At-line multi-angle light scattering detector for faster process development in enveloped virus-like particle purification
Pereira Aguilar, P. (University of Natural Resources and Life Sciences. Department of Biotechnology)
González-Domínguez, Irene (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Schneider, Tobias Amadeus (Austrian Centre of Industrial Biotechnology (ACIB))
Gòdia, Francesc (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Cervera Gracia, Laura (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Jungbauer, Alois (Austrian Centre of Industrial Biotechnology (ACIB))

Date:	2019
Abstract:	At-line static light scattering and fluorescence monitoring allows direct in-process tracking of fluorescent virus-like particles. We have demonstrated this by coupling at-line multi-angle light scattering and fluorescence detectors to the downstream processing of enveloped virus-like particles. Since light scattering intensity is directly proportional to particle concentration, our strategy allowed a swift identification of product containing fractions and rapid process development. Virus-like particles containing the Human Immunodeficiency Virus-1 Gag protein fused to the Green Fluorescence protein were produced in Human Embryonic Kidney 293 cells by transient transfection. A single-column anion-exchange chromatography method was used for direct capture and purification. The majority of host-cell protein impurities passed through the column without binding. Virus-like particles bound to the column were eluted by linear or step salt gradients. Particles recovered in the step gradient purification were characterized by nanoparticle tracking analysis, size exclusion chromatography coupled to multi-angle light scattering and fluorescence detectors and transmission electron microscopy. A total recovery of 66% for the fluorescent particles was obtained with a 50% yield in the main product peak. Virus-like particles were concentrated 17-fold to final a concentration of 4. 45 × 10 particles/mL. Simple buffers and operation make this process suitable for large scale purposes.
Grants:	Ministerio de Educación, Cultura y Deporte FPU16/02555 Agència de Gestió d'Ajuts Universitaris i de Recerca 2017/SGR-898 Agència de Gestió d'Ajuts Universitaris i de Recerca 2014/SGR-1216
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Subject:	Enveloped bionanoparticles ; Fluorescent virus-like particles ; Monoliths ; Nanoparticle tracking analysis
Published in:	Journal of Separation Science, Vol. 42, Num. 16 (2019) , p. 2640-2649, ISSN 1615-9314

DOI: 10.1002/jssc.201900441
PMID: 31169979

11 p, 1.7 MB

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