Ernesto Chico

Perfusion culture of baculovirus-infected BTI-Tn-5B1-4 insect cells : A method to restore cell-specific β-trace glycoprotein productivity at high cell density

The impact of different cultivation-infection strategies on the productivity of baculovirus-infected BTI-Tn-5B1-4 (High Five) cells was investigated. Using beta-trace protein as the recombinant glycoprotein, the effects of multiplicity of infection (MOI) and time of infection (TOI) were studied on growth after infection as well as the degree of infection and recombinant protein productivity in batch culture. The highest productivities were found when infecting Tn5 cells at early exponential growth phase (EGP) (low cell density) using a high MOI. To increase the productive cell density of Tn5 cells after beta-trace-baculovirus infection, we performed studies infecting cells in the range of 1 to 5 x 10(6) cells/mL in fresh medium. Although the protein production was increased twofold, a strong negative cell density effect was still observed when maximal productive cell density exceeded 1 x 10(6) cells/mL. To verify whether the changing cell environment of the batch experiments was responsible for the decrease in protein productivity at increasing cell density at infection, several perfusion experiments were designed by infecting Tn5 cells at cell densities over 2 x 10(6) cells/mL under more steady-state conditions. The use of this experimental setup enabled successful infections at high cell densities with volumetric productivities of up to 1.2 g L(-1) day(-1) of beta-trace protein, which is very high for a glycoprotein expressed with the baculovirus expression vector system (BEVS). The cell specific protein productivity observed after infections at higher cell densities in perfusion mode was the same as in batch experiments at low cell concentrations, which clearly demonstrates that the cell density effect could be completely overcome with perfusion cultivation.

CFD simulation of an internal spin-filter: evidence of lateral migration and exchange flow through the mesh

In the present work Computational Fluid Dynamics (CFD) was used to study the flow field and particle dynamics in an internal spin-filter (SF) bioreactor system. Evidence of a radial exchange flow through the filter mesh was detected, with a magnitude up to 130-fold higher than the perfusion flow, thus significantly contributing to radial drag. The exchange flow magnitude was significantly influenced by the filter rotation rate, but not by the perfusion flow, within the ranges evaluated. Previous reports had only given indirect evidences of this exchange flow phenomenon in spin-filters, but the current simulations were able to quantify and explain it. Flow pattern inside the spin-filter bioreactor resembled a typical Taylor-Couette flow, with vortices being formed in the annular gap and eventually penetrating the internal volume of the filter, thus being the probable reason for the significant exchange flow observed. The simulations also showed that cells become depleted in the vicinity of the mesh due to lateral particle migration. Cell concentration near the filter was approximately 50% of the bulk concentration, explaining why cell separation achieved in SFs is not solely due to size exclusion. The results presented indicate the power of CFD techniques to study and better understand spin-filter systems, aiming at the establishment of effective design, operation and scale-up criteria.

Measurements of Changes in Cell Size Distribution to Monitor Baculovirus Infection of Insect Cells

We have found that the cell size distributions measured by a CASYTM cell counter can be used to follow the infection process of various insect cell lines. After infection, cell size deviates from the typical distribution of exponentially growing cells, shifting to an increased amount of cells withbigger cell diameters. This deviation has proved to be dependent on the MOI as well as time post-infection. A method is proposed to estimate the degree of infection of a population of insect cells based on the cell size distribution (CSD). The potential of using this method for measuring the ratio of infected to non-infected cells is discussed.

Particle image velocimetry (PIV) study of rotating cylindrical filters for animal cell perfusion processes

In the present work, the main fluid flow features inside a rotating cylindrical filtration (RCF) system used as external cell retention device for animal cell perfusion processes were investigated using particle image velocimetry (PIV). The motivation behind this work was to provide experimental fluid dynamic data for such turbulent flow using a high‐permeability filter, given the lack of information about this system in the literature. The results shown herein gave evidence that, at the boundary between the filter mesh and the fluid, a slip velocity condition in the tangential direction does exist, which had not been reported in the literature so far. In the RCF system tested, this accounted for a fluid velocity 10% lower than that of the filter tip, which could be important for the cake formation kinetics during filtration. Evidence confirming the existence of Taylor vortices under conditions of turbulent flow and high permeability, typical of animal cell perfusion RCF systems, was obtained. Second‐order turbulence statistics were successfully calculated. The radial behavior of the second‐order turbulent moments revealed that turbulence in this system is highly anisotropic, which is relevant for performing numerical simulations of this system.

