Recombinant AAV Vector manufacturing follows a linear flow of operations and utilizes disposable, closed bioprocessing circuits. Multiple products can be manufactured simultaneously within the Clinical Manufacturing Facility (CMF) at any given time. Multiple AAV services are provided through the CMF including Process Development, Research Productions, GMP Productions and Quality Control testing. A Formal Project Request Form is filled out for every production in the CMF.
Process Development (PD) – The rAAV Vector plasmid and the associated helper plasmids are evaluated for yield performance in order to determine their suitability for use in preclinical and cGMP production. Downstream purification may be assessed and or developed for novel serotypes as appropriate. For all PD performed a proposal is provided and signed by the client and RINCH to guide the actions within the laboratory. Standard Quality Control (QC) testing with technical review is performed and a final report is delivered to the client for each assessment.
Research Productions – All research productions take place in the Viral Vector Core (VVC).
The VVC is a high throughput facility that provides standard research grade productions as well as large scale productions suitable for IND enabling toxicology studies. The VVC has experience purifying serotypes AAV1, AAV2, AAV2NHB, 4, 5, 6, 8, rh10, rh74 & 9 as well as several novel and proprietary serotypes.
Standard research vectors are produced at three scales: Small Scale, Mid-Scale and Large Scale. Yields may vary greatly depending on the transgene and serotype, however the average yield in DNase Resistant Particles for each scale has been provided below.
- Small Scale: Approximately 2x1012 total DNase Resistant Particles
- Mid-Scale: Approximately 1x1013 total DNase Resistant Particles
- Large scale: Approximately 2x1013 total DNase Resistant Particles
Along with above listed scales, custom scales are available upon discussion with the VVC Operations Manager or Coordinator.
For IND enabling toxicology grade productions a Research Process Plan (RPP) is provided and signed by both the client and RINCH to guide actions within the laboratory. All processes are documented in Research Batch Records (RBRs) followed by a technical review. The scale of the production selected will be dependent on the final yield required by the client. For all VVC productions, standard QC testing with technical review is performed.
All research grade productions utilize the same processes as those used for GMP productions with slight variations to accommodate scale.
Good Manufacturing Practices (GMP) Production – GMP Vectors are produced in the Clinical Manufacturing Facility (CMF) at Nationwide Children’s Hospital using practices consistent with the US Food and Drug Administration’s “Guidance for Industry – cGMP for Phase 1 Investigational Drugs”, July 2008. The manufacturing process includes procedures to ensure the safety, identity, quality, purity and strength of the manufactured drug product. The identified serotypes for GMP Production include AAV1, 2, 5, 8, rh74, and 9, or alternatively generated/modified capsids. Vectors are produced using calcium phosphate mediated triple DNA plasmid transfection into HEK293 cells in Corning HYPERStacks®. Subsequent downstream processes involve disposable, closed bioprocessing circuits where possible (filtration, tangential flow filtration and column chromatography). Following vector purification, the drug product is formulated in a final formulation buffer and sterile filtered through a 0.22 µm filter. Multiple sub-lots may be produced, tested, and pooled to generate the final drug product. The product is manually filled into sterile vials within a certified ISO Class 5 biosafety cabinet within the CMF and stored at ≤ -60°C as a frozen liquid.
The current manufacturing process has produced crude harvest yields up to 3E+16 DNase Resistant Particles. Vector yields may vary depending on transgene and serotype.
The GMP facility is capable of delivering approximately 1 X 1015 total DNase Resistant Particles per lot.
All GMP productions require a Master Production Plan that is reviewed and signed by the client to guide actions in the CMF. Client specific QC testing is performed for all GMP Productions.