High‐throughput screening of chromatographic separations: IV. Ion‐exchange

Abstract

Ion-exchange (IEX) chromatography steps are widely applied in protein purification processes because of their high capacity, selectivity, robust operation, and well-understood principles. Optimization of IEX steps typically involves resin screening and selection of the pH and counterion concentrations of the load, wash, and elution steps. Time and material constraints associated with operating laboratory columns often preclude evaluating more than 20-50 conditions during early stages of process development. To overcome this limitation, a high-throughput screening (HTS) system employing a robotic liquid handling system and 96-well filterplates was used to evaluate various operating conditions for IEX steps for monoclonal antibody (mAb) purification. A screening study for an adsorptive cation-exchange step evaluated eight different resins. Sodium chloride concentrations defining the operating boundaries of product binding and elution were established at four different pH levels for each resin. Adsorption isotherms were measured for 24 different pH and salt combinations for a single resin. An anion-exchange flowthrough step was then examined, generating data on mAb adsorption for 48 different combinations of pH and counterion concentration for three different resins. The mAb partition coefficients were calculated and used to estimate the characteristic charge of the resin-protein interaction. Host cell protein and residual Protein A impurity levels were also measured, providing information on selectivity within this operating window. The HTS system shows promise for accelerating process development of IEX steps, enabling rapid acquisition of large datasets addressing the performance of the chromatography step under many different operating conditions.

High‐throughput screening of chromatographic separations: III. Monoclonal antibodies on ceramic hydroxyapatite

“…1B). A similar relationship has also been noted for AEX resins (Kelley et al, 2008a). Because of this strong correlation between K p and Q 70 , HTS data from the low challenge region and the resulting correlations can be used to predict product binding well in the non-linear region, which would be valuable for cycle development.…”

Recovery and purification process development for monoclonal antibody production

“…76 Agreement with results obtained by chromatographic methods is generally good. Kelley et al demonstrated optimization of a cation exchange capture step for a recombinant human mAb carried out in 8 hrs using 35 mg of protein in total; they evaluated eight different resins and multiple pH and salt conditions for binding and elution.…”

“…Kelley et al demonstrated optimization of a cation exchange capture step for a recombinant human mAb carried out in 8 hrs using 35 mg of protein in total; they evaluated eight different resins and multiple pH and salt conditions for binding and elution. 76 Their results clearly demonstrate the potential for rapid screening of initial operating conditions that can be subsequently defined by conventional packed bed chromatography.…”

High‐throughput screening of chromatographic separations: II. Hydrophobic interaction

“…Since the completion of this study, high-throughput screens have successfully encompassed up to eight sequential elution stages per well (Kelley et al, 2008a). A single-well design is superior to the split-gradient design because twice as many conditions can be explored in a single filterplate, half the protein is required, and the split-gradient design may have caused artifacts in the resulting chromatograms.…”