Refinement of HR Multi-Attribute Method from Sample Preparation to Data Analysis

Significance of the Topic

The high-resolution multi-attribute method (HR MAM) leverages accurate-mass Orbitrap technology to monitor critical quality attributes of biotherapeutics with high specificity and sensitivity. Robust workflows spanning sample preparation to data analysis are essential for reliable characterization, impurity detection, and regulatory compliance in biopharmaceutical development and quality control.

Study Objectives and Overview

This work aims to refine the HR MAM workflow by optimizing each stage from automated protein digestion and chromatographic separation to software-driven peak detection and quantitation. Key focus areas include evaluating digestion protocols, assessing column performance, and enhancing data analysis features.

Methodology and Instrumentation

Sample digestion was automated using low-pH SMART Digest kits on a KingFisher Duo Prime system, reducing preparation time to under one hour. In-solution trypsin digestion and magnetic bead protocols both achieved complete sequence coverage of the NISTmAb reference material. Peptide separations employed a Vanquish Horizon UHPLC with four Thermo Scientific columns (Accucore Vanquish, Accucore Vanquish Phoenix, Acclaim Vanquish, Hypersil GOLD). Mass spectrometry data were acquired on a Q Exactive Plus Hybrid Quadrupole-Orbitrap at 140 000 resolution, processed in Chromeleon CDS v7.2.10 and BioPharma Finder v3.2.

Main Results and Discussion

The SMART Digest workflow halved digestion time compared to traditional in-solution protocols, with minor increases in missed cleavages that can be mitigated by adjusting enzyme loading. All columns delivered retention reproducibility below 2 % RSD for selected peptides; Hypersil GOLD and Acclaim Vanquish showed superior retention of early-eluting hydrophilic peptides. Separation of deamidated species varied by column chemistry, with Acclaim Vanquish PA II eluting deamidates after native forms and Hypersil GOLD resolving most deamidation peaks. Chromeleon CDS’s flexible retention-time windows, component matching algorithms, smoothing filters, and peak detection methods enabled consistent integration of challenging isomeric and modified peptides.

Benefits and Practical Applications

• Automated, rapid digestion reduces hands-on time and sample variability.
• High-resolution LC-MS monitoring ensures accurate quantitation of CQAs and impurity profiling.
• Optimized column selection enhances chromatographic resolution of critical modifications.
• Advanced software algorithms deliver reliable new peak detection and consistent integration.

Future Trends and Opportunities

• Integration of artificial intelligence for adaptive peak detection and accelerated data review.
• Development of novel proteases and digestion platforms for broader protein coverage.
• Emerging column chemistries to improve selectivity for challenging analytes.
• Enhanced automation and data connectivity for streamlined quality control workflows.

Conclusion

Refinement of the HR MAM workflow enhances consistency, throughput, and data quality, facilitating robust monitoring of critical quality attributes throughout biopharmaceutical development and quality control. Continued advances in sample preparation, chromatography, and informatics will further streamline biotherapeutic characterization.

References

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• Rogstad S et al. Multi-attribute method for quality control of therapeutic proteins. Anal Chem. 2019;91:14170.
• Ren D et al. An improved trypsin digestion method minimizes digestion-induced modifications on proteins. Anal Biochem. 2009;392:12.