Compaction and permeability effects with virus filtration membranes

Journal of Membrane Science 254 (2005) 71–79

Compaction and permeability effects with virus filtration membranes David M. Bohonak, Andrew L. Zydney∗ Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, USA Received 8 March 2004; received in revised form 20 December 2004; accepted 23 December 2004 Available online 12 February 2005

Abstract Virus filtration can provide a robust, size-based viral clearance mechanism in the production of biotherapeutics. Successful application of virus filtration requires membranes with both high capacity and high hydraulic permeability. Normal flow filtration experiments were performed with Viresolve 180, DV20, DV50 and Omega 300 membranes using different flow orientations. The normalized flux for the Viresolve 180 membranes declined by nearly 50% in the skin-side up orientation due to fouling by trace levels of submicron-sized particles. These particles could be removed using a small pore size filter placed directly in-line. The flux through the Viresolve membrane with the skin-side down was insensitive to particle fouling since the large pore substructure acts as a depth pre-filter. However, membrane compaction with the skin-side down resulted in a 10–20% reduction in permeability. In contrast, the flux through the DV20 and DV50 membranes increased during filtration due to the time-dependent wetting of the membrane pores. Less dramatic increases in flux were observed with the Omega 300 membrane. Particulate fouling had little effect on the performance of the DV20, DV50 and Omega 300 membranes. These results provide important insights into the hydraulic characteristics of these virus filtration membranes. © 2005 Elsevier B.V. All rights reserved. Keywords: Biotechnology; Compaction; Fouling; Virus filtration; Ultrafiltration

1. Introduction One of the critical issues in the development of therapeutic proteins for human use is the risk of virus contamination. Pathogenic viruses or virus-like particles can enter process streams through contaminated media, in mammalian cell lines or by propagation in fermentors. Virus contamination has been reported in a variety of cell cultures, vaccines and other biotherapeutic products [1–3]. Several outbreaks of hepatitis A [4–7] and hepatitis C [8–10] have been attributed to the use of contaminated blood-derived products. In order to meet federal and international guidelines that assure adequate virus clearance and product safety, biopharmaceutical manufacturers typically employ a series of steps to inactivate and remove viruses from process streams. Heat, radiation or chemical treatment can be used for virus inactivation, while virus removal is typically accomplished by ∗

Corresponding author. Tel.: +1 814 863 7113; fax: +1 814 865 7846. E-mail address: [email protected] (A.L. Zydney).

0376-7388/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2004.12.035

chromatography or membrane filtration. Use of small-pore size virus filtration membranes offers a robust, size-based viral clearance mechanism. Since the presence of only a small number of abnormally large pores will permit excessive virus leakage [11], virus filters must be manufactured so as to eliminate all macro-defects. This has often led to the use of composite (multi-layer) membrane structures that provide the required combination of virus retention and mechanical stability. Virus filters were originally designed for use in tangential flow filtration (TFF) with the feed flowing adjacent to the upper skin layer of the asymmetric membrane [12]. TFF provides high flux by sweeping the membrane surface to reduce concentration polarization and fouling. However, the simplicity and lower capital cost of normal flow filtration (NFF) has led to the widespread use of virus filters specifically designed for NFF. In contrast to TFF, these normal flow filters are typically operated with the more open side of the membrane facing the feed stream [13] allowing protein aggregates and other large foulants to be captured within the

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macroporous substructure thereby protecting the virusretentive skin layer. The sizing of virus filtration systems is governed by two key parameters: the clean membrane permeability, which provides a measure of the initial volumetric filtrate flow rate through the filter, and the capacity, which provides a measure of the maximum volume of feed solution that can be processed before fouling reduces the flux to unacceptably low values. The membrane permeability is determined by the pore size, porosity and overall pore structure of the membrane. Although the permeability is usually assumed to be constant, independent of both time and pressure, there is considerable experimental evidence that microfiltration, ultrafiltration and reverse osmosis membranes can compact under pressure, resulting in significant changes in permeability. However, there have been no published studies of compaction or permeability effects with virus filtration membranes.

