Effect of flux and shear rate on E. coli recovery in tangential flow filtration through a single hollow fiber.

Pathogenic bacteria which enter a viable but non-culturable (VBNC) state impede efforts to reach detectable concentrations required for PCR methods. This motivated a strategy for tangential flow filtration to concentrate bacteria in aqueous samples while maintaining the bacteria in a viable state, maximizing their recovery and achieving high fluxes through a single hollow fiber membrane. Filtrations were carried out for green fluorescent protein (GFP) E. coli at high shear rates (up to 27,000 sec-1 ) through 0.2 µm cut-off polyethersulfone (PES) microfilter membranes or 50 kDa polysulfone (PS) ultrafilter membranes. High shear minimized bacterial attachment on membrane surfaces, which would otherwise occur due to forced convection of the particles to the membrane surface at high flux conditions. Single fiber filter modules were constructed to facilitate concentration of Escherichia coli at fluxes ranging from 55 to 4500 L m-2 h-1 . The effect of high shear rates on bacterial viability was found to be minimal with bacterial losses during filtration caused principally by their accumulation on the membrane surface. Recoveries of 90% were achievable at high shear rates when the average flux was ≤300 L m-2 h-1 . This corresponded to a 3-h filtration time for a 225 mL sample through a single hollow fiber. Detectable bacteria concentrations of 1800 colony-forming unit (CFU)/mL were achieved for starting concentrations of 140 CFU/mL.

Intramodule pressure profiles and protein accumulation during tangential flow filtration.

Tangential flow filtration (TFF) through a 30 kDa nominal molecular weight cut-off (MWCO) ultrafiltration membrane is widely employed to concentrate purified monoclonal antibodies (mAbs) to levels required for their formulation into injectable biologics. While TFF has been used to remove casein from milk for cheese production for over 35 years, and in pharmaceutical manufacture of biotherapeutic proteins for 20 years, the rapid decline in filtration rate (i.e., flux) at high protein concentrations is a limitation that still needs to be addressed. This is particularly important for mAbs, many of which are 140-160 kDa immunoglobulin G (IgG) type proteins recovered at concentrations of 200 mg/mL or higher. This work reports the direct measurement of local transmembrane pressure drops and off-line confocal imaging of protein accumulation in stagnant regions on the surface of a 30 kDa regenerated cellulose membrane in a flat-sheet configuration widely used in manufacture of biotherapeutic proteins. These first-of-a-kind measurements using 150 kDa bovine IgG show that while axial pressure decreases by 58 psi across a process membrane cassette, the decrease in transmembrane pressure drop is constant at about 1.2 psi/cm along the 20.7 cm length of the membrane. Confocal laser scanning microscopy of the membrane surface at the completion of runs where retentate protein concentration exceeds 200 mg/mL, shows a 50 µm thick protein layer is uniformly deposited. The localized measurements made possible by the modified membrane system confirm the role of protein deposition on limiting ultrafiltration rate and indicate possible targets for improving membrane performance.