TY - JOUR
T1 - Multi-dimensional fractionation and characterization of crude protein mixtures
T2 - Toward establishment of a database of protein purification process development parameters
AU - Nfor, Beckley K.
AU - Ahamed, Tangir
AU - Pinkse, Martijn W.H.
AU - van der Wielen, Luuk A.M.
AU - Verhaert, Peter D.E.M.
AU - van Dedem, Gijs W.K.
AU - Eppink, Michel H.M.
AU - van de Sandt, Emile J.A.X.
AU - Ottens, Marcel
N1 - Copyright © 2012 Wiley Periodicals, Inc.
PY - 2012/12
Y1 - 2012/12
N2 - 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.
AB - 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.
KW - Chromatography
KW - Crude mixture of proteins
KW - Hybridoma cell culture supernatant
KW - Monoclonal antibody
KW - Process development parameters
UR - http://www.scopus.com/inward/record.url?scp=84868157621&partnerID=8YFLogxK
U2 - 10.1002/bit.24576
DO - 10.1002/bit.24576
M3 - Article
C2 - 22688729
AN - SCOPUS:84868157621
SN - 0006-3592
VL - 109
SP - 3070
EP - 3083
JO - Biotechnology and Bioengineering
JF - Biotechnology and Bioengineering
IS - 12
ER -