Raman Spectroscopy for Characterizing Monoclonal Antibody Reduction: A Process Analytical Technology Approach for Antibody-Drug Conjugation Process Development.

Authors

David Pople, Zhenshu Wang, Anton Kozyryev, Bhumit Patel, Emmanuel Appiah-Amponsah, Hanzhou Feng

Published in

Pharmaceutical research. Jul 14, 2026. Epub Jul 14, 2026.

Abstract

Controlled disulfide bond reduction is critical in antibody-drug conjugate (ADC) manufacturing, enabling site-specific conjugation and desired drug-to-antibody ratios. Current characterization relies on time-consuming offline capillary electrophoresis, creating bottlenecks in process development where multiple conditions must be rapidly screened. This study evaluates Raman spectroscopy as a process analytical technology (PAT) to elucidate reduction kinetics and accelerate optimization for ADC development.
A Design of Experiments approach investigated TCEP-mediated reduction of an IgG1 antibody under varying TCEP/mAb ratios (5-15) and pH conditions (5.5-7.5). Raman spectra were collected throughout reduction reactions. Principal component analysis (PCA) characterized reduction kinetics, while partial least squares (PLS) regression quantified fragment formation against non-reduced capillary electrophoresis sodium dodecyl sulfate (nrCE-SDS) measurements.
PCA effectively captured reduction kinetics, with PC1 trajectories correlating strongly with heavy chain (HC) formation measured by nrCE-SDS. The analysis revealed pH-dependent TCEP saturation effects: higher pH showed convergent kinetics at elevated ratios while lower pH maintained ratio-dependent rates. PLS models successfully predicted HC formation, demonstrating potential for endpoint detection despite the interchain disulfide bonds representing only ~ 0.2% of total protein mass.
Raman spectroscopy with chemometric analysis provides valuable insights for ADC process development. PCA enables rapid screening of reduction conditions without nrCE-SDS confirmation, reducing analytical burden during early development. Unlike offline nrCE-SDS, Raman offers potential for real-time online PAT implementation. Future work focusing on narrower operating ranges could enhance model performance for manufacturing applications.

PMID:
42449032
Bibliographic data and abstract were imported from PubMed on 15 Jul 2026.

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