Removal of nucleic acid

In some cases it is desirable/necessary to remove/destroy the nucleic acid content of a cell ho-mogenate prior to subsequent purification of the released intracellular protein. Liberation of large amounts of nucleic acids often significantly increases the viscosity of the cellular homoge-nate. This generally renders the homogenate more difficult to process, particularly on an industrial scale. Significant increases in viscosity place additional demands upon the method of cell debris removal employed. Increased centrifugal forces for longer time periods may be required to collect (pellet) cell debris in such solutions efficiently. If a filtration system is employed to remove cellular debris, then the increased viscosity will also adversely affect flow rate and filter performance.

Effective nucleic acid removal is particularly important when purifying a protein destined for therapeutic use. Regulatory authorities generally insist that the nucleic acid content present in the final preparation be, at most, a few picograms per therapeutic dose (see Chapter 7).

Effective removal of nucleic acids during protein purification may be achieved by precipitation or by treatment with nucleases. A number of cationic (positively charged) molecules are effective precipitants of DNA and RNA; they complex with, and precipitate, the negatively charged nucleic acids. The most commonly employed precipitant is polyethylenimine, a long-chain cationic polymer. The precipitate is then removed, together with cellular debris, by centrifugation or filtration. The use of polyethylenimine during purification of proteins destined for therapeutic applications is often discouraged, however, as small quantities of unreacted monomer may be present in the polyethylenimine preparation. Such monomeric species may be carcinogenic. If polyethylenimine is utilized in such cases, then the subsequent processing steps must be shown to be capable of effectively and completely removing any of the polymer or its monomeric units that may remain in solution.

Nucleic acids may also be removed by treatment with nucleases, which catalyse the enzymatic degradation of these biomolecules. Indeed, nuclease treatment is quickly becoming the most popular method of nucleic acid removal during protein purification. This treatment is efficient, inexpensive and, unlike many of the chemical precipitants used, nuclease preparations themselves are innocuous and do not compromise the final protein product.

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