Biomedical Engineering Reference
In-Depth Information
(centrifugation)
Inclusion body
solubilization
(guanidinium chloride)
Homogenization
and centrifugation
Ultrafiltration and
protein refolding
Ultrafiltration
Anion-exchange
chromatography
Ultrafiltration
Lys-chromatography
Ultrafiltration
Excipient addition
and sterile filtration
Gel filtration
chromatography
Final product
Figure 6.2
A likely purifi cation procedure for tPA produced in recombinant
E. coli
cells. The heterologous
product accumulates intracellularly in the form of inclusion bodies. In this particular procedure, an ultrafi ltra-
tion step is introduced on several occasions to concentrate the product stream, particularly prior to applica-
tion to chromatographic columns. Lysine affi nity chromatography (Lys-chromatography) is employed, as tPA is
known to bind immobilized lysine molecules. Adapted with permission from
Bio/Technology
(1993),
11
, 351
Figure 6.3
Photograph illustrating a typical pharmaceutical cleanroom and some of the equipment usually
therein. Note the presence of a curtain of (transparent) heavy-gauge polyethylene strips (most noticeable directly
in front of the operator). These strips box off a grade A laminar fl ow work station. Product fi lling into fi nal product
containers is undertaken within the grade A zone. The fi lling process is highly automated, requiring no direct
contact between the operator and the product. This minimizes the chances of accidental product contamination
by production personnel. Photograph courtesy of SmithKline Beecham Biological Services, s.a., Belgium
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