Identifying Monoclonal Antibody Mutation Sites Using the Agilent 1290 Infinity II 2D-LC Solution with Q-TOF LC/MS

Significance of the Topic

Monoclonal antibodies are central to modern biopharmaceuticals but their large size and heterogeneity demand advanced analytical techniques to ensure safety and efficacy. Patent expirations and regulatory requirements drive the development of biosimilars, which must match originator products in amino acid sequence and structure. Two dimensional liquid chromatography coupled with high resolution mass spectrometry offers the resolving power needed to detect subtle sequence variants.

Objectives and Study Overview

This study aims to compare an infliximab originator with a candidate biosimilar by applying the Agilent 1290 Infinity II two dimensional liquid chromatography solution in combination with quadrupole time of flight LCMS. The goal is to identify any mutations or sequence differences that could affect biosimilarity.

Methodology

Sample preparation involved reduction of disulfide bonds, alkylation of cysteines, and tryptic digestion. Middle up analysis employed reversed phase LC on a C4 column with UV detection and accurate mass MS. Peptide mapping was performed using two orthogonal two dimensional methods: reversed phase×reversed phase at different pH values and strong cation exchange×reversed phase, both with heart cutting and modulation. Data dependent MSMS was acquired on the fly to confirm sequence changes.

Instrumentation

• Agilent 1290 Infinity II High Speed Pumps
• Agilent 1290 Infinity II Multisampler and Multicolumn Thermostat
• Agilent 1290 Infinity II Diode Array Detector
• Agilent 6530 and 6540 Quadrupole Time of Flight LCMS systems
• Agilent OpenLab CDS and MassHunter software
• GC Image LC×LC Edition Software

Key Results and Discussion

Middle up RPLCMS revealed a 99 Da mass increase on the heavy chain of the biosimilar, corresponding to a shift in glycoforms. RPLC×RPLC peptide maps showed disappearance of heavy chain peptides SLSLSPG and SLSLSPGK, replaced by SLSLSPGI, indicating a lysine to isoleucine substitution (+113 Da) and a threonine to serine replacement (−14 Da) at another site. SCX×RPLC mapping confirmed altered chromatographic behavior due to net charge differences. On‐the‐fly MSMS and RT PCR sequencing validated two point mutations (ACC to AGC and AAA to AUA), explaining the observed mass shifts.

Benefits and Practical Applications

This integrated 2D LCMS platform offers high resolution and orthogonality to detect even single amino acid substitutions in monoclonal antibodies. It supports biosimilar development, clone selection, and quality control by ensuring sequence integrity and comparability with originators.

Future Trends and Applications

Ongoing advancements may include faster modulation, novel separation chemistries, and automated data interpretation using machine learning. Expanded applications to other complex biologics and integration with real time process monitoring are anticipated.

Conclusion

The Agilent 1290 Infinity II 2D LC solution with QTOF MS proved effective in detecting and characterizing point mutations in a candidate infliximab biosimilar. This approach enhances analytical confidence in biopharmaceutical comparability assessments.

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