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The authors of this study demonstrate an innovative method that is useful and complements traditional HA assays.
In this study, a complete membrane-based downstream process for the purification of influenza virus-like particles (VLPs) was monitored through transmission electron microscopy (TEM) imaging and customized image analysis using an innovative TEM-based method. The display of hemagglutinin (HA) spikes on the VLPs’ surface was evaluated throughout each step of the process, from initial clarification through a cascade of ultrafiltration/diafiltration steps with different pore sizes. It is demonstrated that this innovative TEM-based method was able to detect the pleomorphic HA spiked structures and unwanted debris. The developed method was also able to monitor the relative density of spikes of the VLPs to make sure that there was no loss of spikes due to downstream operations. The analysis ensured that the HA quantification measurements obtained are derived from HA exposed on the VLP surface and not from free HA protein, HA incorporated in baculovirus, or HA associated with cell membrane-derived debris. This determination is further supported by the finding that the TEM-based image analysis showed that small-sized debris was removed by the purification process. We hence show that this innovative method is a useful tool to complement the traditional HA assays.
Editor's note: Table III has been corrected and a new PDF reflecting the corrected table has been published.
Martin Ryner is strategic development manager; Nina Forsberg is marketing director; and Vanessa Carvalho*, Vanessa.Carvalho@vironova.com, is senior scientist all at Vironova. Cristina Peixoto is head of downstream process development and Sofia B. Carvalho, PhD, is a student at Instituto de Biologia Experimental e Tecnológica (iBET).
*To whom correspondence should be addressed.
Submitted: July 9, 2019
Accepted: Aug. 21, 2019
Vol. 32, No. 11
When referring to this article, please cite it as M. Ryner et al., “Using TEM-Based Image Analysis to Validate the Presence of HA Spikes on Influenza VLPs,” BioPharm International 32 (11) 2019.