A chapter in a book that combines various methods of extending the half-life of proteins. Our chapter deals with PEGylation and describes various aspects achieved by PEGylation. From half-life extension dependent on various PEG shapes and lengths to the proteolytic stability improvement of modified proteins, advantages and potential disadvantages of PEGylation, which are largely related to the potential accumulation of long PEG chains in the body.
Trends in preparation of PEGylated protein drugs strive for simple, fast and cheap processes, resulting in well-defined homogenous products. We investigated the on-column PEGylation of tumor necrosis alpha (TNF-alpha), where purification and conjugation were performed in one step by using Immobilized Metal Affinity Chromatography (IMAC). The same quality of the PEGylated product was obtained by the on-column approach starting from either the crude E. coli protein extract or the purified protein. In comparison to the PEGylation in solution the on-column approach resulted in more homogeneous PEGylated product.
Colloidal interactions between proteins determine the behavior and stability of globular proteins such as monoclonal antibodies (mAbs) against their propensity to cluster formation in solution. We study interactions between these proteins through their dilute solution behavior. Experiments to quantify intermolecular interactions were done using Dynamic and Static Light Scattering (DLS and SLS) in a high-throughput manner in parallel with zeta potential measurements with Laser Doppler Electrophoresis method (M3-PALS). This approach offers a rapid indirect determination of colloidal interactions through their measured second virial coefficient. Electrostatic part of the DLVO interaction was conveniently parameterized via the corresponding surface charge and/or surface potential, while the van der Waals interactions were parameterized via their Hamaker coefficient, both as functions of ionic strength and pH of the bathing solution. This parametrization of protein-protein interactions improves our understanding of mAb assembly and provides a means for its control by solution parameter variation. Additionally, our results also provide a consistency check and validation of applicability of the DLVO theory in mAbs solution assembly processes.
The determination of single high doses of active pharmaceutical ingredients (API) is used mostly to fulfill regulatory demands. Oral LD50 values in animals for over 300 API were compared to the minimal effective therapeutic doses (METD) in humans in order to find a correlation between animal and human data. The highest correlation between human METD and animal LD50 was found for the dog (R = 0.323), the lowest for the rat (0.287). It was determined that acute oral LD50 of rats have poor correlation with the METD, and cannot be used as a classification criteria into official acute toxic categories. Only 13% of API has been classified as fatal if swallowed according to the EU CLP regulation, none of the substances with very low therapeutic dose have been identified as EU CLP acute toxicity category 1. Substances with very low therapeutic doses, which could potentially have toxic effects in humans, are not identified with the use of oral LD50 and current classification system. We propose that the acute toxicity based on rat LD50 dose is not used as a basis for classification of pharmaceuticals, and that the METD is applied as basis for classification.