Large Scale Cultivation of Host Cells for Production of Recombinant Proteins

Recombinant proteins that are genetically engineered in host cells must be cultured in large scale (up to kilograms and sometimes up to kilotons) to meet pharmaceutical needs. During the discovery or research phase, recombinant proteins are produced on a laboratory scale, to produce a few micrograms using one to two liters of cell culture. Many of the methods used for purification and isolation of proteins from these cell cultures cannot be easily scaled or proportionally increased to produce pharmaceutically useful quantities. A different set of instruments and analytical techniques are developed for large-scale cell cultures. The science of growing cells and microbes for production of pharmaceuticals and chemical compounds under well-specified conditions in large quantities is called fermentation.

Most fermentation procedures are systematically optimized in a pilot plant, which typically uses a table-top fermentor of about 30 liter capacity. These fermentors are designed to contain all the ports, valves, controls, and cleaning capabilities essential

Figure 4.10. Schematic representation of the relationship between unit cost and increased product yield or titer as a function of time.

Figure 4.11. A 30 liter bench fermenter that can be scaled for production of recombinant proteins. The bench-top scale configuration contains all the control valves and ports necessary to monitor and control cell cultivation while maintaining sterility of the culture. The stainless steal reaction vessel allows easy cleaning and permits heat and pressure sterilization in place by connecting the vessel to a steam supply. (New Brunswick Bioflo-4500, adapted from the manufacturer's literature with permission)

Figure 4.11. A 30 liter bench fermenter that can be scaled for production of recombinant proteins. The bench-top scale configuration contains all the control valves and ports necessary to monitor and control cell cultivation while maintaining sterility of the culture. The stainless steal reaction vessel allows easy cleaning and permits heat and pressure sterilization in place by connecting the vessel to a steam supply. (New Brunswick Bioflo-4500, adapted from the manufacturer's literature with permission)

1. Embedded Controller with touchscreen interface

2. Viewing Window, allows viewing the culture above and below liquid level

3. Sterilizable-In-Place Vessel. Type 316L stainless steel polished to an internal finish of 15 - 20 Ra eases cleaning and prevents residue build-up

4. Resterilizable Sample

5. ValvePeristaltic Pumps

6. Resterilizable Harvest/Drain Valve, Easy access through removable vessel panel

7. Services for water, air, clean and house steam, water return and drain

8. Steam Traps, multiple stainless-steel 316L traps guarantee the sterilization temperature is maintained in all process lines

9. Ports, multiple 19 & 25 mm Ingold-type ports allow addition of RTD, pH, D.O. and other sensors

10. Thermal Mass Flow Controller provides precise, automatic control of air flow

11. Open Frame Piping facilitates access for cleaning, maintenance and servicing

12. Re-sterilizable Inoculation/Addition Valves

13. Filters, sterilizable-in-place in 316L stainless steel housings accommodate various-brand filter cartridges.

14. Rupture Disk, safety device of 316L stainless steel prevents over-pressurization of vessel; includes discharge tube to convey liquid to bottom of console

15. Automatic Back-Pressure Regulator

16. Exhaust Line with heat exchanger and view glass. Heat exchanger heats gas above dew point and prevents filter clogging

17. Motor, top-drive provides agitation for both microbial and mammalian cell culture

18. Exhaust Condenser

19. Combination Light and Fill Port provided, with sanitary Tri-Clamp fittings

20. Headplate Ports, (7) 28 mm ports (I.D. 19 mm) allow addition of sensors, sampling and addition devices for acceptable quality control and assurance during and throughout the cultivation process (Figure 4.11).They are constructed with a configuration similar to very much larger fermentors. The task of pilot plant engineers is to develop strategies that lead to the production of protein product in fer-mentors with a capacity of 100,000 liters (Table 4.9). A typical pilot scale, table-top fermentor is shown in Figure 4.11. Additional details on design and engineering aspects of fermentation can be found elsewhere [2,3].

Fermentation can be applied only to those cells that can grow in suspension. These cells include most prokaryotes, such as E. coli, and lower eukaryotes, such as yeast. However, only a small fraction of mammalian cells can be grown in suspension and adapted to a fermentation process.

Most recombinant mammalian cells are adherent cells that grow in an anchorage-dependent manner, requiring a surface support to replicate. To provide a large surface area with a minimum of cell culture medium, the adherent mammalian cells are

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