Research Article A systematic study in CIEF: Defining and optimizing experimental parameters critical to method reproducibility and robustness A systematic study of two-step CIEF analysis was completed to identify key components that could be optimized to enhance the performance of mAb analysis by CIEF. Reso- lution between mAb isoforms was increased by selecting a narrow detector aperture, utilizing chemical rather than pressure mobilization, and improving protein solubility by incorporating urea into the carrier ampholyte (CA) solutions. Loss of the extreme pI CAs and sample components by the bidirectional ITP inherent to IEF was avoided by setting the concentration of the phosphoric acid anolyte to 200 mM and sodium hydroxide catholyte to 300 mM and by adding sufficient amounts of an acidic (pIo3) and basic (10opI) sacrificial ampholyte to the CIEF sample solution. Optimization of the concentrations of the sacrificial ampholytes, iminodiacetic acid and arginine to 1.7 and 40 mM, respectively, yielded a stable focused CA train that spanned the 4opHo10 range without sacrificing either mAb isoform resolution or sample throughput. Inter- mediate precision studies performed on the CIEF method with three basic mAbs yielded 0.04 to 0.09% CVs for the estimated pI values and 0.6À3% CVs for isoform group percent composition, indicating that the two-step CIEF method developed meets the rigorous demands of therapeutic mAb analysis. Keywords: CE / IEF / mAbs / Therapeutic protein characterization DOI 10.1002/elps.200800690 1 Introduction During the last two decades, CIEF analysis with single-point detection (CIEF) [1] and full-column imaging CIEF analysis (ICIEF) [2] became widely used alternatives [3] to traditional gel and paper-based IEF analysis [4]. The biopharmaceutical industry has shown great interest in CIEF analysis for characterization of charge heterogeneity in therapeutic mAbs. Determination of charge heterogeneity adds a critical dimension of measurement, useful for establishing the identity, purity, post-translational modifications and stability of a therapeutic protein preparation. However, variability in the CIEF peak profiles of mAbs (and thus variability in the assigned pI values) caused by, e.g. aggregation and precipitation, variations in the type and concentration of the sample buffer(s), anodic and cathodic drift [5], etc. have hindered the implementation of CIEF as a routine analytical tool. In order to develop a robust, reproducible CIEF method for mAb characterization, the influences of both instrument-specific and IEF-specific parameters had to be re-evaluated. The choice of some of the instrumental parameter settings is straightforward. The need for rapid CIEF analysis calls for as short a capillary and as high a separation potential as possible. The total length of the capillary (L t ) and the length to the detector (L d ) are set by the design of the instrument and cannot be varied readily. Joule heat generation limits the maximum IEF current (and conse- quently the maximum separation potential) that can be applied. The need for minimal EOF and minimal interac- tion between mAbs and the capillary wall calls for a hydro- philic polymer layer on the inner wall of the capillary. Commercial availability of these coated capillaries limits one’s choices of the inner diameter of the capillary. The UV absorbance spectra of carrier ampholytes (CAs) and mAbs dictate the selection of 280 nm as the detection wavelength. The smallest detector aperture width preserves best the resolution achieved during the separation, albeit at the expense of reduced signal-to-noise ratio. Some of the CIEF method parameter settings can also be chosen in a straightforward manner as well. With Scott Mack 1 Ingrid Cruzado-Park 1 Jeff Chapman 1 Chitra Ratnayake 1 Gyula Vigh 2 1 Beckman Coulter, Inc., Fullerton, CA, USA 2 Department of Chemistry, Texas A&M University, College Station, TX, USA Received October 22, 2008 Revised August 5, 2009 Accepted August 25, 2009 Abbreviations: ARG, L-arginine; CA, carrier ampholyte; ICIEF, full-column imaging CIEF analysis; IDA, iminodiacetic acid;L d , length to detector;L t , total capillary length; PN, part number; rcf, relative centrifugal force Correspondence: Scott Mack, Senior Development Scientist, Beckman Coulter, Inc., 4300 North Harbor Blvd., Mail Stop D16 E, Fullerton, CA 92834-3100, USA E-mail: stmack@beckman.com Fax: 11-714-773-8993 & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2009, 30, 4049–4058 4049