Measurement of pore size distribution and prediction of membrane filter virus retention using liquid–liquid porometry

Journal of Membrane Science 476 (2015) 399–409

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Measurement of pore size distribution and prediction of membrane filter virus retention using liquid–liquid porometry Sal Giglia n, David Bohonak, Patricia Greenhalgh, Anne Leahy EMD Millipore Corporation, 290 Concord Road, Billerica, MA 01821, United States

art ic l e i nf o

a b s t r a c t

Article history: Received 14 August 2014 Received in revised form 4 November 2014 Accepted 28 November 2014 Available online 8 December 2014

Virus removal using membrane filtration is designed to work by a size exclusion mechanism. Removal is dictated by the pore size distribution of the membrane and the virus size. However, information characterizing the pore size distribution of virus filters, and comparisons of quantitative predictions of virus retention performance from pore size distribution data to measured virus retention performance, is limited. In this work, liquid–liquid porometry (LLP) was used to characterize the pore size distributions of developmental and commercial virus filtration membranes spanning a range of pore size distributions. Measurements were conducted using an automated high resolution LLP test system customized for virus filter membranes. Using pore size distributions determined from LLP, a mechanistic mathematical model grounded on the principle of particle retention by size exclusion closely predicted measured virus retention performance, both as a function of virus size and of membrane pore size. The data and model predictions from this study support the understanding that size exclusion is the primary mechanism for virus retention in membrane filters. An abridged LLP test, which is focused on a critical portion of the pore size distribution, was developed and is utilized at-line during membrane casting to assure consistent membrane pore size and therefore consistent virus retention. & 2014 Elsevier B.V. All rights reserved.

Keywords: Virus filter Virus removal Porometry Pore size Virus membrane

1. Introduction Manufacturers of biotherapeutic proteins frequently use virus filtration membranes to reduce the risk of product contamination and to meet regulatory expectations. Viruses may enter manufacturing processes through contaminated human plasma for plasmaderived products or through contaminated cell lines, cell culture media, and other raw materials for cell-derived products. To assure product safety, it is essential to incorporate explicit virus removal steps in the manufacturing process, along with careful control of the raw materials and testing of the product and product intermediate pools. As part of the overall viral clearance strategy, virus filters provide a robust, size-based removal mechanism which is complementary to other common physical removal techniques (e.g., chromatography) or inactivation methods (e.g., low pH, solvent– detergent, or temperature treatments). Successful virus filtration requires careful control of the membrane pore size distribution. For parvoviruses and other small viruses approximately 20 nm in diameter or larger, greater than 99.99% clearance is commonly targeted for this unit operation. The presence or formation of even a small number of larger pores, often present in membranes used in other applications, such as ultrafiltration, can

n

Corresponding author. Tel.: þ 1 781 533 2564. E-mail address: [email protected] (S. Giglia).

http://dx.doi.org/10.1016/j.memsci.2014.11.053 0376-7388/& 2014 Elsevier B.V. All rights reserved.

permit excessive viral passage [1,2]. On the other hand, the protein product, often only half the size of small viruses, should pass completely through the filter. Due to these highly specific needs, the pore size distribution of commercial membranes manufactured for virus filtration is tightly controlled. For biopharmaceutical manufacturers using virus filtration membranes, understanding the underlying mechanism for virus retention is essential to process development, viral clearance validation, and adoption of quality by design methodologies. A thorough quantitative understanding of the mechanism of virus retention is also of interest to membrane manufactures to enable development of more optimized filters. Although it is widely accepted that virus filtration membranes remove viruses primarily through size-based exclusion [3] and several studies have provided supporting evidence [4–6], common practices reflect perceived uncertainty regarding the size exclusion mechanism. For example, regulatory submissions to the U. S. Food and Drug Administration (FDA) for viral clearance of small virus retentive filters by producers of monoclonal antibodies commonly include data for both small viruses and large viruses [7], even though in principle the demonstration of retention of small viruses indicates at least a comparable level of retention for larger viruses. Understanding the size-based retention mechanism of viruses depends on accurate and relevant determination of the membrane's pore size distribution. Since many virus filters are asymmetric in structure, surface based characterization techniques, such as atomic force microscopy and scanning electron microscopy, are not

