Protein continuous culture

When sufficiently high levels of expression and protein accumulation are achieved, efficient downstream processing protocols must be developed to insure product quality and the economic feasibility of production. As the demand for safe, recombinant pharmaceutical proteins continues to expand, the market potential of plant-produced recombinant proteins is considerable. Molecular farming can produce recombinant proteins at a lower cost than traditional expression systems based on microbial or animal cell culture, and without the risk of contamination with human pathogens. 

Blocking of Continuous Labeling of HaloTag Protein in Cultured Mammalian Cells... 

Information on animal cell metabolism is essential for the formulation of new culture media, as well as for the composition of the most appropriate nutrient feed that may be used in various strategies of batch, semi-batch or continuous culture that may be designed for the production of the targeted protein. As already discussed in detail in Chapter 4 and concisely presented in Figure 5.1, two fundamental substrates for animal cells are glucose and glutamine. 

Fig. 3. Monitoring of the variant formation in a continuous culture of Bacillus stearothermo-philus PV72 (as in Fig. 2) by SDS-PAGE. The organisms differed in the S- (surface) layer proteins. The wild type formed an S-layer protein of 130 kDa apparent molecular mass, the variant S-layer protein appears at 97 kDa molecular mass. The wild-type S-layer protein was quantified relative to the band of the altered protein by densitometry. Numbers on the curve represent samples harvested at distinct points of time during variant formation. The decrease of the wild-type S-layer protein followed the theoretical washout curve in a stirred tank reactor at the set dilution rate of 0.3 h 1 (Reprinted from J. Biotechnol. 54, K. C. Schuster et al, p. 20,1997, with permission from Elsevier Science)...

Because of the need to avoid mutations and maintain the superior qualities of the genetically developed strain, batch or fed-batch operations are used in most applications. Continuous culture operations, however, provide a time-invariant environment that facilitates greatly the study of a biological process in research laboratories. Moreover, some industrial operations employ continuous reactors, such as the single-cell protein facility of ICI in Billingham, England (total reactor volume of about 2,300 m3), all waste treatment processes, and others. It should be noted that it is relatively common to follow a batch process with a period of fed-batch or continuous operation. Also, in most cases batch cultivation is the optimal start-up procedure for continuous or fed-batch cultivation (Yamane et al, 1977). 

Apparently the majority of protein processing pathways known to exist in higher eucaryotic cells also function in baculovirus infected cells. Some of these are list in Table II. Caution is recommended in making such a recommendation since the predominance of data has been generated in only one cell line (Sf9, 2) in continuous culture for many years.The majority of the recombinant proteins exhibit biological or functional properties very similar to the authentic proteins. 

Banik GG, Todd PW Kompala DS (1996) Foreign protein expression from S phase specific promoters in continuous cultures of recombinant CHO cells. Cytotech-nology 22 179-184. 

The yeast produced by continuous culture techniques is separated from the liquid medium and solvent washed by centrifugation or filtration techniques. After drying, a protein supplement is obtained, which contains 65-68% protein and is suitable for addition to animal feeds. This protein content compares very favorably with that of dry fish meal, which contains about 65%, and dry skim milk powder with about 32%. The SCP processes have operated on the thousands of tonne/year scale in the U.K., France, and Italy, but regulatory problems with facilities operating on unpurified gas oil feedstocks have caused some shutdowns [64]. Nevertheless, because of a cell mass doubling time of 2.5-3 hr and the efficient carbon conversion to protein of this technology, these developments deserve to be explored further. 

On the laboratory-scale, batch cell culturing is mainly conducted in flasks, dishes or microtiter plates (Fig. 1), however industrial-scale bioreactors with perfusion (continuous culture) are also available for production of, e.g. antibodies and other proteins. In both laboratory- and industrial-scale systems, a large volume of cell medium is available in comparison with the cell volume to provide the cultured cells with sufficient amount of nutrients. 

The ceU culture process includes inoculum build-up and continuous culture. For initiation of inoculum build-up, a vial of CHO cells from the WCB is thawed, and the cells are grown in a proprietary, protein-free culture medium in a small flask. Upon the attainment of a critical cell density, the cells are passaged to foster loga-... 

The use of plant suspension cells for the production of biopharmaceutical proteins has many advantages, including safety, defined growth conditions, containment, and the ability to use continuous-culture strategies. However, some challenges remain to... 

Figure 3. Strategies for optimization of continuous culture for production of single-cell protein. Top, under continuous culture isotherm with a fixed value for carbon source feed concentration. D is the dilution rate of maximum productivity. Bottom, comparison of isotherm for fixed substrate feed concentration. So, with that for fixed oxygen transfer rate, OTR (curves show the maximum cell concentration for a given OTR and cell yield).

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