Capillary isoelectric focusing with whole column imaging detection for analysis of proteins and peptides

Mao, Qinglu; Pawliszyn, Janusz

doi:10.1016/s0165-022x(99)00006-8

Cited by 78 publications

(56 citation statements)

References 35 publications

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“…Hjertén applied his scanning rotating tube apparatus to IEF [17] and developed scanning equipment for stationary capillaries [18]. More recently, to overcome the drawbacks of single point detection in capillary IEF, instrumentation with optical whole column imaging, including approaches based upon pulling the separation capillary through the detection window of a UV absorbance detector [19], spatial scanning laser fluorescence detection with the capillary mounted on a precision translational stage that moves the capillary through the probe beam [20] and real-time imaging without moving parts along a short (about 5 cm), internally coated capillary [21][22][23][24] or microchannel [25] led to the following of the IEF process of amphoteric solutes. The latter approach with the coated fused-silica capillary and UV absorption detection has been commercialized and is now available as iCE280 instrument (Convergent Bioscience, Toronto, ON, Canada).…”

Section: Introductionmentioning

confidence: 99%

High‐resolution computer simulation of the dynamics of isoelectric focusing of proteins

et al. 2004

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A dynamic electrophoresis simulator that accepts 150 components and voltage gradients employed in the laboratory was used to provide a detailed description of the focusing process of proteins under conditions that were hitherto inaccessible. High-resolution focusing data of four hemoglobin variants in a convection-free medium are presented for pH 3-10 and pH 5-8 gradients formed with 20 and 40 carrier ampholytes/pH unit, respectively. With 300 V/cm, focusing is shown to occur within 5-10 min, whereas at 600 V/cm separation is predicted to be complete between 2.5 and 5 min. The time interval required for focusing of proteins is demonstrated to be dependent on the input protein charge data and, however less, on the properties of the carrier ampholytes. The simulation data reveal that the number of transient protein boundaries migrating from the two ends of the column towards the focusing positions is equal to the number of sample components. Each protein is being focused via the well-known double-peak approach to equilibrium, a process that is also characteristic for focusing of the carrier ampholytes. The predicted focusing dynamics for the hemoglobin variants in pH 3-10 and pH 5-8 gradients are shown to qualitatively agree well with experimental data obtained by whole-column optical imaging.

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Section: Introductionmentioning

confidence: 99%

High‐resolution computer simulation of the dynamics of isoelectric focusing of proteins

et al. 2004

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“…While it does consume significantly more protein, the approach is less laborious, making it more cost effective in the long run. We anticipate the ChromiCE approach may be developed more comprehensively for use in regulated environments, 22 as well as for applications beyond monoclonal antibodies, and it may be particularly beneficial for the analysis of hetero-oligomeric proteins that behave aberrantly on traditional 2D-PAGE.…”

Section: Discussionmentioning

confidence: 99%

“…By taking a non-standard approach to analytical size-exclusion chromatography (SEC), sufficient amounts of reduced and denatured heavy and light chains were separated to subsequently perform capillary IEF with whole column imaging, more commonly known as imaged capillary IEF (icIEF). 22 This novel combination of 2 relatively simple methods, referred to as ChromiCE, has the flexibility to allow for antibody-specific optimization, increases throughput while reducing time and labor, and provides quantitative charge variant data on the individual heavy and light chains. These attributes make ChromiCE an excellent characterization assay for in-process monitoring of charge heterogeneity and detection of changes in product quality during routine storage.…”

Section: Introductionmentioning

confidence: 99%

Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity

Vanam

Schneider

Marlow

2015

mAbs

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To cite this article: Ram P Vanam, Michael A Schneider & Michael S Marlow (2015) Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity, mAbs, 7:6, 1118-1127, DOI: 10.1080DOI: 10. /19420862.2015 An alternative method to traditional 2-dimensional gel electrophoresis (2D-PAGE) and its application in characterizing the inherent charge heterogeneity of chromatographically isolated monoclonal antibody heavy and light chains is described. This method, referred to as ChromiCE, utilizes analytical size-exclusion chromatography (SEC), performed under reducing and denaturing conditions, followed by imaged capillary isoelectric focusing (icIEF) of the chromatographically separated heavy and light chains. Under conditions suitable for the subsequent icIEF analysis, the absolute and relative SEC elution volumes of the heavy and light chains were found to be highly pH dependent, a phenomenon that can be exploited in optimizing chromatographic separation. Compared to 2D-PAGE, the ChromiCE method substantially decreases the time and labor needed to complete the analysis, improves reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown.

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“…Several kinds of whole-column detectors have been developed for CIEF: universal refractive index gradient imaging detector [24], optical absorption imaging detector [9,24,25], and fluorescence imaging detector [26,27]. See reviews on whole column detection methods in [23] and [28]. Some new developments in imaging are discussed below.…”

Section: Methodology Of Ciefmentioning

confidence: 99%

Recent applications of capillary isoelectric focusing

Kilár

2003

Electrophoresis

100

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After the advent of capillary isoelectric focusing (CIEF) in the 80's several approaches have been developed in order to use the technique in routine analyses. The recent years showed an extensive increase in the applications of this technique employing its exceptionally high-resolution power. Methodological improvements, as well as hyphenation with other electrophoretic and chromatographic separation procedures, proved the versatility of CIEF in studies of clinically important proteins, recombinant product, cell lysates and other complex mixtures. The combination of CIEF with mass spectrometry detection is one of the major challenges for studying proteomics. This review collected the recent applications of CIEF including innovations in the experimental setup, remedies for the presence of salts in samples, calibration of the pH gradient, carrier ampholyte-free isoelectric focusing, the progress in micropreparation, two-dimensional separations, etc.

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Capillary isoelectric focusing with whole column imaging detection for analysis of proteins and peptides

Cited by 78 publications

References 35 publications

High‐resolution computer simulation of the dynamics of isoelectric focusing of proteins

High‐resolution computer simulation of the dynamics of isoelectric focusing of proteins

Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity

Recent applications of capillary isoelectric focusing

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