2. Previous work Membrane compaction remains a somewhat controversial topic due to the difficulty in distinguishing true membrane compaction effects from membrane fouling [14]. For example, McGregor [15] reported that the distilled water flux through several different polysulfone ultrafiltration membranes declined by almost a factor of two during the first hour of filtration, a phenomenon that was initially attributed to membrane compaction. However, subsequent studies demonstrated that this flux decline could be completely eliminated by first passing the distilled water through an activated carbon cartridge, a mixed bed ion exchange resin, and then a 0.2-␮m pore size microfilter. Thus, the flux decline seen in the original experiments was actually due to some type of membrane fouling, probably associated with the deposition of particulates or microorganisms that were present at trace (essentially undetectable) levels in the distilled water. Numerous quantitative studies of membrane compaction have been performed with reverse osmosis membranes. The data have typically been correlated using a power law model that describes the change in volumetric flux (Jv ) over time (t):


Jv J0




−m t = t0

(1)

where t0 is a characteristic time for compaction, J0 is the initial flux and the parameter m is a function of the membrane polymer, pore size and porosity. Typical values for m range from 0.03 to 0.1 with t0 = 24 h, indicating that the change in flux occurs slowly over very long periods of time. For example, flux decline data obtained by Baayens and Rosen [16] for asymmetric cellulose acetate membranes were consistent with Eq. (1) over more than 100 h of operation. Similar results were reported by Kimura and Nakao [17] for tubular cellulose acetate membranes.

Ohya [18] correlated the time-dependent compaction of cellulose acetate membranes using a more complex relationship: 1 1 = + at + be−t/τ Lp Lp,0

(2)

where Lp = µJv / P is the hydraulic permeability, µ the solution viscosity, P the transmembrane pressure, Lp,0 the initial permeability, and a, b and τ are constants for a given membrane. Ohya [18] attributed the exponential term to the rapid elastic compression of the membrane substructure, with the linear term representing the gradual increase in thickness of the membrane skin. Belfort et al. [19] used SEM measurements of membrane thickness to demonstrate that the elastic compaction of asymmetric cellulose acetate membranes occurs in less than 15 min and at pressures below 1 MPa. Peterson et al. [20] used ultrasonic time-domain reflectometry to study the viscoelastic behavior of cellulose acetate membranes, with the data suggesting that the dominant effect is due to changes in the macroporous substructure. Studies of compaction with microfiltration and ultrafiltration membranes are somewhat more limited, although there is clear evidence that compaction of these larger pore size membranes can occur at relatively low pressures. For example, Bowen and Gan [21] found a 25% decline in flux for microfiltration membranes after only 40 min of compression at 200 kPa. Tarnawski and Jelen [22] observed significant compaction effects with ultrafiltration membranes at pressures well below 400 kPa, with compaction providing the dominant contribution to the observed flux decline during ultrafiltration of cottage cheese whey. Persson et al. [23] examined the compaction of polyamide, polysulfone and cellulose acetate ultrafiltration membranes by measuring both the membrane permeability and thickness after static compression in a hydraulic press. Compaction caused a significant reduction in membrane thickness, with changes on the order of 100 ␮m after compression at only 300 kPa. Compaction caused a 60% reduction in permeability for the polysulfone membrane but only a 35% reduction in permeability for the cellulose acetate. Persson et al. [23] hypothesized that the very different effects of compaction were related to differences in membrane structure, with the high degree of compaction of the polysulfone membrane arising from the deformation of the macrovoids in the membrane substructure. Bowen and Gan [21] evaluated the time-dependent changes in resistance (Rm = 1/Lp ) of composite polysulfone microfiltration membranes as: Rm = K1 P a + K2 P b t c

(3)

where K1 and K2 are constants describing an initial instantaneous compaction and a slower compression, respectively. Huisman et al. [24] described the compaction of hollow fiber polysulfone ultrafiltration membranes in terms of both a reversible and irreversible compression. The data also

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suggested a hysteresis phenomenon; membranes initially exposed to low pressures gave lower resistance than membranes that had previously been exposed to high pressure. In contrast to these studies, which all report a reduction in permeability (or an increase in resistance) with increasing pressure, Lawson et al. [25] used gas permeation data to show that the permeability of microporous membranes used for membrane distillation can actually increase upon compaction. They hypothesized that this increase in permeability was due to a reduction in membrane thickness, an effect that more than compensated for the simultaneous changes in pore size and porosity. Ovchinnikov et al. [26] also observed increases in permeability with pressure during gas permeation through track-etched polyethylene terephthalate membranes. In addition to these studies of membrane compaction, there have also been investigations of the filtration characteristics of compressible cakes formed during microfiltration of deformable cells and particles. For example, Hwang and Hsueh [27] showed that a compact skin layer forms next to the filter membrane, with this skin providing nearly 90% of the hydraulic resistance to flow even though it occupies less than 20% of the entire cake thickness. This dense skin forms due to the frictional drag on the deformable particles throughout the cake structure [28]. However, it is very difficult to extrapolate these results to the behavior of integral membranes in which gradients in porosity and pore size are formed during membrane casting independent of the filtration process. Despite the growing interest in virus filtration for the preparation of therapeutic proteins, there have been no published studies of compaction effects with any of the commercial virus filtration membranes. Virus filters are typically designed to reduce the virus load by at least a factor of 104 (minimum of 4-log removal), requiring that these membranes have a completely defect-free structure. For example, the Viresolve membranes are cast on a microporous support, yielding a multilayer structure that has a thin skin and a more open supporting layer on top of the microfiltration membrane. The objective of this study was to examine the permeability and compaction of the Viresolve 180, DV20, DV50 and Omega 300 membranes. Particular emphasis was placed on understanding the effect of membrane orientation on the permeability and the degree of compaction since virus filtration membranes are currently used in both the skin-side up (typical for TFF) and skin-side down (typical for NFF) orientations.

