Recombinant protein production and insect cell culture and process

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A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using the cultured insect cells as host for a virus encoding the described polypeptide such as baculovirus. The insect cells can also be a host for viral production.

In vitro cultures of animal cells are hosts for an increasing assortment of recombinant protein products. This is a relatively new phenomenon, beginning in the mid 1980's. In this short period of time, these proteins have made a substantial contribution to the U.S. economic market. In the pharmaceutical industry alone, biotherapies, which are primarily recombinant proteins, generated $1.2 billion in U.S. sales in 1991 and will grow to nearly $8.0 billion in 2001 as more proteins become commercially available. The market size and its capacity for growth demonstrate the importance of recombinant protein production to the fields of biotechnology and bioengineering. Recombinant proteins derived from animal cells are employed in a range of disciplines from agriculture to medicine to basic research. During the past decade in medicine, progress has been made in genetic engineering and animal-cell cultivation to produce an increasing assortment of recombinant proteins in sufficient quantities for the development of new drug therapies. The proteins themselves can serve as therapies, or they can be used to identify and test chemotherapies that regulate protein function in vivo . Recombinant tissue plasminogen activator is an example of a protein therapy. It is given to patients with evolving myocardial infarction to dissolve coronary thrombus and restore blood flow to the ischemic area. In clinical trials, another protein therapy, erythropoietin, has been used to treat anemia in patients with progressive renal failure and sickle cell disease Examples of protein-regulating chemotherapies include enalapril, an inhibitor of angiotensin- converting enzyme, for the treatment of sickle cell disease. A chemotherapy derived from azole regulated cytochrome P-450 dependent testosterone biosynthesis. At present, both procaryotes and eucaryotes are utilized as hosts for commercial production of recombinant proteins. The choice of one over the other is based on the structural complexity of the protein being produced and the desired yield. If a protein can be produced in a biologically active form from either host, procaryotes are preferred: they grow faster and express more protein than animal cells . Doubling times are in hours rather than days. Similarly, yields are in grams of protein per liter media rather than milligrams per liter. Eucaryotes are chosen as hosts when procaryotes are unable to produce functional protein . This typically occurs when the protein requires post-translational modification (e.g., glycosylation, phosphorylation or macromolecular assembly) to be functional. Bacteria cannot perform post- translational modifications at all; simple eucaryotes such as yeast do so to a limited extent; but complex eucaryotes such as animal cells, with few exceptions, perform the entire complement of post-translational modifications.

Commercial production of recombinant proteins from animal cells requires that the production process be reliable, yielding consistent amounts of product with reproducible biological activity. Such stringency has been achieved in vitro from animal cells cultured in a bioreactor which provides a controlled environment for cell growth.