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Charge heterogeneity profiling of monoclonal antibodies using low ionic strength ion-exchange chromatography and well-controlled pH gradients on monolithic columns


Talebi, M and Nordborg, A and Gaspar, A and Lacher, NA and Wang, Q and He, XZ and Haddad, PR and Hilder, EF, Charge heterogeneity profiling of monoclonal antibodies using low ionic strength ion-exchange chromatography and well-controlled pH gradients on monolithic columns, Journal of Chromatography A, 1317 pp. 148-154. ISSN 0021-9673 (2013) [Refereed Article]

Copyright Statement

Copyright 2013 Elsevier B.V. A

DOI: doi:10.1016/j.chroma.2013.08.061


In this work, the suitability of employing shallow pH gradients generated using single componentbuffer systems as eluents through cation-exchange (CEX) monolithic columns is demonstrated for thehigh-resolution separation of monoclonal antibody (mAb) charge variants in three different biopharma-ceuticals. A useful selection of small molecule buffer species is described that can be used within verynarrow pH ranges (typically 1 pH unit) defined by their buffer capacity for producing controlled andsmooth pH profiles when used together with porous polymer monoliths. Using very low ionic strengtheluents also enabled direct coupling with electrospray ionisation mass spectrometry. The results obtainedby the developed pH gradient approach for the separation of closely related antibody species appear tobe consistent with those obtained by imaged capillary isoelectric focusing (iCE) in terms of both resolu-tion and separation profile. Both determinants of resolution, i.e., peak compression and peak separationcontribute to the gains in resolution, evidently through the Donnan potential effect, which is increasedby decreasing the eluent concentration, and also through the way electrostatic charges are distributedon the protein surface. Retention mechanisms based on the trends observed in retention of proteins atpH values higher than the electrophoretic pI are also discussed using applicable theories. Employingmonolithic ion-exchangers is shown to enable fast method development, short analysis time, and highsample throughput owing to the accelerated mass transport of the monolithic media. The possibility ofshort analysis time, typically less than 15 min, and high sample throughput is extremely useful in theassessment of charge-based changes to the mAb products, such as during manufacturing or storage.

Item Details

Item Type:Refereed Article
Keywords:pH gradient, ion exchange, monoclonal antibodies, polymer monolith, imaged capillary isoelectric focussing, liquid chromatography–mass spectrometry
Research Division:Chemical Sciences
Research Group:Analytical chemistry
Research Field:Separation science
Objective Division:Expanding Knowledge
Objective Group:Expanding knowledge
Objective Field:Expanding knowledge in the chemical sciences
UTAS Author:Talebi, M (Dr Mohammad Talebi)
UTAS Author:Nordborg, A (Dr Anna Nordborg)
UTAS Author:Gaspar, A (Dr Andras Gaspar)
UTAS Author:Haddad, PR (Professor Paul Haddad)
UTAS Author:Hilder, EF (Professor Emily Hilder)
ID Code:88249
Year Published:2013
Web of Science® Times Cited:45
Deposited By:Chemistry
Deposited On:2014-01-22
Last Modified:2014-05-13

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