PEGylation Analysis and Method Verification

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
  • Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Purity of PEGylated proteins and the extent of PEGylation
  • Dynamic Light Scattering (DLS)
Hydrodynamic diameter, zeta potential and size distribution of PEGylated species
  • High Performance Size Exclusion Chromatography (HPSEC)
The size of PEGylated proteins
  • Circular Dichroism Measurement (CD)
Secondary and tertiary structural conformation of native and PEGylated proteins.
  • Fourier transform-infrared spectroscopy (FT-IR)
Qualitative assessment of PEGylation relies on the presence of CH2 and C-O-C absorption bands.
  • Zeta Potential Measurement
The surface charge of PEGylated species
  • 13C-Nuclear Magnetic Resonance (13C-NMR)
Qualitative assessment of PEGylation relies on the presence or absence of specific chemical moieties
  • 1H-Nuclear Magnetic Resonance (1H-NMR)
Qualitative assessment of the structural conformations of the lipid PEGylated nanocarriers
  • Transmission Electron Microscopy (TEM)
The morphology of PEGylated nanocarriers
  • Atomic Force Microscopy (AFM)
Identification the density and spatial distribution of PEG polymers on the surface of nanocarriers
  • Scanning Tunneling Microscopy (STM)
Identification the density and spatial distribution of PEG polymers on the surface of nanocarriers
  • Gel Permeation Chromatography (GPC)
Evaluation of the degree of PEG substitution
  • Many More …

Quantitative Assessments

Method Analysis Content
  • High Performance Liquid Chromatography (HPLC)
The amount of PEG associated with colloidal carriers
  • Enzyme-linked Immunosorbent Assay (ELISA)
The concentration of PEGylated proteins in serum samples
  • Colorimetric Assays
The concentration and localization of PEG chains
  • Gas Chromatography (GC)
The amount of attached PEG polymer
  • Mass Spectrometry (MS)
The molecular mass of PEGylated species
  • Liquid Chromatography Mass Spectrometry (LC-MS)
The molecular mass of PEGylated species
  • Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI–MS)
The average molecular weight of PEGylated species and degree of PEGylation
  • Electrospray Ionization Mass Spectrometry (ESI-MS)
The average molecular weight of PEGylated species and degree of PEGylation
  • X-Ray Photoelectron Spectroscopy (XPS)
Changes of elemental composition on the conjugate surface before and after PEGylation
  • Raman Spectroscopy
Determination of PEG content and conformation
  • Thermogravimetric analysis (TGA)
The content of PEG
  • Inductively Coupled Plasma Mass Spectrometry (ICP-MS)
The quantitative measurement of metal-containing PEGylated pharmaceuticals or nanoparticles
  • Many More …

*** 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

  1. 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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