High‐throughput screening of chromatographic separations: I. Method development and column modeling

Abstract

The development of purification processes for protein biopharmaceuticals is challenging due to compressed development timelines, long experimental times, and the need to survey a large parameter space. Typical methods for development of a chromatography step evaluate several dozen chromatographic column runs to optimize the conditions. An efficient batch-binding method of screening chromatographic purification conditions in a 96-well format with a robotic liquid-handling system is described and evaluated. The system dispenses slurries of chromatographic resins into filter plates, which are then equilibrated, loaded with protein, washed and eluted. This paper evaluates factors influencing the performance of this high-throughput screening technique, including the reproducibility of the aliquotted resin volume, the contact time of the solution and resin during mixing, and the volume of liquid carried over in the resin bed after centrifugal evacuation. These factors led to the optimization of a batch-binding technique utilizing either 50 or 100 mL of resin in each well, the selection of an industrially relevant incubation time of 20 min, and the quantitation of the holdup volume, which was as much as one quarter of the total volume added to each well. The results from the batchbinding method compared favorably to chromatographic column separation steps for a cGMP protein purification process utilizing both hydrophobic interaction and anionexchange steps. These high-throughput screening tools can be combined with additional studies on the kinetics and thermodynamics of protein-resin interactions to provide fundamental information which is useful for defining and optimizing chromatographic separations steps.

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

“…A TECAN Genesis 100 workstation was used to dispense resin (100 mL slurry/well) into 96-well polypropylene plates equipped with embedded 0.45 mm polypropylene filters (#F20023; Innovative Microplate, Chicopee, MA). The resin stock suspension was continuously shaken at 400 rpm (Coffman et al, 2008) to maintain homogenous slurry while dispensing into plates. To ensure complete transfer of resin from pipette tip to well, 200 mL phosphate buffer were aspirated into the tip prior to the resin, then used to purge tip contents into the well.…”

“…The resin in each well was incubated in 0.1 N NaOH at 48C overnight, then washed with 300 mL of water. Resin was equilibrated first by incubation with shaking at 1,300 rpm (Coffman et al, 2008) for 5-10 min at RT in 300 mL of 1 M sodium chloride, 100 mM sodium phosphate, pH 7.2, followed by two successive incubations with 300 mL of 100-1,600 mM sodium chloride, 2-32 mM sodium phosphate, 50 mM HEPES, pH 6.5-8.0, as indicated in Table II. Following the last equilibration stage, the supernatant was removed by centrifugation, leaving a damp resin bed that is ready for subsequent contacting with a protein solution at the same pH and solution composition.…”

“…Similar procedures for high-throughput resin screening have been described elsewhere (Charlton et al, 2006;Coffman et al, 2008). Briefly, the plates containing the resin and protein solutions were shaken (1,300 rpm) for 20 min at ambient temperature to establish binding equilibrium (Coffman et al, 2008).…”

“…Briefly, the plates containing the resin and protein solutions were shaken (1,300 rpm) for 20 min at ambient temperature to establish binding equilibrium (Coffman et al, 2008). The solution phases containing unbound protein were collected by stacking the resin plate atop a UV-transparent collection plate followed by centrifugation at 1,200g for 3 min.…”

Industrial bioprocesses: Beyond routine applications of established methodologies

“…-Using CTAB and fractional precipitation, separation of relaxed and denatured plasmid DNA conformational forms from lysate as well as proteins, RNA and endotoxin was accomplished by Merck in 2002 (Lander et al, 2002). -A 96-well format for high-throughput screening of chromatographic separations using robotic liquid handling to survey large parameter spaces within compressed timelines was developed by Wyeth in 2008 (Coffman et al, 2008).…”