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Pharmaceutical Technology BIOLOGICS AND STERILE DRUG MANUFACTURING 2020 27 chromatography. It should be noted that large losses of lentivirus have been reported using ster- ilizing grade (0.2 µm or 0.22 µm) membranes; as such, it is recommended that 0.45 µm or larger be used for intermediate bioburden control filtration. Lentivirus-specific concerns. The majority of depth filtration media was designed for CHO cell pro- duction of mAbs. These mAbs are approximately 15.5 nm long and positively charged in standard cell culture media. Lentiviruses do not share these physical char- acteristics. Like most viruses, lentiviruses have a considerable net negative charge and are between 80–100 nm in size and are relatively fragile in terms of osmotic/pH conditions and interfacial shear. These characteristics presents challenges with modern depth filter media. Diatomaceous earth, or the synthetic silica equivalent, has a specificity for large particles via a range of binding mecha- nisms, making the media excellent for removal of product aggregates; unfortunately, it also binds to large particles such as lentiviruses. Evidence suggests that the binding to the diatomaceous earth silica is the principle mechanism of yield loss within these filters, with the charge interac- tion having a minor contribution. Another shortcoming is that depth filter media are typically held together with a cationic resin binder for structural strength and to assist in re- moval of negatively charged contaminants, such as DNA and some HCP. As lentiviruses are nega- tively charged, this also encourages binding to the media, which can lead to a dramatic reduction in yield when processed on this type of depth filter. As such, if depth filtration is to be utilized suc- cessfully for lentivirus clarification, neutral media components are required. Case study: lentivirus clarification using depth filters In this case study, the authors evaluated the usage of a synthetic depth filter (Clarisolve, Millipore- Sigma, Burlington, MA) for the clarification of a lentivirus-based therapy feed. The depth filters in this case study have a gra- dient density structure specifically designed to the particle size distributions of pretreated feed streams. Clarification of pretreated feeds can be processed in a significantly reduced footprint without the need for a secondary stage of clarifica- tion. This enables the fastest and most efficient way to clarify high-density streams and easily transfer processes from upstream to downstream without the use of centrifugation. Uncharged and polypropylene-based depth filters (Clarisolve 60HX 23 µPods CS60HX01L3 [23 cm 2 ] and Clarisolve 60HX 140 [CS60HX01H1, 135 cm 2 ], MilliporeSigma, Burlington, MA) were used as part of a scouting experiment. The filters were trialed with the suspension-adapted cell line, HEK293Tsa, with cell densities from 4.16 to 5.12 E6 cells/mL and viability from 83.6% to 87.2%. Pressure curves were generated with the different starting conditions. This clarification filter train was then scaled up to 50-L scale successfully, as shown in Figure 2. Normalizing the data against f low rate, a reduc- tion in total turbidity was achieved for each small- scale experiment. All recovery for the actual titer was greater than 60%, which was deemed accept- Different feed streams of higher cell density or lower viability might require a secondary clarification step.