SEC coupled with in-line multiple detectors for the characterization of an oncolytic Coxsackievirus

Figures

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  Figure 1. Assembly of picornavirus particles Five protomers consisting of VP0-VP3-VP1 associate to form pentamers. Twelve pentamers may further assemble either into empty procapsids or may encapsidate the RNA genome to produce provirions. Mature virions are formed after VP0 cleavage into VP2+VP4.

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  Figure 2. SEC chromatograms of V937 DS with size and Mw distribution The left y axis describes the Mw (Da) detected by MALS, and the right y axis represents hydrodynamic radius (Rh [nm]) measured by QELS. The red trace is the UV signal at A280 nm, the black trace is the MALS signal, the cyan trace is QELS, and the blue trace is the RI signal. DS was detected as a peak at ∼19 min, and sample matrix components elute after 24 min. The green line across the peak represents the particle Mw distribution, while the dotted purple line represents the particle Rh distribution.

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  Figure 3. Analysis of viral proteins by SDS-PAGE Viral proteins VP0, VP1, VP2, VP3, and VP4 are indicated in process intermediate samples. VP0 is not detectable in DS. Lane 1: Mw marker; lane 2: blank; lane 3: V937 DS; lane 4: blank; lane 5: process intermediate.

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  Figure 4. Particle number linearity curve The particle number in DS versus the injection volume demonstrates a linear relationship with R2 > 0.9999.

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  Figure 5. Comparison of Rh and Mw for empty, full, and empty/full mixture viral capsids LS signals are normalized by concentration factors. All samples eluted out at the same retention time on the chromatogram. The blue trace represents full virus, the black trace represents empty/full mixture, and the red trace is empty virus. (A) All three virus samples (empty, full, and empty/full mix) have indistinguishable Rh values (∼15 nm), represented by the dotted lines across the peak. (B) The measured Mw from these three samples are different, with 8,200 kDa for full virus (blue line across the peak), 7,195 kDa for empty/full mixture (black line across the peak), and 5,828 kDa for empty virus (red line across the peak).

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  Figure 6. Analysis of V937 DS thermal stability by SEC-UV-(MALS-QELS)-RI and DLS (A) Comparison of samples Stressed at 37°C, 45°C and 60°C on SEC-UV-MALS(QELS)-RI. Red trace is for sample stressed at 60°C, blue is for 45°C stressed sample and green trace is for sample at 37°C. Mw for each species is shown by the lines across the peaks. (B) DLS Temperature Study. Rh (black) and scattering intensity (red) were measured across a temperature gradient from 25 to 65°C in triplicate. A size and scattering increase was observed between 50°C and 60°C.

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  Figure 7. Proposed V937 thermal degradation pathways Mature virions may degrade under thermal stress through RNA escape or/and virus aggregation pathways. At extended exposure to elevated temperatures, the empty capsids and capsid aggregates may further unfold into disassembled subunits that aggregate and/or degrade.