Process development for a novel influenza A virus based therapeutic agent

The annual occurrence of epidemics, and the threat of a serious pandemic, make the influenza A virus (IAV) one of the most important human pathogens. In addition to vaccines, new, broadly effective and readily available antiviral treatment strategies are therefore needed. Influenza-based defective interfering particles (DIP) are a promising candidate for this purpose, as they are replication-incompetent and exhibit antiviral properties by interfering with and suppressing viral replication. The aim of this thesis was to take the first steps towards good manufacturing practice (GMP) manufacturing of the recently engineered “OP7 chimera” DIP (IAV-S1W/OP7), paving the way for clinical trials. Therefore, this work focused on the production of biological starting material and investigation of a fed-batch approach for IAV-S1W/OP7 production. Although the fed-batch mode is commonly used for bioprocessing, it is not established for IAV-DIP production. As multiplicity of infection (MOI) is a key control variable for the biological activity of the IAV-S1W/OP7 DIP construct, comprising a mixed population of OP7-DIP and helper-DIP, its robustness was investigated over three virus passages to cover subsequent GMP manufacturing operations. The optimal MOI was confirmed across a wide range of three orders of magnitude, with maximal interfering efficacy achieved for an MOI of 1E-4 or 1E-5. For process development, two media (4Cell and HyClone) were investigated for virus production of the backbone virus Influenza A/PR/8/34 (IAV-PR8) at increased cell density. While the HyClone medium supported extremely high virus production at low cell density, a steep decline in cell-specific virus productivity (CSVY) was observed at higher viable cell density (VCC). Since feed supplementation was less effective than media exchange strategies for overcoming the so-called cell density effect (CDE), fed-batch development remained unproductive. By contrast, IAV infection in the 4Cell medium exhibited lower CSVY at low cell density, but the decline in CSVY was less steep with increasing VCC. Furthermore, optimized HEK FS feed supplementation was effective in overcoming or limiting the CDE, enabling virus infection at VCCs of up to 10E+6 cells/mL and yielding plaque titers of up to 7E+9 PFU/mL for fed-batch production in a 1 L-stirred-tank bioreactor (STR). Next, fed-batch production in 4Cell medium was compared to a batch reference process, demonstrating a sixfold increase in plaque titer and a fourfold increase in volumetric virus productivity (VVP). Finally, the fed-batch approach was successfully verified for IAV-S1W/OP7 and resulted in a higher virus titer and Seg7-OP7 vRNA level than batch production, leading to increased interfering efficacy. Both the virus banking and fed-batch approaches could serve as a basis for further GMP manufacturing of IAV-S1W/OP7. Furthermore, the fed-batch process provides an efficient, simple and transferable strategy for producing IAV-DIPs, which could facilitate access of clinical-grade investigational medicinal products (IMPs).