Materials and Methods

Bacterial Strains

A wild type B. pertussis strain Tohama-I was used in this study. The strain was procured from the American Type Culture Collection (Manassas, VA) as a freeze-dried ampoule.

Purified pertussis toxin, purified FHA, anti-PT serum (mouse), anti-FHA serum (mouse), anti-PT serum (sheep), and anti-FHA serum (sheep) were obtained from National Institute for Biologicals and Control (Hertfordshire, UK). Anti-mouse polyvalent immunoglobins peroxidase conjugate (GAMHRP) was obtained from Sigma (St. Louis, MO). Sodium-L-glutamate, glutamic acid, and CaCl₂.2H₂O were from Merck. All other chemicals were obtained from Sigma.

Growth Conditions

The B. pertussis Tohama strain was grown and maintained on Bordet-Gengou (B.G.) agar medium containing defibrinated horse blood. The strain was stored in a lyophilized state at 2–8 °C until grown on B.G. agar medium. The solid bacterial growth on B.G. agar slant, incubated for 48 h at 35 °C, was transferred into 500-mL Erlynmeyer flasks containing 200 mL of liquid medium similar to the defined medium described by Stainer and Scholte.⁷ One liter of medium contained 10.7 g of monosodium glutamate, 0.24 g of L-proline, 2.5 g of NaCl, 0.5 g of KH₂PO₄, 0.2 g of KCl, 0.1 g of MgCl₂.6H₂O, 1.5 g of Tris base, 10 g of casamino acids, and 5 g of yeast extract. The medium was supplemented with the following amount of supplements per liter: 0.04 g of L-cysteine monohydrochloride, 0.01 g of FeSO₄.7H₂O, 0.15 g glutathione (reduced), 0.4 g of L-ascorbic acid, 0.004 g of niacin, and 0.02 g of CaCl₂.2H₂O. The supplement was prepared in a concentrated form (100x) and filter sterilized. Cultures were incubated on an orbital shaker (170 rev/min) for 24 h at 35 °C.

This 200-mL culture was transferred to 10-L flask containing 3 L of liquid medium having the same composition as the 200-mL medium. The 3-L culture was incubated on a rotary shaker (190 rev/min) for 24 h at 35 °C. When optical density (OD) at 580 nm of the 3-L culture was more than 1.0, it was transferred into a fermenter containing 30 L of defined medium. Heptakis solution was sterilized by filtration and added into the fermenter at a concentration of 1 g/L of liquid medium. The bacteria were grown in batch mode in a fermenter of 50 L operating volume at 35 °C. The fermentation was maintained at a temperature of 35 °C, a dissolved oxygen level of 25% by cascading with an aeration of 3 L/min to 21 L/min and the agitation rate from 130 rpm to 290 rpm. The pH was controlled by a solution of glutamic acid and FeSO₄.7H₂O in hydrochloric acid. Foaming was controlled by adding antifoam 204 as needed. The culture was harvested after reaching an OD 7.5±0.1.

TFF Membrane and System

The TFF was carried out using a microporous membrane of pore size 0.45 μm. The construction material of the membrane was Durapore, i.e., hydrophilized polvinyledene difluoride (PVDF). The Prostak TFF module (Millipore, MA) consists of pre-assembled, pretested, and prebonded PVDF membranes on polysulfone plates and available in filtration area. This open channel module is designed to operate up to 5.6 bar, is steam sterilizable, and can be regenerated by cleaning.

An automated microfiltration (MF) system (Millipore, Bangalore, India) consisting of a 75-L jacketed tank was used in this study. The fluid to be clarified entered from the tank to a rotary lobe pump of capacity 1.8 m³/h at 4 bar pressure and 750 rpm, after the inlet pneumatic diaphragm valve was opened. The feed temperature and pressure were monitored by transmitters. Differential pressure across the installed device was controlled by throttling the motorized diaphragm valve on retentate (fluid that is retained by the membrane) line and retentate flow was measured by an electromagnetic flow meter. The permeate (fluid that passed through the membrane) line was equipped with a flow meter, a pressure and conductivity transmitter, and a peristaltic pump to control the permeate flow. The total system was steamed-in-place (SIP) and a steam trap was used at drain points to remove the condensate after SIP. The flow rate, volume of fluid at permeate and retentate line were monitored continuously during TFF pressures at the feed, permeate, and retentate line.