International audiencePEGylation has been widely used as a post-production modification methodology for improving biomedical efficacy and physicochemical properties of therapeutic proteins since the first PEGylated product was approved by Food and Drug Administration in 1990. Applicability and safety of this technology have been proven by use of various PEGylated pharmaceuticals for many years. It is expected that PEGylation as the most established technology for extension of drug residence in the body will play an important role in the next generation therapeutics, such as peptides, protein nanobodies and scaffolds, which due to their diminished molecular size need half-life extension. This review focuses on several factors important in the production of PEGylated biopharmaceuticals enabling efficient preparation of highly purified PEG-protein conjugates that have to meet stringent regulatory criteria for their use in human therapy. Areas addressed are PEG properties, the specificity of PEGylation reactions, separation and large-scale purification, the availability and analysis of PEG reagents, analysis of PEG-protein conjugates, the consistency of products and processes and approaches used for rapid screening of pharmacokinetic properties of PEG-protein conjugates

Human granulocyte colony stimulating factor (hG-CSF) was expressed in the methylotrophic yeast Pichia pastoris, using two different constructs which resulted in proteins with different N-terminal sequences. In the first construct, a hexa-histidine tag and enterokinase cleavage site were added to the N-terminus of the protein to achieve one-step separation and exact processing. In the second construct, the gene was fused to the alpha-MF prepro leader at the Lys-Arg processing site (without Glu-Ala spacer). The PCR products were cloned in pPIC9 commercial vector and integrated into the alcohol oxidase region of the host genome. Transformation was done by electroporation or spheroplasting. Selection of good producing clones was performed by immunoblot analyses of the supernatants from shake-flask fermentation. Proper processing of the products was confirmed by N-terminal sequencing of the secreted proteins. With both plasmid constructs, the target proteins, bearing the histidine tag or not, represented majority of the secreted proteins. Although the proteins were present in the soluble form, they were highly aggregated, which interfered with purification. The most efficient way to obtain monomeric, biologically active protein was complete denaturation by guanidine-HCl or urea and subsequent renaturation during gel filtration chromatography.

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Our tumor necrosis factor-alpha (TNF-alpha) analog LK-805 (E107K) exhibited twofold higher specific cytotoxicity on the mouse fibroblast L-929 cell line than its native counterpart. In addition, significantly lowered systemic toxicity was observed in tumor-bearing mouse models treated with this analog. Due to a charge reversal and clustering of three lysines in the exposed tip region of LK-805, we assumed that additional ionic interactions between the positively charged TNF analog and the negatively charged components of the cell surface were created, which might contribute to improved properties of LK-805. To prove this hypothesis, we designed truncated forms of TNF-alpha and analog LK-805 and performed three independent sets of experiments: measurement of cytotoxic activity in the presence of excess heparan sulfate, determination of cytotoxic activity on heparinase-treated L-929 cells, and binding of various TNF-alpha proteins onto the heparin-sepharose affinity column. Cytotoxicity studies of both kinds confirmed the pivotal role of the E107K mutation for interaction with heparan sulfate proteoglycans on the cell surface of L-929 cells. However, heparin-binding studies revealed that intact, full-length N-termini of TNF-alpha or its analogs were necessary for high retention on the heparin affinity column, whereas the three-lysine containing tip of LK-805 by itself was not enough for binding. Obviously, immobilized heparin does not represent an adequate model for membrane-bound heparan sulfate proteoglycans of L-929 cells.

A new PEGylation reagent enabling selective modification of free thiol groups is described in this article. The reagent was synthesized by attaching linear polyethylene glycol (PEG) N-hydroxysuccinimide to selenocystamine. The reaction was very fast, resulting in over 95% conversion yield. The active group of this new PEG-Se reagent is a diselenide, reacting with thiols via thiol/diselenide exchange reaction. Recombinant human granulocyte colony-stimulating factor (rhG-CSF) with an unpaired cysteine at the position 18 (Cys18) was used as a model protein. It was comparatively PEGylated with the new PEG-Se reagent, as well as with commercially available maleimide (PEG-Mal) and ortho-pyridyl disulfide (PEG-OPSS) PEG reagents. The highest PEGylation yield was obtained with PEG-Mal, followed by PEG-OPSS and PEG-Se. The reaction rates of PEG-Mal and PEG-Se were comparable, while the reaction rate of PEG-OPSS was lower. Purified monoPEGylated rhG-CSF conjugates were characterized and compared. Differences in activity, stability, and in vivo performance were observed, although all conjugates contained a 20 kDa PEG attached to the Cys18. Minor conformational changes were observed in the conjugate prepared with PEG-Mal. These changes were also reflected in low in vitro biological activity and aggregate formation of the maleimide conjugate. The conjugate prepared with PEG-Se had the highest in vitro biological activity, while the conjugate prepared with PEG-OPSS had the best in vivo performance.