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To reduce the number of recovery steps during downstream processing and to overcome the limitations of present fusion-based affinity separations, a controllable self-splicing protein element in the form of a mini-intein was used to optimize the recovery of proteins for both batch and flow purification strategies. The ability to recover purified proteins was demonstrated using a tripartite fusion consisting of a maltose binding domain, a truncated intein as a controllable linker molecule, and a protein of interest. To characterize expression level, solubility, cleavage rates, pH and temperature controllability, and protein activity, recombinant human acidic fibroblast growth factor (aFGF) was used as a model protein. A simple mass transport model, based on cleavage reaction-limited mass transfer and constant dispersion, was successfully used to predict product concentration and peak shape in relation to critical process parameters (with no fitting parameters). Insight into the nature of the cleavage reaction and its regulation was obtained via temperature- and pH-dependent kinetic data.

This article provides an overview of the upstream technologies used in the industrial production of therapeutic monoclonal antibodies (mAbs) based on the cultivation of mammalian cells. More specifically, in a first section, after a short discussion of relevant biochemical characteristics of antibodies, we review the cell lines currently employed in commercial production and the methods of constructing and isolating production clones. This is followed with a review of the most current methods of commercial scale production and their associated technologies. Selected references and short discussions pertaining to emerging and relevant technologies have been embedded throughout the text in order to give a sense of the overall direction the field is taking.

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