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26 Pharmaceutical Technology BIOLOGICS AND STERILE DRUG MANUFACTURING 2020 P h a r mTe c h . c o m effective when bioreactor volumes exceed 3000 L, and in fact are often the only economical way of processing such large volumes. Most viral vectors, however, do not require large bioreactor facilities. When operating at volumes less than 3000 L, the disadvantages of centrifugation, such as noise lev- els, relatively poor clarification effectiveness, con- trolling discharge, complex clean-in-place (CIP) operations, and significantly large capital expen- ditures (CAPEX), greatly outweigh the advantages. Tangential flow filtration. Tangential flow filtration (TFF) using microfilters is a proven and robust clarification method for mammalian cell process- ing. Permeates are clear enough to pass through normal f low membrane filtration, thus eliminat- ing the need for further downstream clarification. Gamma-irradiated closed f low paths are also pos- sible to install with current technology. The disadvantage of TFF processes derives from its iterative f low path. The feed stream requires substantial time, often hours, and dozens of pump passes in the TFF system to process. Lentiviruses themselves are very susceptible to interface shear that is difficult to minimize in these systems, and cell viability will suffer in these conditions. This leads to reduced yield and increased contaminant loads fed to the remainder of the downstream process, exacerbated by the lack of adsorptive contaminant removal a depth filter can provide. TFF systems also involve considerable CAPEX and installation lead times. Depth filtration/membrane filtration. Depth filters are the leading technology for clarification of mammalian cell culture, either as a two-stage train for smaller bioreactors (2000 L and below) or as a polishing filter for centrates at larger scales. Depth filters have many advantages over other technologies. If set up correctly, even at process scale, a fully single-use f low path can be estab- lished. CAPEX costs are relatively limited, and installation is rapid with minimal CIP required. Depth filter media are typically adsorptive by design and can remove significant levels of HCP, DNA, aggregates, and other contaminants. Depth filtration has some drawbacks, however, which can limit use in certain applications. Al- though recent advances in multi-layer (4+) depth filter modules and f locculation technology have helped address this challenge, the technology still provides limited capacities with the significantly high cell densities that are more common in fed- batch and perfusion cell culture. Even with a rea- sonable capacity, footprint can also be an issue; the number of filters required at large bioreactor scales can be prohibitive to install and dispose of in clas- sified facilities. Additionally, the organic nature of depth filter media components, such as diatoma- ceous earth and cellulose, means the f lushing re- gime can be operationally limiting. Recently intro- duced synthetic media components are expected to reduce the impact of this issue, however. Despite these challenges, one- or two-stage depth filtration remains the best choice for mammalian cell-culture clarification at volumes of 2000 L bio- reactor or less. Membrane filtration is usually implemented post-clarification as a bioburden reduction step and for final assurance of particle removal before Manufacturing Gamma-irradiated closed flow paths are also possible to install with current technology.