Absolute quantification of proteins by ICP-MS/MS

Protein content determination is a critical quality attribute for biopharmaceutical products, requiring methods that deliver high accuracy, precision, and robustness, and that can be used at different stages of development. However, existing approaches may be limited by the need for reference standards, complex sample preparation, or insufficient sensitivity in challenging matrices.

Since 2015, Quality Assistance has been offering a method based on sulphur-specific ICP-MS/MS detection combined with ³⁴S/³²S isotope dilution quantification. While recognised for its high precision and accuracy, its applicability was initially limited by the relatively large sample quantity required, particularly for early-stage projects.

Building on extensive experience, the method has been optimised to reduce sample uptake by a factor of ten. This application note presents an overview of the optimised method together with a demonstration of its performance through a partial validation study.

The full technical note is available for download below.

Method performance highlights

• Absolute quantification using ³⁴S/³²S isotope dilution ICP-MS/MS, without the need for protein-specific reference standards
• Tenfold reduction in sample uptake compared to the original method
• Demonstrated high precision and accuracy, with %RSD values below 1% and recoveries between 100 and 102% in most conditions
• Excellent method linearity, with correlation coefficients close to 1 across the working range
• Low limit of quantification (0.14 µg S/g), enabling sensitive protein determination

What this means for your projects?

This optimised ICP-MS/MS approach provides a reliable framework for protein quantification in biopharmaceutical development and quality control environments. It addresses key analytical challenges while maintaining high performance standards.

• Enables absolute protein quantification independent of specific reference standards
• Supports early-stage development thanks to reduced sample requirements
• Ensures high reproducibility and robustness across different sample types
• Allows accurate quantification in complex matrices, including those containing various excipients
• Improves confidence in data for quality control and standard qualification

This method contributes to more reliable and consistent protein quantification throughout development workflows.

Applications

• Biological reference standard qualification
• Protein content determination in drug substances and drug products
• Determination of protein molecular extinction coefficients (in combination with UV spectroscopy)
• Analysis of proteins in complex formulation matrices