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Protein activity regulated by interactions with metal ions can be utilized for many different purposes, including biological therapies and bioprocessing, among others. Calcium ions are known to interact with the frequently occurring EF-hand motif, which can alter protein activity upon binding through an induced conformational change. The calcium-binding loop of the EF-hand motif has previously been introduced into a small protein domain derived from staphylococcal Protein A in a successful effort to render antibody binding dependent on calcium. Presented here, is a combinatorial library for calcium-regulated affinity, CaRA, based on this domain. CaRA is the first alternative scaffold library designed to achieve novel target specificities with metal-dependent binding. From this library, several calcium-dependent binders could be isolated through phage display campaigns towards a set of unrelated target proteins (IgE Cε3-Cε4, TNFα, IL23, scFv, tPA, PCSK9 and HER3) useful for distinct applications. Overall, these monomeric CaRA variants showed high stability and target affinities within the nanomolar range. They displayed considerably higher melting temperatures in the presence of 1 mM calcium compared to without calcium. Further, all discovered binders proved to be calcium-dependent, with the great majority showing complete lack of target binding in the absence of calcium. As demonstrated, the CaRA library is highly capable of providing protein-binding domains with calcium-dependent behavior, independent of the type of target protein. These binding domains could subsequently be of great use in gentle protein purification or as novel therapeutic modalities. [Display omitted] • Combinatorial library of calcium-regulated affinity binders. • Phage display selections for novel target specificities with metal-dependent binding. • Highly stable binders with nanomolar calcium-dependent affinities to target of choice. • Conditional binding for multiple purposes such as gentler protein purification. • Switch-like properties for engineering of therapeutic protein domains. [ABSTRACT FROM AUTHOR]

In this study, we demonstrated the first, to our knowledge, integrated continuous bioprocess (ICB) designed for the production of acid‐sensitive monoclonal antibodies, prone to aggregate at low pH, on pilot scale. A high cell density perfusion culture, stably maintained at 100 × 106 cells/ml, was integrated with the downstream process, consisting of a capture step with the recently developed Protein A ligand, ZCa; a solvent/detergent‐based virus inactivation; and two ion‐exchange chromatography steps. The use of a mild pH in the downstream process makes this ICB suitable for the purification of acid‐sensitive monoclonal antibodies. Integration and automation of the downstream process were achieved using the Orbit software, and the same equipment and control system were used in initial small‐scale trials and the pilot‐scale downstream process. High recovery yields of around 90% and a productivity close to 1 g purified antibody/L/day were achieved, with a stable glycosylation pattern and efficient removal of impurities, such as host cell proteins and DNA. Finally, negligible levels of antibody aggregates were detected owing to the mild conditions used throughout the process. The present work paves the way for future industrial‐scale integrated continuous biomanufacturing of all types of antibodies, regardless of acid stability. [ABSTRACT FROM AUTHOR]

Secrets of secreted proteins: Although proteins that are actively secreted from cells constitute a large proportion of the targets of pharmaceutical drugs, we lack a complete understanding of the size and composition of this group of proteins, collectively referred to as the human "secretome." Uhlén et al. analyzed the genes of the Human Protein Atlas that encode proteins that are predicted to be secreted and combined this with data derived from antibody- and mass spectrometry–based analyses of the relative abundances, sites of origin, and final locations of proteins within the body. Their analysis suggests that many proteins that were thought to be secreted are retained intracellularly, and that another group of secreted proteins are retained locally rather than being released into the blood. These data serve as a resource for future research on this important class of proteins. The proteins secreted by human cells (collectively referred to as the secretome) are important not only for the basic understanding of human biology but also for the identification of potential targets for future diagnostics and therapies. Here, we present a comprehensive analysis of proteins predicted to be secreted in human cells, which provides information about their final localization in the human body, including the proteins actively secreted to peripheral blood. The analysis suggests that a large number of the proteins of the secretome are not secreted out of the cell, but instead are retained intracellularly, whereas another large group of proteins were identified that are predicted to be retained locally at the tissue of expression and not secreted into the blood. Proteins detected in the human blood by mass spectrometry–based proteomics and antibody-based immunoassays are also presented with estimates of their concentrations in the blood. The results are presented in an updated version 19 of the Human Protein Atlas in which each gene encoding a secretome protein is annotated to provide an open-access knowledge resource of the human secretome, including body-wide expression data, spatial localization data down to the single-cell and subcellular levels, and data about the presence of proteins that are detectable in the blood. [ABSTRACT FROM AUTHOR]

Therapeutic antibodies are at the forefront of modern medicine where high purity, which is typically obtained by Protein A-based affinity purification, is of utmost importance. In this chapter, we present a method for neutral and selective purification of antibodies by utilizing an engineered affinity ligand, Z Ca , derived from Protein A. This domain displays a calcium-dependent binding of antibodies and has been multimerized and immobilized to a chromatography resin to achieve an affinity matrix with high binding capacity. IgG antibodies can be eluted from the tetrameric Z Ca ligand at pH 7 with the addition of EDTA, or at pH 5.5 with EDTA for purification of monoclonal IgG1, which is significantly milder than the low pH (3-4) required in conventional Protein A affinity chromatography. Here, a protocol for selective capture of IgG with elution at neutral pH from a Z Ca tetramer ligand immobilized on a chromatography resin is described.