Ultrafiltration and Microfiltration Principles

Membrane Selection

Membrane selection should start with the choice of a high quality vendor since robustness, reliability and reproducibility of manufacturing operations are of paramount importance in most bioprocessing applications. Consistent membrane and device characteristics can be as important to product quality, yield and economics as the inherent differences between various membranes and devices. Cellulosic membranes are attractive for many bioprocessing applications because of their low protein adsorption and low fouling characteristics. Synthetic polymers (e.g. poly-sulfone and polyvinylidene fluoride) are also attractive due to their greater chemical and mechanical stability. These polymers are often surface-treated to render them more hydrophilic to reduce protein adsorption and fouling. Membranes used for sterile filtration must be steam-sterilizable, have minimal particle shedding, low extractables and must pass United States Pharmacopoeia (USP) Class VI toxicity testing.

Most manufacturers rate ultrafiltration membranes by their nominal molecular weight cutoff, which is defined as the molecular weight of a solute with a particular retention coefficient:

filtrate/ Cfeed

where Cfiltrate and Cfeed are the solute concentrations in the filtrate solution and feed stream, respectively. Data are typically obtained with a range of model proteins or with polydisperse dextrans. Unfortunately, the procedures used for assigning molecular weight cutoffs, including the choice of solutes, the specific buffer and flow conditions, and the chosen retention value (usually R = 0.9) vary widely throughout the industry. In addition, ultrafiltration systems used in bioprocessing generally require protein retention of at least 99%, and often as high as 99.9%, to minimize loss of high value products through the membrane. Data obtained with solutes having R = 0.9 are often of little value in determining whether a given membrane can provide these high levels of protein retention due to differences in the details of the pore size distributions.

Microfiltration membranes are typically rated by their pore size or their particle retention characteristics using the log reduction value (LRV), defined as the logarithm (base 10) of the ratio of the particle, cell or virus concentration in the feed to that in the filtrate solution. Sterilizing-grade (0.2 |im pore size) filters are currently defined by the Health Industry Manufacturing Association (HIMA) as a filter which produces a sterile filtrate when challenged by 107 colony-forming units of Brevundimonas diminuta (formerly classified as Pseudomonas diminuta) per cm2 of membrane area. This challenge uses the smallest bacteria at a concentration that exposes essentially every pore to the microorganisms. Sterile filters are often thought of as operating via a purely size-based (siev ing) mechanism, although bacteria can also be removed by adsorption on to the membrane surface.

The chemical compatibility of the membrane needs to be verified with the feed, regeneration chemicals and storage solutions. Sodium hypochlorite (NaOCl) is used most extensively for chemical disinfection of membrane systems in bioprocessing applications. Many membrane systems are designed for steam-in-place (SIP) sterilization, with the entire unit exposed to flowing steam as part of the completely assembled filtration system. Minimum requirements for an effective steam sterilization are 15 min exposure to steam at 121°C and 1 atm pressure. Polysulfone membranes tend to have broader chemical and thermal stability than cellulosic membranes but also require harsher chemical treatment for regeneration due to their greater fouling characteristics. Inorganic (ceramic) membranes have the greatest chemical compatibility, but they are much more expensive than polymeric membranes. The mechanical strength of the membrane is important since reverse-pressure spikes can cause membrane delamination and catastrophic yield loss.

Module Design

Dead-end, or normal-flow, filtration (Figure 2A) is used primarily for laboratory-scale separations and for systems in which the retained species are present at very low concentration. For example, dead-end microfiltration cartridges are used extensively for sterile filtration since the retained bacteria are present at very low concentration. Similar modules can be employed for virus removal applications. Almost all large scale commercial ultrafiltration devices use tangential flow filtration, also referred to as a cross-flow configuration, in which the feed flow is parallel to the membrane and thus perpendicular to the filtrate flow (Figure 2B). This allows retained species to be swept along the membrane surface and out of the device exit, significantly increasing the process flux compared to that obtained with dead-end operation.

A number of tangential flow modules have been developed for ultrafiltration and microfiltration

Figure 2 Comparison of (A) dead-end and (B) cross-flow configurations.

Figure 2 Comparison of (A) dead-end and (B) cross-flow configurations.

Table 1 Comparison of different module configurations

Module configuration

Channel spacing (cm)

Packing density

Energy costs (pumping)

Particulate plugging Ease of cleaning

Flat sheet Hollow fibre Tubular Spiral wound

300 1200 60 600

Moderate Low High Low

Moderate

High

Very high

Good Fair

Excellent Poor to fair processes, differing primarily in the size and shape of the feed and filtrate flow channels. Table 1 provides a general summary of the physical characteristics of the most common modules. Detailed descriptions of these modules are available elsewhere.

The small channel spacing in flat-sheet, hollow-fibre and spiral-wound modules provides high membrane-packing densities. In addition, these modules have low hold-up volumes, which facilitates the recovery of high value products. The screens used to define the flow path in spiral-wound modules and many flat-sheet cassettes are susceptible to particle plugging and this may make cleaning more difficult. Hollow-fibre membranes are self-supporting, so they can often be cleaned by simple backflushing. The large-bore tubular membranes can be cleaned by both physical and chemical methods. However, these modules operate in the turbulent flow regime which can cause cell lysis, protein denaturation or aggregation. A variety of enhanced mass transfer modules which exploit flow instabilities have also been developed for bioprocessing applications. Rotating cylinder modules which induce Taylor vortices have very high mass transfer rates, although there are concerns about the moving parts. Another attractive approach is to use helically coiled hollow fibres wrapped around a central core to induce Dean vortices.