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S. Giglia et al. / Journal of Membrane Science 476 (2015) 399–409

appropriate for measurement of the tightest pores which are responsive for retention. For example, Bakhshayeshi et al. [8] found that PP7 bacteriophage was retained within the highly asymmetric Viresolves Pro membrane near the downstream (smaller pore size) surface. For the relatively symmetric Ultipors DV20 membrane these authors found virus deposited as far as 12 μm into the filter depth from the upstream surface. Due to potential membrane asymmetry, as well as potential confounding effects, associated with dead-ended pores, pore characterization techniques based on phase change, such as thermoporosimetry, gas adsorption/desorption, NMR spin relaxation, and others, tend to be inappropriate for characterization of virus filtration membranes. Dextran challenge tests have been adapted specifically to virus filtration membranes, but data interpretation is complicated by internal concentration polarization and peak spreading in the SEC chromatograms [9]. Furthermore, even when fluorescent molecules are used to increase the sensitivity, dextran challenge tests can only measure sieving coefficients as low as approximately 0.001 [10], which is at least an order of magnitude high compared to common virus retention levels. Gas–liquid porometry (GLP) is a useful method for characterizing membrane pore size distribution, and is often used for microfiltration membranes. This technique does not suffer from the complications associated with concentration polarization related to particle challenges and measures the size of active pores at their narrowest point in the flow path through the membrane, ignoring dead-ended pores. These narrowest, active pores represent the portion of the membrane responsible for virus retention. However, for the pore sizes typical of virus filters, the high pressure required to evacuate liquid filled pores with a gas may be impractical using common gas–liquid pairs. For example, the pressure required to evacuate water from a 20 nm water filled pore with air is estimated to be in excess of 10,000 kPa. Liquid–liquid porometry (LLP) offers an alternative to characterize the pore size distribution in virus filters but at much lower pressures compared to GLP. As will be discussed in more detail later, the low pressure operation using LLP is possible owing to the much lower surface tension between selected liquid–liquid pairs compared to gas–liquid pairs. It should be noted that membrane tests such as gas– liquid diffusion and pressure or vacuum decay, while useful for detecting the presence of relatively large defects, will not provide information on membrane pore size distribution. LLP has been used by several authors to characterize parvovirus retentive membranes. Philips and DiLeo [11] described use of the CorrTest™ procedure, which utilizes an aqueous polyethylene glycol/ ammonium sulfate system for the wetting and intrusion fluids, to characterize polyvinylidene fluoride (PVDF) virus filters (Viresolves 180 and Viresolves 70 membranes). This test used a LLP measurement from a single pressure, which was corrected for temperature and then shown to correlate with retention of virus. Similarly, for ultrafiltration membranes Gadam et al. [12] demonstrated that the CorrTest™ procedure can be correlated with protein retention. Peinador et al. [13] characterized polyethersulfone (PES) parvovirus retentive membranes with LLP using mixtures of water and isobutanol. In this study, virus retention was shown to correlate with the largest measured pore size. As other authors have reported swelling of PES membranes when isobutanol/water/methanol mixtures were used [14], it is unclear how the pore size distributions reported by Peinador et al. relate to the actual pore sizes when in use with aqueous systems which are typical for bioprocessing applications. In this work, LLP was used to characterize virus filtration membranes with different pore size distributions, morphologies, and made from different materials. While previous studies have demonstrated correlation between porometry data and virus retention, in this study a mechanistic model based on the principles of size-based exclusion was used to predict the levels of virus retention for several viruses of different sizes. Experimental results using membranes of different pore size distributions and a panel of viruses were compared with

model predictions. In Addition, a customized LLP test method was developed to characterize a subset of the membrane pores in a shorter period of time. This abridged test enables real-time prediction of virus retention during membrane casting processes, facilitating atline process control, and reducing variability in pore size distribution and virus retention.

2. Background 2.1. LLP theory Several methods for determining the membrane pore size distribution using LLP have been reviewed and numerically tested by Morison [15]. The interfacial pore flow model was used for the current work, as it is the most appropriate mode for use with the asymmetric membranes which were evaluated. For this model, the Hagen–Poiseuille equation is used to describe flow through the individual membrane pores. When one liquid phase is present, the volumetric flow rate, Q, through a pore of radius, r, is Q ðrÞ ¼

π U ΔP Ur 4 8UηUδ

ð1Þ

where ΔP is the transmembrane pressure, η is the dynamic viscosity, and δ is the pore length. When two immiscible liquid phases are present, flow only occurs when sufficient pressure exists to overcome the interfacial tension and flush the interface through the pore. Experimentally, the wetting fluid flows through the membrane so as to wet all of the pores, and then the intrusion fluid is introduced at a low pressure. The pressure is increased slowly or incrementally so that the flow at any instant only occurs through those pores which are large enough for the interfacial tension to be overcome. Q ðrÞ ¼

ΔP Z

π U ΔP Ur 4 when 8UηUδ

2 U k U γ U cos θ at some time when the second fluid is present r ð2aÞ

Q ðrÞ ¼ 0 when

ΔP o

2 U k U γ U cos θ at all times when both fluids are present r

ð2bÞ

where k is a shape factor, γ is the surface tension and θ is the contact angle between the interface and the pore wall. For cylindrical pores, the shape factor is equal to unity. If sufficient pressure has not been applied, the wetting fluid remains in the pore and no flow occurs. The analysis of the LLP data was based on the ratio of intrusion fluid flow rate when the wetting fluid was present to the flow rate when the wetting fluid was absent [16]. This analysis has the advantage that the solution viscosity, pore length, and the total number of pores do not need to be known to calculate the pore size distribution. The flow rate in the absence of the wetting fluid results from flow through all of the pores in the membrane. The flow rate when the wetting fluid is present is a function of only those pores which have been intruded (i.e., those pores with nonzero flux according to Eq. (2)). The ratio of these two flows, R can be calculated from Eqs. (1) and (2) as follows: Q 2phases ðΔPÞ RðΔPÞ ¼ ¼ Q Intrusion fluid ðΔPÞ

