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38 Pharmaceutical Technology ® Trends in Formulation 2022 eBook PharmTech.com Triton X-100 was placed on the "substance of ver y high concern list" by European Chemicals Agency under REACH regulations (13). Advancements in novel cell lysis solutions allow for improved efficiency, lower foam, and improved v i ra l vec tor recover y (10,1 4 ,15). One s uc h novel solution is biodegradable and meets the Organiza- tion for Economic Co-operation and Development (OECD) guideline for readily biodegradable material according to OECD 301 (15,16). Prior to t his, none of the lysis methods prevented viral vector loss due to shear stress during processing. As a result, there was a clear need for an easy-to-use cell lysis method t h at i s not on ly ef f ic ient i n lysi ng t he cel l s but also protects the viral vector from damage during processing conditions. Following cell lysis, difficulties are encountered in filtration and purification of viral vectors. Typi- cally, the methods used for small-scale production do not translate to large-scale application. Filter size and type must be optimized for the vector as well, as there are vast differences between AAV and lentivi- ruses. Filtration can be a yield-limiting step, and, as detritus from cell debris is plentiful, filters can be easily clogged (17). Purification is also challenging with current sys- tems resulting in low v ira l y ields. Af f in it y ch ro- matography is employed but cannot differentiate between full and empty viral capsids. Separation of empty capsids from full capsids by a cesium chloride gradient method is not scalable. The alternative ion exchange chromatography method in which high buf fer salt concentrations are necessar y for viral elution are damaging to virus activity. Aggregation, oxidation, deamidation, and proteolysis are all risk factors in the purification process as well, further contributing to the low viral yields produced by cur- rent methodologies (10,11). The final considerations in the viral vector produc- tion are storage and efficacy. Viruses must remain stable and highly effective in vivo. Buffers must be caref ully optimized to the vector, including ionic strength and buffer pH. The packaged final product should also be freeze-thaw resistant, to prevent deg- radation in transit between storage locations and on- site for usage. The primary causes of viral damage in storage are aggregation, genome leak from the capsid, and free-radical oxidation, which should be consid- ered when formulating the final virus solution (17). Mitigating contamination risks To combat dif f iculties in plasmid expansion, cell culture surface area can be increased for adherent cell culture, as long as commensurate contamina- tion risks are mitigated through careful adherence to culture protocols (10,17). Common methods of scal- i ng up ad herent cel l c u lt u re i nclude mu lt i layer systems and cell factories, while the use of biore- actors is becom ing more f requent for improv ing large-scale suspension yield. Multilayer cell factory systems can be up to 40 layers and can drastically re- duce the manual labor time and contamination risks by allowing culture seeding to take place simultane- ously across plates (12,18). Alternatively, bioreactors, which enable large-scale suspension culturing, are growing in popularity as they are closed systems. They reduce the risk of con- tamination and eliminate the need of dedicated clean rooms, resulting in markedly reduced manufactur- ing costs. Further reducing contamination risks, sin- gle-use components can alleviate the manufacturing process by eliminating the need of cleaning valida- tion. Although the scale for bioreactors remains low, early success with scaling from 50 L to 500 L AAV culturing is promising, and some manufactures are scaling up to 5000 L (19). Par tial automation in both approaches can f ur- ther streamline and insulate manufacturing using si ng le-u se component s a nd a sept ic t ra n sfer de- vices, though each pipeline must be tailored to the application, virus, cell type, compatibility, and per- formance necessary. Improving virus stability through excipients Investigation and addition of various excipients that increase virus stability, extend shelf life, and reduce cold dependency will also benefit both production yields and product storage. Excipients range widely in form and can include salts, sugars, surfactants, polymers, and amino acids. Thus, the type of excipi- ent necessary strongly depends on the need, and ex- cipients can be used to stabilize vectors both in solu- tion and in lyophilized form. For clinical applications, excipients must be well-characterized with low im- purities and endotoxins to control degradation rates. This, in turn, increases the safety of the product, as viral products are often stored frozen, and therefore, Advancements in novel cell lysis solutions allow for improved efficiency, lower foam, and improved viral vector recovery.