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Jan 15, 2011 (Vol. 31, No. 2)

Optimizing Downstream Processing of Enveloped Viruses

Case Study Demonstrates Utility of Inventive Strategies

  • Results

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    Process development overview: recovery yields and product quality (total viruses versus infective viruses (TP/IP)).

    Based on purification steps that are easily scalable, a downstream processing workflow was established. The use of membrane processes facilitated the use of disposable formats, which resulted in cleaning and validation cost savings.

    A series of two depth filters integrated downstream of the bioreactor allowed for efficient passage of the recombinant baculoviruses while efficiently removing cells and cell debris. Tangential flow filtration of up to sixfold concentration with a subsequent diafiltration step of two volumes (Figure 1B, Table) was achieved, allowing concentration and primary purification of LMW impurities for shorter times as compared to ultracentrifugation.

    Use of the Sartobind D membrane adsorber as an anion-exchange chromatography step constituted a fast purification protocol with decreased buffer consumption and recovery yields up to 65% (Table).

    To improve performance of the anion-exchange chromatography step, SPR and DLS scale-down methodologies were developed to increase the fundamental understanding of this unit operation.

  • Click Image To Enlarge +
    Figure 2. Comparison of adsorption capacity on DEAE sensor surface of recombinant baculoviruses (rBVi) and: (A) major process-derived impurities; (B) major product-derived impurities (baculoviruses depleted of gp64 protein, rBVgp64-, envelope, rBVenv-, and gp64 protein isolated). (C) Sensor surface geometry of a Biacore sensor chip. (D) Selectivity estimation between rBVi and major product-derived impurities. [A and B reproduced from Journal of Biotechnology, 148:171-181. C from Journal of Chromatography A, 1217:2032-2041 and D from Journal of Chromatography A, 1217:3754-3764 with permission of Elsevier]

    An analytical method for analyzing binding and elution of the viruses over a surface mimicking the ion-exchange matrix was developed by iBET scientists. A sorption rate model capable of quantitatively describing binding and elution of the recombinant baculoviruses and major impurities from dilute to overloaded conditions within a broad range of salt concentrations was subsequently validated (Figure 2A–2B).

    A comparison of the adsorption of the major components of the baculovirus bulk onto an anion-exchange surface was conducted with this methodology. SPR operated as a pseudo-chromatographic tool utilized 1,200-fold less material (Figure 2C) than an analytical membrane adsorber unit for the estimation of adsorption isotherms.

    Determination of the zeta-potential (a measure of the electrostatic equilibria) further elucidated the contribution of the different virus components to the interaction phenomenon during the ion-exchange process. The main species, product (infective virus particle) and product-derived impurities (dsDNA-, glycoprotein-, and envelope-deprived baculovirus particles), were isolated and correspondent zeta-potentials were measured through DLS.

    The overall particle electrostatic interaction energy profile based on a fine-tuned electrokinetic model was able to distinguish the electrostatic properties of the infective virus particle from the major virus-related impurities. Scenarios such as Figure 2D allowed for prediction of the best operating conditions for promoting process selectivity.

    Analytical tools based on SPR and DLS combined with in silico models grounded on fundamental knowledge can serve as relevant process predictors/scale-down models for addressing the useful design space more efficiently. The sensible selection of advanced downstream processes allied with rational design streamlines process development of such viruses and potentially other challenging biopharmaceuticals.


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