologies are presented. The potential for a robust, high yield and generic vaccine affinity-based purification platform is discussed. Relying on a full process economic model independently developed by a customer, the impact of this approach on cost of goods of the drug substance for a current vaccine candidate is demonstrated.

Zhao et al 2015

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Novel Ubiquitin-derived High Affinity Binding Proteins with Tumor Targeting Properties

Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range. Scaffold-based binding molecules are small proteins with high affinity and short systemic circulation. Due to their low molecular complexity, they are well suited for combination with effector molecules as well as half-life extension technologies yielding therapeutics with half-lives adapted to the specific therapy. We have identified ubiquitin as an ideal scaffold protein due to its outstanding biophysical and biochemical properties. Based on a dimeric ubiquitin library, high affinity and specific binding molecules, so-called Affilin molecules, have been selected against the extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues. Extradomain B-binding molecules feature high thermal and serum stability as well as strong in vitro target binding and in vivo tumor accumulation. Application of several half-life extension technologies results in molecules of largely unaffected affinity but significantly prolonged in vivo half-life and tumor retention. Our results demonstrate the utility of ubiquitin as a scaffold for the generation of high affinity binders in a modular fashion, which can be combined with effector molecules and half-life extension technologies.

Published, JBC, January 28, 2014
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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New Binding Mode to TNF-Alpha Revealed by Ubiquitin- Based Artificial Binding Protein

A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a betasheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNFalpha trimer. Using NMR spectroscopy and other analytical methods the 1:3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins – designed ankyrin repeat proteins – without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNFalpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies.

2012 Hoffmann et al.

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NH exchange in point mutants of human ubiquitin

Several point mutants of human ubiquitin (Ub(T9V), Ub(F45W), Ub(F45G), and Ub(A46S)) were prepared by recombinant techniques. The NH exchange rate constants were measured by the NMR diffusion and the MEXICO methods and compared with those in the wild type to examine the influence of structural changes and to improve the understanding of this important reaction in studies of protein folding and denaturation. The observed changes follow qualitatively the polarity and steric alterations caused by the introduced amino acids. Attempts to reproduce quantitatively the observed changes by modeling studies and molecular dynamics simulations were not satisfactory.

© 2011 Elsevier B.V. All rights reserved.

AFFILIN MOLECULES Novel Ligands for Bioseparation

With the success of biotherapeutics the need for new ligands for affinity purification is growing. A novel approach to generate customized ligands is the use of alternative binding proteins. Here, we describe the generation, selection, and the use of specific AffilinTM molecules as small and robust ligands for recombinant protein purification. The AffilinTM molecules were isolated from a complex phage display library based on the randomization of eight surface exposed amino acids of the human eye lens protein g-B-crystallin. Characterization of AffilinTM candidates by surface plasmon resonance (SPR) revealed dissociation constants in the nanomolar range. The E9 AffilinTM variant which was characterized in more detail has nanomolar affinity to the pro-form of human nerve growth factor (proNGF) and was used for matrix coupling to test proNGF recovery. The use of E9 AffilinTM as a ligand in affinity chromatography has demonstrated efficient recovery of its target protein,
proNGF, from complex mixtures, such as spiked CHO supernatant or E. coli crude extract. Further, it has been shown that the E9 AffilinTM ligand can withstand denaturing conditions standard for cleaning processes in affinity chromatography. These findings suggest the application of AffilinTM molecules as potent and versatile ligands for affinity chromatography in the field of bioseparation.

Copyright 2006 Institution of Chemical Engineers
Trans IChemE, Part C, March 2006
Food and Bioproducts Processing, 84(C1): 3–8

Artificial, non-antibody binding proteins for pharmaceutical and industrial applications

Using combinatorial chemistry to generate novel binding molecules based on protein frameworks (‘scaffolds’) is a concept that has been strongly promoted during the past five years in both academia and industry. Nonantibody recognition proteins derive from different structural families and mimic the binding principle of immunoglobulins to varying degrees. In addition to the specific binding of a pre-defined target, these proteins provide favourable characteristics such as robustness, ease of modification and cost-efficient production. The broad spectrum of potential applications, including research tools, separomics, diagnostics and therapy, has led to the commercial exploitation of this technology by various small- and medium-sized companies. It is predicted that scaffold-based affinity reagents will broaden and complement applications that are presently covered by natural or recombinant antibodies. Here, we provide an overview on current approaches in the biotech industry, considering both scientific and commercial aspects.

© 2018 The Authors. Published by Elsevier Inc.