Viral Vector CDMO Excellence with in-house Plasmid Production
Biovian is removing bottlenecks in gene therapy production with true One-Stop-Shop service. As regards Viral Vector production, the services span from in-house GMP plasmid DNA manufacture to Qualified Person-approved release of the final labelled Drug Product.
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 2005.
- 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-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
- 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
- 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 manufacture
- Automated Aseptic Filling line for live Viral Vectors – Filling in vials
- 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
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.
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:
- Transfer plasmid – a plasmid that carries the therapeutic gene.
- Rep/Cap plasmid – a plasmid that contains the protein-coding genes called rep and cap, which are needed for AAV replication and capsid formation.
- Helper plasmid – a plasmid that enables AAV replication in the host cell.
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.
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.
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 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.