3. Experimental methods Experiments were performed with Millipore’s Viresolve 180 membrane (Millipore Corp., Bedford, MA) and with Pall’s DV20, DV50 and Omega 300 membranes (Pall Corp., East Hills, NY). The Viresolve 180, DV20 and DV50 membranes are made from a hydrophilized polyvinylidene fluoride (PVDF). The Viresolve 180 membrane is a composite structure consisting of an asymmetric, skinned ultrafiltration membrane cast on top of a macroporous support structure.

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The DV20 and DV50 membranes have a composite triplelayer structure. The Omega 300 is a more traditional skinned ultrafiltration membrane made from polyethersulfone (PES). The pore structures of the Viresolve and DV50 membranes are shown in the scanning electron micrographs in Fig. 1. The image for the Ultipor DV50 shows only one of the three distinct layers in the DV50 membrane. These images were obtained by first allowing the membranes to dry at room temperature for a minimum of 16 h. The membranes were then freeze-fractured in liquid nitrogen, mounted on specimen studs and sputtered with a thin layer of gold under vacuum using a Bal-tec SCD-050 sputter coater (Techno Trade, Manchester, NH). Images were collected at 10 kV with a JSM 5400 scanning electron microscope (Jeol, Peabody, MA) in the Huck Institutes of Life Sciences at The Pennsylvania State University. Membranes were cut into 25 mm disks for use in a 10 mL ultrafiltration cell (Model 8010, Millipore Corp.). A 25 mm diameter Tyvek spacer (pore size of approximately 30 ␮m) was placed at the bottom of the ultrafiltration cell beneath the membrane to prevent deformation of the membrane structure into the support. The Viresolve and Omega membrane disks were soaked in buffer for at least 20–30 min prior to use to wet the pores. Soaking the DV20 and DV50 membranes in buffer led to the formation of bubbles between the membrane layers, so these membranes were simply placed directly into the stirred cell. The membranes were studied in two different configurations: skin-side up, in which the virus-retentive skin was facing the feed solution and skin-side down, in which the flow passes first through the membrane substructure. The DV20 and DV50 membranes were also used in both flow directions, with the “normal” orientation indicated by the manufacturer’s packaging. Phosphate-buffered saline solution (PBS) was prepared by dissolving 0.03 M KH2 PO4 and 0.03 M Na2 HPO4 ·7H2 O (J.T. Baker, Phillipsburg, NJ) in distilled water that was deionized to a minimum resistivity of 18 M cm, irradiated with ultraviolet light, and filtered through an 0.2 ␮m membrane by a Barnstead water purification system (Barnstead/Thermodyne Co., Dubuque, IA). A small amount of NaOH was added to obtain a pH of 7.4 as determined using a Model 420 Thermo Orion pH meter (Beverly, MA). This buffer and pH are representative of typical conditions encountered in industrial applications of virus filtration, including the preparation of purified proteins from donated plasma. The buffer was then vacuum filtered through a 0.2-␮m pore size Gelman Supor-200 membrane (Gelman Science Inc., Ann Arbor, MI) to remove any particulate matter and undissolved salts. In some experiments, the buffer was further filtered through a Pellicon XL 100 kDa Biomax membrane (Millipore Corp.) used in a cross flow configuration with approximately 50% of the feed removed as permeate in a single pass. Buffer solutions were stored in low-density polyethylene carboys connected to the ultrafiltration cell with Tygon tubing. In-line pre-filters were placed between the reservoir and stirred cell in some experiments to remove any particulate

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Fig. 1. Scanning electron micrographs showing the cross-section of (a) the Viresolve 180 at 750× and 5000×, and (b) the DV50 at 1500× and 5000×.

matter that might have been introduced to the feed solution from residues in the carboy or from the atmosphere. Several different in-line filters were used, including a 0.22-␮m pore size Durapore membrane (Millipore Corp.) as well as 100, 50 and 30 kDa Omega polyethersulfone ultrafiltration membranes (Pall Corp.). Filtration experiments were conducted at constant pressure, which was maintained by adjusting the air pressure used to pressurize the buffer reservoir. The pressure difference across the virus filter was evaluated using a digital pressure transducer (Model PX215, Omega Engineering Inc., Stamford, CT) placed directly in the Tygon tubing immediately before the ultrafiltration cell. Filtrate flux was measured by timed collection using a digital balance (Model AG104, Mettler Toledo, Columbus, OH). All data were obtained at ambient temperature (20–25 ◦ C).

