Identification of Recombinant Chimpanzee Adenovirus C68 Degradation Products Detected by AEX-HPLC.

Physicochemical tests represent important tools for the analytical control strategy of biotherapeutics. For adenoviral modalities, anion-exchange high performance liquid chromatography (AEX-HPLC) represents an important methodology, as it is able to simultaneously provide information on viral particle concentration, product purity and surface charge in a high-throughput manner. During product development of an adenoviral-based therapeutic, an accelerated stability study was performed and showed changes in each of the AEX-HPLC reportable attributes. These changes also correlated with a decrease in product infectivity prompting a detailed characterization of the impurity and mechanism of the surface charge change. Characterization experiments identified the impurity to be free hexon trimer, suggesting that capsid degradation could be contributing to both the impurity and reduced particle concentration. Additional mass spectrometry characterization identified deamidation of specific hexon residues to be associated with the external surface charge modification observed upon thermal stress conditions. To demonstrate a causal relationship between deamidation and surface charge changes observed by AEX-HPLC, site-directed mutagenesis experiments were performed. Through this effort, it was concluded that deamidation of asparagine 414 was responsible for the surface charge alteration observed in the AEX-HPLC profile but was not associated with the reduction in infectivity. Overall, this manuscript details critical characterization efforts conducted to enable understanding of a pivotal physicochemical test for adenoviral based therapeutics.

Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer.

We observed differential infectivity and product yield between two recombinant chimpanzee adenovirus C68 constructs whose primary difference was genome length. To determine a possible reason for this outcome, we characterized the proportion and composition of the empty and packaged capsids. Both analytical ultracentrifugation (AUC) and differential centrifugation sedimentation (DCS, a rapid and quantitative method for measuring adenoviral packaging variants) were employed for an initial assessment of genome packaging and showed multiple species whose abundance deviated between the virus builds but not manufacturing campaigns. Identity of the packaging variants was confirmed by charge detection mass spectrometry (CDMS), the first known application of this technique to analyze adenovirus. The empty and packaged capsid populations were separated via preparative ultracentrifugation and then combined into a series of mixtures. These mixtures showed the oft-utilized denaturing A260 adenoviral particle titer method will underestimate the actual particle titer by as much as three-fold depending on the empty/full ratio. In contrast, liquid chromatography with fluorescence detection proves to be a superior viral particle titer methodology.

Oncolytic Vaccinia Virus Gene Modification and Cytokine Expression Effects on Tumor Infection, Immune Response, and Killing.

Oncolytic vaccinia viruses have promising efficacy and safety profiles in cancer therapy. Although antitumor activity can be increased by manipulating viral genes, the relative efficacy of individual modifications has been difficult to assess without side-by-side comparisons. This study sought to compare the initial antitumor activity after intravenous administration of five vaccinia virus variants of the same Western Reserve backbone and thymidine kinase gene deletion in RIP-Tag2 transgenic mice with spontaneous pancreatic neuroendocrine tumors. Tumors had focal regions of infection at 5 days after all viruses. Natural killer (NK) cells were restricted to these sites of infection, but CD8+ T cells and tumor cell apoptosis were widespread and varied among the viruses. Antitumor activity of virus VV-A34, bearing amino acid substitution A34K151E to increase viral spreading, and virus VV-IL2v, expressing a mouse IL2 variant (mIL2v) with attenuated IL2 receptor alpha subunit binding, was similar to control virus VV-GFP. However, antitumor activity was significantly greater after virus VV-A34/IL2v, which expressed mIL2v together with A34K151E mutation and viral B18R gene deletion, and virus VV-GMCSF that expressed mouse GM-CSF. Both viruses greatly increased expression of CD8 antigens Cd8a/Cd8b1 and cytotoxicity genes granzyme A, granzyme B, Fas ligand, and perforin-1 in tumors. VV-A34/IL2v led to higher serum IL2 and greater tumor expression of death receptor ligand TRAIL, but VV-GMCSF led to higher serum GM-CSF, greater expression of leukocyte chemokines and adhesion molecules, and more neutrophil recruitment. Together, the results show that antitumor activity is similarly increased by viral expression of GM-CSF or IL2v combined with additional genetic modifications.