On-line characterization of monoclonal antibody variants by liquid chromatography–mass spectrometry operating in a two-dimensional format

Alvarez, Melissa; Tremintin, Guillaume; Wang, Jennifer; Eng, Marian; Kao, Yung-Hsiang; Jeong, Justin; Ling, Victor; Borisov, Oleg V.

doi:10.1016/j.ab.2011.07.033

Cited by 82 publications

(86 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An online concentration step was used to analyse dilute fractions to elucidate minor size variants. While unable to give complete peptide mapping of glycoforms, this represents a first step towards monitoring some of the important QbD properties online including glycoform heterogeneity and N-cyclisation [170]. In another recent report, a method for the rapid detection and differentiation of sialic acids by HPLC was developed that is capable of analysing the content of N-acetylneuraminic acid versus N-glycolylneuraminic acid in about five minutes [171] simply by using a shorter column.…”

Section: In-process Analysis Of Glycoproteinsmentioning

confidence: 99%

Application of Quality by Design Paradigm to the Manufacture of Protein Therapeutics

Val¹,

Jedrzejewski²,

Exley³

et al. 2012

Glycosylation

View full text Add to dashboard Cite

Section: In-process Analysis Of Glycoproteinsmentioning

confidence: 99%

Application of Quality by Design Paradigm to the Manufacture of Protein Therapeutics

Val¹,

Jedrzejewski²,

Exley³

et al. 2012

Glycosylation

View full text Add to dashboard Cite

“…Alvarez et al first demonstrated the utility of a generic 2D format for the rapid characterization of mAb charge and size variants. 20 Proteins contained in selected fractions of interest from IEX or SEC separations were identified by trapping and desalting the fractions using a series of trap cartridges containing a RP stationary phase, with subsequent online MS analysis. An online disulfide reduction step was successfully incorporated in the workflow, allowing more detailed characterization of modified mAbs.…”

Section: Introductionmentioning

confidence: 99%

Comparison of originator and biosimilar therapeutic monoclonal antibodies using comprehensive two-dimensional liquid chromatography coupled with time-of-flight mass spectrometry

Sorensen

Harmes

Stoll

et al. 2016

mAbs

View full text Add to dashboard Cite

As research, development, and manufacturing of biosimilar protein therapeutics proliferates, there is great interest in the continued development of a portfolio of complementary analytical methods that can be used to efficiently and effectively characterize biosimilar candidate materials relative to the respective reference (i.e., originator) molecule. Liquid phase separation techniques such as liquid chromatography and capillary electrophoresis are powerful tools that can provide both qualitative and quantitative information about similarities and differences between reference and biosimilar materials, especially when coupled with mass spectrometry. However, the inherent complexity of these protein materials challenges even the most modern one-dimensional (1D) separation methods. Two-dimensional (2D) separations present a number of potential advantages over 1D methods, including increased peak capacity, 2D peak patterns that can facilitate unknown identification, and improvement in the compatibility of some separation methods with mass spectrometry. In this study, we demonstrate the use of comprehensive 2D-LC separations involving cation-exchange (CEX) and reversed-phase (RP) separations in the first and second dimensions to compare 3 reference/biosimilar pairs of monoclonal antibodies (cetuximab, trastuzumab and infliximab) that cover a range of similarity/disimilarity in a middle-up approach. The second dimension RP separations are coupled to time-of-flight mass spectrometry, which enables direct identification of features in the chromatograms obtained from mAbs digested with the IdeS enzyme, or digestion with IdeS followed by reduction with dithiothreitol. As many as 23 chemically unique mAb fragments were detected in a single sample. Our results demonstrate that these rich datasets enable facile assesment of the degree of similarity between reference and biosimilar materials.

show abstract

“…Incorporating IEX separation up-stream of mass spectral analysis has been shown to provide greater detail regarding the molecular origin of charge variants. 19 Intact mass of resolved peaks can reveal differential glycan forms, but additional processing and data analysis (e.g., tryptic peptide mapping) is required to identify the location and nature of specific chemical modifications (e.g., residue specific deamidation). 20,21 Thus, a rapid and simple separation method with greater information content would be highly beneficial.…”

Section: Introductionmentioning

confidence: 99%

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

Vanam

Schneider

Marlow

2015

mAbs

View full text Add to dashboard Cite

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.

show abstract

On-line characterization of monoclonal antibody variants by liquid chromatography–mass spectrometry operating in a two-dimensional format

Cited by 82 publications

References 35 publications

Application of Quality by Design Paradigm to the Manufacture of Protein Therapeutics

Application of Quality by Design Paradigm to the Manufacture of Protein Therapeutics

Comparison of originator and biosimilar therapeutic monoclonal antibodies using comprehensive two-dimensional liquid chromatography coupled with time-of-flight mass spectrometry

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

Contact Info

Product

Resources

About