Product analysis - Pharmaceutical Biotechnology: Concepts and Aapplications - page 203

comprehensive, with follow-up validation studies being undertaken at appropriate time intervals
(e.g. daily, weekly or monthly). It is considered judicious to validate older items of equipment with
increased frequency. Such studies can forewarn the manufacturer of impending equipment failure.
Some validatory studies are straightforward, e.g. validation of weighing equipment simply entails
weighing of standardized weights. Autoclaves may be validated by placing external temperature
probes at various points in the autoclave chamber during a routine autoclave run. Validation stud-
ies should confi rm that all areas within the chamber reach the required temperature for the re-
quired time.
Periodic validation of clean room air (HEPA) fi lters is also an essential part of GMP. After their
installation, HEPA fi lters are subjected to a leak test. Particle counters are also used to validate
cleanroom conditions. A particle counter is a vacuum-cleaner-like machine capable of sucking air
from its surroundings at constant velocity and passing it through a counting chamber. The number
of particles per cubic metre of air tested can easily be determined. Furthermore, passage of the air
through a 0.2 µm fi lter housed in the counter will trap all airborne microorganisms. By placing the
fi lter on the surface of a nutrient-agar-containing Petri dish, trapped microorganisms will grow as
colonies, allowing determination of the microbial load per cubic metre of air.
In addition to equipment, many processes/procedures undertaken during pharmaceutical man-
ufacture are also subject to periodic validation studies. Validation of biopharmaceutical aseptic
fi lling procedures is amongst the most critical. The aim is to prove that the aseptic procedures
devised are capable of delivering a sterile fi nished product, as intended.
Aseptic fi lling validation entails substituting a batch of fi nal product with nutrient broth. The
broth is subject to sterile fi ltration and aseptic processing. After sealing the fi nal product contain-
ers, they are incubated at 30-37

C, which encourages growth of any contaminant microorgan-
isms. (Growth can be easily monitored by subsequently measuring the absorbance at 600 nm.)
Absence of growth validates the aseptic procedures developed.
Contaminant-clearance validation studies are of special signifi cance in biopharmaceutical
manufacture. As discussed in Section 7.6.4, downstream processing must be capable of removing
contaminants such as viruses, DNA and endotoxin from the product steam. Contaminant-clear-
ance validation studies normally entail spiking the raw material (from which the product is to be
purifi ed) with a known level of the chosen contaminant and subjecting the contaminated material
to the complete downstream processing protocol. This allows determination of the level of clear-
ance of the contaminant achieved after each purifi cation step, and the contaminant reduction fac-
tor for the overall process.
Viral clearance studies, for example, are typically undertaken by spiking the raw material with
a mixture of at least three different viral species, preferably ones that represent likely product
contaminants, and for which straightforward assay systems are available. Loading levels of up to
1 × 10 10 viral particles are commonly used. The cumulative viral removal/inactivation observed
should render the likelihood of a single viral particle remaining in a single therapeutic dose of
product being greater that one in a million.
A similar strategy is adopted when undertaking DNA clearance studies. The starting material
is spiked with radiolabelled DNA and then subjected to downstream processing. The level of re-
sidual DNA remaining in the product stream after each step can easily be determined by monitor-
ing for radioactivity.
The quantity of DNA used to spike the product should ideally be somewhat in excess of the
levels of DNA normally associated with the product prior to its purifi cation. However, spiking