filtered through a sterilizing grade 0.2-␮m pore size membrane prior to use in these experiments. The data are plotted as the pressure-normalized flux (or membrane permeability): Lp =

µ Jv P

(4)

to eliminate the effect of any small fluctuations in pressure (which were less than 5% of the mean absolute value). The error bars on the calculated values of the permeability were all within the size of the symbols in Fig. 2 and are thus not shown for clarity. The initial flux for the skin-side down

4. Results and discussion 4.1. Viresolve 180 Fig. 2 shows typical experimental data for the membrane hydraulic permeability as a function of time for two different Viresolve 180 membranes, one used with the skin-side facing the feed and one used with the skin-side down. The data were obtained at a constant transmembrane pressure of 103 kPa (15 psi) using a PBS solution that had been pre-

Fig. 2. Normalized flux as a function of time for the Viresolve 180 membrane in the skin-side up and skin-side down orientations at a constant pressure of 103 kPa (15 psi). The PBS was pre-filtered through a 0.2-␮m pore size membrane.

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Fig. 3. Normalized permeability for the Viresolve 180 membrane in the skin-side up orientation at a pressure of 103 kPa (15 psi) with different prefiltration or inline filters.

orientation (Jv = 1.50 × 10−4 m/s) was slightly larger than that in the skin-up orientation (Jv = 1.40 × 10−4 m/s), although this difference was within the normal variability between membrane samples. The flux declined with time throughout the filtration, with the flux declining by more than 40% in the skin-side up orientation compared to only 15% when the skin-side was facing away from the feed. The significant flux decline seen in these experiments occurred even though the feed solution had previously been filtered through a 0.2 ␮m membrane to remove particulates. In order to explore the origin of the flux decline in more detail, a series of experiments were performed with different pre-filtration steps or inline filters designed to minimize the possibility of particulate fouling. The results are shown in Fig. 3 for filtration in the skin-side up orientation at a constant pressure of 103 kPa (15 psi). The data are presented in terms of the normalized permeability, Lp /Lp,0 , to eliminate the small differences in initial permeability between samples. Placement of a 0.22-␮m filter directly inline between the feed reservoir and the stirred cell had no significant effect on the flux decline, with the flux decreasing by nearly 50% after only 30 min of filtration. This was actually a somewhat larger decline in flux than seen in Fig. 2 without the in-line filter, although this was within the normal variation seen for experiments using these relatively large pore size pre-filters. In contrast, pre-filtering the PBS through a 100 kDa membrane prior to use significantly reduced the magnitude of the flux decline, suggesting that the decline in flux seen previously was due to the presence of sub-micron sized fouling components that were able to pass through the sterilizing grade filter. The use of a 30 or 100 kDa inline filter completely eliminated the flux decline seen with the Viresolve membrane (oriented with the skin-side up), with the flux remaining constant throughout the 30 min filtration. Note that a constant permeability was obtained only when using a very small pore size prefilter placed directly in the feed line just before the stirred cell. The flux decline that occurred when the buffer was prefiltered through a 100 kDa membrane prior to the experiment

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Fig. 4. Normalized permeability for the Viresolve 180 membrane with skinside down at a pressure of 103 kPa (15 psi) using different pre-filters. The data for t > 1800 s were obtained after reducing the applied pressure to zero for 20 min before being reapplied.

– but not through a 100 kDa in-line filter – is likely due to contaminants that entered the system through the atmosphere, from the walls of the polyethylene carboy used to contain the feed solution, or from the air used to pressurize the system. The size and concentration of these submicron contaminants varied considerably, leading to a relatively large variability in the rate of flux decline for experiments performed without a small pore size in-line filter. Corresponding data for the normalized permeability for membranes used in the skin-side down orientation are shown in Fig. 4. In contrast to the results with the skin-side up, the flux decline in Fig. 4 shows no clear dependence on the type of pre-filtration. For example, the flux decline with the 0.2-␮m and 30 kDa in-line filters were essentially identical, with the smallest decline in flux seen with the feed that was pre-filtered through a 100 kDa membrane (off-line). In addition, the use of a 30 or 50 kDa in-line filter was unable to eliminate the flux decline seen in these experiments, with the permeability after 30 min of filtration being about 5–20% smaller than the initial permeability. In order to explore this behavior in more detail, the membrane used with the 50 kDa inline filter was de-pressurized for 20 min (without being removed from the ultrafiltration cell) immediately after completion of the 30 min filtration (denoted by the vertical dashed line in Fig. 4). The pressure was then reapplied, with the resulting data shown in the righthand panel of Fig. 4. The permeability was again normalized by the initial permeability at the start of the original filtration (and not at the start of this second pressurization). The permeability after re-pressurization was nearly 10% larger than that evaluated at the end of the first filtration, recovering about a third of the permeability that had been lost during the first 30 min. The flux rapidly declined to the value obtained at the end of the first filtration, with the final permeability (after 60 min of total filtration) being slightly smaller than the value obtained after 30 min. The increase in flux upon repressurization was not observed for membranes used with the skin-side up orientation—the permeability in this case was

