Highly efficient concentration of lenti- and retroviral vector preparations by membrane adsorbers and ultrafiltration

Abstract

BackgroundLentiviral vectors (LVs) can efficiently transduce a broad spectrum of cells and tissues, including dividing and non-dividing cells. So far the most widely used method for concentration of lentiviral particles is ultracentrifugation (UC).An important feature of vectors derived from lentiviruses and prototypic gamma-retroviruses is that the host range can be altered by pseudotypisation. The most commonly used envelope protein for pseudotyping is the glycoprotein of the Vesicular Stomatitis Virus (VSV.G), which is also essential for successful concentration using UC.ResultsHere, we describe a purification method that is based on membrane adsorbers (MAs). Viral particles are efficiently retained by the anionic exchange MAs and can be eluted with a high-salt buffer. Buffer exchange and concentration is then performed by utilizing ultrafiltration (UF) units of distinct molecular weight cut off (MWCO). With this combined approach similar biological titers as UC can be achieved (2 to 5 × 109 infectious particles (IP)/ml). Lentiviral particles from small starting volumes (e.g. 40 ml) as well as large volumes (up to 1,000 ml) cell culture supernatant (SN) can be purified. Apart from LVs, vectors derived from oncoretroviruses can be efficiently concentrated as well. Importantly, the use of the system is not confined to VSV.G pseudotyped lenti- and retroviral particles and other pseudotypes can also be purified.ConclusionsTaken together the method presented here offers an efficient alternative for the concentration of lenti- as well as retroviral vectors with different pseudotypes that needs no expensive equipment, is easy to handle and can be used to purify large quantities of viral vectors within a short time.

Downstream Processing of Lentiviral Vectors: Releasing Bottlenecks

“…4). In Process C, CIM Ò DEAE monolithic columns improved the purification step yields to 80%, 42% more than the results obtained by Merten et al (2011) (Process A) and Zimmermann et al (2011) (using TSKgel DEAE columns and LentiSelect kits, respectively) and 10% more than obtained by Slepushkin et al (2003) using Mustang Q Ion Exchange Capsules. Furthermore, a faster process was achieved, with overall infectious vector recoveries of 36%, 12% more than obtained in Process A.…”

An optimized method for high-titer lentivirus preparations without ultracentrifugation

“…Several alternative methods have been reported to purify high-titer lentivirus preparations, which bypassed ultracentrifugation 19 20 21 22 23 24 25 26 27 . Ion exchange chromatography was used to purify lentiviral particles based on the fact that lentivirus are retained by the anionic exchange columns and can be eluted subsequently with buffers containing a high concentration of salts 19 25 26 . This method has the advantage of removing more cell debris compared with the ultracentrifugation.…”

Lentiviral Vector Purification Using Genetically Encoded Biotin Mimic in Packaging Cell

“… 3 , 4 , 5 , 6 Purification is then usually achieved by chromatographic means, with anion exchange chromatography (AEX) being the most commonly used in both research and clinical-grade LV applications. 7 , 8 , 9 , 10 , 11 , 12 As AEX results in the co-purification of nucleic acids, which currently represents the major contaminant of LV supernatant, 13 most large-scale downstream processing schemes also include a nuclease treatment step using Benzonase, which in turn needs to be removed from the final product. 5 , 14 , 15 , 16 , 17 Size exclusion chromatography (SEC), which suffers from product dilution and slow flow rates, is then used as a polishing step.…”