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

A high‐throughput screen (HTS) was developed to evaluate the selectivity of various hydrophobic interaction chromatography (HIC) resins for separating a mAb from aggregate species. Prior to the resin screen, the solubility of the protein was assessed to determine the allowable HIC operating region by examining 384 combinations of pH, salt, and protein concentration. The resin screen then incorporated 480 batch‐binding and elution conditions with eight HIC resins in combination with six salts. The results from the screen were reproducible, and demonstrated quantitative recovery of the mAb and aggregate. The translation of the HTS batch‐binding data to lab‐scale chromatography columns was tested for four conditions spanning the range of product binding and selectivity. After accounting for the higher number of theoretical plates in the columns, the purity and recovery of the lab‐scale column runs agreed with the HTS results demonstrating the predictive power of the filterplate system. The HTS data were further analyzed by the calculation of pertinent thermodynamic parameters such as the partition coefficient, KP, and the separation factor, α. The separation factor was used to rank the purification capabilities of the resin and salt conditions explored. Biotechnol. Bioeng. 2008;100: 707–720. © 2008 Wiley Periodicals, Inc.

[1]  B. Kelley,et al.  High-throughput screening of chromatographic separations: I. Method development and column modeling. , 2008, Biotechnology and bioengineering.

[2]  Roger G. Harrison,et al.  Bioseparations Science and Engineering , 2002 .

[3]  F. Rothstein.  Differential precipitation of proteins. Science and technology. , 1994, Bioprocess technology.

[4]  G. Antranikian,et al.  Single-step purification of a recombinant thermostable alpha-amylase after solubilization of the enzyme from insoluble aggregates. , 2000, Journal of chromatography. B, Biomedical sciences and applications.

[5]  P. Gagnon,et al.  Large-scale process development for hydrophobic interaction chromatography. II: Controlling process variation , 1995 .

[6]  Edwin J. Cohn,et al.  THE PHYSICAL CHEMISTRY OF THE PROTEINS , 1925 .

[7]  T. Root,et al.  Refining the description of protein chromatography , 1997 .

[8]  Ranga Godavarti,et al.  Weak partitioning chromatography for anion exchange purification of monoclonal antibodies , 2008, Biotechnology and bioengineering.

[9]  W. Kauzmann.  The Physical Chemistry of Proteins , 1957 .

[10]  T. Root,et al.  Protein retention in hydrophobic interaction chromatography: modeling variation with buffer ionic strength and column hydrophobicity , 1997 .

[11]  D. Hochstrasser,et al.  The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences , 1993, Electrophoresis.

[12]  Gail Sofer,et al.  Handbook of Process Chromatography: A Guide to Optimization, Scale Up, and Validation , 1997 .

[13]  Jon Coffman,et al.  High‐throughput screening of chromatographic separations: IV. Ion‐exchange , 2008, Biotechnology and bioengineering.

[14]  M. Ladisch,et al.  Hydrophobic interaction chromatography. , 1989, Biotechnology advances.

[15]  P. Gagnon,et al.  Large-scale process development for hydrophobic interaction chromatography. I: Gel selection and development of binding conditions , 1995 .