Purification of recombinant adenovirus type 3 dodecahedric virus-like particles for biomedical applications using short monolithic columns

Abstract

Adenovirus type 3 dodecahedric virus-like particles (Ad3 VLP) are an interesting delivery vector. They penetrate animal cells in culture very efficiently and up to 300,000 Ad3 VLP can be observed in one cell. The purification of such particles usually consists of several steps. In these work we describe the method development and optimization for the purification of Ad3 VLP using the Convective Interaction Media analytical columns (CIMac). Results obtained with the CIMac were compared to the already established two-step purification protocol for Ad3 VLP based on sucrose density gradient ultracentifugation and the Q-Sepharose ion-exchange column. Pure, concentrated and bioactive VLP were obtained and characterized by several analytical methods. The recovery of the Ad3 VLP was more than 50% and the purified fraction was almost completely depleted of DNA; less than 1% of DNA was present. The purification protocol was shortened from five days to one day and remarkably high penetration efficacy of the CIMac-purified vector was retained. Additionally, CIMac QA analytical column has proven to be applicable for the final and in-process control of various Ad3 VLP samples.

Novel coronavirus-like particles targeting cells lining the respiratory tract

“…Heparin column purification allowed to remove majority of contaminants, including albumin from HF culture medium, but VLPs copurified with other proteins, most likely originating from BVs. Samples were thus submitted for the second stage of purification using CIM-QA monolith column recently reported to be a useful tool for virus and VLPs preparations [47][48][49]. This step allowed for further fractionation of the material collected from heparin column.…”

Downstream processing and chromatography based analytical methods for production of vaccines, gene therapy vectors, and bacteriophages

“…New generation chromatography media, CIM monolithic columns were used in DSPs of different viruses like influenza [11,13,37] adenovirus [8,29], adeno-associated virus [9,38], lentivirus [10,39], rubella [40], different phages [26,27,28,41,42] and different VLPs [6,43] with good virus/phage/VLP recoveries while reaching high dynamic binding capacities at the same time.…”

Application of monolithic chromatographic supports in virus research

“…Of course, principles proved for virus particles are expected to be valuable in chromatography of VLPs. And this is demonstrated for dodecahedric VLPs assembled exclusively from 12 adenoviral penton bases without participation of hexon and fiber protein . By using monolith chromatography approach and strong anion exchanger (QA), this efficient delivery vector was purified in 1 day.…”