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Abstract: Differential scanning calorimetry was used to identify the thermal stability profile of the replication deficient and protein IX deleted recombinant adenovirus type 5 that contains the p53 transgene (rAd/p53) in phosphate buffered saline (vPBS) or 10% glycerol (TRIS/phosphate buffer). The wildtype adenovirus (Ad/WT) and purified hexon protein (major capsid protein) were also evaluated in 10% glycerol (TRIS/phosphate buffer) as controls. The thermal profile of rAd/p53 revealed three endothermic transitions (T 1, T 2 and T 3) occurring between 25 °C and 90 °C. T 1, which occurred at 46.7 °C in vPBS and 49.4 °C in TRIS/PO4 10% glycerol buffer, was irreversible following repeated scanning and attributed to the degradation of the intact vector. The latter two endothermic transitions, T 2 and T 3, occurring at 69 °C and 78 °C, respectively, corresponded with the two transitions of purified hexon in temperature and amount of heat absorbed. The thermal profile of Ad/WT revealed four endothermic transitions at 51.5 °C (T 1), 70.5 °C (T 2A), 73.6 °C (T 2B), and 77.4 °C (T 3). The higher temperature of degradation as well as additional transition was attributed to the presence of protein IX associated with the hexon. The positions and excess molar heat capacities of the intact rAds were found to be affected by pH, glycerol, vector concentration and the presence or absence of protein IX in the capsid. Irreversibility of T 1 implied that the degradation of the intact virus may follow first-order kinetics. The thermal scan rate dependence of T 1 further confirmed that degradation of the intact virus may be first-order. The apparent activation energies for the degradation of the intact vectors were determined from the scan rate dependence of T 1 and shown to be affected by protein IX in the capsid and solution conditions. Analysis of rAd samples incubated at 45 °C by Field Emission Electron Microscopy (FESEM) confirmed that loss of single particles was first-order. Although aggregates were observed in the samples, degradation appeared to be the dominant reaction leading to disappearance of single virions from the aqueous matrix. Based on thermal and FESEM analysis, an empirical model was proposed that accounted for the disappearance of single rAd particles. At or near T 1, degradation of rAd particles followed a unidirectional, pseudo-first order reaction. However, at lower temperatures, disappearance of single virions resulted from competing irreversible degradation and aggregation reactions. [Copyright &y& Elsevier]

The clinical application of recombinant adenoviruses as vectors for gene therapy brings about the need to develop new analytical methodologies for monitoring the quality of the viral production as well as establishing structure–function relationships. A mass spectrometry-based assay has been developed for the characterization of structural proteins of the recombinant adenovirus type 5 vector encoding human p53 tumor suppressor gene. The fingerprinting of the viral proteome was accomplished by integration of MALDI-MS and/or MALDI-PSD-MS with SDS–PAGE and RP-HPLC, followed by database search using MS-Fit and MS-Tag algorithms. Viral proteins (molecular weights ∼10,000–100,000 Da) corresponding to more than 95% of total protein mass were resolved and identified, which include hexon (II), penton base (III), peripentonal hexon-associated protein (IIIa), minor core protein (V), major core protein (VII), and other hexon-associated proteins (VI and VIII). An important finding of our studies was the identification of some precursor proteins (i.e., pVIII) and propeptides of precursor proteins (pVIII, pX, and pVI) present in the adenovirus sample. The information obtained allows direct and accurate assessment of the quality of recombinant adenoviruses. [Copyright &y& Elsevier]

Recombinant adenoviruses (rAd), widely used as vectors for gene therapy, are generally purified by column chromatography and frequently contain empty capsids and other aberrant forms of virus particles. To determine particle heterogeneity we utilized analytical ultracentrifugation (AUC) in CsCl density gradients. Preparations of three different rAd vectors were assessed. AUC was able to resolve multiple density forms including two empty capsid types in various virus preparations. One unusual density form (form V), was noninfectious and lacked protein VI. AUC was able to quantify empty capsids and monitor their removal during process development. Their relative concentrations were reduced by either addition of an immobilized zinc affinity chromatography (IZAC) step or by extension of the infection time. The Adenovirus Reference Material (ARM), a wild-type Ad5, had 2.2% empty capsids and no other detectable minor particle forms. Finally, AUC was utilized to monitor the thermal instability of the three rAd vectors via the transformations of different density forms. The vector and empty capsids containing protein IX were more stable than those without IX. Together, these results exemplify AUC in CsCl density gradients as a valuable technique for evaluating product particle heterogeneity and stability. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97: 746–763, 2008 [ABSTRACT FROM AUTHOR]

An abstract of the article "Quantification of Empty Capsids in Preparations of Conditionally Replicating Adenovirus," by Sundari Ravindran, Xiaoyu Yang, Suganto Sutjipto, Yan-Hui Liu, and Gary Vellekamp is presented.