Biomedical Engineering Reference
In-Depth Information
6.9 Final product formulation
High-resolution chromatography normally yields a protein that is 98-99 per cent pure. The next
phase of downstream processing entails formulation into fi nal product format. This generally
involves:
•
Addition of various excipients (substances other than the active ingredient(s) which, for exam-
ple, stabilize the fi nal product or enhance the characteristics of the fi nal product in some other
way).
•
Filtration of the fi nal product through a 0.22
m absolute fi lter in order to generate sterile prod-
uct, followed by its aseptic fi lling into fi nal product containers.
µ
•
Freeze-drying (lyophilization) if the product is to be marketed in a powdered format.
The decision to market the product in liquid or powder form is often dictated by how s
table
the
protein is in solution. This, in turn, must be determined experimentally, as there is no way to pre-
dict the outcome for any particular protein. Some proteins may remain s
table
for months (or even
years) in solution, particularly if stabilizing excipients are added and the solution is refrigerated.
Other proteins, particularly when purifi ed, may retain biological activity for only a matter of hours
or days when in aqueous solution.
6.9.1 Some infl uences that can alter the biological activity of proteins
A number of different infl uences can denature or otherwise modify proteins, rendering them less
active/inactive. As all protein products are marketed on an activity basis, every precaution must be
taken to minimize loss of biological activity during downstream processing and subsequent stor-
age. Disruptive infl uences can be chemical (e.g. oxidizing agents, detergents, etc.), physical (e.g.
extremes of pH, elevated temperature, vigorous agitation) or biological (e.g. proteolytic degrada-
tion). Minimization of inactivation can be achieved by minimizing the exposure of the product
stream to such infl uences, and undertaking downstream processing in as short a time as possible.
In addition, it is possible to protect the protein from many of these infl uences by the addition
of sui
table
stabilizing agents. The addition of such agents to the fi nal product is often essential
in order to confer upon the product an acceptably long shelf life. During initial development,
considerable empirical study is undertaken by formulators to determine what excipients are most
effective in enhancing product stability.
A number of different molecular mechanisms can underpin the loss of biological activity of any
protein. These include both covalent and non-covalent modifi cation of the protein molecule, as
summarized in
Table
6.5. Protein denaturation, for example, entails a partial or complete alteration
of the protein's three-dimensional shape. This is underlined by the disruption of the intramolecular
forces that stabilize a protein's native conformation, namely hydrogen bonding, ionic attractions
and hydrophobic interactions (Chapter 2). Covalent modifi cations of protein structure that can
adversely affect its biological activity are summarized below.
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