No abstract

No abstract

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.

During manufacture, therapeutic proteins may be exposed to ultraviolet (UV) radiation. Such exposure is of concern because UV radiation may cause photooxidative damage to proteins, which in turn could lead to physical changes such as aggregation and enhanced immunogenicity. We exposed murine growth hormone (mGH) to controlled doses of UV radiation, and examined the resulting chemical, physical and immunogenic changes in the protein. mGH chemical structure was analyzed by mass spectrometry after UV irradiation. Photooxidation products detected by mass spectrometry included methionine sulfoxide formed at Met[127] and Met[149] residues, and, tentatively assigned by MS/MS analysis, ether cross-links between original Ser[78] and Cys[188], and Cys[206] and Ser[213], and a thioether cross-link between Cys[17] and Cys[78] residues, transformation of Cys[189] into Ala, and various hydrolytic fragments. Physical damage to UV-irradiated mGH was monitored by infrared spectrometry, chromatographic analyses, and particle counting by microflow imaging. UV radiation caused mGH to aggregate, forming insoluble microparticles containing mGH with non-native secondary structure. When administered subcutaneously to Balb/c or Nude Balb/c mice, UV-irradiated mGH provoked antibodies that cross-reacted with unmodified mGH in a fashion consistent with a T-cell dependent immune response. In wildtype Balb/c mice, titers for anti-mGH IgG1antibodies increased with increasing UV radiation doses.

Evaluating a particle profile for parenteral drug products is a well-known challenge due to inevitable variability of results with limited accuracy to actual particle levels present in the product, especially in the subvisible particulate (SbVP) range. It is important to understand the appropriate SbVP counting/ characterization technology, methodology capability, and the particle source (intrinsic or extrinsic). Elastomeric closures are prevalent in many types of drug product container closure systems and are a known source of particle contribution. These components need to be considered when establishing a drug product particle profile. In this work, we describe available particle extraction methodology and its applicability in the analysis of elastomeric closure components using multiple detection technologies. Optimum sample preparation and analytical techniques were established to evaluate submicron particle and SbVP loads from elastomeric closure components. In addition, the impact of stopper siliconization and polysorbate 80 interaction on the degree of SbVPs in the final drug product was assessed.