Manufacturing recombinant adeno-associated viral vectors from producer cell clones.

A commercial rAAV manufacturing process needs to provide a safe product at high yield, be easily scalable, regulatory-compliant, and have reasonable cost of goods. Considerations for process development include not only product quantity and quality, but also ease of obtaining equipment, performing validation, and demonstrating control. In these regards, it is usually efficient to make use of proven technologies for more established areas of manufacturing, such as cell culture and purification methods used by the recombinant protein/monoclonal antibody industry. We have focused on stable mammalian producer cell lines with adenovirus type 5 helper virus as a means of achieving these goals. This review describes our current approach for generating producer cell clones and designing a scalable, regulatory-compliant vector production and purification process that addresses any product safety concerns relating to helper virus. To date, a producer cell line-based manufacturing process has been implemented at the 250-liter production scale, with no foreseeable impediments to scaling up to commercial vector manufacturing in 2000-liter bioreactors or larger.

Characterizing clearance of helper adenovirus by a clinical rAAV1 manufacturing process.

Recombinant adeno-associated viral vectors (rAAV) are being developed as gene therapy delivery vehicles and as genetic vaccines, and some of the most scaleable manufacturing methods for rAAV use live adenovirus to induce production. One aspect of establishing safety of rAAV products is therefore demonstrating adequate and reliable clearance of this helper virus by the vector purification process. The ICH Q5A regulatory guidance on viral safety provides recommendations for process design and characterization of viral clearance for recombinant proteins, and these principles were adapted to a rAAV serotype 1 purification process for clinical vectors. Specific objectives were to achieve overall adenovirus clearance factors significantly greater than input levels by using orthogonal separation and inactivation methods, and to segregate adenovirus from downstream operations by positioning a robust clearance step early in the process. Analytical tools for process development and characterization addressed problematic in-process samples, and a viral clearance validation study was performed using adenovirus and two non-specific model viruses. Overall clearance factors determined were >23 LRV for adenovirus, 11 LRV for BVDV, and >23 LRV for AMuLV.

Secretion of a TNFR:Fc fusion protein following pulmonary administration of pseudotyped adeno-associated virus vectors.

This study evaluated and compared delivery of the tumor necrosis factor alpha receptor (TNFR)-immunoglobulin G1 (IgG1) Fc fusion (TNFR:Fc) gene to the lung by single and repeat administrations of multiple pseudotyped adeno-associated virus (AAV) vectors as a means for achieving systemic distribution of the soluble TNFR:Fc protein. A single endotracheal administration of AAV[2/5]cytomegalovirus (CMV)-TNFR:Fc vector (containing the AAV2 inverted terminal repeats and AAV5 capsid) to the rat lung resulted in long-term, high levels of serum TNFR:Fc protein that gradually declined over a period of 8 months. Endotracheal delivery of AAV[2/1]CMV-TNFR:Fc resulted in serum TNFR:Fc protein levels that were detectable for at least 4 months but were 10-fold lower than that of the AAV[2/5] vector. In contrast, secretion of the TNFR:Fc protein following pulmonary delivery of AAV[2/2]CMV-TNFR:Fc vector was very inefficient, and the protein was detected in the blood only when an airway epithelial cell-specific promoter, CC10, was substituted for the CMV enhancer/promoter to control transgene expression. In the context of AAV[2/5], the CC10 promoter was as efficient as CMV enhancer/promoter in generating similar levels of systemic TNFR:Fc protein, suggesting that this protein is secreted primarily from the airway epithelium. In mice, comparable long-term secretion of TNFR:Fc protein was demonstrated after AAV[2/2] and AAV[2/5] delivery, although the kinetics of transduction appeared to be different. All pseudotyped AAV vectors elicited serum anti-AAV capsid-neutralizing antibody responses, but these did not prevent lung transduction and efficient secretion of TNFR:Fc protein to the circulation following readministration with AAV[2/5]. These results highlight the potential utility of AAV vectors containing serotype 5 capsid to deliver and redeliver genes of secreted proteins to the lung to achieve long-term systemic protein expression.

Production of recombinant adeno-associated type 5 (rAAV5) vectors using recombinant herpes simplex viruses containing rep and cap.

We developed a scaleable production system for adeno-associated virus type 5 (AAV5)-based vectors using a replication-defective recombinant herpes simplex type 1 virus (rHSV) containing the rep and cap genes of AAV5. Multiple rHSV isolates containing AAV5 rep and cap with normal or altered p5 promoter elements were constructed and tested in vector production. Compared with rAAV5 vector yields obtained by plasmid transfection, yields of rAAV5 using any of the rHSV isolates were low. Evidence suggests that the low vector yields are a consequence of the extensive and early cytopathology induced by the rHSV isolates. In addition, we found a correlation between the amount of Rep52 or Rep40 proteins and the amount of vector produced by each rHSV isolate, suggesting that packaging of vector DNA into virus particles is rate-limiting when using rHSV to generate rAAV5 vectors.