Protein synthesis and secretion are essential to cellular life. Although secretory activities may vary in different cell types, what determines the maximum secretory capacity is inherently difficult to study. Increasing protein synthesis until reaching the limit of secretory capacity is one strategy to address this key issue. Under highly optimized growth conditions, recombinant CHO cells engineered to produce a model human IgG clone started housing rod-shaped crystals in the endoplasmic reticulum (ER) lumen. The intra-ER crystal growth was accompanied by cell enlargement and multinucleation and continued until crystals outgrew cell size to breach membrane integrity. The intra-ER crystals were composed of correctly folded, endoglycosidase H-sensitive IgG. Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG, and the crystallization was reproduced in vitro by exposing a high concentration of IgG to a near neutral pH. The striking cellular phenotype implicated the efficiency of IgG protein synthesis and oxidative folding exceeded the capacity of ER export machinery. As a result, export-ready IgG accumulated progressively in the ER lumen until a threshold concentration was reached to nucleate crystals. Using an in vivo system that reports accumulation of correctly folded IgG, we showed that the ER-toGolgi transport steps became rate-limiting in cells with high secretory activity.Immunoglobulins continue to serve as an important model secretory cargo for investigating biochemical processes of oxidative protein folding and subunit assembly in the ER 2 lumen (1). Although immunoglobulins are indispensable as research tools, their potential as human therapeutics has attracted significant interest in recent years in the manufacture of human IgG at large scale (2, 3). Therapeutic human IgGs are often recombinantly produced in variants of CHO cells that were adapted to propagate in suspension culture format. Mammalian cell hosts are often preferred for biopharmaceutical production not only just to achieve desired co-translational and post-translational modifications (4) but also to exploit the stringent protein quality control mechanisms that only allow the secretion of properly folded and correctly assembled proteins (5, 6).To achieve high recombinant protein expression levels in mammalian cells, various cis-acting exogenous nucleotide elements have been engineered into transgene expression cassettes to enhance transcription efficiency, extend message halflife, and increase translation initiation frequency (7,8). Exogenous nucleotide elements also enabled strategies to increase transgene copy number by gene amplification and to suppress epigenetic silencing (9 -12). Despite the success in boosting protein expression per se through these expression vector engineering approaches, such enhancements did not translate into higher glycoprotein secretion partly because post-translational events such as protein folding/assembly and intracellular vesicular transport steps along the secreto...

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The need for purification of biomolecules extends to larger bioparticles as well. For example, virus purification is required for production of many vaccines and gene delivery vectors, and understanding virus removal in porous media is also important in downstream processing of therapeutic proteins and in purification of water in soils. A convective entrapment mechanism for retention of large bioparticles is discussed here based on retention of large bioparticles in pore constrictions at high enough flow rates, even under non-binding conditions. A simple equation to predict whether such entrapment is expected to occur in a given system is derived based on a Péclet number that is proportional to the flow rate and to the cube of the bioparticle diameter. To test the theory, adenovirus was spiked onto chromatographic beds. As expected from the theory, under non-interacting conditions a progressively larger amount of virus becomes trapped with increasing flow rate. The entrapment is reversible upon flow rate reduction, which, within the proposed model, is based on the possibility of diffusive escape from pore constrictions. This mechanism can be exploited for virus purification or removal, and the theory also explains the anecdotal evidence that monoliths and membranes are more difficult to clean than conventional chromatographic beds, especially at high flow rates.

Crystallization holds the potential to be used for protein purification and low-viscosity drug substance and drug product formulations. Twenty-two different proteins (20 monoclonal antibodies and two Fc-fusions) were examined to determine the breadth of applicability of crystallization to these therapeutic proteins. Vapor diffusion technique and an evaporative screening method were used to identify crystallization conditions using around a 100 initial conditions based on reagents that are generally regarded as safe (GRAS). Of 16 IgG2 s examined, at least four formed diffraction-quality crystals and four others formed crystal-like particles. At least three of the IgG2 s that crystallized well were also crystallized under the same set of operating conditions using inexpensive GRAS reagents. The crystals were formed to high-yields in a few hours and were dissolved quickly without impacting product quality. Although only a fraction of the proteins examined crystallized, all exhibited liquid-liquid phase separation (LLPS), which could be used for their concentration or possibly purification. One of the Fc-fusions, for example, was concentrated by LLPS to a self-buffering solution at 150 g/L. Crystallization and LLPS in the salting-in region were shown to be feasible.