The fundamental hypothesis of in silico scale-up and scale-down of chromatography methods is that only the fluid dynamics outside the pore system change. Once a sample component enters the pore system, pore diffusion, adsorption, and desorption are assumed to follow the same mechanism in a filter plate as in a production column. An adsorption isotherm determined at one scale is thus transferable to any other scale.
Isotherm determination by filter plate experiments is favorable because of its simplicity. In comparison to column experiments, fluid dynamic effects such as axial dispersion and film transfer as well as binding kinetics can be neglected due to the long incubation time.
Identification of filter plate phase ration
To obtain adsorption isotherm parameters for column modeling, the batch measurements are typically corrected using an “equivalent column volume” factor. In a per-well capacity study of filter plates prepared with a ResiQuot device, considerable well-to-well differences could be found and, most importantly, deviations from the expected equivalent column volume that result in wrong predictions of column experiments.
To solve this, we present a modified method for fitting batch isotherms to mechanistic model equations that relies only on the applied and measured supernatant concentrations. An assumption on the resin amount in the well is not needed anymore. To this, the isotherm equation is reformulated to include the liquid-to-solid ratio (L/S) as model parameter. Using this method, the average L/S in a 96-well plate filled with SP Sepharose FF could be determined from a single isotherm at constant buffer salt concentration.
Successful prediction of column experiments
A manual calculation of the bound protein concentration using assumptions of the phase ratio is not needed anymore. The resulting binding capacity follows the same trend as correcting each well individually using ionic capacity measurements. Finally, the obtained isotherm parameters could be used successfully to predict a breakthrough curve on a lab-scale column. This confirms the initial hypothesis that protein adsorption follows the same mechanisms in batch and column chromatography and that isotherm model parameters are transferable from one scale to another.
This case study was presented at
- The Fourth International Conference devoted to high-throughput process development (HTPD); 10/2017; Toledo, Spain
- ACS Spring Meeting; 03/2018; New Orleans, USA
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