Biacore T200
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Determination of adenovirus concentration using Biacore T200 Intellectual Property Notice: The Biopharma business of GE Healthcare was acquired by Danaher on 31 March 2020 and now operates under the Cytiva™ brand. Certain collateral materials (such as application notes, scientific posters, and white papers) were created prior to the Danaher acquisition and contain various GE owned trademarks and font designs. In order to maintain the familiarity of those materials for long-serving customers and to preserve the integrity of those scientific documents, those GE owned trademarks and font designs remain in place, it being specifically acknowledged by Danaher and the Cytiva business that GE owns such GE trademarks and font designs. cytiva.com GE and the GE Monogram are trademarks of General Electric Company. Other trademarks listed as being owned by General Electric Company contained in materials that pre-date the Danaher acquisition and relate to products within Cytiva’s portfolio are now trademarks of Global Life Sciences Solutions USA LLC or an affiliate doing business as Cytiva. Cytiva and the Drop logo are trademarks of Global Life Sciences IP Holdco LLC or an affiliate. All other third-party trademarks are the property of their respective owners. © 2020 Cytiva All goods and services are sold subject to the terms and conditions of sale of the supplying company operating within the Cytiva business. A copy of those terms and conditions is available on request. Contact your local Cytiva representative for the most current information. For local office contact information, visit cytiva.com/contact

Determination of adenovirus concentration using Biacore™ T200 This application note demonstrates the performance of two assays for determination of adenovirus concentration based on surface plasmon resonance (SPR). In one assay, virus particles were allowed to bind via the fiber protein (spikes extruding on the virus capsid) to recombinant human coxsackie adenovirus receptor (CAR) protein immobilized on a Biacore sensor chip. In a second assay, Factor X (FX) protein was immobilized on a Biacore sensor chip to allow binding of virus particles via the virus hexon protein (dominating capsid...

Fig 2. Principle of the two Biacore assays for determination of adenovirus concentration, where virus particles injected in a flow system bind to (A) the CAR receptor via the virus fiber protein or (B) FX via the virus hexon protein. In the separate assays, CAR or FX protein is immobilized on Sensor Chip CM5, and bound virus is detected using the Biacore T200 system. Materials and methods Adenovirus production HEK293 suspension cells (HEK-293.2sus, ATCC) were infected with E1/E3-deleted recombinant AdV5 coding for the GFP reporter protein. Cell growth and adenovirus production as well as...

Results and discussion Performance of the CAR and FX assays are shown in Figure 3. The calibration curves demonstrate the stability of the assay. As shown by the near identical standard curves, with 27 samples runs in between, the assay can be used repeatedly with reproducible results (Fig 3B). This is further demonstrated in Figure 4 where calculated concentrations of 16 samples (stored at -20°C) from different steps in the process are shown. The same samples were thawed and diluted at two different occasions, with five days in between, and analyzed using the CAR assay. The same...

Virus titer (log vp/mL) Fig 4. Determination of virus concentration using the CAR assay for 16 process samples (stored at -20°C) analyzed on the same CAR sensor surface at two different occasions five days apart. Purification qPCR Biacore CAR Virus titer (log vp/mL) Eluat Capto Q ImpRes Flowthrough Capto Core 700 Fig 5. Comparison of virus titer determination using Biacore CAR assay, Biacore FX assay, and qPCR for samples from different steps of the adenovirus production process at 10 L scale. Figure 6 shows results from calculation of virus recovery in different fractions of the adenovirus...

10 000 Virus titer (log vp/ml) Signal intensity (log volume) Fig 6. (A) Step elution from ReadyToProcess Adsorber Q membrane. (B) Western blot analysis of fractions from flowthrough, wash, and elution peaks. Fiber protein signal comparison between (C) fluorescent Western blot and (D) Biacore CAR assay. Virus titer (log vp/mL) Fig 7. Calculated adenovirus concentrations in purified and concentrated bulk product, using different methods. IN Cell assay is showing infectious virus titer, whereas qPCR and the Biacore assays show total virus titer. Infections virus titer is expected to be lower...

Ordering information The Biacore assays for determination of adenovirus concentration were robust, provided reproducible results, and correlated well with qPCR, while showing lower variation. The Biacore assays were shown to be significantly less laborious than established methods. Particularly, minimal assay and sample preparation is required, and the standard and samples are directly diluted into the running buffer before analysis. This in contrast to, for example, qPCR that contains laborious and time-consuming sample preparation steps. The use of assays for detection of fiber or hexon...

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