METHOD TRANSFER AND ROUTINE ANALYSIS OF PROTEIN AND PEPTIDE-BASED DRUG PRODUCTS USING A BIOCOMPATIBLE UHPLC SYSTEM

Importance of the Topic

Biologics represent a rapidly growing segment of the pharmaceutical market, driving demand for reliable analytical methods in development and quality control. Legacy HPLC workflows, while established, may limit resolution, throughput and robustness when handling complex protein and peptide-based drug products. Upgrading to biocompatible UHPLC platforms can significantly improve separation performance and system longevity under challenging mobile phase conditions.

Objectives and Study Overview

This work aimed to transfer established protein and peptide separations from conventional HPLC to modern biocompatible UHPLC and UPLC systems while demonstrating method equivalency, performance gains and routine applicability. A range of chromatographic modalities—reversed-phase LC, ion exchange (IEX), hydrophobic interaction (HIC), size-exclusion (SEC) and subunit analysis—were evaluated across multiple instrument platforms.

Methods and Instrumentation

A preserved reversed-phase peptide standard method was run on three systems using identical column chemistries to isolate the impact of instrument design on separation quality. Key steps included:

• Transfer of an IEX method for infliximab charge variant analysis
• HIC profiling of protein standard mixtures
• SEC evaluation of monoclonal antibody fragments
• Subunit RPLC analysis from a NIST mAb digested standard
• Peptide mapping of trastuzumab with complementary mass detection

Instrumentation Used

The study compared:

• Agilent 1260 Infinity I Bio-inert Quaternary HPLC System
• Waters ACQUITY Arc Bio UHPLC System
• Waters H-Class Bio UPLC System
• Columns including Protein-Pak Hi Res SP, XBridge BEH C18, BioResolve RP mAb, Protein-Pak Hi Res HIC, XSelect CSH C18 and XBridge Protein BEH SEC
• QDa single-quadrupole mass detector for peptide map confirmation

Results and Discussion

Biocompatible UHPLC platforms delivered enhanced chromatographic performance and reliable method transfer:

• Resolution of key peptide pairs improved from 1.7 on HPLC to 2.7 and 3.1 on UHPLC and UPLC, while average peak capacity rose from 118 to 170 and 196.
• IEX charge variant profiles for infliximab exhibited retention time deviations of <0.05 and maintained relative peak order between systems.
• HIC separations showed retention time standard deviation <0.007 across six replicates, confirming high repeatability under high salt conditions.
• SEC of trastuzumab fragments achieved retention time RSD <0.004 for all three peaks, demonstrating robust size-exclusion performance.
• Subunit RPLC peak areas were conserved within <0.3% between HPLC, UHPLC and UPLC systems, confirming method equivalency.
• Peptide mapping with integrated QDa mass detection provided complementary mass confirmation, enabling confident identification of CDR peptides.

Benefits and Practical Applications of the Method

Modern biocompatible UHPLC systems offer multiple advantages for routine bioanalytical laboratories:

• Improved resolution, sensitivity and throughput for complex protein and peptide samples
• Corrosion-resistant flow paths that withstand high salt concentrations and extreme pH mobile phases
• Seamless transfer of legacy methods without extensive reoptimization
• Unified column particle size for subunit analysis across platforms
• Enhanced confidence in results through orthogonal optical and mass detection

Future Trends and Applications

Emerging directions include:

• Integration of high-resolution mass spectrometry for deeper characterization of biotherapeutics
• Automation and digital workflows to streamline method transfer and validation
• Development of novel bio-inert materials and stationary phases for challenging separations
• Expansion of UHPLC applications in real-time release testing and continuous manufacturing environments

Conclusion

This study demonstrates that upgrading legacy HPLC methods to biocompatible UHPLC and UPLC platforms yields significant improvements in resolution, peak capacity and robustness, while maintaining method equivalency and repeatability. Such modern systems support reliable routine analysis of protein and peptide-based drug products and position laboratories for future advancements in biopharmaceutical quality control.

References

• ICH Q10 Pharmaceutical Quality System. 2008.