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Fig. 5. Normalized permeability for the Viresolve 180 membrane in the skin-side down orientation at different applied pressures.

identical to that measured before the depressurization, irrespective of the magnitude of the flux decline during the initial filtration. The data in Figs. 3 and 4 indicate that: (1) the flux through the Viresolve 180 is much less sensitive to the presence of sub-micron size foulants in the feed stream when the membrane is oriented with the skin-side facing away from the feed and (2) the flux decline with the skin-side down is probably not due to some type of particulate fouling but is instead related to membrane compaction. The absence of any significant fouling when the membrane is used with the skin-side down, even under conditions in which there is substantial particle fouling with the skin-side up, is a direct result of the multilayer structure of the Viresolve membranes. When the skin-side is downstream, particles will tend to deposit on the external (upper) surface of the membrane support, but these particles will cause little decline in flux since the fluid will be able to flow under and around this surface blockage through the highly interconnected pores in the membrane support. This phenomenon has been clearly demonstrated by Ho and Zydney [29] for microfiltration membranes, with a significant flux decline occurring only after more than 50% of the external surface is covered by particles. In addition, the membrane substructure can act as a microporous depth filter, with small particles captured throughout the entire depth of the substructure [13]. The substructure thus protects the small pores of the virus-retentive skin layer from fouling by small particulates present in the feed solution. In contrast, when the membrane is oriented with the skin-side up particle fouling will tend to obstruct the membrane pores, causing a significant increase in the resistance to fluid flow and a corresponding reduction in the filtrate flux. The flux decline in the skin-side down orientation is examined in more detail in Fig. 5. Data are shown for three separate membranes, each used at a constant transmembrane pressure with a 100 kDa (or smaller) in-line filter placed directly before the ultrafiltration cell. The normalized permeability at 6 and 34 kPa declines by approximately 10% over the course of the filtration and a somewhat larger flux decline (approximately 20%) is seen at the highest pressure (104 kPa = 15 psi). The greater flux decline seen at the highest pressure is consistent

Fig. 6. Steady-state flux as a function of pressure for Viresolve 180 membrane in the skin-side up and down orientations. Best fit lines (—) with the skin-side up Jv [m/s] = 1.4 × 10−9 P [Pa] (R2 = 0.998) and the skin-side down Jv [m/s] = 2.3 × 10−15 P2 + 8.8 × 10−10 P [Pa] (R2 = 0.999).

with a compaction of the membrane in response to the applied pressure. The effect of the transmembrane pressure on the filtrate flux is shown explicitly in Fig. 6. These experiments were again performed using a 100 kDa in-line filter to eliminate the effects of particle fouling. Data are presented for the steady-state values of the filtrate flux for one membrane used with the skin-side up orientation and one membrane used with the skin-side down. In each case, the flux was evaluated after a sufficiently long period of time to fully compress the membrane at the given pressure (typically 30 min for the skin-side up orientation and 60 min for initial compaction with the skin-side down). Data were taken with both increasing and decreasing pressure, with no evidence of hysteresis effects for either orientation. The filtrate flux data with the skin-side up are highly linear, with a correlation coefficient of R2 = 0.998, indicating a constant value of the hydraulic permeability over this range of pressures. In contrast, the filtrate flux data obtained with the skin-side down is slightly concave up, corresponding to an increase in hydraulic permeability with increasing pressure. This effect was small but statistically significant, with Lp = 0.92 ± 0.02 × 10−12 m for pressures between 25 and 35 kPa compared to Lp = 1.22 ± 0.02 × 10−12 m for pressures between 140 and 170 kPa. Additional experiments were performed in which the retentate pressure was controlled by placing a clamp on the outlet tubing to increase the absolute pressure. The filtrate flux and hydraulic permeability evaluated in these experiments were only a function of the transmembrane pressure difference, independent of the actual values of the hydrostatic pressure on the two sides of the membrane. Although the results in Fig. 6 were reproducible for the given membrane samples, there was considerable variability in flux data for different membrane samples. In some cases, the filtrate flux was greater for membranes with the skinside up (as in Fig. 6), but in other cases the flux (at a given pressure) was higher with the skin-side down. Additionally, the magnitude of the increase in permeability with increasing transmembrane pressure for the skin-side down orientation

