Journal: Vaccine

Article Title: Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components

doi: 10.1016/j.vaccine.2019.04.056

Figure Lengend Snippet: Comparison of anion exchange media. Chromatograms from small-scale anion exchange experiments are shown. In all cases, solid lines indicate A 280 (left axis) and dashed lines indicate conductivity (right axis). A 260 data was also collected, with results paralleling the A 280 data, but is omitted from graphs for clarity. In panels A-I, each column of graphs represents data from a single type of media, as per column captions. Panels A-F show chromatograms from experiments in which previously purified ChAdOx2 RabG was loaded on Mustang Q Acrodisc, NatriFlo® HD-Q Recon Mini, and CIM-QA 1 mL capsules. In panels A-C. breakthrough was observed and hence dynamic binding capacity was calculated for the Mustang Q and NatriFlo® capsules; breakthrough was not observed after loading 4x10 13 VP on the (larger) CIM-QA column. Panels D-F show the continuations of the above experiments, in which virus was eluted using a linear gradient of increasing conductivity to allow estimation of elution conditions; the conductivity at the peak of virus elution is shown. Panels G-I show chromatograms from experiments in which diafiltered lysate containing ChAdOx2 RabG was loaded on Mustang Q Acrodisc, NatriFlo® HD-Q Recon Mini, and CIM-QA 1 mL capsules, followed by step elution. Panel J presents DBC, virus recovery and HCP reduction data from the experiments shown in Panels A-F. In the case of the CIM-QA column, breakthrough was not reached despite loading 3x10 13 VP on the 1 mL device. Panels K-L show chromatograms from experiments in which previously purified ChAd63 ME-TRAP and ChAdOx1 RVF were loaded on Mustang Q Acrodisc capsules and eluted with a linear conductivity gradient, as above.

Article Snippet: To ensure that all product-contact components were disposable, single-use liners (Merck) were used with the Pellicon® 2 Mini TFF cassettes, and chromatography monitoring employed single-use pressure, conductivity and UV spectrometry sensor flow cells (Pendotech) (data shown in B).

Techniques: Comparison, Purification, Capsules, Binding Assay, Virus

Journal: Vaccine

Article Title: Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components

doi: 10.1016/j.vaccine.2019.04.056

Figure Lengend Snippet: Downstream process overview and results. Panel A shows an overview of the downstream process steps. Panel B shows conductivity and A 280 traces obtained for at-scale chromatography for each of the three vaccines, as indicated in captions for each plot. In each case, loading occurs over the first ∼ 600 mL (a degree of A 280 deflection from impurity flow-through is visible) and the largest A 280 peak represents the eluted virus which was collected. Note for ChAdOx2 RabG that the pre-elution wash and the post-elution high-salt wash produce relatively small A 280 peaks (predominantly impurity, with some virus, as assessed by SDS-PAGE); this data was obtained using UV and conductivity sensors of the Akta Purifier. For ChAd63 and ChAdOx1, data was obtained using the complete single-use chromatography rig (i.e. with single-use UV and conductivity sensors [Pendotech]) and no post-elution wash was performed. For ChAd63, due to the lower conductivity at which this vaccine eluted, no pre-elution wash was used. Panel C shows samples from each stage of the downstream process on a Coomassie-stained SDS-PAGE gel. Samples shown are from a single process run with the ChAdOx2 RabG vaccine, but are typical of runs with all three investigated vaccines.

Article Snippet: To ensure that all product-contact components were disposable, single-use liners (Merck) were used with the Pellicon® 2 Mini TFF cassettes, and chromatography monitoring employed single-use pressure, conductivity and UV spectrometry sensor flow cells (Pendotech) (data shown in B).

Techniques: Chromatography, Vaccines, Virus, SDS Page, Staining