Scaleable Purification of Adenovirus Vectors

Konz, John; Pitts, Lee; Sagar, Sangeetha L.

doi:10.1007/978-1-60327-248-3_2

Cited by 7 publications

(5 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Suspension HEK293 cells are commonly used in batch-mode stirred-tank upstream processes [11], although the highest volumetric yields have been reported in perfusion-based processes in PERC6 cells [4,12]. Downstream processes have commonly made use of bead-based anion exchange resins, often with an additional polish step such as size exclusion, hydrophobic interaction or multi-modal core-shell (eg CaptoCore Ò ) chromatography [13][14][15][16][17]. More recently, membrane-based and monolith-based chromatography have been used [18,19].…”

Section: Introductionmentioning

confidence: 99%

Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components

Fedosyuk¹,

Merritt²,

Peralta-Álvarez³

et al. 2019

Vaccine

View full text Add to dashboard Cite

a b s t r a c tA variety of Good Manufacturing Practice (GMP) compliant processes have been reported for production of non-replicating adenovirus vectors, but important challenges remain. Most clinical development of adenovirus vectors now uses simian adenoviruses or rare human serotypes, whereas reported manufacturing processes mainly use serotypes such as AdHu5 which are of questionable relevance for clinical vaccine development. Many clinically relevant vaccine transgenes interfere with adenovirus replication, whereas most reported process development uses selected antigens or even model transgenes such as fluorescent proteins which cause little such interference. Processes are typically developed for a single adenovirus serotype -transgene combination, requiring extensive further optimization for each new vaccine.There is a need for rapid production platforms for small GMP batches of non-replicating adenovirus vectors for early-phase vaccine trials, particularly in preparation for response to emerging pathogen outbreaks. Such platforms must be robust to variation in the transgene, and ideally also capable of producing adenoviruses of more than one serotype. It is also highly desirable for such processes to be readily implemented in new facilities using commercially available single-use materials, avoiding the need for development of bespoke tools or cleaning validation, and for them to be readily scalable for later-stage studies.Here we report the development of such a process, using single-use stirred-tank bioreactors, a transgene-repressing HEK293 cell -promoter combination, and fully single-use filtration and ion exchange components. We demonstrate applicability of the process to candidate vaccines against rabies, malaria and Rift Valley fever, each based on a different adenovirus serotype. We compare performance of a range of commercially available ion exchange media, including what we believe to be the first published use of a novel media for adenovirus purification (NatriFlo Ò HD-Q, Merck). We demonstrate the need for minimal process individualization for each vaccine, and that the product fulfils regulatory quality expectations. Cell-specific yields are at the upper end of those previously reported in the literature, and volumetric yields are in the range 1 Â 10 13 -5 Â 10 13 purified virus particles per litre of culture, such that a 2-4 L process is comfortably adequate to produce vaccine for early-phase trials. The process is readily transferable to any GMP facility with the capability for mammalian cell culture and aseptic filling of sterile products.

show abstract

Section: Introductionmentioning

confidence: 99%

Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components

Fedosyuk¹,

Merritt²,

Peralta-Álvarez³

et al. 2019

Vaccine

View full text Add to dashboard Cite

show abstract

“…Detergents commonly used to lyse the host cells include Triton X-100, nonyl phenoxypolyethoxylethanol (NP-40), Tween 20, Brij-58, etc. 20 , 21 , 22 , 23 , 24 The removal of these detergents added in biological products is highly imperative in their processing. The detergents can be removed in a variety of ways.…”

Section: Discussionmentioning

confidence: 99%

The Effect of Residual Triton X-100 on Structural Stability and Infection Activity of Adenovirus Particles

et al. 2020

Molecular Therapy - Methods & Clinical Development

View full text Add to dashboard Cite

To ensure the high purity and biological activity of the adenovirus vector to be used for clinical applications, a stable and linearly scalable preparation method is highly imperative. During the adenovirus-harvesting process, the Triton X-100-based lysis method possesses the advantages of higher efficiency as well as easier linearization and amplification. Most Triton X-100 can be removed from the adenovirus sample by chromatographic purification. However, there is no report that a small amount of residual Triton X-100, present in adenovirus sample, can affect the particle integrity, infectivity, and structure of adenoviruses. Here, we found that although residual Triton X-100 affected the short-term stability, purity, infectivity, and structure of adenoviruses at 37°C, it did not hamper these properties of adenoviruses at 4°C. This study suggests that although the Triton X-100-based lysis method is a simple, efficient, and easy-to-scale process for lysing host cells to release the adenovirus, the storage conditions of adenovirus products must be taken into consideration.

show abstract

“…These include detergent cell lysis (Peixoto et al, 2006), DNA digestion (Shabram et al, 1997), depth filter clarification (Goerke et al, 2005;Peixoto et al, 2008), Ad capture by anion exchange (Duffy et al, 2005;Peixoto et al, 2008) and Ad Characterization of a representative lot of Ad using the assays described in Materials and Methods Section. concentration and buffer exchange by TFF (Albinana-Gimenez et al, 2009;Goerke et al, 2005;Konz et al, 2008;Peixoto et al, 2008). Two column processes are described in the literature.…”

Section: Discussionmentioning

confidence: 99%

Development of a platform process for adenovirus purification that removes human set and nucleolin and provides high purity vector for gene delivery

Riske

Berard²,

Albee³

et al. 2012

Biotech & Bioengineering

View full text Add to dashboard Cite

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.

show abstract

Scaleable Purification of Adenovirus Vectors

Cited by 7 publications

References 15 publications

Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components

Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components

The Effect of Residual Triton X-100 on Structural Stability and Infection Activity of Adenovirus Particles

Development of a platform process for adenovirus purification that removes human set and nucleolin and provides high purity vector for gene delivery

Contact Info

Product

Resources

About