Results and Discussion
There are different options for clarification and acellular pertussis antigen recovery. Centrifugation is the most commonly
used tool in primary clarification. It can handle considerably high concentrations of insoluble material in the feed. A critical
drawback of centrifugation is that cell disruption can occur because of shear, resulting in the generation of smaller particles,
which cannot be separated by a centrifuge efficiently. The precipitation efficiency of a centrifuge decreases with increasing
deposition of solid sludge in the bowl resulting in frequent discharge, which in turn, decreases product yield and increases
process time. Buffer or water used for bowl flush can create osmotic differences between culture broth and flush fluid, resulting
in cell lysis and further downstream complication. Dead end or normal flow filtration is unable to take high particle load
and the residue on the filter is not recoverable, and hence, is not a feasible option for the clarification of B. pertussis fermentation broth, where the whole cells (containing PRN) and the filtrate containing PT and FHA are the products of interest.
In this microfiltration study, recirculation is accomplished in a closed loop (Figure 1) using a TFF module. The feed pump
allows the product present in the feed tank into the TFF module tangentially across the membrane. Materials smaller than the
pore size of the membrane are able to pass through the membrane (B. pertussis fermentation media containing the antigens PT and FHA), and are collected aseptically. The membrane retains the larger particles
(retentate) containing B. pertussis, cells from where PRN is recovered.
Figure 1. A schematic diagram of microfiltration flow pattern
The retained components do not build up at the surface of the membrane. Instead, they are swept along by the tangential flow.
This feature of TFF makes it an ideal process for finer sized-based separations.
The pump at the permeate line is used to control polarization across the membrane by offering permeate flow control and low
pressure from the permeate side. This type of control ensures that the flux and TMP are low and stable across theflow channel
and enables microfiltration to be carried out causing a very low shear to the cells.
Throughout the TFF experiment, pressure at the feed (P1), permeate (P2), and retentate (P3) were monitored and the volume
of permeate collected per unit time (VP), retenate (Qr) was noted. The permeate flux was calculated by measuring the permeate flow rate per unit membrane area. Details of the process
parameters are presented in Table 1.
Table 1. Microfiltration process parameters
The inverse relationship of flux and TMP is commonly observed in a microporous TFF operation. Higher TMP causes greater compaction
of the cake layer deposition against the membrane, resulting in a higher resistance to filtrate flow rate and a lower flux.
Therefore, the flux and TMP control offered by TFF system in this investigation helps to optimize the flux while also preventing
cell damage because of low shear, and thereby, potentially maximizing the passage of soluble FHA and PT components through
Table 2. Recovery of antigens
Flux and TMP were constant throughout the MF operation, with a high yield (Table 2) of cell mass and FHA, PT antigens in
the permeate (results were mean value of five studies). The initial high flux was because of ramping up of the pump to appropriate
feed flow rate, which was stabilized after 20 min. The flux remained steady and the average flux obtained in the experiments
was 37.75 L/m2/h at an average TMP of 0.135 bar. The relationship of Flux and TMP is shown in Figure 2.
Figure 2. Flux and TMP curve during the unit operation
The optimal maintenance of cross flow (retentate flow rate) and well regulation of TMP enhanced and maintained consistent
recovery in all the five experiments. The next goal was the establishment of an effective cleaning regime ensuring repeated
usage of the membrane to reduce the downstream cost. Twenty liters of WFI was recirculated for 5 min and then drained off.
WFI recirculation was done twice. The system was sanitized with sodium hypochlorite solution (200 ppm of chlorine), recirculated
for 30 min and drained off. Normalized water permeability (NWP) was calculated before and after cleaning. After each experiment,
the NWP was restored to its original value, thereby establishing the cleaning effectiveness.
The B. pertussis fermentation broth must be clarified quickly to increase the recovery of the acelullar pertussis vaccine antigenic components.
Conventional methods like centrifugation are cumbersome, time consuming, and have limited scalability. Membrane-based technology
in TFF mode can ensure easy and conventional scale-up with a high recovery percentage. An effective protocol suitable for
scale-up is presented in this article. Because of the same channel geometry in different modules of Prostak, i.e., 2, 4, 10,
and 20 stacks of effective filtration area 0.17, 0.33, 0.84, and 1.7 m2 respectively, it provides a suitable technology for manufacturing at each scale. After a clarification step, the Prostak
modules can be regenerated and sanitized, providing reusability with validated steam sterilization capacity for 20 cycles.
The higher recovery is mainly because of the low hold up volume of the system, the low protein binding nature of the membrane,
and also because the operation takes palce under low shear condition. The Prostak module's design allows aseptic processing
without cross contamination or aerosol generation.