Unified and Versatile Multiplex Platform for Expedited Method Development and Comprehensive Characterization of...

Area of Science:

• Analytical Chemistry
• Pharmaceutical Science

Background:

• Peptide drug development requires agile and comprehensive analytical methods for rapid characterization.
• Current methods can be time-consuming and may not cover all critical quality attributes simultaneously.

Purpose of the Study:

• To develop a unified, versatile multiplex platform for expedited method development and routine simultaneous determination of multiple peptide quality attributes.
• To evaluate the platform's performance on various therapeutic peptide constructs, including macrocyclic and disulfide-constrained peptides.

Main Methods:

• Integration of automated size exclusion chromatography (SEC), reversed-phase liquid chromatography-reversed-phase liquid chromatography (RPLC-RPLC), and hydrophilic interaction liquid chromatography-charged aerosol detection (HILIC-CAD).
• Systematic study of mobile phase effects on impurity profiles using different SEC columns.
• Comprehensive impurity profiling using RPLC-RPLC with varied columns and mobile phases.
• Development of a HILIC-CAD method for trifluoroacetic acid (TFA) quantification.

Main Results:

• A prototype MaxPeak Premier SEC column enabled impurity separation (>2.0% area), outperforming a standard BEH diol column.
• RPLC-RPLC facilitated expedited method development and comprehensive impurity profiling across 12 conditions.
• A HILIC-CAD method was successfully developed for TFA quantification in peptide products.

Conclusions:

• The developed multiplex platform significantly expedites analytical method development for peptide drug candidates.
• The platform enables simultaneous, routine determination of multiple quality attributes, enhancing characterization efficiency.
• This approach supports the development of complex peptide therapeutics by providing robust impurity profiling and quantification.