Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Industrial Protein Synthesis Using Recombinant Microorganisms
Cell Harvesting

To purify a Fermentation product, the Cells must first be separated from the culture medium. Genetically modified and parental, untransformed cells can be harvested using the same Methods. However, transformed cells often possess different physiological properties (such as a different size or the synthesis of extracellular Polysaccharides); As a result, the optimal conditions for harvesting untransformed cells may not be suitable for cells synthesizing a foreign protein.

To isolate cells from large volumes of culture medium, high-speed centrifugation is often used. For this purpose, special semi-continuous high-speed centrifuges have been designed. The Cell suspension is continuously fed into the bowl of the operating centrifuge, where the cells are concentrated, and the clarified medium is discharged. Once the bowl is filled with the sedimented cells, the centrifuge is stopped and the cells are harvested. The main drawback of this method is the need to stop and restart the process. Other disadvantages include the high cost of the equipment and its energy consumption, the risk of releasing microorganisms into the environment, and the inability to completely remove all cells from the medium.

Class="center">Table 16.1. Synthesis of a recombinant protein containing the Insulin B peptide as one of its components during batch fermentation and fed-batch fermentation^1,2)

¹⁾ Data from Gosset et al., Appl. Microbiol. Biotechnol. 39: 541—546, 1993.

²⁾ Biomass is measured in grams of dry weight per liter of culture. The "Batch fermentation + Trp" Procedure indicates that 0.1 g of Tryptophan was added to the culture. In the fed-batch fermentation, 0.1 g of tryptophan was added to the medium every 2 h—for a total of five times over 10 h. Adding larger amounts of tryptophan did not increase either the biomass or The amount of synthesized protein.

An alternative method for harvesting cells from the culture medium is Membrane filtration. Unfortunately, during conventional filtration, cells eventually clog the pores of the membrane filter and accumulate on its surface, rapidly decreasing the filtration rate (Fig. 16.7, A). Filtration can be accelerated by applying pressure, but this is only a temporary solution; cells will still accumulate on the membrane surface, and under pressure, they form a denser and less permeable layer.

Fig. 16.7. Filtration methods used for cell harvesting. A. Filtration with irreversible filter clogging. B. Cross-flow filtration of the cell suspension. Arrows indicate the direction of flow.

To solve this problem, the cell suspension is pumped at high velocity parallel to the membrane surface (Fig. 16.7, B), so that only a small fraction of the circulating liquid passes through the membrane in a single pass. The remainder sweeps the membrane clean of accumulated cells (see figure), and as a result, the filtration rate does not decline as rapidly as it does during irreversible filter clogging. After numerous filtration cycles, almost all of the culture medium passes through the membrane. This method is currently used only in the laboratory; Industrial processes rely on centrifugation for cell harvesting.

Subsequent steps depend on The Nature and localization of the product. If the product is a protein present in the culture medium, the medium is concentrated, and the protein is purified by chromatographic or other methods. If the product is a low-molecular-weight compound in the culture medium, appropriate extraction methods are used. Finally, if the product has an intracellular localization, the cells are disrupted prior to purification.