title: Analytical characterization of complex, biotechnological feedstocks by pH gradient ion exchange chromatography for purification process development.
abstract: The accelerating growth of the market for proteins and the growing interest in new, more complex molecules are bringing new challenges to the downstream process development of these proteins. This results in a demand for faster, more cost efficient, and highly understood downstream processes. Screening procedures based on high-throughput methods are widely applied nowadays to develop purification processes for proteins. However, screening highly complex biotechnological feedstocks, such as complete cell lysates containing target proteins often expressed with a low titre, is still very challenging. In this work we demonstrate a multidimensional, analytical screening approach based on pH gradient ion exchange chromatography (IEC), gel electrophoresis and protein identification via mass spectrometry to rationally characterize a biotechnological feedstock for the purpose of purification process development. With this very simple characterization strategy a two-step purification based on consecutive IEC operations was rapidly laid out for the purification of a diagnostic protein from a cell lysate reaching a purity of ∼80%. The target protein was recombinantly produced using an insect cell expression system.
authors.affiliation: Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
authors.forename: Frieder
authors.initials: F
authors.lastname: Kröner
authors.forename: Alexander T
authors.initials: AT
authors.lastname: Hanke
authors.forename: Beckley K
authors.initials: BK
authors.lastname: Nfor
authors.forename: Martijn W H
authors.initials: MW
authors.lastname: Pinkse
authors.forename: Peter D E M
authors.initials: PD
authors.lastname: Verhaert
authors.forename: Marcel
authors.initials: M
authors.lastname: Ottens
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hubbuch
mesh_terms: D000818:Animals; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007313:Insecta; D010750:Phosphoproteins / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D016601:RNA-Binding Proteins / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification; D000097796:Nucleolin
chemical_list: D010750:Phosphoproteins; D011506:Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000097796:Nucleolin
keywords: Downstream process development; Insect cell expression system; Mass spectrometry; PH gradient ion exchange chromatography; Protein identification; Protein purification