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...
The observed melts are characteristic of crystalline substances and suggest that the sorbitol crystallizes over time. During freezing, the excipient must remain in the same phase as the protein to ensure protein stability. By crystallizing, the sorbitol is phase-separated from the protein, which leads to protein aggregation.
We report the first application of oscillatory shear rheology to study phase behavior of IgG2 in a sucrose-containing formulation and its correspondence with physical stability not explained by glass transition (Tg'). We provide a mechanism and data suggesting that protein instability occurs at the ice/water interface.
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