Process Configurations

Protein concentration can be carried out using either batch or fed-batch operation (Figure 3). In a batch process, the entire feed volume is contained within the recycle tank. Tank design is critically important to ensure adequate mixing while avoiding air entrain-ment and excessive foaming. Batch operation uses a minimum of hardware and allows simple manual or automatic control. The flux rates are also higher in batch processes since the bulk concentration follows a more dilute path in going from initial to final concentration. Disadvantages of the batch configuration include less flexibility in using the same system for multiple processes, greater difficulty in designing a well-mixed system, and difficulties in obtaining high concentration factors.

The fed-batch configuration utilizes an additional tank to feed into the recycle tank (Figure 3). Fed-batch configurations are commonly used to obtain high concentration factors and to provide well-mixed, low-hold-up, retentate reservoirs. These systems also provide flexibility for use in multiple processes. The disadvantages of the fed-batch system include greater process time and greater number of passes of the retentate through the pumps and valves in the recycle line. The latter can lead to excessive cell lysis, protein denaturation or aggregation.

Diafiltration is commonly used for buffer exchange (for products in the retentate) and to enhance yield (for products in the filtrate). The diafiltration system looks similar to the fed-batch configuration shown in Figure 3 except that the feed tank contains a buffer solution which is added to the recycle tank. The most common approach is constant retentate volume diafiltration in which the buffer is added at the same rate as filtrate removed.

The yield and purification obtained in ultrafiltration and microfiltration processes can be evaluated from simple mass balances on the product and impurity assuming constant rejection coefficients. The final product concentration (CF) at the end of a batch concentration process is given as:

where VF is the final retentate volume, V0 is the initial retentate volume and S is the product sieving coefficient (equal to one minus the rejection coefficient). The analogous expression for a fed-batch process is:

The final concentration after a constant retentate volume diafiltration is:

(B) Filtrate

Figure 3 Comparison of (A) batch and (B) fed-batch processes for protein concentration.

(B) Filtrate

Figure 3 Comparison of (A) batch and (B) fed-batch processes for protein concentration.

where the number of diavolumes (N) is given by:

where VD is the diafiltration buffer volume. Even very small sieving coefficients may result in substantial product loss when a large number of diavolumes are required in diafiltration processes. For example, a diafiltration process with a product sieving coefficient of S = 0.01 will result in a 10% product loss during a 10 diavolume buffer exchange.

Concentration Polarization

One of the critical factors determining the overall performance of tangential flow filtration devices is the rate of solute/particle transport in the bulk solution adjacent to the membrane. The filtrate flow causes an accumulation of partially (or completely) retained components at the upstream surface of the membrane, a phenomenon referred to as concentration polarization. The concentration thus varies from its maximum value at the membrane surface (Cw) to its bulk value (Cb) over the thickness of the concentration boundary layer (¿). Most analyses of concentration polarization have employed the simple stagnant film model in which:

where J is the filtrate flux (typically in Lm~2h_1) and k is the solute mass transfer coefficient in the particular membrane device. The accumulation of particles/solutes at the membrane surface increases the overall resistance to filtrate flow through the formation of a particle cake or gel layer and it can reduce the effective pressure driving force through the osmotic pressure of the retained solutes. At high transmembrane pressures, the wall concentration approaches a maximum value determined by the close-packed concentration of the particles or cells, the protein solubility limit or the concentration at which the osmotic pressure of the retained solutes is essentially equal to the applied transmembrane pressure. The net result is that the flux attains a nearly constant pressure-independent value that increases with decreasing bulk concentration and increasing feed flow rate. The dependence on feed flow rate is determined by the module characteristics: approximately 1/3 power for laminar flow in hollow fibres and open channels, 1/2 power for screened channels, and 0.8 power for turbulent flow in tubular modules. The dependence on feed flow rate for cellular suspensions is typically greater than that for protein solutions due to shear-induced particle diffusion and inertial lift effects.

Process Control

Ultrafiltration and microfiltration processes have traditionally been performed at constant transmembrane pressure. Constant-pressure processes are very simple to control. The feed rate is ramped up to the set point and the retentate valve is then partially closed to obtain the desired transmembrane pressure. The transmembrane pressure should be gradually increased to minimize fouling. In some applications it may not be possible to maintain constant transmembrane pressure without severe reductions in filtrate flux over the course of the process due to membrane fouling. This is particularly true for cell microfiltration where the high initial flux leads to very rapid deposition of cells and cell debris on the membrane surface. Several studies have shown that higher overall throughput can often be obtained in these applications by operating at constant filtrate flux. The flux is controlled by regulating the retentate pressure control valve or by using a pump on the filtrate line.

A third method of process control that is very attractive for bioprocessing applications is to vary the filtrate flux so that the wall concentration of retained species (evaluated from eqn [6]) remains constant during the process. Control is performed using a proportional-integral-derivative (PID) loop that measures flux and controls the transmembrane pressure or filtrate flow rate to maintain a constant wall concentration throughout the process. The benefits of constant Cw control are that product yield is maximized, product quality is ensured, membrane area is minimized and process time is consistent and independent of variations in membrane permeability.

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