Direct Quantification of PEGylation for Intact Bioconjugates and Nanoparticles by the Colorimetric Barium/Iodide...

Area of Science:

• Bioconjugation Chemistry
• Analytical Chemistry
• Materials Science

Background:

• Accurate determination of PEGylation degree is crucial for protein and peptide modification.
• Existing methods for quantifying methoxy poly(ethylene glycol) (mPEG) chains can be complex or lack versatility.
• A simple, multiplexable assay for PEGylation extent is highly desirable.

Purpose of the Study:

• To validate the barium-iodide-PEG complex assay for determining the PEGylation degree of protein bioconjugates.
• To assess the assay's utility for quantifying PEGylation on intact nanoparticles.
• To analyze the influence of mPEG properties on assay performance.

Main Methods:

• Utilized a colored barium-iodide-PEG complex for quantitative titration and gel staining.
• Applied the assay to a library of 54 mPEG-protein conjugates.
• Tested the assay on 10 different polymeric nanoparticle formulations.

Main Results:

• Successfully validated the barium-iodide-PEG assay for direct determination of PEGylation extent on protein bioconjugates.
• Demonstrated the assay's effectiveness for quantifying PEGylation on nanoparticles.
• Evaluated the impact of mPEG molecular weight, functional groups, and architecture on assay accuracy.

Conclusions:

• The barium-iodide-PEG complex assay offers a simple, versatile, and accurate method for determining PEGylation degree.
• This validated assay expands the application of PEGylation analysis to protein bioconjugates and nanoparticles.
• Practical guidelines and artifact discussion are provided to improve assay reproducibility and accuracy.