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Viral Vector GMP Production

Biovian is a well-established CDMO partner in Viral Vector projects that supports gene therapy and immuno-oncology applications including vaccines. We offer process development and GMP-grade production of AAV and adenovirus vectors and other biosafety level 2 (BSL2) class viruses. Since 2004 Biovian has provided Viral Vector CDMO services to clients in the EU and the USA. Biovian holds an EMA license for manufacturing Viral Vector products for clinical trials as well as for commercial use. Viral Vector manufacturing experience of Biovian ranges from clinical batch manufacturing up to Phase III to the manufacturing of commercial validation batches.

Viral Vector CDMO Excellence with in-house Plasmid Production

Biovian is removing bottlenecks in Viral Vector production with the One-Stop-Shop service concept. Our services span from in-house GMP plasmid DNA manufacturing to Qualified Person-approved release of the final labelled Drug Product. We can provide access to some of the newest production platforms through our partnerships. Biovian is licensed by the European Medicines Agency for GMP production of both investigational medicinal drugs and commercial gene therapy products. In this way, Biovian supports clients as they take their valuable Viral Vectors through the development and onto the market.

  • Oncolytic viruses
  • Viral Vectors for gene therapy
  • Viral Vector vaccines
Download a Viral Vector flyer
The strength of Biovian lies in their expertise in adenovirus, their flexibility for manufacturing slots and in competitive pricing. I find people at Biovian pleasant and trustworthy. For us one of the reasons for choosing Biovian was their expertise in Adenovirus and I would gladly recommend Biovian to someone looking for an adenovirus CDMO player.
Vice President of CMC

Gene Therapy CDMO Facilities

The Viral Vector production facility of Biovian allows for flexible contract development and production scenarios. The state-of-the-art BSL2 production facility is equipped with e.g. a 200 litre, single-use bioreactor. This enables efficient manufacturing of Viral Vectors on a large scale for advanced clinical trials and for the support of commercial strategies.

Track Record in Viral Vector Development and Manufacturing

  • Biovian has been developing Viral Vector processes since 2004.
  • Over 40 clinical GMP batches have been manufactured, including batches for clinical phases up to Phase III as well as commercial validation batches.
  • Longest-lasting customer relationship >10 years and still active.
One of the reasons for choosing Biovian as a partner was their expertise in oncolytic Adenovirus. Biovian has a holistic view on future scenarios of the program. Because of their experience, they can guide their clients through all the steps of the project. I appreciate the fact that Biovian keeps to the deadlines. Their project management is very good. The regulatory documentation of Biovian is also excellent.
Regulatory CMC Manager

One-Stop-Shop CDMO Services in Viral Vector Production

Cell Banks and Virus Seed Stocks

  • Research Cell Bank
  • Master Cell Bank and Working Cell Bank
  • Master Viral Seed Stock and Working Seed Stock

Upstream Processes

  • Suspension Cell Culture in single-use bioreactors – up to 200 L
  • Adherent Cell Culture in multilayer flasks, packed-bed bioreactor and single-use bioreactors on microcarriers

Downstream Processes

  • Ultracentrifugation-based Downstream Processes
  • Chromatography-based Downstream Processes
  • Comprehensive purification solutions – chromatography, membrane processes, Tangential Flow Filtration processes (TFF)

Aseptic Fill and Finish

  • Formulation and final Drug Product manufacturing
  • Automated Aseptic Filling line for live Viral Vectors
  • Typical batch sizes 200 – 1000 vials

Quality Assurance and Quality Control

  • Product-specific assays
  • Impurity analysis
  • Cell-based assays
  • Microbiological QC and safety assays (sterility, bioburden, endotoxin etc.)
  • QP Certification and full GMP documentation

Viral Vector Process Development Services

Biovian provides comprehensive Viral Vector process development services that are fully integrated with process analytics. Our process development services support the Quality-By-Design (QBD) approach from the earliest possible stage in order to enable a straightforward transition to GMP production.

  • Upstream Process development
  • Downstream Process development
  • Analytical development
  • Development and setting up of in-process control
  • Formulation development
The building of Viral Vector processes from almost any starting point is the expertise of Biovian. Our Process Development team can start the Viral Vector project from a single vial of the virus or from a small volume of plasmids.
Pirjo MerilahtiProcess Development Manager

Read a blog about Viral Vector Process Development

Viral Vector Process Development - Activity Diagram
Viral Vector Process Development – Flow Chart

AAV vectors

AAV vectors are considered attractive vehicles for gene therapy because of their safety profile and effective gene delivery to a broad range of dividing and non-dividing cells. The production of AAV vectors requires access to quality plasmids of three types for triple transfection. Biovian provides GMP-grade production of both plasmids and AAVs under the same roof and can save you the work and effort with plasmid sourcing and quality requirements.

