Chapter 12
Upstream and Downstream Processes for Viral Nanoplexes
as Vaccines
Keven Lothert, Gregor Dekevic, Daniel Loewe, Denise Salzig,
Peter Czermak, and Michael W. Wolff
Abstract
The increasing medical interest in viral nanoplexes, such as viruses or virus-like particles used for vaccines,
gene therapy products, or oncolytic agents, raises the need for fast and efficient production processes. In
general, these processes comprise upstream and downstream processing. For the upstream process, efficiency is mainly characterized by robustly achieving high titer yields, while reducing process times and costs
with regard to the cell culture medium, the host cell selection, and the applied process conditions. The
downstream part, on the other hand, should effectively remove process-related contaminants, such as host
cells/cell debris as well as host cell DNA and proteins, while maintaining product stability and reducing
product losses. This chapter outlines a combination of process steps to successfully produce virus particles in
the controlled environment of a stirred tank bioreactor, combined with a platform-based purification
approach using filtration-based clarification and steric exclusion chromatography. Additionally, suggestions
for off-line analytics in terms of virus characterization and quantification as well as for contaminant
estimation are provided.
Key words Upstream processing, Downstream processing, Viral nanoplexes, Virus, Virus-like particles, Mammalian cells, Stirred tank bioreactor, Polyethylene glycol, Membrane chromatography, Steric
exclusion
1 Introduction
Viruses, membrane fractions of viruses, viral surface proteins, or
virus-like particles are frequently used in modern medicine as ingredient in prophylactic vaccines [1–3]. Most recently, viruses have
also been used as gene therapy vectors to treat patients with genetic
deficiencies or cancer [4, 5]. For the latter, oncolytic viruses which
selectively infect and eliminate cancer cells are also used [6–
9]. Viruses and virus-like particles are commonly produced in
eukaryotic host cells (mammalian, avian- and insect cells)
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021
Keven Lothert and Gregor Dekevic shared first authorship.
217
Upstream and Downstream Processes for Viral Nanoplexes
as Vaccines
Keven Lothert, Gregor Dekevic, Daniel Loewe, Denise Salzig,
Peter Czermak, and Michael W. Wolff
Abstract
The increasing medical interest in viral nanoplexes, such as viruses or virus-like particles used for vaccines,
gene therapy products, or oncolytic agents, raises the need for fast and efficient production processes. In
general, these processes comprise upstream and downstream processing. For the upstream process, efficiency is mainly characterized by robustly achieving high titer yields, while reducing process times and costs
with regard to the cell culture medium, the host cell selection, and the applied process conditions. The
downstream part, on the other hand, should effectively remove process-related contaminants, such as host
cells/cell debris as well as host cell DNA and proteins, while maintaining product stability and reducing
product losses. This chapter outlines a combination of process steps to successfully produce virus particles in
the controlled environment of a stirred tank bioreactor, combined with a platform-based purification
approach using filtration-based clarification and steric exclusion chromatography. Additionally, suggestions
for off-line analytics in terms of virus characterization and quantification as well as for contaminant
estimation are provided.
Key words Upstream processing, Downstream processing, Viral nanoplexes, Virus, Virus-like particles, Mammalian cells, Stirred tank bioreactor, Polyethylene glycol, Membrane chromatography, Steric
exclusion
1 Introduction
Viruses, membrane fractions of viruses, viral surface proteins, or
virus-like particles are frequently used in modern medicine as ingredient in prophylactic vaccines [1–3]. Most recently, viruses have
also been used as gene therapy vectors to treat patients with genetic
deficiencies or cancer [4, 5]. For the latter, oncolytic viruses which
selectively infect and eliminate cancer cells are also used [6–
9]. Viruses and virus-like particles are commonly produced in
eukaryotic host cells (mammalian, avian- and insect cells)
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021
Keven Lothert and Gregor Dekevic shared first authorship.
217