Multi-Attribute Methods for Biopharmaceutical Analysis

Significance of topic

Multi‐attribute methods (MAM) use LC‐MS to directly quantify multiple product quality attributes (PQAs) and detect impurities in biotherapeutics. Compared to legacy chromatographic assays, MAM offers enhanced sensitivity, specificity, and throughput, making it vital for Quality by Design (QbD), process monitoring, and regulatory compliance.

Objectives and overview of studies

This collection of application notes demonstrates end‐to‐end workflows for peptide‐based and subunit‐level MAM across Waters platforms. Key topics include:

• Peptide MAM using high‐resolution MS and waters_connect informatics
• ACQUITY QDa‐based multi‐attribute assays in Empower CDS
• Comparative peptide mapping of innovator and biosimilar mAbs on the Xevo G3 QTof
• Subunit MAM on BioAccord and Vion systems with waters_connect/UNIFI
• INTACT Mass™ App for high‐throughput intact mass confirmation and purity profiling
• Fully automated Protein A capture and IdeS/DTT subunit prep using Andrew+ robot

Methodology and instrumentation

Sample preparation:

• Denaturation, reduction, alkylation, tryptic digest for peptide MAM
• FabRICATOR (IdeS) digestion and DTT reduction for subunit MAM
• Protein A magnetic bead purification from cell culture media
• Automated pipetting on Andrew+ for high‐throughput prep

Chromatography and detection:

• UPLC systems: ACQUITY UPLC I‐Class/Plus, Premier, H‐Class; BioAccord LC
• Columns: CSH C18, BEH C4, Protein BEH C4, RP mAb Polyphenyl
• Detectors: ACQUITY RDa, ACQUITY QDa, Xevo G3 QTof, Vion IMS QTof, TUV

Data processing:

• waters_connect platform: UNIFI App, Peptide MAM App, INTACT Mass™ App, LC‐MS Toolkit
• Empower CDS for QDa‐based workflows

Main results and discussion

• Peptide MAM (BioAccord/HRMS): Integrated workflows delivered product ID, targeted quantification, and new peak detection with <10% RSD and >3 orders dynamic range.
• ACQUITY Premier (HPS surfaces): Metal‐sensitive acidic peptides showed 2× recovery gains, improved peak shapes, and lower RSD in oxidation and deamidation quantitation.
• Biosimilar mAb mapping (Xevo G3 QTof): >95% sequence coverage; quantified oxidation, deamidation, C‐term Lys clipping, and glycoform profiles in innovator vs three biosimilars.
• QDa‐based multi‐attribute assays (Empower): Single LC run monitored CDR peptides, oxidation, deamidation, and glycopeptides via derived channels and SIRs.
• Subunit MAM (BioAccord/Vion): 15‐min LC‐MS of IdeS subunits enabled robust glycosylation, glycation, sequence‐variant, and oxidation profiling with <8% RSD.
• INTACT Mass App: Automated intact mass screening of 6 mAbs in 48‐well plates (2.5‐min desalting method) yielded <10 ppm mass errors and rapid purity assessment; subunit standard deconvolution at 5 ppm.
• Automated subunit prep (Andrew+): Protein A capture and IdeS/DTT digestion directly from spent cell culture media in 96‐well format followed by 4.5‐min LC‐MS subunit screening; matched manual prep and control samples.

Benefits and practical applications

• Streamlined sample‐to‐report workflows for both peptide and subunit MAM in development, manufacturing, and QC.
• High reproducibility across instruments, <5%–10% RSD for low‐abundance attributes.
• High throughput: LC gradients of 2.5–15 min, parallel processing in waters_connect, and automated sample prep.
• Accessible to non‐MS experts through SmartMS, simplified method creation, and compliance‐ready informatics.
• Enables direct monitoring of CQAs and real‐time process decisions, reducing assay bottlenecks.

Future trends and potential uses

• Wider adoption of MAM in regulated QC labs as orthogonal or replacement assays for optical methods.
• Integration with PAT and real‐time bioprocess monitoring for adaptive control.
• Extension to advanced formats: ADCs, bispecifics, fusion proteins, and glycoengineered antibodies.
• Further automation of upstream sample prep, LC‐MS acquisition, and AI‐assisted data analysis.
• Data‐driven predictive analytics for process development and stability modeling.

Conclusion

Advanced LC‐MS multi‐attribute workflows, combining peptide and subunit MAM strategies, high‐performance separations, robust MS platforms, and integrated informatics, deliver comprehensive, reproducible, and high‐throughput analysis of biotherapeutic attributes across the product lifecycle.

Instrument section

• BioAccord LC‐MS System
• ACQUITY UPLC I‐Class/Plus and Premier
• ACQUITY RDa and QDa Detectors
• Xevo G3 QTof
• Vion IMS QTof
• Andrew+ Pipetting Robot (Protein A automation)

Reference

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