Single-use concepts are widespread in all unit operations of the biopharmaceutical industry. Although single-use technology is rapidly advancing and considered to be highly advantageous in many regards (1–3), in some cases it cannot (yet) compete with classical manufacturing systems. Processes with a demanding character (e.g., high cell densities, high titers, high turbidities, increased particle/contaminant loads) especially can bring disposables to their limit of technical feasibility, especially in product harvesting (4–6). Here we focus on that step, which is defined as a removal of cells, debris, and (ideally) typical contaminants such as host-cell proteins (HCPs) and DNA from process f luid. In conventional (multiuse) facilities, a typical harvested is a multistep procedure:• a centrifugation step for removal of cells and solids >1 μm• a depth filtration step for removal of smaller particles, colloids, and contaminants• a final filtration step to remove/reduce bioburden. To perform similar tasks in a single-use facility, different approaches have been necessary because single-use centrifuges were not available until only recently. The most convenient way was to replace the centrifugation step by direct filtration of process f luid through a cascade of depth filters. In general, a significant turbidity reduction beyond those filters with partial removal of contaminants would be achievable with such an approach (7)
Although such filtration results may be promising, depth filtration does have several disadvantages. Depth-filtration system capabilities alone are often insufficient. Flow rates can drop dramatically due to an early pressure increase (5, 8). Problems can occur: e.g., filter blocking and turbidity breakthrough. Depth filtration is the only remaining filtration technology that requires pref lushing of filter material, which is costly and laborious. Finally, harvesting of 1,000-L to 2,000-L volumes (currently the upper limit in single-use bioreactors) using this approach can be economically questionable because of relatively low filtration capacities (per m2 of filter). All those disadvantages have led some people to search for different methods that can be operated in a single-use set-up without a centrifuge: sedimentation (5), tangential-f low filtration (9), f locculation (10, 11), and body-feed filtration (12). And several attempts have been made to remove the clarification step and directly bind target molecules to an adsorptive material (13). One possibility is settling cells based on gravity alone — or with addition of substances that can enhance settling velocity by aggregation (e.g., Chitosan or DEAE). Although sedimentation does not require additional technical equipment, concerns arise regarding settling time and reproducibility of the process, which could adversely affect product quality. Substances added to enhance settling must be removed from the process f luid downstream, which adds complexity to a purification process (10, 11). Those alternative methods have been shown to apply with specific process conditions and products, but universal applicability cannot be claimed. Meanwhile, single-use centrifuges has become commercially available (2). Their application remains limited with respect to removal capacity, achievable g forces, and scalability. We evaluated filtration performance of a new single-use module operated in body-feed mode using diatomaceous earth (DE) as a filter aid in combination with a dead-end filter. The filter aid is crucial. Over 10,000 different species of extinct and living diatom algae have been described (14). Their skeletons consist of an inert SiO2structure that is unlikely to affect product properties. Of the US diatomite production of 0.82 million metric tons per year, 75% is produced for filtration applications (15). The Celpure DE (Advanced Minerals Corporation) that we used is different from common DE used in the food and beverage industry. It is highly purified (96–99% SiO2 with very low contaminants) and certified according to USP-NF requirements (16). During Celpure production, impurities are removed before calcination and fusion onto a diatom surface (17). Consequently, the product can be used in biopharmaceutical production without regulatory restrictions. We decided to test different types (e.g., particle size and permeability) and concentrations of DE as well as different cell lines and starting conditions (cell concentration, viability) to develop a robust method with general applicability. To improve filtration performance, we tested different pH reduction approaches. To enlarge particle size and prevent release of submicron particles, it has been proposed in literature to cause particle precipitation by a pH shift (to pH 4.3–5.5) before clarification process (18, 19). Moreover, pH reduction can lead to DNA/HCP precipitation in the final filtrate (20). Despite the many different f locculants for cell harvest such as pDADMAC, Chitosan, DEAE described in the literature (21, 22), we focused on low-pH precipitation alone. Finally, we compared DBF filtration with a pH-reduced DBF filtration with direct filtration through a depth filter. Filtration using other depth-filter modules with an equivalent process f luid have led to capacities of ~50 L m2. Subsequently we also scaled up the reduced-pH DBF to 600 L to demonstrate practicability and robustness at larger scales. During all experiments, we monitored important parameters such as filtration capacity; trending pressures and f low rates; turbidity; lactate dehydrogenase (LDH) content; and IgG1, HCP, and DNA contents.