title: Development of a platform process for adenovirus purification that removes human SET and nucleolin and provides high purity vector for gene delivery.
abstract: 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.
authors.affiliation: Department of Purification Development, Genzyme a Sanofi Company, Framingham, Massachusetts 01710, USA. frank.riske@genzyme.com
authors.forename: Frank
authors.initials: F
authors.lastname: Riske
authors.forename: Nicole
authors.initials: N
authors.lastname: Berard
authors.forename: Karen
authors.initials: K
authors.lastname: Albee
authors.forename: Peng
authors.initials: P
authors.lastname: Pan
authors.forename: Mike
authors.initials: M
authors.lastname: Henderson
authors.forename: Kris
authors.initials: K
authors.lastname: Adams
authors.forename: Simon
authors.initials: S
authors.lastname: Godwin
authors.forename: Sherri
authors.initials: S
authors.lastname: Spear
mesh_terms: D000256:Adenoviridae / Q000302:isolation & purification*; D004268:DNA-Binding Proteins; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000302:isolation & purification*; D056488:Histone Chaperones / Q000302:isolation & purification; D006801:Humans; D010750:Phosphoproteins / Q000302:isolation & purification; D016601:RNA-Binding Proteins / Q000302:isolation & purification; D013678:Technology, Pharmaceutical / Q000379:methods*; D014157:Transcription Factors / Q000302:isolation & purification; D000097796:Nucleolin
chemical_list: D004268:DNA-Binding Proteins; D056488:Histone Chaperones; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D000097796:Nucleolin; C513473:SET protein, human