Producer cell lines

Non-neuronal transplants such as adrenal chromaffin cells have been tried but do not survive although some L-dopa-producing cell lines (e.g. PC 12) or glomus cells of the carotid body do produce DA in vivo and may provide the equivalent of a continuous infusion of dopa (and DA) directly into the brain. Expression of tyrosine hydroxylase to promote dopa and DA synthesis in striatal cells by direct gene transfer in vivo or in cultures for subsequent transplanting, may also be possible. (See Dunnett and Bjorklund 1999 for a review of these approaches.)... 

The upstream processing element of the manufacture of a batch of biopharmaceutical product begins with the removal of a single ampoule of the working cell bank. This vial is used to inoculate a small volume of sterile media, with subsequent incubation under appropriate conditions. This describes the growth of laboratory-scale starter cultures of the producer cell line. This starter culture is, in turn, used to inoculate a production-scale starter culture that is used to inoculate the production-scale bioreactor (Figure 5.7). The media composition and fermentation conditions required to... 

Figure 5.7 Outline of the upstream processing stages involved in the production of a single batch of product. Initially, the contents of a single ampoule of the working cell bank (a) are used to inoculate a few hundred millilitres of media (b). After growth, this laboratory-scale starter culture is used to inoculate several litres/tens of litres of media present in a small bioreactor (c). This production-scale starter culture is used to inoculate the production-scale bioreactor (d), which often contains several thousands/tens of thousands litres of media. This process is equally applicable to prokaryotic or eukaryotic-based producer cell lines, although the bioreactor design, conditions of growth, etc., will differ in these two instances...

Exact nutrient requirements of producer cell line to maximize cell growth and product production. 

Overall, therefore, the routine manufacture of a biopharmaceutical product is initiated by large-scale culture of its producing cell line (upstream processing). Subsequent to this, the product is recovered, purified and formulated into final product format. These latter operations are collectively termed downstream processing and are described in Chapter 6. 

Hankins, W.D., Chin, K., Dons, R. and Sigounas, G. (1989) Erythropoietin-dependent and erythropoietin-producing cell lines. Implications for research and for leukemia therapy. Annals of the New York Academy of Sciences, 554, 21-28. 

Resuspend the pellet in 0.5 mL of antibody incubation buffer and 25 pL of monoclonal antibody. The dilution of monoclonal antibody will vary according to the preparation (see Notes 5 and 6) The monoclonal antibody used in the author s studies is derived directly from the supernatant from the antibody producing cell line Incubate the tubes for 1 h at room temperature with occasional mixing (see Note 7)... 

The disadvantages of retroviruses include the limited number of cells that are transduced by virus due to dilute retrovirus preparations and their ability to transduce only the dividing cells. To overcome these problems, retrovirus-producing cell lines are directly administered into the tumors. Although there have been no reports from the clinical trials, the mutagenicity after integration of the retrovirus into the host genome has been of concern. 

Hybridoma production can be broken down into four processes, immunization of donor animals, cell fusion, cell selection, and expansion. Each of these stages is important for the quality of the final product. Antigens used to immunize animals must be representative of the target substance (see Note 3) or the likelihood of producing cell lines with the correct specificity is remote. Cell... 

Mathews, L. C., Gray, J. T., Gallagher, M. R. and Snyder, R. O. (2002). Recombinant adeno-associated viral vector production using stable packaging and producer cell lines. Methods Enzymol. 346, 393-413. 

Cell-protecting additives, such as Pluronic F-68, are commonly included as a media component to reduce cell damage in gas-sparged, agitated cultures. Despite the value of Pluronic F-68 in protecting cells, there is a potential problem in that Pluronic may have some cytosolic effects it may reduce the yield of the producer cell line in culture and there is a possibility of complexation or co-purification with a cell product. The replacement of Pluronic with fatty acids, as well as being positive for the half-lives and production of cultured cell lines, provides an alternative method to protect producer cells in agitated cultures (Butler et al., 1999). 

Wagner R, Ryll T, Krafft H, Lehmannm J (1988), Variation of amino acids concentrations in the medium of HU [3-IFN and HU IL-2 producing cell lines, Cytotech-nology 1 145-150. 

The production of heterologous proteins for therapeutic use requires selection of the producer cell line, based on yield, monoclonality (for proteins), product quality, stability, and absence of contaminants like bacteria, molds, mycoplasmas, and viruses. Progress in the production of biopharmaceuticals by cell culture is due mainly to the use of diploid cells and continuous cell lines, together with the maintenance of cells by cryo-preservation. It is important to guarantee that the expression system chosen is able to generate the product in a consistent and economically feasible way (Levine and Castillo, 1999). 

Mycoplasma can be eliminated from cell culture by treatment with immune serum (Pollock and Kenny, 1963) and passage through an animal is often effective in removing mycoplasma from tumour producing cell lines. 

Welsh, N., 1996, Interleukin-l-induced ceramide and diacylglycerol generation may lead to activation of the c-Jun NH2-terminal kinase and the transcription factor ATF2 in the insulin-producing cell line RINm5F. J. Biol. Chem. 271 8307-8312. 

Other approaches to increase desired product yield include optimizing culture conditions, treatment with precursors, or the use of elicitors to induce or increase biosynthesis. The strategies of selection and screening for high producing cell lines and optimization of media did not prove entirely successful (9) therefore, the use of precursors and elicitors seems to be a more promising route. 

Overproduction of berberine in C. japonica cell suspension cultures was achieved by selection of a high-producing cell line (34) with reported productivity of berberine reaching 7 g/L (35). This overproduction is one of the first demonstrations of production of a benzylisoquinoline alkaloid in cell culture at levels necessary for economic production. This cell line has facilitated greatly the identification of the biosynthetic enzymes. 

The mechanism of action of GLP-l(7-37) and GLP-1 (7-36)-NH2 has been the subject of in vitro study. Both peptides increase c-AMP levels in insulin-producing cell lines in a dose-dependent manner [128, 141, 142] and functional receptors have been identified on insulin- and somatostatin-producing cell lines [142-144]. 

DiPaolo JA, Nelson RL, Donovan PJ (1969) Sarcoma-producing cell lines derived from clones transformed in vitro by benzo[a] pyrene. Science 165 917-918... 

---