Advances in industry and medicine have led to the engineering of complex “designer” proteins, such as antibodies in targeted therapeutics and enzymes in process development. The ability to easily generate an almost infinite number of variants at the DNA level has increased the demand for improved protein expression methodologies to fully capture what can be produced genetically. Often, the protein of interest is eukaryotic in origin and may require posttranslational modifications specific to its native host or may be toxic to the host cells expressing them. Cell-free protein expression systems have allowed us to step beyond the limits of traditional in vivo expression methodologies by decoupling protein expression from host cell viability. Furthermore, the ability to produce complex proteins using cell-free transcription/translation systems uniquely enables high-throughput directed evolution and protein engineering efforts. Several cell-free protein expression systems have been developed in the last decade with recent advances focusing on special folding or assembly environments. Equally as important is the capability to transition from the in vitro system to larger-scale in vivo expression, while maintaining activity of the target protein…
Tag: <span>post-translational modification</span>
Glycosylation drives protein quality and therapeutic benefits in protein-based therapies. Recently, there has been a push in the pharmaceutical industry to improve the consistency and quality of the glycan patterns on therapeutic proteins like monoclonal antibodies. Post-translational modification begins in the endoplasmic reticulum but is finished in the Golgi where more complex glycans are added. In this study, the addition of lipids via a novel mechanism provided by the medium supplement, Cell-Ess®, improves the consistency in glycan patterns so that they are more reproducible between product batches. The effect of media supplementation with Cell-Ess on the variation of glycan patterns was measured in two different media formulations across two separate experiments. Supplementation with Cell-Ess resulted in a statistically significant reduction in the variation of glycoforms when measured by the standard error of the mean. In addition, to improved consistency, there were increased higher glycoforms or galactosylation. There was also significantly more total galactosylation and significantly fewer lower glycoforms for antibodies produced by CHO cells supplemented with Cell-Ess. These data taken together suggest that the addition of lipids via Cell-Ess results in a more functional Golgi and an associated improvement of protein quality and consistency…