Biomedical subjects

J G Aunins

Publications and source records attributed to J G Aunins.

9 recordsLinked to original sources

PubMed2000

Computational and experimental investigation of flow and fluid mixing in the roller bottle bioreactor.

The fully three-dimensional velocity field in a roller bottle bioreactor is simulated for two systems (creeping flow and inertial flow conditions) using a control volume-finite element method, and validated experimentally using particle imaging velocimetry. The velocity fields and flow patterns are described in detail using velocity contour plots and tracer particle pathline computations. Bulk fluid mixing in the roller bottle is then examined using a computational fluid tracer program and flow visualization experiments. It is shown that the velocity fields and flow patterns are substantially different for each of these flow cases. For creeping flow conditions the flow streamlines consist of symmetric, closed three-dimensional loops; and for inertial flow conditions, streamlines consist of asymmetric toroidal surfaces. Fluid tracers remain trapped on these streamlines and are unable to contact other regions of the flow domain. As a result, fluid mixing is greatly hindered, especially in the axial direction. The lack of efficient axial mixing is verified computationally and experimentally. Such mixing limitations, however, are readily overcome by introducing a small-amplitude vertical rocking motion that disrupts both symmetry and recirculation, leading to much faster and complete axial mixing. The frequency of such motion is shown to have a significant effect on mixing rate, which is a critical parameter in the overall performance of roller bottles.

Bioreactors↗

PubMed2000

Phosphate feeding improves high-cell-concentration NS0 myeloma culture performance for monoclonal antibody production.

Phosphorus depletion was identified in high-cell-concentration fed-batch NS0 myeloma cell cultures producing a humanized monoclonal antibody (MAb). In these cultures, the maximum viable and total cell concentration was generally ca. 5 x 10(9) and 7 x 10(9) cells/L, respectively, without phosphate feeding. Depletion of essential amino acids, such as lysine, was initially thought to cause the onset of cell death. However, further improvement of cell growth was not achieved by feeding a stoichiometrically balanced amino acid solution, which eliminated depletion of amino acids. Even though a higher cell viability was maintained for a longer period, no increase in total cell concentration was observed. Afterwards, phosphorus was found to be depleted in these cultures. By also feeding a phosphate solution to eliminate phosphorus depletion, the cell growth phase was prolonged significantly, resulting in a total cell concentration of ca. 17 x 10(9) cells/L, which is much greater than ca. 7 x 10(9) cells/L without phosphate feeding. The maximum viable cell concentration reached about 10 x 10(9) cells/L, twice as high as that without phosphate feeding. Apoptosis was also delayed and suppressed with phosphate feeding. A nonapoptotic viable cell population of 6.5 x 10(9) cells/L, as compared with 3 x 10(9) cells/L without phosphate feeding, was obtained and successfully maintained for about 70 h. These results are consistent with the knowledge that phosphorus is an essential part of many cell components, including phospholipids, DNA, and RNA. As a result of phosphate feeding, a much higher integral of viable cell concentration over time was achieved, resulting in a correspondingly higher MAb titer of ca. 1.3 g/L. It was also noted that phosphate feeding delayed the cell metabolism shift from lactate production to lactate consumption typically observed in recombinant NS0 cultures. The results highlight the importance of phosphate feeding in high-cell-concentration NS0 cultures.

Amino Acids↗

PubMed1997

Large-scale mammalian cell culture.

Mammalian cell culture continues to draw major research efforts. A great deal of progress has recently been made in cellular physiology, especially in factors adversely affecting cell growth or viability. Through molecular genetic manipulation, cells are more readily cultivated in a medium free of animal proteins. Achieving a high cell concentration and high viability continue to be common themes in engineering research. The need to implement a control policy for fed-batch and perfusion cultures has prompted increased efforts in process monitoring and control. Integrating these advances will be beneficial for ensuring product quality and process consistency.

Animals↗

PubMed1994

Studies of baby hamster kidney natural cell aggregation in suspended batch cultures.

Microcarrier cultures of animal cells of industrial relevance are known to shed aggregates into the suspension phase. For a BHK cell line, which is known to be prone to aggregate naturally, microcarrier and aggregate forms of culture are compared in spinner culture. In microcarrier cultures, it is shown that increasing initial microcarrier concentration yields decreasing concentration of smaller aggregates in suspension; roughly equivalent concentrations of total cells and single cells in suspension are obtained. In the absence of Cytodex 3, aggregate final size is hydrodynamically controlled in batch and semicontinuous suspension culture. Rate of agitation is the main variable controlling aggregate size in batch cultures. The range of agitation rates studied (20 to 70 rpm in 250 mL spinner flasks) produced aggregates with maximum sizes of 200 microns. Necrotic centers were not observed; this was confirmed by Trypan blue viability measurements after mechanical dissociation of aggregates and also by the constant productivity obtained from different aggregate sizes. Comparing aggregate and microcarrier culture conditions, it is shown that at 100 rpm maximum total cell concentration is larger in the absence of microcarriers; dead cell concentrations, most of which exist in suspension, are slightly larger in microcarrier culture. Total viable cell concentrations in aggregate, hydrodynamically controlled culture, are almost one order of magnitude higher than in microcarrier cultures. These results suggest that there might be advantages in using aggregate cultures under hydrodynamic control of aggregate size in lieu of microcarrier cultures for naturally aggregating cell lines.

