Development, validation, and implementation of a robust and quality control-friendly focused peptide mapping method for monitoring oxidation of co-formulated monoclonal antibodies

Xu, Chengdong; Khanal, Sumee; Pierson, Nicholas A.; Quiróz, Jorge A.; Kochert, Brent A.; Yang, Xiaoyu; Wylie, David T.; Strulson, Christopher A.

doi:10.1007/s00216-022-04366-z

Cited by 9 publications

(2 citation statements)

References 31 publications

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“…In the case of emerging therapies, such as viral vector‐based gene therapies, an alternative protease may be required (Guapo et al., 2022). Other frequently used proteases are chymotrypsin (Yang et al., 2010), Lys‐C (Li et al., 2022; Xu et al., 2022), Asp‐N or Glu‐C (Buettner et al., 2020). In all cases, it is important to obtain a reproducible digest with low artificially induced modifications (Jakes et al., 2021).…”

Section: Introductionmentioning

confidence: 99%

Multi‐Attribute Method (MAM) Analytical Workflow for Biotherapeutic Protein Characterization from Process Development to QC

Millán‐Martín,

Jakes,

Carillo

et al. 2023

Current Protocols

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The multi‐attribute method (MAM) has emerged significantly in recent years to support biotherapeutic protein characterization from process development to the QC environment. MAM is a liquid chromatography mass spectrometry (LC‐MS) based peptide mapping approach, which combines the benefits from liquid chromatography coupled to high resolution accurate mass mass spectrometry (LC‐HRAM MS), enabling direct assessment of protein sequence and product quality attributes with site specificity. These product quality attributes may impact efficacy, safety, stability, and process robustness. MAM is intended to replace conventional analytical approaches as it offers a more streamlined strategy for parallel monitoring of multiple attributes in a single analysis with high sensitivity and confidence, and ultimately supports more robust Quality by Design (QbD) approaches and faster decision cycles for biotherapeutic development. MAM consists of three main stages. The first stage is sample digestion, which typically entails proteolytic digestion of the protein. The second stage is reversed‐phase chromatographic separation of the generated peptides and detection by HRAM MS in two phases. During MAM Phase I (discovery phase), data‐dependent acquisition (DDA) MS/MS is performed to enable confident identification of peaks and development of a peptide workbook. During MAM Phase II (monitoring phase), full MS acquisition is only carried out for the monitoring of predefined product quality attributes (PQAs). The third stage is data processing, which entails analysis and reporting for each of the two phases including evaluation of sequence coverage, assessment of PQAs and peptide workbook creation during phase I, and targeted monitoring of predefined product attributes and new peak detection (NPD) during phase II. The latter is a comparative analysis that uses a base peak alignment algorithm to determine any non‐monitored differences between the LC‐MS chromatograms of a test sample and a reference standard. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: In‐solution sample digestionAlternate Protocol: Automated sample digestionBasic Protocol 2: Reversed‐phase chromatographic separation and detection by HRAM‐MS (RPLC‐HRAM MS)Basic Protocol 3: Data processing and reporting

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Section: Introductionmentioning

confidence: 99%

Multi‐Attribute Method (MAM) Analytical Workflow for Biotherapeutic Protein Characterization from Process Development to QC

Millán‐Martín,

Jakes,

Carillo

et al. 2023

Current Protocols

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“…Given that oxidation can occur at various stages of antibody development, it is crucial to establish a sensitive and reliable method for monitoring oxidative variants. Several physicochemical methods are available for determining oxidative variants, including size exclusion chromatography (SEC), , cation exchange chromatography (CEX), − liquid chromatography–mass spectrometry (LC-MS), − reversed-phase high-performance liquid chromatography (RP-HPLC), and hydrophobic interaction chromatography (HIC). RP-HPLC is commonly used for separating oxidized variants, , but it is a denaturing method and cannot provide native oxidation variants for functional studies.…”

mentioning

confidence: 99%

In-Depth Characterization for Methionine Oxidization in Complementary Domain Region by Hydrophobic Interaction Chromatography

Tang,

Geng,

Zhang

et al. 2024

ACS Pharmacol. Transl. Sci.

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The oxidation of the complementarity-determining region (CDR) in monoclonal antibodies (mAbs) is a critical quality attribute that can affect the clinical efficacy and safety of recombinant mAb therapeutics. In this study, a robust hydrophobic interaction chromatography (HIC) method was developed to quantify and characterize CDR oxidation variants in mAb-A by using a Proteomix Butyl-NP5 column. The HIC analysis revealed oxidation variants that eluted earlier than the main species with weaker hydrophobicity. It was found that Met 105 in the CDR was more susceptible to oxidation. Additionally, it was noted that the oxidation of Met 105 on a single heavy chain resulted in elution at a distinct position compared to the oxidation on two heavy chains. This observation led to the fractionation and enrichment of the oxidized variants for further evaluation of their biofunction. The study also demonstrated that the oxidation of Met 105 did not impact the antigen-binding capacity but significantly reduced the PD-1/PD-L1 blockade activity of mAb-A. The HIC method, which was employed to quantify CDR oxidation, underwent validation and was subsequently utilized for stability studies as well as for assessing the similarity between mAb-A and its reference product.

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An improved workflow for the development of MS-compatible liquid chromatography assay purity and purification methods by using automated LC Screening instrumentation and in silico modeling

Manheim,

Singh,

Aggarwal

et al. 2024

Anal Bioanal Chem

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Development, validation, and implementation of a robust and quality control-friendly focused peptide mapping method for monitoring oxidation of co-formulated monoclonal antibodies

Cited by 9 publications

References 31 publications

Multi‐Attribute Method (MAM) Analytical Workflow for Biotherapeutic Protein Characterization from Process Development to QC

Multi‐Attribute Method (MAM) Analytical Workflow for Biotherapeutic Protein Characterization from Process Development to QC

In-Depth Characterization for Methionine Oxidization in Complementary Domain Region by Hydrophobic Interaction Chromatography

An improved workflow for the development of MS-compatible liquid chromatography assay purity and purification methods by using automated LC Screening instrumentation and in silico modeling

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