Application of a Best Practice Approach Using Resonant Mass Measurement for Biotherapeutic Product Characterization.

Characterizing and quantifying subvisible particles in protein drug products is critical to ensuring product quality. A variety of analytical methods are used to detect and make meaningful measurements of subvisible particles. Resonant mass measurement (RMM) is a novel technology that characterizes the subvisible particle content of samples on a particle-by-particle basis. The technology presents great promise in the study of therapeutic protein products. As an emerging tool in the biopharmaceutical field, the best practices and limitations of RMM for protein products have not been well established. One key challenge of particle analysis is producing robust and reliable data, with high precision and accuracy, for particle characterization. In this study, we develop a set of possible best practices for RMM using a model protein system. We test the effects of these practices on the repeatability and reproducibility of particle measurements. Additionally, we present the data collected under a rigorously controlled set of operating conditions at 3 collaborating sites as well as a summary of the resulting optimal practices. In employing these practices, we successfully obtained improved relative standard deviation values and achieved high reproducibility and repeatability in both sizing and concentration measurement results over a broad range of sample volumes.

UV photodegradation of murine growth hormone: chemical analysis and immunogenicity consequences.

During manufacturing, 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 micro-flow 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 IgG1 antibodies increased with increasing UV radiation doses.

Glass particles as an adjuvant: a model for adverse immunogenicity of therapeutic proteins.

Unwanted immune responses to parenterally administered therapeutic proteins pose serious safety and economic risks, but the mechanism(s) by which these responses are generated are unknown. We measured immune responses to aggregates of recombinant murine growth hormone (mGH) formed by agitation or freeze-thawing, two pharmaceutically relevant stresses, as well as to mGH adsorbed on microscopic glass or alum particles. Insoluble aggregates, even at levels below the detection limits of size-exclusion high-performance liquid chromatography analysis (<1%), induce immune responses when administered subcutaneously. Furthermore, we show that application of high hydrostatic pressures (200 MPa) reduces aggregate levels to 0.02 ng/dose and eliminates immunogenicity.

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.

Immunogenicity of aggregates of recombinant human growth hormone in mouse models.

Aggregation of recombinant therapeutic protein products is a concern due to their potential to induce immune responses. We examined the immunogenicity of protein aggregates in commercial formulations of recombinant human growth hormone produced by freeze-thawing or agitation, two stresses commonly encountered during manufacturing, shipping and handling of therapeutic protein products. In addition, we subjected each preparation to high-pressure treatment to reduce the size and concentration of aggregates present in the samples. Aggregates existing in a commercial formulation, as well as aggregates induced by freeze-thawing and agitation stresses enhanced immunogenicity in one or more mouse models. The use of high-pressure treatment to reduce size and concentrations of aggregates within recombinant human growth hormone formulations reduced their overall immunogenicity in agreement with the "immunon" hypothesis.