One of the most broadly used harvest technologies for animal cell cultures is cross-flow or tangential-flow filtration (TFF). Unlike normal-flow filtration where the feed and filtrate flows are perpendicular to the filter media, in TFF operations the feed flows tangentially along the filter surface. The filtrate is forced through the membrane by applying a pressure drop across the surface while the cells and cellular debris are retained by the membrane (retentate) and flow back to the bioreactor. The shear caused by the feed flow across the membrane surface helps prevent formation of a concentration polarization layer which enables TFF operations to process volumes at higher fluxes than can NFF methods. Since the filtrate is continually removed, the solids are concentrated in the bioreactor until the desired end point. This concentration phase is typically followed by a diafiltration phase in which buffer enters the feed stream at the same rate as filtrate is removed to wash the cells of any residual product-containing fluid. During diafiltration, the volume of buffer used to exchange the entire volume remaining in the bioreactor (retentate) is called a diavolume. The theorectical product yield can be calculated as a function of the concentration factor and the number of diavolumes using (Cheryan 1986):
where Y is yield, CF is concentration factor, R is retention and DV is number of diavolumes. Retention is calculated using R = 1 — Cfilt/Cfeed where Cfilt and Cfeed are the product concentrations in the filtrate and feed, respectively. Therefore products that completely pass through the filter have a retention equal to 0.
According to equation (4), a process with CF = 10, R = 0 and DV = 1 achieves a theoretical yield of 96 %. Increasing the DV to 3 improves the yield to 99.5 %.
Tangential-flow filtration is not only used as a cell harvest method, but also as a method for protein concentration and buffer exchange. Application of TFF for cell harvest is presented in this section and its application for protein concentration is presented in Chapter 17. Cell harvesting via TFF uses microfiltration membranes sized to retain intact cells and some portion of the cell debris. Since this method returns the cells to the bioreactor and removes the filtrate, TFF can be used to harvest both perfusion and batch bioreactors. Regardless of the mode of bioreactor operation, the principle of operation, membrane selection and key process development goals are similar.
Microfiltration membranes are made from a variety of polymers, including cellulose nitrate and acetate, polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polyethylene,
polypropylene, polysulfone, polyethersulfone (PES), polycarbonate, polyvinylchoride (PVC) and polyamide (Zeman & Zydney, 1996). These polymers are cast into a thin layer and are bonded onto a support to form the membrane. Often the membrane surface is chemically modified to improve the performance. Unlike depth filters, microfiltration membranes have more defined pore structures with effective pore sizes ranging from 0.1 to 5 |im.
The membranes are then formed into hollow fibre, flat sheet or spiral wound modules from which tangential-flow devices of various filtration areas are constructed (Figure 16.8). These three geometries have all been implemented for harvesting biological solutions and the features of each will be described in this section. In addition, researchers are continually experimenting with new designs aimed at increasing the mass transfer rates by applying external fields, centrifugal forces or motion. These contributions to the traditional microfiltration approach will be presented later in this chapter.
One of the earliest devices manufactured for microfiltration was the flat plate or cassette device. Flat plate modules consist of layers of membrane, retentate (feed side) and filtrate (or permeate) channels. These layers are either held together with gaskets and fitted into a holder that is compressed to form a seal, or their edges are glued or heat-sealed using polyurethane or other hard polymer into a cassette. Cassettes can then be stacked together into large stainless steel holders for production-scale operations.
Cassettes are typically rectangular with the feedstock entering channels on one side, flowing across the membrane and exiting via retentate channels on the other side. The filtrate flows through the membrane and exits through filtrate channels. Figure 16.8 shows the flow paths through a flat plate TFF device. Most TFF device manufacturers offer cassettes with or without turbulence-promoting screens inserted into the feed and filtrate channels. These screens promote mixing by creating turbulent flow through the channel, improving the mass transfer rates without increasing the pumping requirements. Alternatively, unscreened or open-channel devices are noted for producing lower shear on the feedstock and are often the preferred choice for cell harvest operations.
Flat plate microfiltration systems can be used to harvest large (>10 000 l) production cultures as shown in Figure 16.9. This industrial-scale system contains 2000 ft2 (186 m2)of membrane area capable of harvesting 10 000 l of cell culture fluid in approximately 3 hours.
MICROFILTRATION MEMBRANES AND DEVICES
MICROFILTRATION MEMBRANES AND DEVICES
188.8.131.52b Hollow fibre
Another microfiltration device widely used for cell harvesting is the hollow-fibre module. Hollow-fibre devices group together many small diameter fibres that are packed together and bound on either end such that the inner diameters of the tubes are exposed. Usually, for microfiltration applications, the feed flows through the inner diameter of the tubes while the filtrate flows through the membrane into the 'shell' side. Gaskets on either end separate the feed and filtrate flow paths. Hollow-fibre devices are noted for their lower plant space requirements per-surface area, lower shear rates due to the open feed flow path and laminar flow through the inner fibre diameters, and ability to scale from bench to production harvest operations. Drawbacks to hollow-fibre systems include lower filtrate rates (or higher pumping requirements) relative to flat plates, fewer choices in membrane chemistries, and a greater tendency to foul when harvesting cells with higher debris loads.
Spiral-wound microfiltration devices are constructed by rolling a membrane layer and support screen around a hollow cylinder. The feed flows in at the top of the cylinder, along the membrane surface, and the retentate exits at the other end, similar to the design in a hollow-fibre module (Figure 16.8). The filtrate flows radially inward to the centre tube and is collected from one end of the module. Benefits of spiral-wound devices include high packing densities and relatively high filtrate fluxes, due to the incorporation of turbulence-promoting screens. Spiral-wound devices are not as easy to implement for production-scale operations due to their inherent inability to be scaled up linearly. Spiral-wound modules are also more susceptible to fouling and are more difficult to clean due to inaccessible regions within the module.
