HPCE methods for the identification and quantitation of antibodies, their conjugates and complexes

Krull, Ira S.; Liu, X.; Dai, Jingquan; Gendreau, Christina; Li, G.

doi:10.1016/s0731-7085(97)00071-x

Cited by 27 publications

(10 citation statements)

References 40 publications

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“…The various DAR species of an ADC can potentially exhibit very different pharmacokinetic properties. ,,,, As such, the DAR and often the distribution are deemed as being critical quality attributes of an ADC. At the intact level, analyses of these species are often accomplished using separation techniques such as hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC), reverse phase liquid chromatography (RPLC), capillary electrophoresis (CE), or capillary isoelectric focusing. ,,,− These approaches are coupled to absorbance-based detection techniques and are generally incompatible with mass spectrometry due to the high salt content required for analysis. RPLC can be interfaced with a mass spectrometer as a desalting technique to generate an infusion-like spectrum of the intact ADC sample, but separation of variants does not occur. ,− Recently, several studies have been published using native spray ion mobility spectroscopy (IMS) with MS detection for characterizing ADCs. − With this approach, DAR and distribution can be determined from the mass spectrum and some resolution between DAR species is obtained during the IMS separation.…”

mentioning

confidence: 99%

Characterization of Intact Antibody Drug Conjugate Variants Using Microfluidic Capillary Electrophoresis–Mass Spectrometry

Redman¹,

Mellors

Starkey

et al. 2016

Anal. Chem.

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In this work, we utilize capillary electrophoresis-mass spectrometry (CE-MS) in an integrated microfluidic platform to analyze an intact, lysine-linked antibody drug conjugate (ADC) in order to assess post translational modifications and drug load variants. The initial charge heterogeneity of the unconjugated IgG-2 monoclonal antibody (mAb) was assessed by separating intact charge variants. Three main charge variants were resolved in the CE dimension. These variants were attributed to pyroglutamic acid formation and decarboxylation on the primary structure of the mAb through characteristic mass shifts and changes in electrophoretic mobility. Additionally, glycoforms of the antibody charge variants were identified in the deconvoluted mass spectra. The observed glycoforms and their distribution compared favorably to a released N-glycan analysis performed on the mAb. After conjugation, the ADC was analyzed using the same microchip CE-MS method. The addition of a drug load resulted in a decrease in mobility and an increase in mass of 3145 Da. Five main species that differed in their respective drug-to-antibody ratios (DAR) were fully resolved in the CE separation, with each DAR displaying the same variant population observed on the unconjugated mAb. A DAR range of 0-4 was observed with an average of 1.7 drug loads. The DAR distribution generated from the microfluidic CE-MS data compared favorably to results from infusion-ESI-MS and imaging CE (iCE) analysis of the ADC, techniques commonly used for intact mAb and ADC characterization.

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mentioning

confidence: 99%

Characterization of Intact Antibody Drug Conjugate Variants Using Microfluidic Capillary Electrophoresis–Mass Spectrometry

Redman¹,

Mellors

Starkey

et al. 2016

Anal. Chem.

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“…The role of CE as analytical tool for recombinant proteins has been extensively reviewed [132][133][134][135][136]. One major application is the characterization of glycoprotein variants [137,138].…”

Section: Capillary Electrophoresismentioning

confidence: 99%

Chromatographic and electrophoretic characterization of protein variants

Ahrer

Jungbauer

2006

Journal of Chromatography B

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“…These reviews have provided information based upon the individual capillary electrophoresis (CE) techniques such as capillary zone electrophoresis (CZE, [6][7][8]), capillary isoelectric focusing (clEF, [9,10]), capillary electrophoresis using sodium dodecyl sulfate (CE-SDS, [11]) and micellar electrokinetic chromatography (MEKC, also called micellar electrokinetic capillary chromatography [MECC], [12]). Reviews have also been written for the analysis of recombinant proteins in general [13][14][15], antibodies [16], and glycoproteins [17,18].…”

Section: Capillary Electrophoresis Reviewsmentioning

confidence: 99%

“…Krull et al [16] reviewed the existing literature on the CE methods for the identification and quantitation of antibodies, conjugates, and complexes. The authors described the analysis of antibodies using the major modes of CE and the major application of the respective modes, including clEF, MEKC, and CE-SDS.…”

Section: S-12mentioning

confidence: 99%

Capillary Electrophoresis in the Development of Recombinant Protein Biopharmaceuticals

Chen¹,

Canova-Davis²

2001

CE in Biotechnology: Practical Applications for Protein and Peptide Analyses

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The understanding of the physicochemical properties of proteins in solution is required to understand the separations taking place in the capillary. For example, during isoelectric focusing, it is important to ensure the lack of protein precipitation during fOCUSing and mobilization otherwise artifacts occur and the true resolution of charge variants is not achieved. Similarly, buffer conditions, including additives, composition of buffer, and pH can influence the resolution of product variants. With diligent efforts one can achieve desired separations. As knowledge of buffer conditions and use of these techniques increase, the methods will be improved.MECC can be useful for the characterization of recombinant proteins and monoclonal antibodies. However, the relatively long analyte migration times may be a deterrent for routine use in quality control situations. Pharmaceutical companies will employ new techniques which provide advantages over current technology. CE has advantages in certain applications. Applications which use slab gel techniques, IEF and SDS-PAGE can be replaced by the CE methods for formulation development, characterization of charge isoforms, and for purity determinations. CZE and clEF can be used for rapid identity tests which do not require peptide mapping. CZE has a tremendous advantage in analyzing basic proteins, and CZE and CE-SDS can be used to quantify product in the presence of interfering excipients. It is the responsibility of the analytical chemist to determine applications which are appropriate for CEo It is apparent that these are currently being defined for the biotechnology industry, and the usage of CE should continue to increase.

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HPCE methods for the identification and quantitation of antibodies, their conjugates and complexes

Cited by 27 publications

References 40 publications

Characterization of Intact Antibody Drug Conjugate Variants Using Microfluidic Capillary Electrophoresis–Mass Spectrometry

Characterization of Intact Antibody Drug Conjugate Variants Using Microfluidic Capillary Electrophoresis–Mass Spectrometry

Chromatographic and electrophoretic characterization of protein variants

Capillary Electrophoresis in the Development of Recombinant Protein Biopharmaceuticals

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