Integrated Process for Capture and Purification of Virus-Like Particles: Enhancing Process Performance by Cross-Flow Filtration.

Nils Hillebrandt,Philipp Vormittag,Jürgen Hubbuch,Nicolai Bluthardt,Annabelle Dietrich

doi:10.3389/fbioe.2020.00489

Nils Hillebrandt, Philipp Vormittag + Show 3 more

Open Access

https://doi.org/10.3389/fbioe.2020.00489

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Abstract

Virus-like particles (VLPs) are emerging nanoscale protein assemblies applied as prophylactic vaccines and in development as therapeutic vaccines or cargo delivery systems. Downstream processing (DSP) of VLPs comes both with challenges and opportunities, depending on the complexity and size of the structures. Filtration, precipitation/re-dissolution and size-exclusion chromatography (SEC) are potent technologies exploiting the size difference between product and impurities. In this study, we therefore investigated the integration of these technologies within a single unit operation, resulting in three different processes, one of which integrates all three technologies. VLPs, contained in clarified lysate from Escherichia coli, were precipitated by ammonium sulfate, washed, and re-dissolved in a commercial cross-flow filtration (CFF) unit. Processes were analyzed for yield, purity, as well as productivity and were found to be largely superior to a reference centrifugation process. Productivity was increased 2.6-fold by transfer of the wash and re-dissolution process to the CFF unit. Installation of a multimodal SEC column in the permeate line increased purity to 96% while maintaining a high productivity and high yield of 86%. In addition to these advantages, CFF-based capture and purification allows for scalable and disposable DSP. In summary, the developed set-up resulted in high yields and purities, bearing the potential to be applied as an integrated process step for capture and purification of in vivo-assembled VLPs and other protein nanoparticles.

Highlights

Vaccination has reduced morbidity and mortality worldwide, especially since the introduction of the World Health Organization’s Expanded Program on Immunization (Greenwood, 2014)
Hepatitis B core antigen (HBcAg) concentrations in all three experiments (Figure 4B) were comparable, except for the region between 100 and 150 mM (NH4)2SO4, where supernatant HBcAg concentrations during precipitation from non-dialyzed lysate dropped significantly at 100 mM (NH4)2SO4, while the dialyzed samples remained at comparably constant HBcAg concentrations from 0 to 100 mM (NH4)2SO4
To validate that precipitation incubation time is sufficient at larger scale, HBcAg concentration in the supernatant was investigated in 10 min intervals at the previously determined

Summary

Introduction

Vaccination has reduced morbidity and mortality worldwide, especially since the introduction of the World Health Organization’s Expanded Program on Immunization (Greenwood, 2014). Especially VLP downstream processing (DSP) faces major challenges, such as low yields and the lack of platform processes or rapid analytical techniques. This is due to the complexity of the product and the associated processes, resulting in high development and production costs (Ladd Effio and Hubbuch, 2015). Incorporation of foreign epitopes into VLP-forming viral structural proteins results in so-called chimeric VLPs (Pumpens and Grens, 2001). The size difference between VLPs and host cell proteins (HCPs) as well as other smaller contaminants can be exploited for DSP of VLPs (Ladd Effio and Hubbuch, 2015)

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