In the last decades numerous proteins and peptides have been proven as promising human therapeutics for treatment of various pathogenic states. Although very effective and safe, these macromolecules share a number of properties, which do not classify them as optimal drugs. These, so called first-generation biopharmaceuticals, which are protein drugs mimicking the natural protein molecules, frequently display low stability and solubility, short elimination half-life, susceptibility to proteolytic enzymes, immunogenicity, toxicity and other properties that can be improved by covalent attachment of polyethylene glycol chain(s) to a protein, usually called PEGylation. Although pegylation of protein is usually accompanied by decrease of biological activity (measured ''in vitro''), this drawback is successfully compensated ''in vivo'' by improved pharmacokinetic profile. Extension of elimination half life is due to increased hydrodynamic radius of pegylated conjugate.
We have prepared a set of well defined pegylated conjugates of interferon alpha-2b (expressed in E. coli) with various PEGs attached to the protein N-terminus and we used prepared conjugates in various studies. Use of N terminal site specific conjugation enabled us to avoid numerous positional isomers with different physiochemical and biological properties and we were able to study conjugates regarding only PEG size and shape. Specificity of the attachment was confirmed by peptide mapping and mass spectroscopy. For characterization, various methods have been used, not only common methods for estimating molecular weights and sizes, such as size exclusion chromatography (SE-HPLC), electrophoretic mobility in polyacrylamide gel (SDS-PAGE) and dynamic light scattering (DLS), but also cation exchange (CE-HPLC) and reverse phase chromatographies (RP-HPLC). Our SE-HPLC experiments have not revealed any size differences among linear and branched PEGs of the same nominal weight. Our DLS experiments have shown smaller hydrodynamic radii for branched conjugates, which are indicative of more compact structure. CE-HPLC, which is based on ion interactions, has revealed linear relationship between the retention time and logarithm of molecular weight for linear IFN-PEG conjugates. Theoretically all our isolated conjugates have the same overall net charge, but obviously increased PEG size and branched structure have distinct influence on retention on cation exchange column. This is probably caused by PEG which is shielding the protein surface and lowers interaction of conjugate with matrix. Pegylated proteins are expected to be very hydrophilic due to relative high binding of water by PEG moiety; however RP-HPLC analysis resulted in increased retention of conjugates. Retention time of the conjugates depended only on PEGs´ size.
In the second part of our research we studied conjugates´ relationship between their structure and function focused on the influence of molecular weight and shape of PEGylated conjugates on relative biological activity ''in vitro'' as well as on pharmacokinetic behaviour in animal model (rats). ''In vivo'' study on rats showed that pharmacokinetic profiles for Pegasys® (non selectively pegylated interferon α-2a with 40 kDa branched PEG) and our conjugate with 45 kDa branched PEG are comparable. Distinctive linear relationship between mentioned analytical methods and ''in vitro'' biological activity for N-terminally pegylated IFN was established only for CE-HPLC. This finding shows that shielding of protein surface is the main cause that affects ''in vitro'' biological activity. Another interesting conclusion is that CE-HPLC is a useful tool for prediction of ''in vitro'' biological activity for a set of site specific monopegylated conjugates.
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