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

• Published in: Biotechnology and bioengineering Vol. 100; no. 4; pp. 605 - 618
• Main Authors: Coffman, Jonathan L, Kramarczyk, Jack F, Kelley, Brian D
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-07-2008; Wiley; Wiley Subscription Services, Inc
• Subjects: batch binding; Binding sites; Biological and medical sciences; Biotechnology; Chromatography; Chromatography, Affinity - instrumentation; Chromatography, Affinity - methods; Chromatography, Ion Exchange - instrumentation; Chromatography, Ion Exchange - methods; column modeling; Filtration - instrumentation; Filtration - methods; Fundamental and applied biological sciences. Psychology; high-throughput screening; Immunoglobulin Fc Fragments - analysis; Immunoglobulin Fc Fragments - genetics; Immunoglobulin G - analysis; Immunoglobulin G - genetics; Ion Exchange Resins - chemistry; Kinetics; method development; Optimization; Proteins; Proteomics - methods; Recombinant Fusion Proteins - analysis; Research - instrumentation; Research Design; Resins; Thermodynamics; High throughput screening; batch binding; column modeling; Separation; Batchwise; high-throughput screening; method development; Method; Modeling; Chromatography
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.21904

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 μL of resin in each well, the selection of an industrially relevant incubation time of 20 min, and the quantitation of the hold-up volume, which was as much as one quarter of the total volume added to each well. The results from the batch-binding method compared favorably to chromatographic column separation steps for a cGMP protein purification process utilizing both hydrophobic interaction and anion-exchange 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. Biotechnol. Bioeng. 2008;100: 605-618.