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. 2021 Nov 4:9:753480.
doi: 10.3389/fbioe.2021.753480. eCollection 2021.

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

Elise K Mullins  1 , Thomas W Powers  1 , Jim Zobel  1 , Kory M Clawson  1 , Lauren F Barnes  2 , Benjamin E Draper  3 , Qin Zou  1 , Joseph J Binder  4 , Stanley Dai  5 , Kun Zhang  1 , Olga Friese  1 , Herbert A Runnels  1 , Martin F Jarrold  2 , Lawrence C Thompson  1
Affiliations

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

Elise K Mullins et al. Front Bioeng Biotechnol. .

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.

Keywords: AEX-HPLC; adenovirus; analytical ultracentrifugation; charge detection mass spectrometry; differential centrifugation sedimentation; low density viral particles; non-human primate; viral particle titer.

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Conflict of interest statement

Authors EKM, TWP, JZ, KMC, QC, KZ, OF, HAR, LCT and JJB were employed by Pfizer Inc. Author BED was employed by Megadalton Solutions. Author SD was employed by Nektar Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Representative Analytical Ultracentrifugation analyses of AdC68 construct #1 (Panel A) and #2 (Panel B) showing detection at both the absorbance (260 nm) and interference channels.
FIGURE 2
FIGURE 2
Analysis of preparative ultracentrifugation bands for AdC68 #1 and #2. Panel A is a representative picture of the CsCl preparative fractionation. Panels B and C are DCS overlays of the unfractionated and low/high density fractions for AdC68 #1 and #2, respectively. Panel D is a Reversed Phased High Performance Liquid Chromatography stacked-plot of the proteome content for AdC68 #1 unfractionated and low density/high density fractions. Panel E and Panel F are Charge Detection Mass Spectrometry spectra of the AdC68 #1 low/high density fractions showing the experimentally determined molecular masses, respectively.
FIGURE 3
FIGURE 3
Anion Exchange High Performance Liquid Chromatography overlays of the fluorescence (FLD) and 260 nm absorbance (A260) channels for both Human Ad5 Reference Material (Panels A & B) and mixtures of AdC68 low/high density viral particle fractions (Panels C & D). The Human Ad5 Reference Material data is a serial dilution and shows a similar response factor for both channels. The AdC68 mixtures give an increasing response factor on the 260 nm channel for samples of the similar particle # but differing particle density while that for the fluorescence channel is consistent among all samples.
FIGURE 4
FIGURE 4
Overlay of Differential Centrifugation Sedimentation data for an AdC68 #1 (Panel A) and AdC68 #2 (Panel B) accelerated stability time course.
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