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

Abstract

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.

Analysis of Recombinant Adenovirus Vectors by Ion Trap Charge Detection Mass Spectrometry: Accurate Molecular Weight Measurements beyond 150 MDa

“…The peak at 129.6 MDa is attributed to incomplete particles that have not packaged a genome (i.e., empty particles). , Light and heavy particles (where heavy particles are the mature virions) are separable by gradient centrifugation. CDMS measurements for gradient-separated light and heavy recombinant chimpanzee adenovirus C68 have confirmed the assignment of the peaks at around 130 and 156 MDa to empty and full particles, respectively . The broad distribution centered on around 90 MDa is attributed to misassembled or trapped intermediates.…”

“…CDMS measurements for gradient-separated light and heavy recombinant chimpanzee adenovirus C68 have confirmed the assignment of the peaks at around 130 and 156 MDa to empty and full particles, respectively. 82 The broad distribution centered on around 90 MDa is attributed to misassembled or trapped intermediates. Table 2 shows the components of mature HAdV5, along with the masses derived from the sequences (accounting for truncations).…”

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

“…Two AdC68 constructs were manufactured within Pfizer. AdC68 material was formulated in A195 buffer ( Xiang et al, 2002 ): 10 mM Tris, 75 mM NaCl, 5% w/v sucrose, 0.02% (w/v) 80 polysorbate 80, 1.0mM MgCl2, 0.1 mM EDTA, 0.5% (v/v) ethanol, 10 mM L-histidine, pH 7.4 ( Mullins et al, 2021 ).…”

“…The denatured capsid proteins were chromatographically separated on a Phenomenex Jupiter C4, 5 µm, 2 × 150 mm, 300Å analytical column (Part #00F-4167-B0) using an Agilent 1200 HPLC system similar to what was described previously ( Vellekamp et al, 2001 ; Mullins et al, 2021 ) with the following alterations. The column was held at a temperature of 5 0°C with a gradient consisting of 20–100% acetonitrile with trifluoroacetic acid (TFA) over 90 min with a flow rate of 0.2 ml/min.…”

“…Non-human primate adenoviral vectors are attractive for use in human vaccine and immuno-oncological applications as they maintain many of the attributes that make adenovirus appealing as a therapeutic vector while having reduced host pre-existing antivector immunity ( Capone et al, 2013 ; Cheng et al, 2015 ; Zhang et al, 2017 ; Zhao et al, 2018 ; Mullins et al, 2021 ). Recent successes using these constructs include modified chimpanzee adenovirus type 3–vectored ebolavirus vaccine (cAd3-EBO) ( Stanley et al, 2014 ; Ledgerwood et al, 2017 ) and replication-deficient simian adenoviral vectored vaccine ChAdOx1 nCoV-19 against SARS-CoV-2 ( Folegatti et al, 2020 ; Ewer et al, 2021 ; Putter, 2021 ).…”