Multi-dimensional fractionation and characterization of crude protein mixtures: toward establishment of a database of protein purification process development parameters.
A multi-dimensional fractionation and characterization scheme was developed for fast acquisition of the relevant molecular properties for protein separation from crude biological feedstocks by ion-exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and size-exclusion chromatography. In this approach, the linear IEX isotherm parameters were estimated from multiple linear salt-gradient IEX data, while the nonlinear IEX parameters as well as the HIC isotherm parameters were obtained by the inverse method under column overloading conditions. Collected chromatographic fractions were analyzed by gel electrophoresis for estimation of molecular mass, followed by mass spectrometry for protein identification. The usefulness of the generated molecular properties data for rational decision-making during downstream process development was equally demonstrated. Monoclonal antibody purification from crude hybridoma cell culture supernatant was used as case study. The obtained chromatographic parameters only apply to the employed stationary phases and operating conditions, hence prior high throughput screening of different chromatographic resins and mobile phase conditions is still a prerequisite. Nevertheless, it provides a quick, knowledge-based approach for rationally synthesizing purification cascades prior to more detailed process optimization and evaluation.
Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
Beckley K
BK
Nfor
Tangir
T
Ahamed
Martijn W H
MW
Pinkse
Luuk A M
LA
van der Wielen
Peter D E M
PD
Verhaert
Gijs W K
GW
van Dedem
Michel H M
MH
Eppink
Emile J A X
EJ
van de Sandt
Marcel
M
Ottens
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification*; D001709:Biotechnology / Q000379:methods*; D002845:Chromatography / Q000379:methods*; D045424:Complex Mixtures / Q000032:analysis / Q000737:chemistry*; D016208:Databases, Factual; D004591:Electrophoresis, Polyacrylamide Gel; D006825:Hybridomas; D057927:Hydrophobic and Hydrophilic Interactions; D008954:Models, Biological; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
D000911:Antibodies, Monoclonal; D045424:Complex Mixtures; D011506:Proteins