Mary S. Rosendahl

Recombinant Murine Growth Hormone from E. coli Inclusion Bodies: Expression, High-Pressure Solubilization and Refolding, and Characterization of Activity and Structure

We expressed recombinant murine growth hormone (rmGH) in E. coli as a cost‐effective way to produce large quantities (gram scale) of the protein for use in murine studies of immunogenicity to therapeutic proteins. High hydrostatic pressure was used to achieve high solubility and high refolding yields of rmGH protein produced in E. coli inclusion bodies. A two‐step column purification protocol was used to produce 99% pure monomeric rmGH. Secondary and tertiary structures of purified rmGH were investigated using circular dichroism and 2D‐UV spectroscopy. The purified rmGH produced was found to be biologically active in hypophysectomized rats.

Site-Specific PEGylation Enhances the Pharmacokinetic Properties and Antitumor Activity of Interferon Beta-1b

Interferon beta (IFN-β) is widely used to ameliorate disease progression in patients with Multiple Sclerosis. IFN-β has a short half-life in humans, necessitating frequent administration for optimum effectiveness. Covalent modification of IFN-β with polyethylene glycol (PEG) improves the pharmacokinetic properties of the protein, but can adversely affect the proteins in vitro bioactivity. Random modification of lysine residues in IFN-β with amine-reactive PEGs decreased the in vitro bioactivity of the protein 50-fold, presumably due to modification of lysine residues near critical receptor binding sites. PEGylated IFN-β proteins that retained high in vitro bioactivity could be obtained by selective modification of the N-terminus of the protein with PEG. Here we use site-specific PEGylation technology (targeted attachment of a cysteine-reactive-PEG to an engineered cysteine residue in IFN-β) to identify several additional amino acid positions where PEG can be attached to IFN-β without appreciable loss of in vitro bioactivity. Unexpectedly, we found that most of the PEG-IFN-β analogs showed 11- to 78-fold improved in vitro bioactivities relative to their unPEGylated parent proteins and to IFN-β-1b. In vivo studies showed that a lead PEG-IFN-β protein had improved pharmacokinetic properties compared to IFN-β and was significantly more effective than IFN-β at inhibiting growth of a human tumor xenograft in athymic mice.

PEGylation Improves the Pharmacokinetic Properties and Ability of Interferon Gamma to Inhibit Growth of a Human Tumor Xenograft in Athymic Mice

Interferon gamma (IFN-γ) is a 28 kDa homodimeric cytokine that exhibits potent immunomodulatory, anti-proliferative, and antiviral properties. The protein is used to treat chronic granulomatous disease and malignant osteopetrosis, and it is under investigation as a treatment for a variety of cancer, fungal and viral diseases. IFN-γ has a short circulating half life in vivo, which necessitates frequent administration to patients. An unusual feature of IFN-γ is that the protein contains no native cysteines. To create a longer-acting and potentially more effective form of the protein, we introduced a cysteine residue into the IFN-γ coding sequence at amino acid position 103, which is located in a surface-exposed, non-helical region of the protein. The added cysteine residue served as the site for targeted modification of the protein with a cysteine-reactive polyethylene glycol (PEG) reagent. The recombinant protein was expressed in bacteria, purified and modified with 10, 20, and 40 kDa maleimide PEGs. The purified, PEGylated proteins had in vitro bioactivities comparable to IFN-γ, as measured using an in vitro cell growth inhibition assay. The PEGylated proteins displayed 20- to 32-fold longer half lives than IFN-γ in rats, and they were significantly more effective than IFN-γ at inhibiting growth of a human tumor xenograft in athymic mice.