Biology Reference
In-Depth Information
Chapter 14
Canine Adenovirus Downstream Processing Protocol
Meritxell Puig, Jose Piedra, Susana Miravet, and María Mercedes Segura
Abstract
Adenovirus vectors are efficient gene delivery tools. A major caveat with vectors derived from common
human adenovirus serotypes is that most adults are likely to have been exposed to the wild-type virus and
exhibit active immunity against the vectors. This preexisting immunity limits their clinical success. Strategies
to circumvent this problem include the use of nonhuman adenovirus vectors. Vectors derived from canine
adenovirus type 2 (CAV-2) are among the best-studied representatives. CAV-2 vectors are particularly
attractive for the treatment of neurodegenerative disorders. In addition, CAV-2 vectors have shown great
promise as oncolytic agents in virotherapy approaches and as vectors for recombinant vaccines. The rising
interest in CAV-2 vectors calls for the development of scalable GMP compliant production and purification
strategies. A detailed protocol describing a complete scalable downstream processing strategy for CAV-2
vectors is reported here. Clarification of CAV-2 particles is achieved by microfiltration. CAV-2 particles are
subsequently concentrated and partially purified by ultrafiltration-diafiltration. A Benzonase
®
digestion
step is carried out between ultrafiltration and diafiltration operations to eliminate contaminating nucleic
acids. Chromatography purification is accomplished in two consecutive steps. CAV-2 particles are first
captured and concentrated on a propyl hydrophobic interaction chromatography column followed by a
polishing step using DEAE anion exchange monoliths. Using this protocol, high-quality CAV-2 vector
preparations containing low levels of contamination with empty viral capsids and other inactive vector
forms are typically obtained. The complete process yield was estimated to be 38-45 %.
Key words
CAV-2 vectors, Purification, Membrane filtration, Chromatography
1
Introduction
A number of viruses have been used for gene delivery purposes.
Among them, human adenovirus vectors are the most commonly
used vectors in clinical trials (
http://www.wiley.com//legacy/
wileychi/genmed/clinical/
)
. Adenovirus vectors possess several
key advantages including their high production titers, high trans-
duction efficiency in both replicating and differentiated cells, large
cloning capacity and non-integrative nature. However, their clini-
cal utility is severely compromised by the fact that most patients are
likely to have been exposed to wild-type human adenoviruses and
possess active immunity against common adenovirus vectors