Device selection does impact the process robustness and performance that can be achieved from tangential-flow microfiltration harvest systems. Key process development goals may only be met with one device format, given the feedstock characteristics and system constraints such as plant space, process time, operating costs, membrane lifetime and reuse. The primary design components for a tangential-flow microfiltration harvest system are system size (membrane area), feed rate, filtrate rate, filtrate control, transmembrane pressure (TMP), membrane pore size and permeability, concentration factor and number of diavolumes. Compared to depth filtration, TFF systems are more sophisticated and contain more equipment and instrumentation. A typical TFF system contains the membrane unit, feed and filtrate pumps, bioreactor, diafiltration buffer tank, downstream polishing and sterilizing-grade filter housings, and filtrate collection tank. Use of an automated control system at production scale permits process parameter control, data acquisition, electronic batch reporting and improved reproducibility from run to run.
TFF harvest process development is facilitated by the availability of small-scale devices whose performance is predictive of their large-scale counterparts. The benefits of linearly scaleable systems have been described by van Reis et al. (1997). As with depth filtration, small-scale testing with comparable feedstocks is an absolute requirement so that production-scale systems can be implemented with minimal risk. Unfortunately, process development efforts for TFF are considerably more time-consuming than for depth filtration, since flux is not the only parameter to evaluate and an efficient screening method such as Vmax does not exist. Nevertheless, since TFF is primarily a size-based solid-liquid separation method, it is possible to develop standardized TFF harvest processes for multiple products expressed by similar cells.
Process development for TFF means determining the optimum feed and filtrate rates, membrane area, process time, amount of diafiltration, and downstream filtration requirements. Practical considerations such as collection tank volume and turn-around time typically dictate total process times and maximum diafiltration volumes. Once these constraints are in place, experiments are conducted with various membrane chemistries, pore sizes and device formats to assess separation performance. Selection of membrane chemistry may be driven as much by cleanability and compatibility with storage solutions as by permeability and cell retention. Selection of membrane pore size also depends on downstream filtration options. More open (^0.65 |im) membranes have higher fluxes but may require significant downstream depth filtration areas to protect the final sterilizing grade filters. Tighter (^0.2 |im) membranes may not require downstream filtration, but could foul more easily with higher density cultures.
During development, experiments with small-scale devices are conducted with representative feedstocks to determine the optimum feed and filtrate rates and the maximum TMP that can be tolerated by the system and the product. The filtrate rate is controlled using a pump or valve to ensure an adequate cross-flow rate and to minimize rapid membrane fouling during start-up. Ideally the goal is to maximize the filtrate flux and evaluate the filtrate clarity, product retention, TMP and cell lysis at various feed and filtrate rates. While higher feed rates lead to higher filtrate rates at the same TMP, the shear rate and number of pump passes also increase, possibly contributing to higher cell lysis and poorer product quality. Once the optimum filtrate rate and feed rate are determined for a given device, chemistry and pore size, the required membrane area for production scale can be determined.
Membrane area selection depends on the process volume and the desired process time. Larger systems reduce process times, but require higher capital and membrane costs, have higher volumetric hold-up and require more plant space. Too long a process time risks bi-oburden contamination due to longer non-sterile operation, and less efficient use of plant capacity. Once the TFF processing conditions are established, effluent from the TFF harvest system is used to size downstream depth and sterile filters. These filters are sized using the same Pmax and Vmax methods described previously. Since the filtrate is continually changing during the course of the microfiltration harvest process, it is important to test the downstream filters in-line with the TFF system by diverting some of the filtrate into small-scale housings. By extending the TFF and normal-flow testing beyond the expected volume at production scale, a safety margin can be added to the predicted filter area requirements.
Throughout the testing, consideration must be given both to the concentration of solids in the bioreactor and to cell lysis that occurs through multiple passes through the feed pump and system. For example, cell lysis measurements taken during multiple TFF harvests at production scale showed that the harvesting caused as much as 47 % additional cell lysis (George 1994). Another concern is if feed solids become too concentrated to pump through the feed channels at a reasonable pressure, the harvest operation could terminate early due to high pressures, resulting in low product yields. Although the maximum solids volumes can be experimentally determined for each culture, in general, solids volumes approaching 20-30 % become challenging for even the more open TFF membranes. As starting solids volumes increase, the researcher must evaluate the trade-off between concentration factor and the amount of diafiltration required to achieve a high (>95 %) yield. As the concentration factor decreases, the amount of diafiltration increases, the processing time increases and, importantly, the final filtrate volume increases.
While microfiltration harvest systems are reliable and relatively easy to maintain, there are some key factors to consider when installing an industrial-scale system. First, the associated equipment, particularly the feed pump and piping, can be quite large and require significant volumes of cleaning chemicals. The capital costs of an industrial scale TFF system are far greater than those of a depth filtration harvest system. Second, the cost of the membranes encourages reuse, which must be well documented and validated to show that the first and last runs of the membranes perform identically. Finally, a separate set of membranes must be used for each product at each scale manufactured in a given facility. For example, harvests at 400 l, 5000 l and 10 000 l scale will require three complete TFF harvest systems, including pumps and other auxiliary equipment. Nevertheless, TFF continues to be used reliably for harvesting animal cell cultures and is a good option for processing large volumes of lower cell population density cultures. As the cell population densities increase, harvest methodologies that do not require concentration and recycling of the cell mass become more feasible than tangential-flow microfiltration.
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