title: Adaptation of the pore diffusion model to describe multi-addition batch uptake high-throughput screening experiments.
abstract: 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.
authors.affiliation: Biologics Process Development, Bristol-Myers Squibb, Hopkinton 01748, MA, USA.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Traylor
authors.affiliation: Biologics Process Development, Bristol-Myers Squibb, Syracuse, NY 13057, USA. Electronic address: Xuankuo.Xu@bms.com.
authors.forename: Xuankuo
authors.initials: X
authors.lastname: Xu
authors.affiliation: Biologics Process Development, Bristol-Myers Squibb, Hopkinton 01748, MA, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
authors.affiliation: Biologics Process Development, Bristol-Myers Squibb, Syracuse, NY 13057, USA.
authors.forename: Mi
authors.initials: M
authors.lastname: Jin
authors.affiliation: Biologics Process Development, Bristol-Myers Squibb, Hopkinton 01748, MA, USA; Biologics Process Development, Bristol-Myers Squibb, Syracuse, NY 13057, USA.
authors.forename: Zheng Jian
authors.initials: ZJ
authors.lastname: Li
mesh_terms: D000837:Anion Exchange Resins / Q000737:chemistry; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D004058:Diffusion; D007700:Kinetics; D008956:Models, Chemical; D016062:Porosity; D011506:Proteins / Q000737:chemistry
chemical_list: D000837:Anion Exchange Resins; D011506:Proteins
keywords: Fc-fusion proteins; High-throughput screening; Ion-exchange chromatography; Multi-addition batch uptake; Pore diffusion model; Uptake kinetics