Novavax reported that, in 28 days following public release of the avian influenza A/Anhui/1/2013 (H7N9) virus gene sequences, its recombinant DNA and baculovirus-Spodoptera frugiperda cell culture-based technology was used to produce a virus-like particle vaccine to avian influenza A(H7N9) virus, and murine animal challenge studies were initiated. This report describes Novavax’s manufacturing process and the coordinated timing of critical activities necessary to produce and release a clinical batch of avian influenza A(H7N9) virus virus-like particle vaccine, under current good manufacturing practices, within three months from the time that the virus genomic sequences for this potential pandemic influenza virus were reported…
Tag: <span>protein expression</span>
ExpreS2ion, established in January 2010, is located in the Hørsholm Science Park, north of Copenhagen. It was formed as a spin-out from Affitech A/S (Affitech) which holds an equity position in the company. ExpreS2ion operates as a contract research organization (CRO) offering services related to vector and cell line development, cloning, upstream development, optimization, and production of GLP material using its S2 (Drosophila Schneider 2) cell-based ExpreS2 platform. Two of the company’s founders, Drs. de Jongh and Dyring, and early employees of ExpreS2ion were colleagues for many years at Affitech and Pharmexa A/S (Pharmexa) before the two companies combined. It was during this period that they developed and optimized a S2 expression system for use in the production of therapeutic vaccines. ExpreS2ion’s proprietary protein expression platform, ExpreS2, consists of high-yielding expression vectors, a S2 cell line that grows to higher cell densities than standard S2 cells, an optimized culture media, and a highly efficient transfection reagent specifically optimized for S2 cells…
The pitched-blade system is traditionally the choice for stirred tank bioreactors in large-scale production of animal cells in suspension culture. The packed-bed basket technology developed by New Brunswick Scientific provides a shear-free environment for large-scale (up to 100 L) production of animal cells. At present, little information is available on the utility of this system for the production of secreted proteins, especially in perfusion mode of operation. The perfusion process provides a homeostatic environment for optimal cell growth similar to that experienced by cells in vivo. In contrast, the batch culture approach does not appropriately model this homeostatic environment due to the depletion of nutrients and accumulation of waste products in the culture system. Thus, the objective of this study was to compare the growth and productivity of alkaline phosphatase (ALKP)-secreting Chinese hamster ovary (CHO) cells cultured in these two bioreactor types: pitched-blade bioreactors operated in batch mode versus packed-bed bioreactors operated in perfusion mode. CHO cells cultured in the packed-bed bioreactor, operated in perfusion mode, produced greater amounts of ALKP compared to cells cultured in the pitched-blade system run in batch mode. These observations suggest that continuous exposure of cells to fresh culture media and the shear-free culture environment provided by the Fibra-Cel growth matrix disks offered more favorable growth conditions for CHO cells, allowing for either greater cell proliferation (higher density) or greater protein production on a per-cell basis. Overall, the results of this comparison study suggest that packed-bed bioreactors provide significant advantages for moderate-scale production of cells. The benefits of this bioreactor system may translate to large-scale cell culture for generating secreted protein products useful in medical applications…
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…
In general, yeasts offer advantages for recombinant protein expression because their intracellular environment is suitable for the correct folding of recombinant proteins and grow very high cell densities in defined fermentation media. Within the yeast kingdom, Pichia pastoris has been successfully used for expressing several recombinant proteins. The genome of this yeast contains two copies of the alcohol oxidase (AOX) gene, where the AOX1 promoter regulates 85% of the alcohol oxidase activity and drives the recombinant protein expression into the cell. One of the most successfully recombinant proteins expressed in Pichia pastoris is the hepatitis B surface antigen (HBsAg). The current manufacturing process of the active pharmaceutical ingredient (HBsAg) of the Cuban hepatitis B vaccine (HeberBiovac™ HB) starts with the expression of the HBsAg in Pichia pastoris….
Heterologous expression of membrane proteins remains a bottleneck for structural characterization by x-ray crystallography. Such proteins represent approximately 30% of the proteome and are not sufficiently represented in the Protein Data Bank (PDB). G-protein-coupled receptors (GPCRs) are an area of particular interest as it is estimated that one third of current FDA approved drugs act through this class of receptors. We have been studying rhodopsin with an interest in determining the conformational change that leads to signal transduction in this class of receptors. Although there has been some success in expressing select members of the large GPCR family in bacterial systems, the best characterized expression systems have generally been in mammalian tissue culture…
Incorporating a genomic regulatory sequence element can help to develop a robust expression vector, leading to improved recombinant protein expression in cultured cells. Such elements include the Expression Augmenting Sequence Element (EASE), scaffold- or matrix-attachment regions (S/MAR elements), Insulators, or the Universal Chromatin Opening Element (UCOE). However, generating a robust vector further requires having the relevant vector components in a context-optimized manner…
Expression vector and cell line engineering is the basis for expression and industrial production of biopharmaceuticals. The ultimate goal is to obtain clonal cell lines that secrete the protein of interest with high cell-specific productivity, and at consistently high levels over an extended number of cell generations, allowing for scale-up and cost-efficient large-scale manufacturing. Productivity and stability of expression are thus the prerequisites for developing commercially viable processes…
Membrane proteins such as hERG (human Ether-a-go-go Related Gene) and GPCRs (G-protein-coupled receptors) have been widely used as favorite targets for discovery of therapeutic drugs to treat cardiac arrhythmia, diabetes, epilepsy, cancer, glaucoma and many other indications. They are also widely used in cell-based assays to test new pharmaceuticals for safety in the early stages of drug discovery…
In order to unravel new protein activities and functions, we have expressed and purified a large number of human proteins. We have chosen to study secreted proteins and the extra-cellular domains of putative single transmembrane domain-containing proteins. In order to retain the natural protein characteristics as far as possible, we have used a mammalian expression system. Human embryonic kidney (HEK293) cells were chosen as they have been shown to possess a high protein-secretory potential. The secreted proteins were expressed with a carboxy-terminal tag and purified by affinity chromatography. Each protein was produced at a routine scale from 500 ml cell cultures, and the secreted protein was purified from the culture supernatant…