Animals↗

PubMed1994

Changes in animal cell natural aggregates in suspended batch cultures.

Some anchorage-dependent animal cells can form natural aggregates in stirred tanks. Baby hamster kidney (BHK) natural aggregates are described and characterized. Total cell concentration and viability could be obtained after aggregate mechanical dissociation, with negligible cell lysis and no change in cell membrane permeability. During a normal batch run, aggregates were formed immediately after inoculation, a few spherical aggregates increasing in size during the initial growth phase. At the end of the growth phase, an increase in aggregate concentration was observed, without a considerable increase in aggregate diameter. At the end of the batch run, 160 h after inoculation, aggregates disintegrated into smaller, non-spherical units, following a sharp viability decrease. Cell concentrations of 1.2 x 10(6) cells/ml were obtained, with 60% of the total cells being in aggregates; the cell concentration in aggregates achieved 5 x 10(8) cells/ml, with a porosity of 55%. Viability was consistently in the range 85-90%, both for aggregate and suspended cells.

Animals↗

PubMed1994

Repeated-batch cultures of baby hamster kidney cell aggregates in stirred vessels.

Natural aggregates of Baby Hamster Kidney cells were grown in stirred vessels operated as repeated-batch cultures during more than 600 hours. Different protocols were applied to passaging different fractions of the initial culture: single cells, large size distributed aggregates and large aggregates. When single cells or aggregates with the same size distribution found in culture are used as inoculum, it is possible to maintain semi-continuous cultures during more than 600 hours while keeping cell growth and viability. These results suggest that aggregate culture in large scale might be feasible, since a small scale culture can easily be used as inoculum for larger vessels without noticeable modification of the aggregate characteristics. However, when only the large aggregates are used as inoculum, it was shown that much lower cell concentrations are obtained, cell viability in aggregates dropping to less than 60%. Under this 'selection' procedure, aggregates maintain a constant size, larger than under batch experiments, up to approximately 400 hours; after this time, aggregate size increases to almost twice the size expected from batch cultures.

Alkaline Phosphatase↗

PubMed1985

Dissolution of carboxylic acids. III: The effect of polyionizable buffers.

The dissolution behavior of three carboxylic acids of variable aqueous solubility but with approximately equal pKa values into aqueous buffered solutions has been studied as a function of pH and of buffer properties. The dissolution from constant-surface-area compressed disks of benzoic acid, 2-naphthoic acid, and indomethacin into solutions of constant ionic strength (mu = 0.5 with potassium chloride) and constant pH (maintained by pH stat) at 25 degrees C using a rotating disk apparatus was evaluated. Models for dissolution of these weak acids into diprotic and triprotic buffering media are developed to predict the flux of the acid as a function of bulk solution pH and the physical and chemical properties of the buffer and acid. The models assume that mass transfer can be represented by a single second order diffusive term and that instantaneous equilibrium between all reactive species exists. Values of flux and pH at the solid-liquid interface are calculated and the fluxes compared to experimentally determined values. Reasonable correlation was found between values predicted by the models and experimental flux values. Major influences on model accuracy are the Ka and physical properties of the buffer.

Benzoates↗

PubMedDate not supplied

Experimental collision efficiencies of polymer-flocculated animal cells.

The determination of particle collision kinetics is useful to decouple the effects of process parameters on individual events in flocculation. This paper discusses the effects of flocculation conditions on the collision efficiency of ATCC strain CRL 1606 hybridomas flocculated with poly-L-histidine. Experimental determinations of the collision efficiency of cells in Couette flow are presented over a range of experimental conditions. The collision efficiency correlates with the cell zeta potential to the -2.4 power at high surface coverage, consistent with literature results in latex systems. At low coverage, accounting for the distribution of polymer on the cells corrects for deviation from the high-coverage behavior. Collision is dependent on the hydrodynamic environment as well. At high surface coverage, collision efficiency is weakly dependent on hydrodynamic conditions and follows a dependency on the shear rate and viscosity to the -0.32 power. This is consistent with ionic coagulation theory. At low surface coverage, the collision efficiency is strongly dependent on the viscous fluid forces. The results versus both dose and shear rate over the entire range of surface coverages are consistent with weak intercell bonding. Collision kinetics in the presence of high molecular weight dextrans show steric hindrance to cell collision.

Animals↗

PubMedDate not supplied

Monoclonal antibody process development using medium concentrates.

A fed-batch process using concentrated medium was evaluated for its ability to improve cell culture longevity and final monoclonal antibody (MAb) titers for two monoclonal antibody producing cell lines. It was found to result in up to 7-fold increases in final antibody titers compared to batch culture controls. Although the development cell line specific fed-batch protocols is critical to the development of cost-efficient large-scale production processes, the use of complete medium concentrates provided us with a quick and simple method for producing large quantities of antibodies in the early stages of process development, thus accelerating early work on purification process development, analytical development, biochemical characterization, and safety studies. Insights gained from the concentrated medium fed-batch approach were valuable for the development of refined, cell line specific feeding strategies yielding final MAb titers on the order of 1-2 g/L. Process development data on the effects of inhibitory growth byproducts, medium osmolarity, and the mode of nutrient feed addition on culture longevity and MAb production and information on culture metabolic behavior were successfully incorporated in the development of the optimized fed-batch protocols.

Amino Acids↗