The technical challenge in combining the SP chromatography step with the 20-nm nanofiltration step comes from matching flow
requirements and pressure capabilities of the chromatography process with the flow and capacity requirements of the nanofiltration
technology. At the same time, the nanofiltration technology must be used at ultra-large scale because the batch volumes of
product at this stage reach 10,000 L. Flow and capacity capabilities of 20-nm nanofiltration technologies currently available
vary significantly because of the use of different membrane types and pore geometries, or different filtration modes (dead
end versus tangential flow).
Therefore, initial screening studies were undertaken to select the most appropriate 20-nm technology for this application.
Nanofilters from three suppliers were used in the screening process; each meets the technical requirements for a large-scale
disposable. The screening of these technologies was based on the following parameters:
Overall capacity in L/m2
Volumetric flow in L/m2/h
- Virus reduction of PPV.
The screening study involved spiking a sample of the material to be nanofiltered with a preparation of PPV, at a virus concentration
recommended by each supplier. Using a lab-scale nanofilter from each supplier, attempts were made to filter a volume equivalent
to the required full-scale manufacturing volume of 400 L/m2.
Table I outlines data from the non-GLP screening study conducted at Catalent Pharma Solutions (formerly Cardinal Health).
Table I: Results of PPV screening studies at Catalent Pharma Solutions.
For filter B, a volume of 250 mL was obtained using a "run–spike–run" method in which only the last 50 mL was spiked. All
other filtrate volumes were achieved using a "spike–run" method in which the full volume was spiked. The screening studies
demonstrated that a 20-nm polyethersulphone (PESU)-based membrane (Sartorius Stedim Biotech, Aubagne, France) provided the
best flowrate and capacity and met the virus clearance requirements. Once this nanofilter was selected, more detailed analysis
of the virus clearance capability of the Sartorius virus-filter technology ("Virosart CPV") was initiated.
The first goal was to determine the capability of the filter to retain viruses throughout the filter lifetime. Because it
was not feasible to spike the product with viruses at manufacturing scale, the filtration process had to be accurately scaled
down to ensure that good laboratory practice (GLP) spiking studies were representative of the full-scale process.
The first phase of screening studies was carried out using bacteriophage PP7 as a model virus for small nonenveloped viruses.
The PP7 bacteriophage assay has previously been described (4), and the use of PP7 as a model virus for mammalian viruses has
been shown in previous studies (5, 6).
The screening studies were performed using "Virosart CPV Minisart" filters at the Virology Department of Sartorius Stedim
Biotech GmbH in Germany. Eight runs were performed, each using a 2.1 × 107/mL spike concentration of PP7 bacteriophage particles. All runs showed the same log reduction, and the results of a typical
run are summarized in Table II.
Table II: Results of PP7 spiking run.
The loading values are presented in Table II as L/m2 to reflect the volume to be filtered at manufacturing scale (maximum 400 L/m2 of filter area is required to meet the full batch volume). The results demonstrated that a good log reduction could be achieved
throughout the filtration process, and this was replicated in all scaled-down PP7 runs.