Separation of product associating E. coli host cell proteins OppA and DppA from recombinant apolipoprotein A-I(Milano) in an industrial HIC unit operation.
| 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. |
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| Authors: | Hunter AK; Pfizer Global Biologics, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA. alan.k.hunter@pfizer.com; Wang X; Suda EJ; Herberg JT; Shell RE; Thomas KE; Dufield RL; Gustafson ME; Mozier NM; Ho SV |
| Source: | Biotechnology progress [Biotechnol Prog] 2009 Mar-Apr; Vol. 25 (2), pp. 446-53. |
| Publication Type: | Evaluation Study; Journal Article |
| Language: | English |
| Journal Info: | Publisher: Wiley-Blackwell Country of Publication: United States NLM ID: 8506292 Publication Model: Print Cited Medium: Internet ISSN: 1520-6033 (Electronic) Linking ISSN: 15206033 NLM ISO Abbreviation: Biotechnol Prog Subsets: MEDLINE |
| Imprint Name(s): | Publication: : Hoboken, NJ : Wiley-Blackwell; Original Publication: [New York, N.Y. : American Institute of Chemical Engineers, c1985- |
| MeSH Terms: | Industrial Microbiology*; Apolipoprotein A-I/*isolation & purification ; Carrier Proteins/*isolation & purification ; Chromatography, Liquid/*methods ; Escherichia coli Proteins/*isolation & purification ; Lipoproteins/*isolation & purification ; Periplasmic Binding Proteins/*isolation & purification; Apolipoprotein A-I/genetics ; Apolipoprotein A-I/metabolism ; Carrier Proteins/genetics ; Carrier Proteins/metabolism ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Escherichia coli Proteins/genetics ; Escherichia coli Proteins/metabolism ; Lipoproteins/genetics ; Lipoproteins/metabolism ; Periplasmic Binding Proteins/genetics ; Periplasmic Binding Proteins/metabolism ; Recombinant Proteins/genetics ; Recombinant Proteins/isolation & purification ; Recombinant Proteins/metabolism ; Gene Expression ; Hydrophobic and Hydrophilic Interactions |
| 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.; ((c) 2009 American Institute of Chemical Engineers Biotechnol.) |
| Substance Nomenclature: | 0 (Apolipoprotein A-I); 0 (Carrier Proteins); 0 (Escherichia coli Proteins); 0 (Lipoproteins); 0 (OppA protein, E coli); 0 (Periplasmic Binding Proteins); 0 (Recombinant Proteins); 0 (apolipoprotein A-I Milano); 134215-14-8 (dppA protein, E coli) |
| Entry Date(s): | Date Created: 20090318 Date Completed: 20090603 Latest Revision: 20191210 |
| Update Code: | 20260130 |
| DOI: | 10.1002/btpr.106 |
| PMID: | 19291803 |
| Database: | MEDLINE |
Evaluation Study; Journal Article