Interference‐free mass spectrometric detection of capillary isoelectric focused proteins, including charge variants of a model monoclonal antibody

Abstract

CIEF represents an elegant technique especially for the separation of structural similar analytes, whereas MS is a state-of-the-art instrumentation for the identification and characterization of biomolecules. The combination of both techniques can be realized by hyphenating CIEF with CZE-ESI-MS applying a mechanical valve. During the CZE step, the remaining ESI-interfering components of the CIEF electrolyte are separated from the analytes prior to MS detection. In this work, a multiple heart-cut approach is presented expanding our previous single heart-cut concept resulting in a dramatical reduction of analysis time. Moreover, different sample transfer loop volumes are systematically compared and discussed in regard to peak width and transfer efficiency. With this major enhancement, model proteins (1.63-9.75 mg/L), covering a wide pI range (5-10), and charge variants from a deglycosylated model antibody were analyzed on intact level. The promising CIEF-CZE-MS setup is expected to be applicable in different bioanalytical fields, e.g. for the fast and information rich characterization of therapeutic antibodies.

Recent advances in protein analysis by capillary and microchip electrophoresis

“…162, 163 For compatibility with small transfer volumes and cIEF high voltages a multi-port valve and sample loop system was implemented, 163 and an evaluation of sample transfer loop volumes for efficiency and peak width was done. 162 IEF separation for p I range of 5–10 is applied to the analysis of charge variants of mAbs. In 3 h up to 6 analytes were transferred from cIEF to CZE-MS with the ability to detect a mass differences of 2 Da between mAb variants.…”

Determination of protein charge variants with (imaged) capillary isoelectric focusing and capillary zone electrophoresis

“…for the separation of charge variants . There are also multidimensional approaches, for example CE‐MS , HPLC‐MS , or CE‐CE . These procedures offer a lot of analytical information, but are expensive and time consuming.…”

“…CEC is a hybrid technique between CE and HPLC and was recently used by Zhang et al for the separation of charge variants [37]. There are also multidimensional approaches, for example CE-MS [38,39], HPLC-MS [40][41][42][43][44], or CE-CE [45]. These procedures offer a lot of analytical information, but are expensive and time consuming.…”

Coupling imaged capillary isoelectric focusing with mass spectrometry using a nanoliter valve

“…The evident benefits of CIEF and MS coupling regarding the high resolving power of p I ‐based separations and the unequivocal MS identification of individual peaks have promoted several investigations . Our research group has previously shown the value of two‐dimensional systems using standard CE instrumentation for CIEF, CZE‐MS as second dimension and a nanoliter valve for the coupling . The four‐port valve (VICI AG International, Schenkon, Switzerland) allows the direct coupling of two capillary‐based techniques with high flexibility .…”

“…When the iCIEF peak is in the sample loop, the valve is switched to position B where the loop is in the second dimension. PVA‐coated capillaries are connected to the valve for the second dimension. In this work, two different ways to transport the peaks from the sample loop to the MS were tested: flow injection (FI) (Fig.…”