Downstream processing - Pharmaceutical Biotechnology: Concepts and Aapplications - page 138

in numerous collisions between the microbial cells and the glass beads. It also results in the
grinding of cells between the rotating beads. These forces promote effi cient disruption of most
microbial cell types. Operational parameters, such as ratio of cells to beads and the rate/duration
of agitation, may be adjusted to achieve optimum disruption of the particular cells in question.
Laboratory systems can homogenize several grams of microbial cells in minutes. Industrial-
scale bead-milling systems can process in excess of 1000 l of cell suspension per hour. Cooling
systems minimize protein inactivation by dissipating the considerable heat generated during this
process.
Upon completion of the homogenization step, cellular debris and any remaining intact cells can
be removed by centrifugation or by microfi ltration. As mentioned previously, these techniques are
also used to remove whole cells from the medium during the initial stages of extracellular protein
purifi cation.
6.4 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 purifi cation of the released intracellular protein. Liberation of
large amounts of nucleic acids often signifi cantly increases the viscosity of the cellular homoge-
nate. This generally renders the homogenate more diffi cult to process, particularly on an indus-
trial scale. Signifi cant 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 effi ciently. If a fi ltration system is employed to
remove cellular debris, then the increased viscosity will also adversely affect fl ow rate and fi lter
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
fi nal preparation be, at most, a few picograms per therapeutic dose (see Chapter 7).
Effective removal of nucleic acids during protein purifi cation 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 poly-
mer. The precipitate is then removed, together with cellular debris, by centrifugation or fi ltration.
The use of polyethylenimine during purifi cation 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 pop-
ular method of nucleic acid removal during protein purifi cation. This treatment is effi cient, inex-
pensive and, unlike many of the chemical precipitants used, nuclease preparations themselves are
innocuous and do not compromise the fi nal protein product.