The energetics of mammalian cell growth.
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Adenosine Triphosphate↗
Biomedical subjects
D G Kilburn
Publications and source records attributed to D G Kilburn.
Rapid immunoassay technique for process monitoring of animal cell fermentations.
A calibration and quality control technique suited to process monitoring with immunoassay is demonstrated. The particle concentration fluorescence immunoassay (PC-FIA) is shown to provide a sensitive and rapid method for the quantification of specific biomolecules in cell cultures. Smoothing of linear calibration parameters is performed by forming weighted averages of standard points as the run progresses. These estimates are then used to determine slope and intercept values for improved calibration. The nonuniformity of the fluorescent signal variance is also considered, and a weight model is developed to describe the relationship between signal fluorescence and signal variance for weighted linear curve fitting. Pooling calibration results over the process run improves overall assay performance as determined by using standard control chart analysis. This method is suitable for semicontinuous monitoring of animal cell fermentations and has been used here to measure cell-associated and culture supernatant concentrations of monoclonal antibody (Ab) from hybridoma cells. The cell-associated Ab concentration correlates with cell-specific production rate. Assay times on the order of 10 min for supernatant and 25-30 min for cell-associated Ab concentrations can be achieved, making this procedure suitable for process monitoring and control. Under these conditions the assay has a detection limit of approximately 10 ng/mL, providing a sensitive and specific method for the quantification of cell culture constituents.
Animals↗
Processing of fusion proteins with immobilized factor Xa.
Factor Xa, with a cellulose-binding domain (CBD) fused to the C-terminus of the heavy chain (FXa-CBD), is active in solution and when immobilized on cellulose. A second derivative of factor Xa in which a hexahistidine tail is fused to the C-terminus of the heavy chain (FXa-H6) also retains activity when immobilized, in this case on Ni(2+)-NTA agarose. The stabilities and activities of of FXa-CBD and FXa-H6 immobilized on cellulose and Ni(2+)-NTA agarose, respectively, are similar. Immobilized factor Xa derivatives can be used to remove affinity tags from appropriate fusion proteins without contaminating the desired product with factor Xa.
Affinity Labels↗
Batch and semicontinuous aggregation and sedimentation of hybridoma cells by acoustic resonance fields.
Ultrasound was used to enhance the sedimentation of hybridoma cells from medium in a 75 mL resonator chamber. Forces in the acoustic standing waves aggregated the cells, and the aggregates were then rapidly sedimented by gravity. Cell separation increased with acoustic treatment time and cell concentration. The separation efficiency was over 97% for cell concentrations between 10(6) and 10(7) cells/mL. During acoustic treatment at 180 W/L, the medium temperature increased at a rate of 1.3 degrees C/min. Ultrasonic exposures up to 220 W/L did not influence the viability or subsequent growth and antibody production of the cells. A decrease in cell viability was observed at a power level of 260 W/L. Batch separation efficiencies were as high as 98%. Acoustic separation was tested under semicontinuous operation, and above 90% separation efficiency was achieved at a flow rate of 0.7 L/h.
Acoustics↗