Infoscience

Thesis

Characterization of the mechanism of action of spin-filters for animal cell perfusion cultures

The growing demand for high levels of recombinant proteins of medical and pharmaceutical interest stimulates the development of cell bioprocess technology. Spin-filter technology is employed in order to reach high levels of such compounds. The aim of this thesis was to characterize the mechanisms of cell retention, as well as filter fouling during animal cell spin-filter perfusion cultures. A good understanding of these mechanisms would allow a good optimization of spin-filter parameters and culture conditions in order to achieve high cell density cultures at large scale operation and for long-time and thus increase proteins productivity. The first part of the thesis was focused on the study of particle retention as a function of four main parameters: filter pore size, filter rotation speed, perfusion rate and particle concentration, during perfusion simulations of agarose beads of 13 μm in diameter. Bead retention by filters with pore sizes of 13 and 14.5 μm, larger than the mean particle diameter was found to be dependent mainly on the filter rotation velocity and filter pore size. Filter retention followed a saturation dynamics with an initial direct correlation with respect to filter rotation rate. A plateau was reached above a filter tangential velocity of 0.45 m/s and 0.87 m/s for filters with pore size of 13 and 14.5 μm respectively. The lower the filter velocity was, the greater the influence of perfusion rate on bead retention, whereas the retention was slightly improved when the particle concentration was increased. The presence of a draft tube around open spin-filters was observed to lower the retention, with the effect being greater for non-porous than for porous draft tubes. In the second part of this work, a prediction of radial particle migration near the surface of rotating filter was developed. The lift force was demonstrated to be important in the spin-filter system since it contributes to particle removal from the filter surface. Competition between centrifugal sedimentation, lift forces, Stokes drag and perfusion forces were found to be responsible for determining particle motion relative to the filter. At certain filter rotation rates, centrifugation and lift forces are sufficiently high as to balance perfusion flow and result in the movement of particles away from the filter, a situation that experimentally was found to correspond to maximum particle retention. The model also revealed that filter acceleration is the key parameter to be conserved from small to large scale in order to achieve similar retention rates. This hypothesis has been confirmed experimentally. Then spin-filter cell retention was modeled using response surface methodology. A second-order polynomial model was used to predict the effects of the filter pore size, cell concentration, perfusion capacity and filter acceleration on cell retention. The retention rates obtained experimentally during two different spin-filter perfusion cultures of CHO SSF3 agreed with those predicted by the model, indicating the applicability of the model to animal cell perfusion culture. In the last part of this work the study of filter fouling during long-term perfusion simulations with CHO animal cells was investigated. It was demonstrated that at low filter acceleration, below 6.2 m/s2, a high perfusion rate of 25 cm/h induced rapid filter pore, whereas increasing the filter acceleration to 25 m/s2 increased filter longevity eight times, for filters with a pore size of 8.5 μm. Increasing the filter pore size to 14.5 μm improved filter longevity by 84% and revealed less viable and dead cell deposits on filter surface. Ultrasonic technology was used to reduce filter fouling. Filter vibration, induced by a piezo actuator, improved filter longevity by 113% during real CHO perfusion culture. This work allowed a better understanding of the mechanism of action of spin-filters. The cell retention model developed in this study permits to choose the optimal acceleration at which the filter, of a certain pore size has to be operated in order to achieve similar retention rates for small scale as well as for large-scale processes. The ultrasonic technology through the use of piezoactuators was demonstrated to be a powerful technique for the on-line reduction of filter-fouling, during animal cell perfusion cultures.

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