A conventional procedure to reduce Asn deamidation artifacts during trypsin peptide mapping

Kori, Yekaterina; Patel, Rekha; Neill, Alyssa; Liu, Hongcheng

doi:10.1016/j.jchromb.2015.12.009

Cited by 15 publications

(8 citation statements)

References 43 publications

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“…This method adopted an ultrafast trypsin digestion protocol we developed for assays for glycan analysis 44 and site-specific oxidation, 45 and avoided the assay artifacts of oxidation, deamidation, and isomerization induced during the cLC-MS method. 8,[34][35][36][37]39 With uLC-MS available for comparison, assay artifacts for Asn deamidation and Asp isomerization at certain sites were clearly shown in the longer digestion procedure. For example, deamidation at Asn316 of mAb A was »120-fold overestimated by an 18-hour digestion in the cLC-MS compared to the 5-min digestion procedure (60% vs 0.5%).…”

Section: Discussionmentioning

confidence: 99%

“…To increase the throughput and minimize assay artifacts, we developed uLC-MS by using an easy and fast digestion strategy that was employed for fast glycan profiling and for mAb oxidation analysis. 44,45 In this method, mAbs are first denatured and reduced in 4-8 M urea/10 mM DTT bicarbonate buffer at 70 C for 3 min, and then directly digested by trypsin at 37 C for 5 min. The alkylation and buffer exchange steps used in cLC-MS were eliminated.…”

Section: Comparison Of Different Digestion Conditionsmentioning

confidence: 99%

“…[31][32][33] The treated proteins are digested with trypsin or other proteases, and the peptides generated are separated by RP-HPLC for MS. The sample digestion step for the method is a time consuming process that could take up to 24 hours at 37 C, a step that could potentially induce artifacts 34 such as asparagine deamidation, 8,[35][36][37] N-terminal glutamine cyclization, 38 and methionine oxidation. 39,40 In addition, the long preparation time hampers the high throughput analysis for a large number of samples in forced degradation studies and clone and media selection.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Comparison Of Different Digestion Conditionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…The stability of deamidated and oxidated peptides have been a technical challenge for peptide mapping based MS analysis. 4,19,[25][26] The baseline level of those PTMs are generally low (< 2%).…”

Section: Mam Methods Validationmentioning

confidence: 99%

“…19 The low-pH digestion approach together with other digestion parameters (such as different buffer components and addition of ACN) were reported to help control the stability of deamidations. [25][26]…”

Section: Mam Methods Validationmentioning

confidence: 99%

Qualification of Identity, Quality-Attribute Monitoring and New Peak Detection in An Improved Multi-Attribute Method with Lys-C Digestion for Characterization and Quality Control of Therapeutic Monoclonal Antibodies

Pierson

Hua

et al. 2022

Preprint

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The use of Multi-attribute method (MAM) for identity and purity testing of biopharmaceuticals offers the ability to complement and replace multiple conventional analytical technologies with a single mass spectrometry (MS) method. Method qualification and phase-appropriate validation is one major consideration for the implementation of MAM in a current Good Manufacturing Practice (cGMP) environment. We developed an improved MAM workflow with optimized sample preparation using Lys-C digestion for therapeutic monoclonal antibodies. In this study, we qualified the enhanced MAM workflow for mAb-1 identity, product quality attributes (PQAs) monitoring and new peak detection (NPD). The qualification results demonstrated the full potential of the MAM for its intended use in mAb-1 characterization and quality control in regulated labs. To the best of our knowledge, this is the first report of MAM qualification for mAb identity, PQA monitoring, and new peak detection (NPD) in a single assay, featuring 1) the first full qualification of MAM using Lys-C digestion without desalting using a high-resolution MS, 2) a new approach for mAb identity testing using MAM, and 3) the first qualification of NPD for MAM. The developed MAM workflow and the approaches for MAM qualification may serve as a reference for other labs in the industry.

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CE–MS Methods for the Characterization of Monoclonal Antibodies

Reinert¹,

Beck²,

Houzé³

et al. 2022

Capillary Electrophoresis‐Mass Spectrometry for Proteomics and Metabolomics

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A conventional procedure to reduce Asn deamidation artifacts during trypsin peptide mapping

Cited by 15 publications

References 43 publications

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

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

Qualification of Identity, Quality-Attribute Monitoring and New Peak Detection in An Improved Multi-Attribute Method with Lys-C Digestion for Characterization and Quality Control of Therapeutic Monoclonal Antibodies

CE–MS Methods for the Characterization of Monoclonal Antibodies

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