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Encyclopedia of Physical Science and Technology EN014J-683 July 30, 2001 20:3

652 Separation and Purification of Biochemicals

protein coded by its gene, in a host cell culture. The
recombinant protein is identical to the one produced in
the native source.
SOP Standard operating procedures are an important part
of Quality Assurance and GMP in order to assure prod-
uct quality and consistency.

BIOLOGICALS and especially the products of the mod-
ern biopharmaceutical industry come in many forms and
from many sources. They can be molecules of various
sizes ranging from small organic metabolites (ethanol,
citric acid, antibiotics) to peptides and proteins (insulin,
antibodies, enzymes) that are amino acid based, as well
as most recently nucleotide-based molecules (antisense
DNA, RNA, plasmid DNA). They may be metabolites
of microorganisms, plant, or animal cells; they may be
FIGURE 1 Relationship between the product concentration in the
the product of an enzymatic reaction or a chemical pep-
starting material and the selling price. [From Dwyer, J. L. (1984).
tide synthesis; or they may simply be found in a natural
Biol. Technol. With permission.]
source such as blood, milk, or plant material. If, for exam-
ple, the source is a microorganism, the desired substance
may be excreted in the culture medium, may be enriched the final product, i.e., the maximum acceptable level and
in the cyto- or periplasma, or may form inclusion bod- the chemical nature of impurities or contaminants. The re-
ies (large aggregates of the denatured protein found inside quirements regarding purity for biologicals depend highly
the bacterium). Product concentrations may range accord- on the intended use of the product; they will be highest
ingly from several hundred grams per liter as in the case for human therapeutics and preventive drugs, somewhat
of ethanol or citric acid, down to a few milligrams per less for a number of diagnostics, and often considerably
liter in the case of recombinant antibodies, and even mi- lower for industrial enzymes. In the case of therapeutic
crograms per liter as in the case of blood coagulation fac- proteins, requirements also depend on the administration
tors. Given the present trend (mainly driven by econom- dose, frequency, and route. Injectables have the highest
ical considerations) the percentage of biopharmaceutical requirements. Example values are given in Table I for a
products such as recombinant proteins and antibodies for preventive injectable protein.
therapeutic use is expected to increase steadily over the Regulatory issues and environmental considerations
next years. While the isolation/purification of a highly (GMP,variousnationallaws,etc.)arealsoimportant,espe-
concentrated, low value product such as ethanol is rela- cially in the case of industrial downstream processing. The
tively straightforward and based on standard operations
in chemical engineering, the isolation of biologicals such
as blood factors, monoclonal antibodies and recombinant TABLE I Example of Regulatory Requirements for a
Therapeutic Recombinant Protein a
proteins is much more demanding. The set of complex
isolation and purification steps involved in the recovery
Purity >99%
of the product is generally referred to as the downstream
Aggregates <1%
process. In general, the contribution of downstream pro-
Host cell proteins (HCP) <1 ppm
cessing to the overall production cost depends on the up-
Nucleic acids (mostly DNA) <100 pg per dose
stream product concentration. It may vary from less than b
Endotoxins <5EU per kg of body weight
10% in the case of citric acid to more than 90% in the case and per maximal dose per kg.
of some of the above mentioned high value products. As Viruses >12 log reduction
a result, a direct relationship has been observed between Cells and particles Not detectable
the selling price, respectively the production costs, and the Leachables <1 ppm
product concentration in the original feed, as illustrated by
a Final purity requirements depend highly on the intended use of the
Fig. 1.
product, diagnostic, preventive or therapeutic. No definite values can be
In the design of the process leading to product recov-
given for therapeutic biomolecule, as they also vary depending on the
ery, the nature of the starting material is a key parame- administration frequency and route.
ter. Equally important, however, is the desired quality of b EU = Endotoxin units (see FDA, 12/1987).