The feed is a non- limiting source of carbon such as glucose or glycerol that is aseptically fed directly into the bioreactor vessel. A typical recipe would be 2XYS containing 40%
glucose and 20 gm/L sodium phosphate. Due to the high glucose level, this feed source is fi ltered rather than
autoclaved. If autoclaved, the glucose at this high concentration will caramelize and the carbon feed will become unpredictable as to how many single glucose molecules are available for metabolic activity by the cell population. The phosphate is added to increase the growth potential of the culture [33].
These two carbon sources feed directly into the glycolytic pathway at different points. Glucose enters into the beginning of the Embden-Meyerhof pathway of glucose metabolism as it is converted to glucose 6-phosphate. This pathway yields 2 pyruvate molecules, 2 ATP and 2 NADH molecules for every molecule of glucose. One mmole of glucose, completely oxidized through the TCA cycle, makes 25 to 30 moles of ATP. Alternatively, glycerol fi rst has to be converted to glyceraldehyde 3-phosphate. This is at a cost of 1 ATP but also generates 1 molecule of FADH2. The GA3P is then converted to pyruvate before entering the TCA cycle. This generates 1 pyruvate molecule, 1 ATP and 1 NADH for a net generation of 0 ATP, 1 NADH and 1 FADH2 molecule.
As discussed in Chapter 1 , the feed strategies developed to deliver the glucose or glycerol carbon source are paramount to the successful development of a recombinant fermentation process. The simplest method of feeding is to feed the culture in a batch mode where the media contains a given amount of carbon source and once that carbon source is exhausted the cells are harvested. This method is simple and straightforward but is not likely to be used in large- scale production, due to the lack of feeding control and limits in total production yields.
A fed- batch strategy is just like it sounds, a process of feeding the cell culture a carbon source in a controlled fashion. The fed- batch method can be separated into two different strategies:
1. fi xed volume fed- batch; and 2. variable volume fed- batch.
In the fi xed volume scheme, the carbon source is fed without dilution of the culture. This can be achieved by feeding the limiting substrate at high concentrations, so as to negate any dilution affects or through dialysis of the substrate directly into the media. In some instances, once the cells have slowed their metabolism, due to a lack of limiting substrate (glucose), the cells are harvested, centrifuged and brought up again in fresh culture media and the fermentation continues. The substrate feeding is then re- initiated until volume addition limits are reached and the cells are processed as before. This cycle continues until suffi cient product yields are attained, at which time the fed- batch fermentation is harvested for product recovery. The variable volume feeding strategy does not control the culture volume and adds the carbon substrate (formulated in the initial culture media) directly to the culture. This can be added in dilute form or concentrated form, depending on the needs of the operator and limitations of the bioreactor itself.
Furthermore, a fed- batch strategy has many advantages over batch cultures. The amount of a substrate, such as glucose, that is added to the growing culture can be rate limiting and thus the production of by- products that are generally associated with excessive amounts of residual glucose can be avoided. This overloading of the oxidative capacity of the cells (the Crabtree effect) causes inhibition of the glycolytic pathway while generating an excess amount of acetate that can affect the metabolic effi ciency of the cell culture as a whole. In a recombinant E. coli system that is being used for the GMP production of a marketed product, a reduction in the carbon fl ux can have a signifi cant impact on cell density and fi nal product formation and therefore should be at least minimized, if not avoided completely.
Controlling the substrate is also important due to catabolic repression. Catabolite repression is a global control system where the bacteria, such as E. coli , adapt quickly to the presence of a preferred carbon and energy source. In the presence of glucose, the lactose operon system is repressed until all glucose has been exhausted. With the extended feed of glucose, the fed- batch fermentation system keeps the cells focused on a single carbon source (glucose), instead of alternating between carbon sources (lactose/glucose) and thus having two different catabolic pathways operating at the same time. Since this fed- batch method usually permits the extension of the operating time, high cell densities can be achieved and thereby maximize productivity.
When using recombinant strains, fed- batch fermentations are performed in the presence of an antibiotic. The antibiotic selection guarantees the presence of the plasmid containing the recombinant gene of interest. Since the growth can be regulated by the carbon feed, considering that in many cases a high growth rate can decrease the expression of recombinant products, the possibility of having different feed rates and substrates makes fed- batch an extremely fl exible tool for control. An additional advantage of fed- batch culture is that the productive late exponential phase of a process may be extended under controlled feeding conditions. The controlled periodic shifts in growth rate provide an opportunity to optimize product synthesis, particularly if the product of interest is a secondary metabolite and/or induced recombinant protein, whose maximum production takes place under discontinuous feed strategies.
Residual glucose or glycerol concentrations are usually measured on a bio- analyzer, but can be measured using over- the-counter glucose test strips such as AcuCheck. One of the advantages of using a bio- analyzer is that it gives the researcher the opportunity to monitor other important
residual components and/or parameters of the fed- batch fermentation. Glucose, acetate and phosphate residual levels are important to monitor, as they can be indicators of the health of the culture. Following this, it has been shown that the maintenance of adequate levels of phosphate is important to the growth of the culture and fi delity of the induced protein product in a recombinant system [31]. High residual glucose and acetate levels ( Chapter 1 ) can be indicative of a potential metabolic overfl ow state [36] and if left uncorrected can lead to slower growth and lower productivity of the recombinant protein product [35]. Parameters such as pH and osmolality can also be monitored during the growth and induction of the culture.