Biomedical Engineering Reference
In-Depth Information
Incoming
potable water
Depth filtration
Organic trap
Activated charcoal
Anion exchanger
Water for
injections
(WFI)
Distillation
(or reverse osmosis
in some cases)
Filtration
Cation exchanger
Purified water
Figure 6.4 Overview of a generalized procedure by which purifi ed water and WFI are generated in a
pharmaceutical facility. Refer to text for specifi c details
6.2 Initial product recovery
The initial step of any downstream processing procedure involves recovery of the protein from its
source. The complexity of this step depends largely upon whether the product is intracellular or
extracellular ( Figure 6.1). In general, animal cell-culture-derived biopharmaceuticals are secreted
into the media (i.e. are produced as extracellular proteins), whereas the product accumulates intra-
cellularly in many recombinant prokaryotic producer cell types. In both cases, upstream processing
is followed by initial collection (harvest) of the cells. This is normally achieved by centrifugation,
or sometimes microfi ltration. If the product is an extracellular one, then the cell paste is generally
inactivated (e.g. by autoclaving) and discarded, whereas the product-containing extracellular fl uid
is subject to further processing. In the case of intracellular product, cell recovery is followed by cel-
lular disruption, in order to release the intracellular contents, including the protein of interest.
6.3 Cell disruption
Disruption of microbial cells is rendered diffi cult due to the presence of the microbial cell wall.
Despite this, a number of very effi cient systems exist that are capable of disrupting large quantities
of microbial biomass ( Table 6.1). Disruption techniques, such as sonication or treatment with the
enzyme lysozyme, are usually confi ned to laboratory-scale operations, due either to equipment
limitations or on economic grounds.
Protein extraction procedures employing chemicals such as detergents are effective in many
instances, but they suffer from a number of drawbacks, not least of which is that they often induce
protein denaturation and precipitation. This obviously limits their usefulness. Furthermore, even
if the chemicals employed do not adversely affect the protein, their presence may adversely affect
a subsequent purifi cation step (e.g. the presence of detergent can prevent proteins from binding to
a hydrophobic interaction column). In addition, the presence of such materials in the fi nal prepara-
tion, even in trace quantities, may be unaccep table for medical reasons.
Disruption of microbial cells (and, indeed, some animal/plant tissue types) is most often
achieved by mechanical methods, such as homogenization or by vigorous agitation with abrasives.
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