Clarification of Fermentation Harvest
The TFF system was installed with a 0.45-μm open channel Prostak device (PVDF membrane, Millipore). The MF filtration module
consisted of four stacks with total filtration area 0.34 m2. The system, fitted with the membrane, was flushed with 20-L water for injection (WFI). Integrity of the MF membrane and
installation was checked by a diffusion test using an automated integrity testing machine (IT4, Millipore). The system was
then drained. SIP of the system was conducted, steam pressure inside the system was maintained at 1.2 bar and 123 °C for 30
min, and then the system was left at a positive pressure of 1 bar. The next day, it was observed that the system was at a
positive pressure of 0.4 bar at 28 °C.
Thirty litres of B. pertussis culture was transferred aseptically into the system tank and clarification was carried out at constant transmembrane pressure
(TMP). A secondary peristaltic pump in the permeate side was used for permeate flow control. The feed was concentrated 10
times (30 to 3 L). Temperature, pressure, and volume were monitored at the feed, permeate, and retentate line during the operation.
The PT and FHA were quantized by ELISA with purified antigens as standards. Microtiter plates (Nunc Maxisorp, Roskilde, Denmark)
were coated with either sheep anti-PT antibody (PT ELISA) at a dilution of 1:4,000 or sheep anti-FHA antibody (FHA ELISA)
at dilution of 1:12,000 in 50-mM sodium carbonate buffer (pH 9.6). Plates were incubated overnight at 2–8 °C. Volumes of 0.1
mL were used in all steps and the plates were washed four times between incubations by using 100-mM phosphate-buffered saline
containing 0.05% (w/v) Tween 20. The plates were blocked for 1 h at 37 °C with 2% (w/v) bovine serum albumin (BSA) in phosphate-buffered
saline (PBS; pH 7.4). The plates were washed and culture supernatants were serially diluted and added to the wells, and then
the plates were incubated at 37 °C for 1 h. Mouse anti–PT at dilution of 1:1,000 and mouse anti-FHA at dilution of 1:500 in
1% (w/v) BSA in phosphate-buffered saline (PBS; pH 7.4) were added to the plates. The plates were incubated for 1 h at 37
°C. Bound antigen was detected by using goat antimouse horse reddish peroxidase conjugate with a dilution of 1:7,000 (PT ELISA)
and 1:10,000 (FHA-ELISA) in 1% BSA in phosphate-buffered saline. The plates were incubated for 1 h at 37 °C and were washed
and developed with tetramethylbenzidine and 0.006% hydrogen peroxide in 100 mM citrate-phosphate buffer, pH 5.0. The reaction
was stopped by adding 2NH2SO4. The enzyme substrate reaction that developed at 37 °C was measured at 450 nm on a Universal Microplate reader (EL-800 with
KC4 data analysis software, BioTek Instruments, Inc., Vermont, MA).