Managing integrated continuous bioprocesses in real time: Deviations in product quality.

The N-mAb case study was produced by the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) to support teaching and learning for both industry and regulators around adoption of advanced manufacturing process technologies such as integrated continuous bioprocesses (ICB) for monoclonal antibodies (mAbs). N-mAb presents the evolution of an integrated control strategy, from early clinical through process validation and commercial manufacturing with a focus on elements that are unique to ICB. The entire N-mAb case study is quite comprehensive, therefore this publication presents a summary of the chapter on managing deviations from a state of control in real time.

A common framework for integrated and continuous biomanufacturing.

There is a growing application of integrated and continuous bioprocessing (ICB) for manufacturing recombinant protein therapeutics produced from mammalian cells. At first glance, the newly evolved ICB has created a vast diversity of platforms. A closer inspection reveals convergent evolution: nearly all of the major ICB methods have a common framework that could allow manufacturing across a global ecosystem of manufacturers using simple, yet effective, equipment designs.

End-to-end collaboration to transform biopharmaceutical development and manufacturing.

An ambitious 10-year collaborative program is described to invent, design, demonstrate, and support commercialization of integrated biopharmaceutical manufacturing technology intended to transform the industry. Our goal is to enable improved control, robustness, and security of supply, dramatically reduced capital and operating cost, flexibility to supply an extremely diverse and changing portfolio of products in the face of uncertainty and changing demand, and faster product development and supply chain velocity, with sustainable raw materials, components, and energy use. The program is organized into workstreams focused on end-to-end control strategy, equipment flexibility, next generation technology, sustainability, and a physical test bed to evaluate and demonstrate the technologies that are developed.

Economic Analysis of Batch and Continuous Biopharmaceutical Antibody Production: A Review.

Purpose: There is a growing interest in continuous biopharmaceutical processing due to the advantages of small footprint, increased productivity, consistent product quality, high process flexibility and robustness, facility cost-effectiveness, and reduced capital and operating cost. To support the decision making of biopharmaceutical manufacturing, comparisons between conventional batch and continuous processing are provided.

Methods: Various process unit operations in different operating modes are summarized.

Membrane insertion by anthrax protective antigen in cultured cells.

The enzymatic moieties of anthrax toxin enter the cytosol of mammalian cells via a pore in the endosomal membrane formed by the protective antigen (PA) moiety. Pore formation involves an acidic pH-induced conformational rearrangement of a heptameric precursor (the prepore), in which the seven 2beta2-2beta3 loops interact to generate a 14-strand transmembrane beta-barrel. To investigate this model in vivo, we labeled PA with the fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) at cysteine residues introduced into the 2beta2-2beta3 loop.

Clostridium sordellii lethal toxin is maintained in a multimeric protein complex.

Clostridium sordellii lethal toxin (TcsL) is distinct among large clostridial toxins (LCTs), as it is markedly reduced in its rate of intoxication at pH 8.0 yet is cytotoxic at pH 4.0.

Mutational analysis of the enzymatic domain of Clostridium difficile toxin B reveals novel inhibitors of the wild-type toxin.

Toxin B (TcdB), a major Clostridium difficile virulence factor, glucosylates and inactivates the small GTP-binding proteins Rho, Rac, and Cdc42. In the present study we provide evidence that enzymatically inactive fragments of the TcdB enzymatic domain are effective intracellular inhibitors of native TcdB. Site-directed and deletion mutants of the TcdB enzymatic region (residues 1 to 556), lacking receptor binding and cell entry domains, were analyzed for attenuation of glucosyltransferase and glucosylhydrolase activity.

Clostridium difficile toxin B activates dual caspase-dependent and caspase-independent apoptosis in intoxicated cells.

Clostridium difficile toxin B (TcdB) inactivates the small GTPases Rho, Rac and Cdc42 during intoxication of mammalian cells. In the current work, we show that TcdB has the potential to stimulate caspase-dependent and caspase-independent apoptosis. The apoptotic pathways became evident when caspase-3-processed-vimentin was detected in TcdB-treated HeLa cells.