Prepping Biosimilars

Significance of the Topic

Monoclonal antibody biosimilars are an emerging class of therapeutics offering cost-effective alternatives to innovator biologics. Their complex structure and sensitivity to manufacturing changes demand robust analytical strategies to ensure high similarity in structure, efficacy, and safety. Comprehensive characterization workflows can accelerate development while satisfying regulatory requirements for biosimilarity.

Study Objectives and Overview

This eBook investigates analytical approaches for characterizing trastuzumab and its biosimilar candidates. It aims to outline key analytical techniques—ranging from affinity chromatography to high-resolution liquid chromatography and mass spectrometry—that support clone selection, glycan and charge variant profiling, and overall comparability assessment.

Methods

The workflow begins with Protein A affinity chromatography to isolate mAbs directly from Chinese hamster ovary cell culture supernatant and measure antibody titer. Subsequent steps include

• Reversed-phase liquid chromatography (RPLC) for intact protein mass determination and subunit analysis
• Peptide mapping via RPLC-MS/MS to confirm sequence, modifications, and sequence variants
• Hydrophilic interaction chromatography (HILIC) for fluorescently labeled N-glycan profiling
• Ionic exchange chromatography (IEX) for quantifying charge variants and deamidation products
• Size exclusion chromatography (SEC) for monitoring aggregation and low molecular weight fragments

Used Instrumentation

The study leverages industry-standard platforms including:

• Agilent 1290 Infinity UHPLC system
• Agilent 6540 Accurate-Mass Q-TOF LC/MS
• Bio-Monolith Protein A monolithic columns
• ZORBAX RRHD 300SB-C8 and AdvanceBio RP-mAb columns
• AdvanceBio Peptide Mapping and glycan mapping columns with sub-2 micron and superficially porous particles

Main Results and Discussion

Affinity chromatography enabled rapid determination of mAb titer with high precision and linearity across relevant concentrations. RPLC-QTOF mass analysis revealed glycoform distribution differences between originator and biosimilar, including undergalactosylation in select clones. Peptide mapping identified minor variants such as deamidation and lysine truncation. Charge variant analysis by WCX distinguished deamidated and sialylated species, and SEC detected differences in aggregate levels under stress conditions. Mirror plots facilitated direct visual comparison of molecular profiles.

Benefits and Practical Applications

These integrated analytical workflows support informed clone selection, media optimization to control glycosylation patterns, and robust comparability assessment. High-throughput, high-resolution methods reduce development timelines and provide confidence for regulatory submissions.

Future Trends and Possibilities

Emerging trends include increased automation of sample preparation, single-cell analysis for clone screening, real-time process monitoring, and incorporation of advanced bioinformatics and machine learning for data interpretation. Continued improvements in column technologies and MS detectors will further enhance throughput and sensitivity.

Conclusion

A multidimensional analytical strategy combining affinity capture, high-resolution chromatography, and accurate mass spectrometry is essential for comprehensive biosimilar characterization. These approaches enable detailed structural, glycosylation, charge, and aggregation profiling, thus ensuring biosimilar quality, safety, and efficacy.