Screening of Polysorbate-80 Composition by High Resolution Mass Spectrometry with Rapid H/D Exchange

Area of Science:

• Analytical Chemistry
• Biopharmaceutical Science

Background:

• Polysorbate (PS) is crucial for stabilizing protein drugs in biotherapeutics.
• Commercial PS is a complex mixture, and its heterogeneity impacts drug stability.
• Characterizing PS composition is analytically challenging.

Purpose of the Study:

• To develop a high-throughput screening protocol for profiling polysorbate-80 (PS-80) heterogeneity.
• To utilize high-resolution mass spectrometry (HRMS) coupled with hydrogen/deuterium (H/D) exchange for detailed PS-80 analysis.
• To identify and differentiate PS-80 species, including isomers, based on their structural characteristics.

Main Methods:

• High-throughput screening protocol employing HRMS.
• Rapid H/D exchange in deuterated methanol.
• Utilizing accurate mass measurements and H/D exchange-induced mass shifts for species identification.
• Automated searching against a comprehensive PS-80 molecular mass database.
• Kendrick Mass Defect plots for data visualization.

Main Results:

• Successfully profiled over 180 PS-80 species in a high-throughput manner.
• Confirmed molecular identities and differentiated isomers using H/D exchange mass shifts.
• Eliminated the need for chromatographic separation or tandem mass spectrometry for initial profiling.
• Demonstrated the protocol's efficiency in characterizing complex PS-80 mixtures.

Conclusions:

• The developed HRMS and H/D exchange protocol provides an efficient method for characterizing PS-80 heterogeneity.
• This approach enhances confidence in molecular identity assignment and isomer differentiation.
• The high-throughput nature of the protocol facilitates comprehensive analysis of polysorbate excipients.
• This method can aid in understanding the impact of PS composition on biotherapeutic stability.