Method Development Tools for More Efficient Screening of Biopharmaceutical Method Conditions Using the ACQUITY Arc Bio SystemPart 1 of 2: Screening Multiple RPLC Columns for High Throughput Analysis

Importance of the Topic

Reversed-phase liquid chromatography (RPLC) remains a cornerstone in biopharmaceutical analysis, yet selecting the optimal column chemistry and mobile phase conditions can be laborious. Efficient screening of multiple chemistries accelerates method development for peptide mapping, impurity profiling, and quality control of complex biomolecules.

Objectives and Study Overview

This study demonstrates how the Waters ACQUITY Arc Bio System streamlines high-throughput screening of four RPLC columns in a single sample queue. Using a peptide standard mix and a NIST monoclonal antibody (mAb) tryptic digest, the work evaluates chromatographic performance under two common mobile phase additives—trifluoroacetic acid (TFA) and formic acid (FA)—and monitors targeted peptides via an integrated mass detector.

Methodology

The protocol employs column switching valves to cycle through four 4.6×150 mm RPLC columns: BEH C18 XP, CSH C18 XP, Peptide HSS T3, and CORTECS C18. Two gradients were used: a 20-minute ramp (0.5–45% acetonitrile) for a MassPREP peptide mixture and a 60-minute ramp for the NIST mAb digest. Each condition was tested with 0.1% TFA and 0.1% FA in both aqueous and organic phases. Peak capacity was calculated at 4σ to compare chromatographic resolution, and extracted ion chromatograms (XICs) tracked selectivity differences among eight peptide targets.

Instrumentation Used

• ACQUITY Arc Bio System with CM-A column manager and CM-Aux auxiliary module for four-column switching
• Quaternary pump for solvent delivery (water and acetonitrile with 0.1% TFA or FA)
• 2489 UV/Visible detector at 214 nm
• ACQUITY QDa Mass Detector (ESI+ full scan, 300–1250 Da)
• Empower 3 Chromatography Data Software for automated valve switching and data analysis

Results and Discussion

Average peak capacities were consistently higher in TFA across all four chemistries, with the highest values observed on HSS T3 and lowest on CORTECS. FA yielded increased MS sensitivity but reduced chromatographic resolution. Selectivity trends revealed coelution of key peptides (e.g., L10/H26) under FA on all columns, while TFA separated these species effectively. Certain peptides (enolase T37 and melittin) reversed elution order between TFA and FA, and some chemistries like CORTECS showed coelution in FA, underscoring the need for tailored column selection based on target analytes.

Benefits and Practical Applications

By integrating column switching and mass detection, the workflow:

• Reduces manual intervention and conditioning time by screening four columns in one injection series
• Enables rapid identification of the optimal column/additive combination for targeted peptide separation
• Combines UV and MS data to balance chromatographic resolution and detection sensitivity

Future Trends and Potential Applications

Advances may include expansion of column managers to accommodate additional chemistries, integration of higher-resolution mass detectors for full sequence coverage, and automated buffer preparation for pH and ionic strength screening. Applying similar workflows to size-exclusion and ion-exchange chromatography can further accelerate overall biopharmaceutical method development.

Conclusion

The ACQUITY Arc Bio System with column switching and QDa detection offers a streamlined approach for high-throughput RPLC method screening. By evaluating peak capacity and selectivity under TFA and FA, analysts can make data-driven decisions on column and additive selection, significantly expediting peptide mapping workflows.

References

Koza SM, Chambers EE. Selecting a Reversed-Phase Column for the Peptide Mapping Analysis of a Biotherapeutic Protein. Waters Application Note 720005924EN; 2017.