Biovian’s platform approach to AAV production addresses many of the important steps in AAV manufacturing. The optimized AAV production platform of Biovian is designed to generate AAV preparations of high purity, enriched for capsids with full vector genomes while minimizing the number of empty capsids. Appropriate analytical methods are provided throughout the process for the demonstration of factors that are essential for product safety.  

Production of AAV vectors

AAV vectors are produced in mammalian producer cell lines, typically HEK293 or HEK293T, using either adherent or suspension culture.  We at Biovian provide assistance with the selection of the producer cell line as well as culture type, based on the AAV serotype and other factors and needs. The benefit of suspension cultures is that they can more easily be scaled up because cell growth is limited by the concentration of cells in the medium and not by the surface area, which is the case with adherent cell cultures. If needed, suspension cells can be cultured in serum-free media and are thus free from bovine-derived impurities such as TSE and BSE.  On the other hand, suspension cell cultures require daily cell counts and viability determination for growth monitoring, whereas adherent cell cultures can be inspected visually under the microscope. Also, there are AAV constructs that do not produce good yields in suspension cultures.

Triple transfection

The most common approach to AAV production is the triple transfection strategy, also called transient transfection. Three types of plasmids are needed to produce functional recombinant AAVs:

  1. Transfer plasmid – a plasmid that carries the therapeutic gene.
  2. Rep/Cap plasmid – a plasmid that contains the protein-coding genes called rep and cap, which are needed for AAV replication and capsid formation.
  3. Helper plasmid – a plasmid that enables AAV replication in the host cell.   
AAV triple transfection

Production of AAV plasmids

Harvesting and testing of AAV vectors

Intracellular AAVs are released into the culture media by disrupting the cultured mammalian host cells using chemical lysis. The composition of the lysis buffer depends on the type of culture. Nuclease enzymes such as Benzonase can be added during or after cell lysis in order to digest residual nucleic acids of the producer cells and plasmids. The subsequent clarification process involves the removal of cellular debris from the viral supernatant and is done using depth filtration. At Biovian the harvest is analyzed with qPCR or ddPCR for accurate quantification and characterization of AAV vectors.

AAV vector production – Upstream Processes

Purification of AAV vectors

Biovian’s multi-step platform purification process is compatible with the multiple AAV serotypes. This optimized method generates AAV preparations of high purity, enriched for capsids with full vector genomes while minimizing the number of empty capsids.

The purification process starts with affinity chromatography where the resin and column size will be selected based on the virus load. AAV capsids effectively bind to antibody fragments on the affinity chromatography resin. Empty capsids are subsequently removed with anion exchange chromatography, AIEX based on the difference in charge between the two populations. Anion exchange chromatography also effectively removes host cell proteins and host cell DNA contaminants. An additional polishing step using cation exchange chromatography, CIEX, can be added for further purification if required.

Final AAV concentration and formulation

The final concentration of the AAV product and the exchange to formulation buffer is performed using tangential flow filtration, TFF. The goal of the formulation optimization is to enhance the chemical and physical stability and thus prevent degradation of the AAV gene delivery system. The optimized formulation may also enhance the transduction efficiency and thereby the effectiveness of the AAV vector in vivo.

AAV vector production – Downstream Processes

Quality Control assays for AAV vectors

Purification intermediates are analyzed to confirm the presence and identity of AAV vectors and to measure viral titers and purity. Biovian provides clients with full access to process analytics and documentation packages that demonstrate the quality attributes mandatory for GMP Viral Vector batches. An overview of the suggested quality control methods and the documentation package is shown in the tables below:

Overview of of the suggested AAV vector QC package
Overview of the AAV vector QA documentation package:
  • TSE/BSE certificate
  • Certificate of Analysis
  • Certificate of GMP compliance
  • Comprehensive production summary report

AAV vector Fill and Finish

Fill and Finish of AAV vectors is performed in dedicated rooms that fulfil BSL 2 requirements. Fill and Finish of AAVs is one of the core competences of Biovian. Our automated aseptic filling line for AAVs is validated for up to 1000 vials per batch. The finished AAV vials may either be kept in intermediate storage at Biovian or be directly shipped to the clinic, once the batch has been released by one of our qualified persons.

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GMP-Grade Plasmid DNA Production

Biovian manufactures plasmid DNA for a variety of client projects.

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Antti Nieminen
Director, Business Development and Projects
Magnus Gustafsson_Biovian
Magnus Gustafsson
Head, Global Business Development
Eero Mustalahti
Manager, Sales and Business Development