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

Authors

  • Jack F. Kramarczyk,

    1. Purification Process Development, Wyeth BioPharma, 1 Burtt Road, Andover, Massachusetts 01810; telephone: 978 247 2176; fax: 978 247 2604
    Search for more papers by this author
  • Brian D. Kelley,

    1. Purification Process Development, Wyeth BioPharma, 1 Burtt Road, Andover, Massachusetts 01810; telephone: 978 247 2176; fax: 978 247 2604
    Current affiliation:
    1. Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080.
    Search for more papers by this author
  • Jonathan L. Coffman

    Corresponding author
    1. Purification Process Development, Wyeth BioPharma, 1 Burtt Road, Andover, Massachusetts 01810; telephone: 978 247 2176; fax: 978 247 2604
    • Purification Process Development, Wyeth BioPharma, 1 Burtt Road, Andover, Massachusetts 01810; telephone: 978 247 2176; fax: 978 247 2604.
    Search for more papers by this author

Abstract

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.

Ancillary