The small-scale studies described above were carried out to support the manufacture of full-scale batches. A large-scale nanofiltration
rig capable of handling as much as 5000 L of product was used. The filter rig was designed so that it can be directly connected
to the outlet of the chromatography rig. This has enabled the entire setup to be controlled and monitored by GE Healthcare
BioProcess equipment and Unicorn control software, and has combined the SP Sepharose and nanofiltration steps into a one-unit
The filters remain in the rig for integrity testing, which reduces the manual handling requirements and therefore risk of
damage to the filters. Integrity test scenarios for such large-scale systems have been presented (12) and include the use
of test systems capable of handling multiple filters simultaneously in a cleanroom environment. Such test systems enable the
operator to test more than 20 filter elements independent of the pore size in less than 1 h.
With respect to the overall process design for the large-scale manufacture of this ovine Fab fragment, the disposable approach
is applied wherever possible. Sterilizing-grade filters, prefilters, product collection bags, and the majority of tubing is
irradiated and disposable. In addition, all processing solutions are manufactured in single-use disposable bags.
Design-space studies performed by the process development group at Protherics UK provided the parameters within which the
SP Sepharose chromatography step could be operated at process scale. A flow rate of ?1000 L/h was consistently achieved during
validation runs, without exceeding the maximum column pressure. This in turn delivered a pressure well within the validated
nanofiltration pressure of 30 psi.
The challenge in handling such a large amount of liquids in a Class 10,000 environment exists in finding disposable filter
and bag technologies that will not only meet process flow requirements but also meet cost requirements and provide a realistic
level of manual handling. The nanofiltration filtrate was therefore collected into 1000 L bags, each having an in-line sterilizing-grade
0.2 µm filter to ensure bioburden control.
The integration of large-scale chromatography with nanofiltration can be effectively achieved using disposable technologies
wherever possible. The use of disposable tubing and bag assemblies plus the integration of the nanofiltration operation with
the chromatography step all help reduce the amount of cleaning validation.
The direct connection of the nanofiltration set up to the chromatography system offers the benefit of using the same software
to control and monitor both operations. With accurate process control, the integration of two technology platforms has given
a robust, convenient, and combined process step.
The 20-nm nanofiltration step has been proven to provide robust and efficient virus clearance data for all model viruses tested
and acts as the cornerstone of the virus clearance strategy used for this ovine-derived biopharmaceutical product.
Small-scale PPV and PP7 scouting studies are a useful part of the development process; having a good understanding of the
optimal parameters before the GLP study can save time and money. Use of a chromatography-purified virus for GLP studies allows
for significantly better filter performance without compromising infectivity.
Aline Denton* is a compliance manager, and Carl Jones is a production manager (scale-up), both at Protherics UK, Blaenwaun, Ffostrasol, Llandysul, Ceredigion, Wales, UK, SA44
5JT, tel. +44 1239 851 122, Aline.Denton@protherics.com
. Klaus Tarrach is a senior product manager of purification technologies at Sartorius Stedim Biotech.
*To whom all correspondence should be addressed
Submitted: June 4, 2008. Accepted July 8, 2008.
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