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

Riske, Frank; Berard, Nicole; Albee, Karen; Pan, Peng; Henderson, M. G.; Adams, Kris; Godwin, Simon; Spear, Sherri

doi:10.1002/bit.24742

Cited by 7 publications

(4 citation statements)

References 34 publications

(56 reference statements)

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“…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

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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.

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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

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“…Similar approaches have been applied to HCP analysis of other viral vectors. Riske et al identified human SET and nucleolin proteins in adenovirus (Ad) produced in HEK293 cells by N-terminal sequencing (nucleolin) and in-gel tryptic digestion followed by LC/MS/MS [62]. They showed that a three step chromatography purification strategy was required for 'robust' reduction of HCPs.…”

Section: Rumachik Et Al Have Reported An Extensive Analysis Of the Hc...mentioning

confidence: 99%

Analytics of host cell proteins (HCPs): lessons from biopharmaceutical mAb analysis for Gene therapy products

Bracewell

Smith²,

Delahaye

et al. 2021

Current Opinion in Biotechnology

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“…Tangential flow filtration (TFF) methods have been used to concentrate and purify influenza virus, a retrovirus vector, and AAV using size exclusion as the main mechanism of separation with recoveries up to 100%. Adsorption mechanisms, ranging from chromatography beads to monoliths are also often applied to viral particles. The recovery from adsorption methods is usually lower than filtration methods, with a typical range between 30 and 68%.…”

Section: Introductionmentioning

confidence: 99%

A generalized purification step for viral particles using mannitol flocculation

Heldt

Saksule

Joshi

et al. 2018

Biotechnology Progress

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Vaccine manufacturing has conventionally been performed by the developed world using traditional unit operations like filtration and chromatography. There is currently a shift in the manufacturing of vaccines to the less developed world, requiring unit operations that reduce costs, increase recovery, and are amenable to continuous manufacturing. This work demonstrates that mannitol can be used as a flocculant for an enveloped and nonenveloped virus and can purify the virus from protein contaminants after microfiltration. The recovery of the virus ranges from 58 to 96% depending on virus, the filter pore size, and the starting concentration of the virus. Protein removal of 80% was achieved for the small nonenveloped virus using a 0.1 µm filter because proteins were not flocculated with the virus and flowed through the filter. It is hypothesized that mannitol dehydrates the viral surface by controlling the water structure surrounding the virus. Without the ability to become compact, as occurs with proteins, the virus aggregates in the presence of osmolytes and proteins do not. Osmolyte flocculation is a scalable process using high flux microfilters. It has been applied to both an enveloped and nonenveloped virus, making this process friendly to a variety of vaccine and gene therapy products. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1027-1035, 2018.

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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

Cited by 7 publications

References 34 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

Analytics of host cell proteins (HCPs): lessons from biopharmaceutical mAb analysis for Gene therapy products

A generalized purification step for viral particles using mannitol flocculation

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