Organizations developing biopharmaceuticals are often faced with the challenge of developing, as rapidly as possible, a production system for a recombinant protein or antibody intended for use in clinical trials. For expression of antibodies and other proteins with complex post-translational modifications, Chinese hamster ovary (CHO) cells are often the host of choice. However, isolation of CHO cell lines producing even moderate levels of a protein of interest is usually a lengthy process due to the need for at least one and usually several gene amplification steps. Gene amplification, which is usually accomplished through the dihydrofolate reductase (dhfr)/methotrexate system, is a requirement for most CHO expression vectors because the absolute expression level from each copy of an integrated expression plasmid is generally very low…
Tag: <span>recombinant proteins</span>
More than 130 drug and vaccine approvals for 95 entities over the last 20 years have generated roughly $30 billion in revenue for the biotech industry. The vast majority of this revenue comes from 30 proteins that have manufacturing bottlenecks resulting from the complexities of consistent protein production. The lag times involved in constructing mammalian cell fermentation facilities keeps supply of immensely successful high-volume drugs like Enbrel, Rituxan, and Remicade well below estimated demand. In other cases, the complexities of peptide synthesis threaten the potential of soon-to-be-launched or recently approved drugs like Fuzeon. The Pharmaceutical Research and Manufacturers of America (PhRMA) has documented more than 371 new biotech drugs in development, supporting the view that demand for many biopharmaceuticals will continue to outstrip supply. That number does not include the multitude of biotech drugs still in research stages…
The Gel Microdrop (GMD) Secretion Assay involves encapsulating cells within a biotinylated agarose matrix, followed by capture and detection of cell-secreted molecules with fluorescent markers. This technology differs from other encapsulation methods in that the small size of the microdrop (<50 ?m diameter) creates a defined microenvironment around the cell without impeding the fusion of nutrients, antibodies, or nucleic acid probes into the GMDs, or the diffusion of secreted products out of the GMDs. Large numbers of GMDs can be readily analyzed using flow cytometry, and sub-populations of rare or high-secreting cells, as small as 0.1%, can be detected and recovered in one day. This assay format is a rapid alternative to limited dilution cloning (LDC)...
The baculovirus-insect cell system consists of a recombinant baculovirus vector and its host, which may be a lepidopteran insect larvae or an established lepidopteran insect cell line. Hundreds of different recombinant proteins have been produced using the baculovirus-insect cell system, facilitating biomedical research on protein structure, function, and the roles of various proteins in disease. In addition, many biotechnology companies are using this system to produce recombinant proteins for potential clinical use as vaccines, therapeutics, or diagnostic reagents…
It is well known that the characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. There are numerous selective and adaptive culture processes, in addition to genetic instability, that may promote phenotypic changes in cell growth, virus susceptibility, gene expression, et cetera. Similar detrimental effects of long term passaging of insect cells have also been reported for continuous cell lines. In this paper, we describe the isolation of cell clones from low passage BTI Tn5B1-4 cells (High FiveTM Cells), and report their growth characteristics and high level of recombinant protein production…
The insect cell/baculovirus expression system typically results in more rapid expression and higher concentrations of recombinant proteins than what can be achieved with other animal cell culture systems. The lack of complex glycosylation in the proteins produced by this system, however, limits its use in the commercial-scale production of therapeutics. Complex glycosylation is required in many cases for adequate protein activity and pharmokinetic characteristics. In contrast to the protein’s primary structure, which is encoded by the genetic material and is constant regardless of the host utilized, the extent of glycosylation is determined by the host, and by the protein itself. Even cells from different tissues of the same organism provide different glycosylation profiles. In addition, culture conditions and the cellular metabolic state can also influence protein glycosylation…
The baculovirus expression vector system (BEVS) is one method utilized for the production of recombinant proteins, and typically works without significant difficulties. However, some proteins are produced in insoluble forms, and degradation can occur. This article will focus on this degradation issue, and present a method to stabilize a protease-sensitive protein that has been produced at the 40-liter scale…
The baculovirus expression system promises to revolutionize the production of recombinant proteins for use as clinical products. The technology is robust, efficient, and low-cost when compared to other cell based systems. The technique may also present an advantage in producing safer products versus the equivalent materials made with mammalian cells. Proteins can be produced in insect cells without animal supplements such as fetal calf serum. In the current climate of concerns over Bovine Spongiform Encephalopathies, and bovine viral risks, this method offers a significant safety, as well as cost advantage, over other production methods…
Baculovirus expression technology, or BEVS, gained its first broad industry exposure in the early 1980s, primarily through the many papers published by students and post-doctoral fellows in Dr. Max Summers’ laboratory at Texas A&M University (College Station, Texas). This technology fostered popular appeal because of its simplicity and high protein expression capabilities. As more work was done, it became even more evident that this was a very rapid, and relatively inexpensive method for producing proteins. It was also postulated that BEVS would offer a valuable means of producing recombinant proteins for use in human therapy, especially since baculovirus was considered non-infectious to human cells. It was thought that any problems with post-translational modifications of the manufactured proteins could be worked out, and fully functional glycoproteins could be manufactured…