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Fig. 7. Scanning electron micrograph showing the Virsolve 180 membrane after filtration in the skin-side up orientation at 103 kPa for 30 min.

varied from sample to sample, ranging from as little as 6.5% to more than 35% over pressures from 10 to 180 kPa. The permeability with the skin-side up was independent of the pressure with variations less than 5% in this pressure range. The pore structures of the compacted membranes were examined by scanning electron microscopy. Cross-sectional images were obtained for membranes used in both the skin-side up and skin-side down orientations after filtration of buffer for 30 min through a 30 kDa inline pre-filter. Results are shown in Fig. 7 for a membrane used with the skin-side up at a pressure of 103 kPa for 30 min. There was no measurable change in the total membrane thickness, and the overall pore structure seems nearly identical to that of the uncompacted membrane (Fig. 1). However, there did appear to be some reduction in thickness of the dense layer near the skin, from approximately 7 to 6 ␮m. Similar behavior was seen when the membrane was used in the skin-side down orientation. Thus, it was not possible to detect differences in the pore size/structure of the membranes used in the different orientations at the resolution of these scanning electron micrographs. 4.2. DV20 and DV50 Fig. 8 shows the change in hydraulic permeability of the DV20 and DV50 membranes as a function of time at a constant transmembrane pressure of 155 kPa (22.5 psi). The feed solution was pre-filtered through first a 0.2-␮m and then a

Fig. 8. Normalized flux for the DV20 and DV50 membranes at a constant pressure of 155 kPa (22.5 psi) through a 30 kDa inline filter.

100 kDa pore size membrane and the system was operated with an inline 30 kDa molecular weight cutoff membrane. Data are shown with the membrane used in both the orientation recommended by the manufacturer (Pall Corp.) and in the reverse orientation. The average steady permeabilities of the DV20 (Lp = 2 × 10−15 ) and DV50 (Lp = 8 × 10−14 ) were more than an order of magnitude smaller than that for the Viresolve 180, reflecting the very different morphology of the Pall DV membranes (Fig. 1). In contrast to the data obtained with the Viresolve membranes, the normalized flux for the DV20 and DV50 membranes increased during filtration, obtaining a steady-state value after 2 h. The data in Fig. 8 are replotted in Fig. 9 as the normalized permeability, using the steady-state value of the permeability for the normalization, as a function of the cumulative filtrate volume. When the membranes were in the reverse orientation, the permeability increased rapidly to its steady-state value, requiring the filtration of only about 2.5 × 10−6 m3 (corresponding to 6 L/m2 ) of PBS. In contrast, the normalized permeability in the recommended orientation does not attain a steady value until filtration of about 7 × 10−6 m3 of buffer (17 L/m2 ) with similar behavior seen for both membranes. The increase in flux seen with the DV20 and DV50 membranes is likely due to the wetting of the membrane pores. It was not possible to pre-wet these membranes by simply soaking the membranes in buffer since this led to the forma-

Fig. 9. Normalized permeability as a function of filtrate volume for the DV20 and DV50 membranes at a constant pressure of 155 kPa (22.5 psi) through a 30 kDa inline filter.

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tion of bubbles between the layers of the multilayer structure. Attempts to wet the membranes using 70% isopropyl alcohol at a pressure of 172 kPa (25 psi) were unsuccessful; no significant flux (less than 3 × 10−7 m/s) was observed after wetting with isopropyl alcohol for at least 1000 s. The volume of PBS buffer required to wet the membranes was approximately 160 times the pore volume (assuming an average membrane porosity of 50%). Once the pores became fully wetted, the filtrate flux remained constant out to periods of at least 3 h with no evidence of any fouling. This constant permeability was evident even during experiments run without any inline filter (data not shown). The hydraulic permeability was also independent of the applied pressure, with a value of 2.2 ± 0.1 × 10−14 m for the DV20 over a range from 35 to 162 kPa. In addition, scanning electron micrographs obtained after a 2-h filtration showed no observable changes in pore structure or thickness. The steady-state permeabilities of the DV20 and DV50 membranes were also independent of the membrane orientation, although the normalized flux increased more rapidly when the membrane was used in the reverse orientation. Experiments in which the membrane was carefully removed from the stirred cell and then returned to the device with the opposite orientation gave identical values of the permeability. 4.3. Omega 300 membrane Fig. 10 shows experimental results for the Omega 300 membrane oriented with the skin-side up at a constant pressure of approximately 103 kPa (15 psi). In all cases, the buffer was first pre-filtered through a 0.2-␮m membrane. For the experiments using an in-line filter, the buffer was also prefiltered through a 100 kDa membrane prior to use and then through a 30 kDa in-line filter placed immediately before the ultrafiltration cell. The flux initially increased by 10–20% over the first several minutes and then rapidly achieved a steady value. This small increase in flux is likely due to removal of glycerin used as a wetting agent in the pores of the Omega 300 membranes. The permeability of the Omega

Fig. 10. Normalized flux as a function of time for the Omega 300 membrane at a constant pressure of 103 kPa (15 psi) in the skin up and skin down orientations.

