Fast Characterization of Fc-Containing Proteins by Middle-Down Mass Spectrometry Following IdeS Digestion

Liu, Tao; Guo, Haipeng; Zhu, Lei; Zheng, Yingxin; Xu, Jin; Guo, Qingcheng; Zhang, David; Qian, Weizhu; Dai, Jing; Guo, Yajun; Hou, Sheng; Wang, Hao

doi:10.1007/s10337-016-3173-2

Cited by 9 publications

(9 citation statements)

References 47 publications

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“…IdeS is an enzyme modified from Streptococcus pyogenes that is frequently used for the characterization of antibodies and ADCs. , It specifically cleaves human IgG1 antibody at the two consecutive glycine positions and produce F(ab) 2 fragments with all the drug linking positions and Fc with glycans. The F(ab) 2 fragments were further reduced by TCEP to generate free LC and Fd species.…”

Section: Resultsmentioning

confidence: 99%

Characterization of Positional Isomers of Interchain Cysteine Linked Antibody−Drug Conjugates by High-Resolution Mass Spectrometry

Lin²,

Shi

et al. 2019

Anal. Chem.

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Interchain cysteine linked antibody–drug conjugates (ADCs) are emerging therapeutic products that antagonize cancers. The toxic payloads are selectively linked to the interchain cysteines and generate heterogeneous mixtures of positional isomers. These positional isomers might contribute differently to the therapeutic efficacy because of the variation in conjugation stability, and thus they need to be well characterized. However, the characterization of the positional isomers of interchain cysteine linked ADCs is very challenging, mainly because of the high similarity between those isomers. In this research, we developed a novel mass spectrometry method for the characterization of positional isomers of interchain cysteine linked ADCs. The subunit analysis and the bottom-up analysis provided abundant information about the drug numbers and drug linking positions on each chain. Because the method can provide accurate data on drug linking numbers and positions on each chain, it will be very useful for researchers in cancer drug development and cancer treatment.

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Section: Resultsmentioning

confidence: 99%

Characterization of Positional Isomers of Interchain Cysteine Linked Antibody−Drug Conjugates by High-Resolution Mass Spectrometry

Lin²,

Shi

et al. 2019

Anal. Chem.

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“…Since attempts toward the separation of mAb oxidation variants by IP-RP-HPLC at the intact protein level were not successful, we implemented proteolysis with IdeS in order to obtain smaller protein entities. − Digestion with IdeS generated two mAb subunits: Fc/2 and F(ab′) 2 (see Figure a). Subsequent reduction of inter- and intramolecular disulfide bonds with TCEP yielded three antibody subunits, namely, two pairs of heavy chain (HC) fragments: Fc/2 and Fd′ and a pair of light chains (LC), all comprising a molecular mass of around 25 kDa (Figure a).…”

Section: Resultsmentioning

confidence: 99%

“…The latter can be achieved using the streptococcal cysteine protease IdeS which cleaves human IgG antibodies specifically in the hinge region . IdeS has previously been applied to study glycosylation, deamidation, and oxidation of the Fc domain. − Recently, Sokolowska et al presented a fast method for quantification of oxidation upon IdeS digestion, deglycosylation with EndoS and disulfide reduction, employing IP-RP-HPLC coupled to a quadrupole time-of-flight (Q-TOF) mass spectrometer . Even though the method is generic for IgG mAbs, oxidized Fc variants remained unseparated, restricting quantification to mass spectrometry data.…”

mentioning

confidence: 99%

A Generic HPLC Method for Absolute Quantification of Oxidation in Monoclonal Antibodies and Fc-Fusion Proteins Using UV and MS Detection

et al. 2017

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Oxidation of biopharmaceuticals may affect their bioactivity, serum half-life, and (bio)chemical stability. The Fc domain of IgG monoclonal antibodies (mAbs) contains two methionine residues which are susceptible to oxidation. Here, we present a middle-down approach employing the cysteine protease IdeS under reducing conditions to obtain three mAb subunits of approximately 25 kDa: Fc/2, Fd', and LC. These subunits were separated by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) and detected by UV spectroscopy as well as Orbitrap mass spectrometry (MS), as well as MS upon all-ion fragmentation (AIF-MS). We evaluated the feasibility of three strategies for absolute quantification of oxidation in the Fc region of hydrogen peroxide-stressed Rituximab, using a single, commercially available software platform both for data acquisition and evaluation: UV spectroscopy, full-scan MS, and monitoring of product ions obtained by AIF-MS. UV spectroscopy showed the lowest limits of quantification (LOQ) (0.96 ng μL) and featured the lowest relative process standard deviation (V%) of 7.2% compared to MS and AIF-MS with LOQs of 1.24-4.32 ng μL and relative process standard deviations of 9.0-14%, respectively. Our approach is generic in that it allows monitoring and quantification of oxidation in the Fc regions of fully human and humanized IgG1 mAbs as well as of Fc-fusion proteins. This is exemplified by limits of detection of 1.2%, 1.0%, and 1.2% of oxidation in drug products containing the biopharmaceuticals Rituximab, Adalimumab, and Etanercept, respectively. The presented method is an attractive alternative to conventional time-intensive peptide mapping which is prone to artificial oxidation due to extensive sample preparation.

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“…In 2016, we reported the development of a sensitive and high-throughput subunit-based MAM for monitoring the protein fragment ( Liu et al, 2016 ). IdeS of 1 μg was added to each 50 μg of protein.…”

Section: Methodsmentioning

confidence: 99%

Identification, Efficacy, and Stability Evaluation of Succinimide Modification With a High Abundance in the Framework Region of Golimumab

Liu

Guo

et al. 2022

Front. Chem.

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Succinimide (Asu) is the intermediate for asparagine deamidation in therapeutic proteins, and it can be readily hydrolyzed to form aspartate and iso-aspartate residues. Moreover, Asu plays an important role in the protein degradation pathways, asparagine deamidation, and aspartic acid isomerization. Here, Asu modification with a high abundance in the framework region (FR) of golimumab was first reported, the effect of denaturing buffer pH on the Asu modification homeostasis was studied, and the results revealed that it was relatively stable over a pH range of 6.0–7.0 whereas a rapid decrease at pH 8.0. Then, the peptide-based multi-attribute method (MAM) analyses showed that the Asu formation was at Asn 43 in the FR of the heavy chain. Meanwhile, the efficacy [affinity, binding and bioactivity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cell-mediated cytotoxicity (ADCC) activity] and stability of the Asu modification of golimumab were evaluated, and the current results demonstrated comparable efficacy and stability between the Asu low- and high-abundance groups. Our findings provide valuable insights into Asu modification and its effect on efficacy and stability, and this study also demonstrates that there is a need to develop a broad-spectrum, rapid, and accurate platform to identify and characterize new peaks in the development of therapeutic proteins, particularly for antibody drugs.

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Fast Characterization of Fc-Containing Proteins by Middle-Down Mass Spectrometry Following IdeS Digestion

Cited by 9 publications

References 47 publications

Characterization of Positional Isomers of Interchain Cysteine Linked Antibody−Drug Conjugates by High-Resolution Mass Spectrometry

Characterization of Positional Isomers of Interchain Cysteine Linked Antibody−Drug Conjugates by High-Resolution Mass Spectrometry

A Generic HPLC Method for Absolute Quantification of Oxidation in Monoclonal Antibodies and Fc-Fusion Proteins Using UV and MS Detection

Identification, Efficacy, and Stability Evaluation of Succinimide Modification With a High Abundance in the Framework Region of Golimumab

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