Purification of HIV-1 gag virus-like particles and separation of other extracellular particles.
Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification of VLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scale able purification processes. We developed a chromatographic procedure for purification of HIV-1 gag VLPs produced in CHO cells. The clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. The majority of host cell impurities passed through the column, whereas the VLPs were eluted by a linear or step salt gradient; the major fraction of DNA was eluted prior to VLPs and particles in the range of 100-200nm in diameter could be separated into two fractions. The earlier eluted fraction was enriched with extracellular particles associated to exosomes or microvesicles, whereas the late eluting fractions contained the majority of most pure HIV-1 gag VLPs. DNA content in the exosome-containing fraction could not be reduced by Benzonase treatment which indicated that the DNA was encapsulated. Many exosome markers were identified by proteomic analysis in this fraction. We present a laboratory method that could serve as a basis for rapid downstream processing of enveloped VLPs. Up to 2000 doses, each containing 1×10(9) particles, could be processed with a 1mL monolith within 47min. The method compared to density gradient centrifugation has a 220-fold improvement in productivity.
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria.
Petra
P
Steppert
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria.
Daniel
D
Burgstaller
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria.
Miriam
M
Klausberger
ACIB GmbH, Vienna, Austria.
Eva
E
Berger
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria.
Patricia Pereira
PP
Aguilar
ACIB GmbH, Vienna, Austria.
Tobias A
TA
Schneider
BIA Separations d.o.o., AjdovšĿina, Slovenia.
Petra
P
Kramberger
Icosagen AS, Tartumaa, Estonia.
Andres
A
Tover
VetCore Facility for Research, University of Veterinary Medicine Vienna, Vienna, Austria.
Katharina
K
Nöbauer
VetCore Facility for Research, University of Veterinary Medicine Vienna, Vienna, Austria.
Ebrahim
E
Razzazi-Fazeli
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria; ACIB GmbH, Vienna, Austria. Electronic address: alois.jungbauer@boku.ac.at.
Alois
A
Jungbauer
D000818:Animals; D016466:CHO Cells; D002499:Centrifugation, Density Gradient; D006224:Cricetinae; D003412:Cricetulus; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D058425:Vaccines, Virus-Like Particle / Q000096:biosynthesis / Q000302:isolation & purification* / Q000648:ultrastructure; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011994:Recombinant Proteins; D058425:Vaccines, Virus-Like Particle; D054301:gag Gene Products, Human Immunodeficiency Virus
Chromatography; Density gradient centrifugation; Extracellular particles; Host cell DNA; Host cell protein; Vaccines