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Development of a platform process for adenovirus purification that removes human set and nucleolin and provides high purity vector for gene delivery
Author(s) -
Riske Frank,
Berard Nicole,
Albee Karen,
Pan Peng,
Henderson Mike,
Adams Kris,
Godwin Simon,
Spear Sherri
Publication year - 2013
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.24742
Subject(s) - nucleolin , viral vector , bioreactor , genetic enhancement , computational biology , downstream processing , recombinant dna , virus , process development , chemistry , biology , gene , chromatography , microbiology and biotechnology , virology , process engineering , biochemistry , nucleolus , engineering , organic chemistry , cytoplasm
The manufacturing of virus occurs at a modest scale in comparison to many therapeutic proteins mainly because a gene therapy dose is typically only µg of vector. Although modest in scale the generation of high purity virus is challenging due to low viral expression levels and the difficulties in adequately characterizing such a large and complex molecule. A 100 L bioreactor might produce only 100 mg of virus that must be separated from host and process impurities that are typically greater by several orders of magnitude. Furthermore, in the later purification stages the main milieu component is often virus at low concentration (µg/mL) which may non‐specifically adsorb to purification surfaces resulting in a lowered virus recovery. This study describes our approach to develop a scalable, manufacturable robust process for an Adenovirus (Ad) gene therapy vector. A number of analytical tools were developed to guide the purification design. During process development, two human proteins, SET and nucleolin, were identified in viral preparations. To our knowledge, this is the first time that SET and nucleolin have been described in Ad. In this report we detail a process for their removal and the robust removal of all process, product and host cell impurities. Biotechnol. Bioeng. 2013; 110: 848–856. © 2012 Wiley Periodicals, Inc.