300 membrane was two to three times larger than that for the Viresolve 180 and nearly 100 times larger than that for the DV20. However, it should be noted that the Omega 300 membrane provides less viral clearance than the specially designed Viresolve and DV-series filters [30]. There was no evidence of any membrane compaction or particulate fouling with the Omega 300 membranes. However, the steady state permeability for the membrane with the skin-side up (Lp = 2.5 ± 0.3 × 10−12 m) was consistently about 25% smaller than that determined with the skin oriented downstream. This could be due to the deformation of the skin layer into the substructure when flow occurs through the skin first. Alternatively, it is possible that the difference arises from the different hydrodynamics for the diverging flow out of, or the converging flow into, the small pores in the skin. However, the Reynolds number for these experiments was less than 10−5 (based on the pore radius of approximately 10 nm), which should result in a fully reversible, and direction-independent, fluid flow. Additional studies would be required to identify the cause of this orientation effect.

5. Conclusions Previous investigations have documented the ability of virus filters to remove viral particles, but there has been relatively little work on the flow properties of these membranes. The data obtained in this study clearly demonstrate that the filtrate flux through the Viresolve 180 membrane operated with the skin-side down declined by 20% within 30 min due to compaction of the membrane. This compaction behavior had both reversible and irreversible components, with a small degree of compaction observed at pressures as low as 6 kPa. This time-dependent compaction was only observed when the membrane was oriented with the skin-side down, which may be due to the deformation of the substructure into the back of the skin layer in this orientation. It was not possible to observe any change in pore structure in the SEM images, although this may simply be a result of the “decompression” of the membrane during drying and sample preparation in combination with the limited resolution of the scanning electron micrographs. However, there did appear to be a small reduction in the thickness of the dense layer near the skin, although this change was evident in both membrane orientations. In contrast to the behavior of the Viresolve 180 membrane, there was no evidence of any compaction for the DV20, DV50 or Omega 300 membranes. Instead, the permeability of these membranes increased with time as the pores became fully wetted, with this effect being most pronounced for the DV series membranes. No changes in the DV50 membrane structure were evident in SEM micrographs. The results also demonstrated that fouling by small particles could cause a significant decline in flux even when the feed solution had been pre-filtered through a small pore size membrane. This effect was particularly pronounced for the Viresolve 180 membranes operated with the skin-side up.

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This type of particle fouling could be eliminated by using a small pore size inline filter, although this approach would be impractical for commercial applications of virus filtration. The effects of particle fouling were also eliminated by operating the membrane with the skin side down since the particles are captured on the external surface or within the pores of the macroporous substructure and thus have minimal effect on the flux due to the highly interconnected pores in this layer. Acknowledgements The authors would like to acknowledge Millipore Corp. for donation of the Viresolve membranes used in these experiments. References [1] R. Harasawa, T. Sasaki, Sequence analysis of the 5 untranslated region of pestivirus RNA demonstrated in interferons for human use, Biologicals 23 (1995) 263. [2] R.L. Garnick, Raw materials as a source of contamination in largescale cell culture, Dev. Biol. Standardization 93 (1998) 21. [3] H. Rabenau, V. Ohlinger, J. Anderson, B. Selb, J. Cinatl, W. Wolf, J. Frost, P. Mellor, H.W. Doerr, Contamination of genetically engineered CHO-cells by epizootic haemorrhagic disease virus (EHDV), Biologicals 21 (1993) 207. [4] M.A. Kedda, M.C. Kew, R.J. Cohn, S.P. Field, R. Schwyzer, E. Song, F. Fernades-Costa, Hepatitis A virus infection in tamarins: experimental transmission via concentrated factor VIII concentrates, Hepatology 22 (1995) 1363. [5] Z. Johnson, L. Thornton, A. Tobin, E. Lawlor, J. Power, I. Hillary, I. Temperley, An outbreak of hepatitis A among Irish haemophiliacs, Int. J. Epidemiol. 24 (1995) 821. [6] P.M. Mannucci, S. Gdorin, A. Gringeri, M. Colombo, A. Mele, N. Schinaia, N. Ciavarella, S.U. Emerson, R.H. Purcell, Transmission of hepatitis A to patients with hemophilia by factor VIII concentrates treated with organic solvent and detergent to inactivate viruses. The Italian Collaborative Group, Ann. Intern. Med. 120 (1994) 1. [7] J. Vermylen, K. Peerlinck, Review of the hepatitis A epidemics in hemophiliacs in Europe, Vox Sang. 67 (88–11) (1994) 24. [8] J.J. Burckhardt, Assessment of needs of plasma for fractionation in Europe, Biologicals 27 (1999) 337. [9] CDC, Epidemiologic notes and reports outbreak of hepatitis C associated with intravenous immunoglobulin administration-United States, October 1993–June 1994, Morbidity and Mortality Weekly Report (MMWR), vol. 43, 1994, p. 505. [10] A. Berger, H.W. Doerr, I. Scharrer, B. Weber, Follow-up of four HIV-infected individuals after administration of hepatitis C virus and GBV-C/hepatitis G virus contaminated intravenous immunglobulin: evidence for HCV but not for GBV-C/HGV transmission, J. Med. Virol. 53 (1997) 25.