Rotating cylindrical filters used in perfusion cultures: CFD simulations and experiments.

The particle and fluid dynamics in a rotating cylindrical filtration (RCF) system used for animal cell retention in perfusion processes was studied. A validated CFD model was used and the results gave numerical evidence of phenomena that had been earlier claimed, but not proven for this kind of application under turbulent and high mesh permeability conditions, such as bidirectional radial exchange flow (EF) through the filter mesh and particle (cells) lateral migration. Taylor vortices were shown to cause EF 10‐100 times higher than perfusion flow, indicating that EF is the main drag source, at least in early stages of RCF operation. Particle lateral migration caused a cell concentration reduction (CCR) near the filter surface of approximately 10%, contributing significantly to cell separation in RCF systems and giving evidence that the mesh sieving effect is not the sole phenomenon underlying cell retention in RCF systems. Filter rotation rate was shown to significantly affect both EF and CCR. A higher separation efficiency (measured experimentally at 2,000‐L bioreactor scale) and an enhanced CCR (predicted by the numerical simulations) were found for the same rotation rate range, indicating that there is an optimal operational space with practical consequences on RCF performance. Experimental data of a large‐scale perfusion run employing the simulated RCF showed high cell viabilities for over 100 days, which is probably related to the fact that the computed shear stress level in the system was shown to be relatively low (below 20 Pa under all tested conditions).

Study of NS0 Cell Line Metabolism in Lipid Supplemented Protein Free Media

NSO cells has become increasingly used as host for production of therapeutics at large scale, therefore the adaptation to Serum and protein free media has been a priority for manufacturers. Gorfien et al, 2000, demonstrated that NS0 cells are unable to grow in CD hybridoma medium unless supplemented with cholesterol containing lipids emulsion. However, Keen and Steward, 1995, demonstrated that it is possible to adapt NS0 cells to protein free medium without cholesterol containing lipid supplementation and animal derived component, however the medium used it is quite complex for preparing and its use become almost impossible for industrial setting. Our results show that NS0 cell line can be adapted to grow in different commercially available protein free and animal component free media. Additionally, lipid supplementation was evaluated in order to know the impact of these on main metabolic parameter, specific MAb rate and the Viability Index.

Metabolic Demands of Bti-TN-5B1-4 (High FiveTM) Insect Cells During Growth and After Infection with Baculovirus

High Five cells are characterized by a very active metabolism, by which Asn and Gln are consumed at very high rates. The use of feeding strategies, aimed to keep the concentrations of these amino acids in a region of lower qAsn and qGln, is strongly recommended to avoid nutrient limitations without extensive medium exchange. Due to the high specific oxygen demand of High Five cells, the growth of this cell line up to high cell densities could be limited by the transfer capabilities of current animal cell bioreactor systems. An additional increase in the oxygen demand must be expected during the first 30 hours post infection.

CFD Study of the Fluid and Particle Dynamics in a Spin-Filter Perfusion Bioreactor

In this work, simulations of a spin-filter bioreactor were carried out using computational fluid dynamics (CFD). Bi-directional radial exchange of fluid through the filter mesh and lateral migration were observed in the simulations. The fluid exchange might explain the observations of cell accumulation also on the internal side of the mesh. Moreover, lower cell concentrations were observed in the vicinity of the filter mesh, indicating that lateral migration could be related to the ability of spin-filters to delay clogging.

Characterization of growth and recombinant protein production of the insect cell line BTI-Tn-5Bl-4 (High Five™) in suspension culture

In this work, the insect cell line BTI-Tn-5Bl-4 (High Five™), normally anchorage dependent, was adapted to grow in suspension by a subculturing procedure without supplementation of heparin. Comparative testing of different serum-free media were carried out in batch suspension culture using spinner flasks. Oxygen, asparagine and glutamine were identified as the limiting nutrients in the growth of this cell line in ExCell 401 medium. A recombinant baculovirus, encoding for a human immunoglobulin G, was used for infection of the High Five™ cells during the study of the cell line production capability.