Coupling Reactions, Conjugation
PEGylation Analysis and Method Verification
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BOC Sciences provides comprehensive PEGylation analysis services in terms of qualitative assessment, quantitative assessment and in vivo & in vitro characterization.
After PEGylation, conjugates will show changes in physiochemical properties, such as surface charge, hydrodynamic diameter, hydrophilicity and protein binding capability. By monitoring changes in these physiochemical properties, PEGylation can be confirmed.
Qualitative Assessments
| Method | Analysis Content |
|---|---|
| Purity of PEGylated proteins and the extent of PEGylation |
| Hydrodynamic diameter, zeta potential and size distribution of PEGylated species |
| The size of PEGylated proteins |
| Secondary and tertiary structural conformation of native and PEGylated proteins. |
| Qualitative assessment of PEGylation relies on the presence of CH2 and C-O-C absorption bands. |
| The surface charge of PEGylated species |
| Qualitative assessment of PEGylation relies on the presence or absence of specific chemical moieties |
| Qualitative assessment of the structural conformations of the lipid PEGylated nanocarriers |
| The morphology of PEGylated nanocarriers |
| Identification the density and spatial distribution of PEG polymers on the surface of nanocarriers |
| Identification the density and spatial distribution of PEG polymers on the surface of nanocarriers |
| Evaluation of the degree of PEG substitution |
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Quantitative Assessments
| Method | Analysis Content |
|---|---|
| The amount of PEG associated with colloidal carriers |
| The concentration of PEGylated proteins in serum samples |
| The concentration and localization of PEG chains |
| The amount of attached PEG polymer |
| The molecular mass of PEGylated species |
| The molecular mass of PEGylated species |
| The average molecular weight of PEGylated species and degree of PEGylation |
| The average molecular weight of PEGylated species and degree of PEGylation |
| Changes of elemental composition on the conjugate surface before and after PEGylation |
| Determination of PEG content and conformation |
| The content of PEG |
| The quantitative measurement of metal-containing PEGylated pharmaceuticals or nanoparticles |
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In addition to using the analytical measurement mentioned above alone, we are also able to utilize the combined techniques, such as combination of ELSD and LC-MS, NIR spectroscopy coupled with principal component analysis (PCA) for characterization of hydrophilic/hydrophobic balance of PEGylated nanoparticles.
In Vitro and In Vivo Characterizations
- In vitro methods include measurement of protein adsorption, measurement of complement consumption, macrophage uptake studies, and the analysis of release rate or content stability.
- The most general in vivo assessments include pharmacokinetic profiling (Radiolabeling, Fluorescence labeling, Bioactivity Assay), tissue distribution studies and vascular permeability studies.
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Reference
- Santos, J. H. P. M.; Torres-Obreque, K. M.; et al. Protein PEGylation for the design of biobetters: from reaction to purification processes. Brazilian Journal of Pharmaceutical Sciences 2018, 54 (SPE).
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