312. Clearance of Helper Adenovirus by an AAV1 Clinical Vector Manufacturing Process

Abstract

The use of live adenovirus (Ad) to supply essential helper functions provides a readily scaleable method for producing recombinant adeno-associated viral vectors (rAAV). One of the primary objectives for recovery and purification operations in an Ad-based manufacturing process is therefore effective and robust clearance of this raw material. For clinical vector, absence of infectious adenovirus is an important quality attribute of final product, and the ICH Q5A guidance on viral safety provides a framework for meeting regulatory expectations.A clinical manufacturing process for rAAV1 pseudotyped vectors was developed based on Ad/AAV-hybrid production technology1,2. Multiple orthogonal separation methods were incorporated into the harvest and purification processes to achieve overall log reduction values (LRV) for adenovirus significantly greater than the inputlevels of Ad. Analytic challenges for assessing clearance include identifying and quantifying matrix interference effects in cell-basedassays.The first major clearance step provides 6 LRV and occurs in the vector production room. This provides a layer of process control by physically segregating Ad from rAAV1 purification, which occurs in a separate dedicated room. Spike-and-remove studies were performed to characterize adenovirus clearance by various subsequent operations. From ICH Q5A, clearance of a ‘relevant virus’ should include both removal and inactivation steps. For Ad inactivation, the relative thermostability of AAV and Ad were exploited. Operational tolerances for both temperature and time were defined in the relevant matrix to develop a robust heat inactivation unit operation. Data characterizing and quantitating removal of Ad from AAV, as well as approaches suitable for routine in process monitoring of Ad clearance, will be discussed.1Liu et al (1999) Gene Therapy 6(2): 2932Gao et. al. (1998) Human Gene Therapy 9(16): 2353 The use of live adenovirus (Ad) to supply essential helper functions provides a readily scaleable method for producing recombinant adeno-associated viral vectors (rAAV). One of the primary objectives for recovery and purification operations in an Ad-based manufacturing process is therefore effective and robust clearance of this raw material. For clinical vector, absence of infectious adenovirus is an important quality attribute of final product, and the ICH Q5A guidance on viral safety provides a framework for meeting regulatory expectations. A clinical manufacturing process for rAAV1 pseudotyped vectors was developed based on Ad/AAV-hybrid production technology1,2. Multiple orthogonal separation methods were incorporated into the harvest and purification processes to achieve overall log reduction values (LRV) for adenovirus significantly greater than the inputlevels of Ad. Analytic challenges for assessing clearance include identifying and quantifying matrix interference effects in cell-basedassays. The first major clearance step provides 6 LRV and occurs in the vector production room. This provides a layer of process control by physically segregating Ad from rAAV1 purification, which occurs in a separate dedicated room. Spike-and-remove studies were performed to characterize adenovirus clearance by various subsequent operations. From ICH Q5A, clearance of a ‘relevant virus’ should include both removal and inactivation steps. For Ad inactivation, the relative thermostability of AAV and Ad were exploited. Operational tolerances for both temperature and time were defined in the relevant matrix to develop a robust heat inactivation unit operation. Data characterizing and quantitating removal of Ad from AAV, as well as approaches suitable for routine in process monitoring of Ad clearance, will be discussed. 1Liu et al (1999) Gene Therapy 6(2): 293 2Gao et. al. (1998) Human Gene Therapy 9(16): 2353