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Separation of product associating E. coli host cell proteins OppA and DppA from recombinant apolipoprotein A‐I Milano in an industrial HIC unit operation
Author(s) -
Hunter Alan K.,
Wang Xing,
Suda Eric J.,
Herberg John T.,
Shell Robert E.,
Thomas Kristin E.,
Dufield Robert L.,
Gustafson Mark E.,
Mozier Ned M.,
Ho Sa. V.
Publication year - 2009
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.106
Subject(s) - recombinant dna , host (biology) , product (mathematics) , chemistry , apolipoprotein b , microbiology and biotechnology , biology , biochemistry , genetics , mathematics , gene , geometry , cholesterol
We have shown how product associating E. coli host cell proteins (HCPs) OppA and DppA can be substantially separated from apolipoprotein A‐I Milano (apo A‐I M ) using Butyl Sepharose hydrophobic interaction chromatography (HIC). This work illustrates the complex problems that frequently arise during development and scale‐up of biopharmaceutical manufacturing processes. Product association of the HCPs is confirmed using co‐immunoprecipitation and Western blotting techniques. Two‐dimensional gel electrophoresis and mass spectrometry techniques are used to confirm the identity of OppA and DppA. In this example, clearance of these difficult to separate HCPs decreased significantly when the process was scaled to a 1.4 m diameter column. Laboratory‐scale experimentation and trouble shooting identified several key parameters that could be further optimized to improve HCP clearance. The key parameters included resin loading, peak cut point on the ascending side, wash volume, and wash salt concentration. By implementing all of the process improvements that were identified, it was possible to obtain adequate HCP clearance so as to meet the final specification. Although it remains speculative, it is believed that viscosity effects may have contributed to the lower HCP clearance observed early in the manufacturing campaign. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009