title: Separation of product associating E. coli host cell proteins OppA and DppA from recombinant apolipoprotein A-I(Milano) in an industrial HIC unit operation.
abstract: 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.
authors.affiliation: Pfizer Global Biologics, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA. alan.k.hunter@pfizer.com
authors.forename: Alan K
authors.initials: AK
authors.lastname: Hunter
authors.forename: Xing
authors.initials: X
authors.lastname: Wang
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Suda
authors.forename: John T
authors.initials: JT
authors.lastname: Herberg
authors.forename: Robert E
authors.initials: RE
authors.lastname: Shell
authors.forename: Kristin E
authors.initials: KE
authors.lastname: Thomas
authors.forename: Robert L
authors.initials: RL
authors.lastname: Dufield
authors.forename: Mark E
authors.initials: ME
authors.lastname: Gustafson
authors.forename: Ned M
authors.initials: NM
authors.lastname: Mozier
authors.forename: Sa V
authors.initials: SV
authors.lastname: Ho
mesh_terms: D016632:Apolipoprotein A-I / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D057927:Hydrophobic and Hydrophilic Interactions; D007218:Industrial Microbiology; D008074:Lipoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D016632:Apolipoprotein A-I; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; C499290:OppA protein, E coli; D033902:Periplasmic Binding Proteins; D011994:Recombinant Proteins; C080631:apolipoprotein A-I Milano; C066768:dppA protein, E coli