A new tool for monoclonal antibody analysis

An, Yan; Zhang, Ying; Mueller, Hans-Martin; Shameem, Mohammed; Chen, Xiaoyu

doi:10.4161/mabs.28762

Cited by 121 publications

(94 citation statements)

References 64 publications

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“…[5][6][7][8][9] When a separation technique is coupled online to electrospray ionization (ESI)-MS, the standardized migration or elution position can also be used for glycospecies assignment. In principle, the MS-based methods for this application can be sub-divided into 3 categories: 1) top-down or middle-down analysis by characterizing the IgG molecule with ESI-MS either after reduction of the disulfide bonds or after digestion with a proteolytic enzyme such as IdeS protease (FabRICATOR Ò , from Streptococcus pyogenes) [10][11][12] 2) bottom-up analysis by proteolytic digesting the IgG molecule, e.g., with trypsin, and measuring the glycopeptides either with or without prior separation, using ESI-MS or matrix-assisted laser desorption ionization (MALDI)-MS detection, respectively;…”

Section: Introductionmentioning

confidence: 99%

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 2: Mass spectrometric methods

Reusch¹,

Haberger

Falck³

et al. 2015

mAbs

113

101

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(2015) Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles-Part 2: Mass spectrometric methods, mAbs, 7:4, 732-742, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 To monitor the Fc glycosylation of therapeutic immunoglobulin G in bioprocess development, product characterization and release analytics, reliable techniques for glycosylation analysis are needed. Several analytical methods are suitable for this application. We recently presented results comparing detection methods for glycan analysis that are separation-based, but did not include mass spectrometry (MS). In the study reported here, we comprehensively compared MS-based methods for Fc glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods investigated were compared with respect to precision, accuracy, throughput and analysis time. Emphasis was put on the detection and quantitation of sialic acid-containing glycans. Eleven MS methods were compared to hydrophilic interaction liquid chromatography of 2-aminobenzamide labeled glycans with fluorescence detection, which served as a reference method and was also used in the first part of the study. The methods compared include electrospray MS of the heavy chain and Fc part after limited digestion, liquid chromatography MS of a tryptic digest, porous graphitized carbon chromatography MS of released glycans, electrospray MS of glycopeptides, as well as matrix assisted laser desorption ionization MS of glycans and glycopeptides. Most methods showed excellent precision and accuracy. Some differences were observed with regard to the detection and quantitation of low abundant glycan species like the sialylated glycans and the amount of artefacts due to in-source decay.

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Section: Introductionmentioning

confidence: 99%

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 2: Mass spectrometric methods

Reusch¹,

Haberger

Falck³

et al. 2015

mAbs

113

101

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“…6 The processing control on critical quality attributes forms the basic principle of quality by design (QbD) in biopharmaceutical development. 21 Deamidation of Asn on mAbs is normally analyzed by ion exchange chromatography and liquid chromatography-mass spectrometry (LC-MS), 22 and isomerization of Asp is almost exclusively characterized by LC-MS. Oxidation of Met and Trp residues can be determined by hydrophobic interaction chromatography (HIC), 23 reverse phase-HPLC coupled with Fabricator digestion, 24 and mixed mode size-exclusion chromatography (SEC) using Sepax SEC-300MK 25 and Waters BEH 200. 26 However, the most common method to characterize site-specific oxidation, deamidation, and isomerization in proteins is bottom-up LC-MS, which is a very effective method for identifying and quantitating PTMs in protein molecules.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous monitoring of oxidation, deamidation, isomerization, and glycosylation of monoclonal antibodies by liquid chromatography-mass spectrometry method with ultrafast tryptic digestion

Wang

Liu

et al. 2016

mAbs

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Monoclonal antibodies are subjected to a wide variety of post-translational modifications (PTMs) that cause structural heterogeneity. Characterization and control of these modifications or quality attributes are critical to ensure antibody quality and to define any potential effects on the ultimate safety and potency of antibody therapeutics. The biopharmaceutical industry currently uses numerous tools to analyze these quality attributes individually, which requires substantial time and resources. Here, we report a simple and ultrafast bottom-up liquid chromatography-mass spectrometry (uLC-MS) method with 5 min tryptic digestion to simultaneously analyze multiple modifications, including oxidation, deamidation, isomerization, glycation, glycosylation, and N-terminal pyro-glutamate formation, which can occur during antibody production in mammalian cell culture, during purification and/or on storage. Compared to commonly used preparation procedures, this uLC-MS method eliminates assay artifacts of falsely-increased Met oxidation, Asp isomerization, and Asn deamidation, a problem associated with long digestion times in conventional LC-MS methods. This simple, low artifact multi-attribute uLC-MS method can be used to quickly and accurately analyze samples at any stage of antibody drug development, in particular for clone and media selection during cell culture development.

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“…This can be accomplished by specific cleavage of the antibody to yield a limited number of smaller protein fragments. IdeS protease (Immunoglobulin G-degrading enzyme of Streptococcus pyogene ) has recently been commercialized and utilized to structurally characterize antibodies [4], since it has superior selectivity for cleavage in a region nearby the main pepsin cleavage site, resulting in the generation of a F(ab′) 2 plus an Fc fragment, similar to those generated by pepsin, but with greater specificity and little, if any, non-selective cleavage.…”

Section: Discussionmentioning

confidence: 99%

Structural characterization of expressed monoclonal antibodies by single sample mass spectral analysis after IdeS proteolysis

Kirley

Greis

Norman

2016

Biochemical and Biophysical Research Communications

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Simple and rapid methods for analysis of monoclonal antibody structure and post-translational modifications are increasingly needed due to the explosion of therapeutic monoclonal antibodies and monoclonal antibody applications. Mass spectral analysis is a powerful method for characterizing monoclonal antibodies. Recent discovery and commercialization of the Immunoglobulin G-degrading enzyme of Streptococcus pyogene (IdeS protease) has facilitated and improved the generation of antibody fragments of suitable size to allow characterization of the structure of the entire antibody molecule via analysis of just a few fragments. In this study, we coupled IdeS fragmentation and simultaneous reduction and alkylation of the resultant fragments using tributylphosphine and iodoacetamide to prepare samples in about 2 hours. Following simple introduction of a single, unseparated mixture of alkylated fragments into a mass spectrometer, detailed structural information is obtained, covering the entire antibody molecule. The large majority of the glycoforms present on the single, conserved N-linked glycosylation site of the heavy chain is elucidated, although some of the very low abundance glycoforms are not determined by this protocol. The ease, simplicity, speed, and power of this method make it attractive for analysis of the developmental stages and production batches of therapeutic monoclonal antibodies.

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A new tool for monoclonal antibody analysis

Cited by 121 publications

References 64 publications

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 2: Mass spectrometric methods

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 2: Mass spectrometric methods

Simultaneous monitoring of oxidation, deamidation, isomerization, and glycosylation of monoclonal antibodies by liquid chromatography-mass spectrometry method with ultrafast tryptic digestion

Structural characterization of expressed monoclonal antibodies by single sample mass spectral analysis after IdeS proteolysis

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