New versatile approach for analysis of PEG content in conjugates and complexes with biomacromolecules based on FTIR spectroscopy.

Abstract

Here we report a new approach based on FTIR-spectroscopy for determining the degree of PEGylation in biomolecules. We show that the PEG COC peak (at 1089cm−1) is the main analytically valuable band in IR spectra of PEG-containing systems: it is narrow and highly intense, it is well distinguished from absorption bands of other principal functional groups of proteins and other biopolymers (carbonyl, amide, hydroxyl etc), and therefore is easily identified in the IR spectra. The proposed method is therefore “reagent-free” and allows for fast and accurate determination of the PEGylation degree of biomolecules as well as the structural characteristics of bioconjugates from a single FTIR spectrum. The measurement is not dependent on PEG polymerization degree or branching and can be applied in a wide pH range, making it a convenient replacement of laborious and unreliable methods. The developed approach was successfully used to study the range of PEG-containing covalent conjugates with chitosan and non-covalent complexes with anionic liposomes. The composition of PEG-chitosan conjugates as well as their storage stability was determined by the method based on FTIR-spectroscopy. In the case of non-covalent complexes, not only PEG content, but also the binding constants of PEG-containing ligands to the liposome membrane were evaluated with this approach. The results obtained by the FTIR method were confirmed by DLS and zeta-potential experiments where the formation of electrostatic complex was monitored by the increase in the particle radius (from 80nm to 105nm) and zeta-potential neutralization (from −20mV to −12mV). Direct comparison of the results of FTIR approach with that of TNBS or OPA titration methods shows very good agreement between the measurements, with the FTIR method showing much lower deviation.