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PharmTech.com Trends in Formulation 2022 eBook Pharmaceutical Technology ® 37 Biologics non-proliferative cells, thus emphasizing their poten- tial for use in gene therapy. While versatile and robust, there are some inherent weaknesses in the production of both AAV and lenti- virus vectors, and, as a result, production is not well optimized. There are frequently failures in producing robustly active virions, with lentivirus yields seen as low as three to 10 virus per producing cell compared to 10 3 /cell in wild HIV-1 infections (8). Production of viral vectors is a complex process and requires innovative approaches to meet safety and efficacy requirements, clinical and market demands, and cost of goods targets. Preparing stable viral vec- tors, preventing their degradation during manufactur- ing, handling and storage, and maintaining their long- term stability and efficacy are all major challenges for the viral vector manufacturer. The viral vector manufacturing process includes sev- eral upstream, downstream, and fill/finish unit oper- ations. The plasmid deliverable must first be carefully engineered to contain the genetic material of interest, complete with necessary regulatory elements for ap- propriate tissue and temporal expression. Next, cell cul- tures (typically human embryonic kidney 293 [HEK293] cells) are expanded to achieve desired cell density for subsequent plasmid transfection. The viral vectors pro- duced from the transfected cell populations must then be purified by removing cell fragments and debris prior to chromatographic purification, and this is complicated during AAV production, which requires the transfected cell populations be lysed to release the vector. Purified viral vectors are then formulated to meet Quality Target Product Profile (QTTP) and desired shelf-life (9). Challenges exist throughout the manufacturing process, including low titer and yield, complex down- stream process, and viral vector physical and chemi- cal degradation, such as unfolding, aggregation and precipitation, oxidation, etc., which can cause loss of therapeutic efficacy and result in undesirable immune responses (10). Environmental obstacles that cause viral vectors degradation and efficacy loss need to be better understood and managed by carefully optimiz- ing buffer, pH, and excipients. Viral vector manufacturing is complicated by the scal- ability of each stage of development. Where the work- flow used pre-clinical studies, at an upstream reactor scale as small as 1 L, the workflow must be iteratively developed to platforms that support commercial-scale production of 500 L or more. Although each manufac- turing step comes with inherent hurdles, significant improvements have been developed. Stressors in production and storage that lead to degradation Complications in the vector production pipeline begin upstream of viral transfection. While highly effective at expanding plasmid content, Escherichia coli (E. coli) fermentation is inherently variable in plasmid yield and variability, both due to individual culture fluctu- ation and effects of the plasmid itself. Consistent production of current good manufac- turing practice (CGMP)-grade plasmids with purity greater than 95% is also a struggle (11). There are ad- ditional considerations regarding the type of culture used, as cells may be adherent or nonadherent; adher- ent cells require a tissue surface area, while nonadher- ent cells must be suspension-adapted (12). The scale-up process for each type of culture comes with additional variability. In adherent cell cultures, size and contamination risk are compounded by cul- ture management difficulties, such as regulation of culture conditions across parallel culture substrates. Conversely, suspension cultures are easily scalable, but carry inherently lower yields than adherent cul- tures. Plasmid levels within the culture can also be more variable and unpredictable. Upon t ra n sfec t ion, f u r t her obst acles must be cleared to achieve desired viral yield and qualit y, i nclud i ng cel l lysis (A AV produc t ion), f i lt rat ion, and purification, where there is a lack of an effec- tive and reproducible platform for the separation of empt y f rom t he f u l l capsid s. W h i le t here a re severa l cel l lysis met hods (bot h mecha n ica l a nd chemical) that are used in viral vector production, not all methods are appropriate for large-scale pro- duction. This eliminates traditional methods, such as repeated cel l f reeze/t haw c ycling fol lowed by gent le cent r if ugat ion (wh ich is dif f icult to sca le up) and mechanical French press homogenization (which is scalable but results in undesirable levels of product loss). Chemical lysis with a surfactant, such as Triton X-100 (Thermo Fisher Scientif ic), is cur- rently an acceptable option in terms of scalabilit y and yield; however, research has shown that Triton X-100 causes acute oral toxicity, eye damage, skin irritation, and chronic aquatic toxicity. As a result, While versatile and robust, there are some inherent weaknesses in the production of both AAV and lentivirus vectors.