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摘要
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.
- 出版日期2014-11-14
