کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1202765 | 1493558 | 2014 | 7 صفحه PDF | دانلود رایگان |
• A novel multi-addition batch uptake technique was developed to measure protein uptake kinetics using plate-based high-throughput approach.
• The pore diffusion model was adapted to describe the multi-addition batch uptake results.
• The proposed approach was validated for an Fc-fusion protein and compared favorably with traditional batch uptake measurements.
• Model sensitivity analysis was performed to evaluate the effects of changes in input parameters on the fitted diffusivity.
Equilibrium isotherm and kinetic mass transfer measurements are critical to mechanistic modeling of binding and elution behavior within a chromatographic column. However, traditional methods of measuring these parameters are impractically time- and labor-intensive. While advances in high-throughput robotic liquid handling systems have created time and labor-saving methods of performing kinetic and equilibrium measurements of proteins on chromatographic resins in a 96-well plate format, these techniques continue to be limited by physical constraints on protein addition, incubation and separation times; the available concentration of protein stocks and process pools; and practical constraints on resin and fluid volumes in the 96-well format. In this study, a novel technique for measuring protein uptake kinetics (multi-addition batch uptake) has been developed to address some of these limitations during high-throughput batch uptake kinetic measurements. This technique uses sequential additions of protein stock to chromatographic resin in a 96-well plate and the subsequent removal of each addition by centrifugation or vacuum separation. The pore diffusion model was adapted here to model multi-addition batch uptake and was tested and compared with traditional batch uptake measurements of uptake of an Fc-fusion protein on an anion exchange resin. Acceptable agreement between the two techniques is achieved for the two solution conditions investigated here. In addition, a sensitivity analysis of the model to the physical inputs is presented and the advantages and limitations of the multi-addition batch uptake technique are explored.
Journal: Journal of Chromatography A - Volume 1368, 14 November 2014, Pages 100–106