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[11] T. Urase, K. Yamamoto, S. Ohgaki, Effect of pore structure of membranes and module configuration on virus retention, J. Membr. Sci. 115 (1996) 21. [12] A.J. DiLeo, A.E. Allegrezza, S.E. Builder, High resolution removal of virus from protein solutions using a membrane of unique structure, Bio/Technol. 10 (1992) 182. [13] H. Brough, C. Antoniou, J. Carter, J. Jakubik, Y. Xu, H. Lutz, Performance of a novel Viresolve NFR virus filter, Biotechnol. Prog. 18 (2002) 782. [14] L.J. Zeman, A.L. Zydney, Microfiltration and Ultrafiltration: Principles and Applications, Marcel Dekker, New York, 1996. [15] W.C. McGregor, Selection and use of ultrafiltration membranes, in: W.C. McGregor (Ed.), Membrane Separations in Biotechnology, Marcel Dekker, New York, 1986. [16] L. Baayens, S.L. Rosen, Hydrodynamic resistance and flux decline in asymmetric cellulose acetate reverse osmosis membranes, J. Appl. Polym. Sci. 16 (1972) 663. [17] S. Kimura, S.-I. Nakao, Fouling of cellulose acetate tubular reverse osmosis modules treating the industrial water in Tokyo, Desalination 17 (1975) 267. [18] H. Ohya, An expression method of compaction effects on reverse osmosis membranes at high pressure operation, Desalination 26 (1978) 163. [19] G. Belfort, G. Alexandrowicz, B. Marx, Artificial particulate fouling of hyperfiltration membranes, Desalination 19 (1976) 127. [20] R.A. Peterson, A.R. Greenberg, L.J. Bond, W.B. Krantz, Use of ultrasonic TDR for real-time noninvasive measurement of compressive strain during membrane compaction, Desalination 116 (1998) 115. [21] W.R. Bowen, Q. Gan, Microfiltration of protein solutions at thin film composite membranes, J. Membr. Sci. 80 (1993) 165. [22] V.R. Tarnawski, P. Jelen, Estimation of compaction and fouling effects during membrane processing of cottage cheese whey, J. Food Eng. 5 (1986) 75. [23] K.M. Persson, V. Gekas, G. Tr¨ag˚ardh, Study of membrane compaction and its influence on ultrafiltration water permeability, J. Membr. Sci. 100 (1995) 155. [24] I.H. Huisman, B. Dutr´e, K.M. Persson, G. Tr¨ag˚ardh, Water permeability in ultrafiltration and microfiltration: viscous and electroviscous effects, Desalination 113 (1997) 95. [25] K.W. Lawson, M.S. Hall, D.R. Lloyd, Compaction of microporous membranes used in membrane distillation. I. Effect on gas permeability, J. Membr. Sci. 101 (1995) 99. [26] V.V. Ovchinnikov, V.D. Seleznev, V.V. Surguchev, V.I. Kuznetsov, Controllable changes in the porous structure of polymeric nuclear track membranes, J. Membr. Sci. 55 (1991) 299. [27] K.-J. Hwang, C.-L. Hsueh, Dynamic analysis of cake properties in microfiltration of soft colloids, J. Membr. Sci. 214 (2003) 259. [28] W.-M. Lu, K.-L. Tung, S.-M. Hung, J.-S. Shiau, K.-J. Hwang, Constant pressure filtration of mono-dispersed deformable particle slurry, Sep. Sci. Technol. 36 (11) (2001) 2355. [29] C.-C. Ho, A.L. Zydney, Effect of membrane morphology on the initial rate of protein fouling during microfiltration, J. Membr. Sci. 155 (1999) 261. [30] J. Carter, H. Lutz, An overview of viral filtration in biopharmaceutical manufacturing, Eur. J. Parenter. Sci. 7 (3) (2002) 72.