R1

2 U k U γ U cos θ=ΔP f n ðrÞ U r R1 4 0 f n ðrÞ Ur Udr

4

Udr

ð3Þ

S. Giglia et al. / Journal of Membrane Science 476 (2015) 399–409

R1 RðΔPÞ ¼

2 U k U γ U cos θ=ΔP f Q ðrÞ Udr R1 0 f Q ðrÞ U dr

ð4Þ

where QIntrusion fluid is the total flow rate through all of the membrane pores when only the intrusion fluid is present, Q2 phases is the total flow rate when both phases are present, fn is the number-weighted pore size distribution, and fQ is the flow-weighted pore size distribution. Differentiating Eq. (4) with respect to ΔP, recognizing that the right-hand denominator is equal to unity, and rearranging reveals   d RðΔPÞ ΔP 2 f Q ðrÞ ¼   U ð5Þ 2 U k U γ U cos ðθÞ d ΔP A similar analysis could be performed on Eq. (3) to reveal the number-weighted pore size distribution, although it should be recognized that the denominator of this expression is not equal to unity. In that case, the final expression for the number-weighted pore size distribution must be normalized so that its integral over the entire range of pore sizes is equal to unity. In either case, the pore size distribution can be calculated from data for R and ΔP determined experimentally using a LLP test. 2.2. Virus retention by pores Several models were examined to describe the size-based retention of viruses by the membrane. These models describe the virus and membrane pores as spherical particles and cylindrical pores, respectively. Each model assumes that there are no significant long-range interactions between the particle and pore walls, such as electrostatic interactions. The models also assume that during normal flow filtration, which is commonly employed during virus filtration, retained particles are captured by the membrane and are not free to diffuse or otherwise be transported. Thus, there is no opportunity for the formation of a polarized layer of concentrated virus. Bungay and Brenner [17] developed an analytical expression for particle retention based on hydrodynamic calculations, accounting for the steric exclusion of particle from the membrane pores. Particle sieving is described as follows: 8 0 λZ1 C f iltrate <  2   2  S¼ ð6Þ ¼ U K s =ðK t U 2Þ λ o 1 : 1λ U 2 1λ C f eed where S is the sieving coefficient, Cfiltrate is the particle concentration in the filtrate, and Cfeed is the particle concentration in the feed. The term λ is the ratio of the particle size to the pore size.

λ¼

r virus r

ð7Þ

where rvirus is the radius of the particle or virus. Kt and Ks can be expressed as follows: pffiffiffi

2 h 7  n i 9 π2 U 2 n3 þ ∑ an U λ Kt ¼ U  ð8Þ 5=2 U 1 þ ∑ an U 1  λ 4 1λ n¼1 n¼3 pffiffiffi

2 h 7  n i 9 π2 U 2 n3 þ ∑ bn U λ Ks ¼ U  U 1 þ b U 1  λ ∑ n 5=2 4 1λ n¼1 n¼3

ð9Þ

The coefficients an and bn are given by Zeman and Zydney [16]. A simpler expression was proposed by Zeman and Wales [18] as an approximation to the approach of Bungay and Brenner. As an alternative to Eqs. (8)–(10), the following expression can be used: 8 0 C f iltrate <    λ Z1 2   2  2 S¼ ¼ U exp  0:7146 U λ λ o1 : 1λ U 2 1λ C f eed ð10Þ

401

As listed, Eqs. (6) and (10) assume that viral transport through the membrane is dominated by convection, with negligible contributions due to diffusion. The validity of this assumption for the virus filters used in this study can be confirmed by estimating the membrane Peclet number, Pem. The membrane Peclet number represents the relative contributions of solute convection and diffusion to passage through the membrane. It can be calculated as  v U δ ϕ U Kc Pem ¼ U ð11Þ D1 ϕ U K d where v is the fluid velocity, D1 is the solute diffusion coefficient,

ϕ is partition coefficient between the pore and the surrounding solution, Kc is the convective transport hindrance factor, and Kd is the diffusive transport hindrance factor. By assuming Poiseuille flow in the pores and using the model of Bungay and Brenner [17] to calculate the bracketed term, Eq. (11) can be expressed for an individual membrane pore as "  # ΔP U r 2 þ2 Ur Ur virus  r virus 2 Pem ¼ ð12Þ UK s 96 U π U μ UD1 Using Eq. (12) it can be shown that the lowest Peclet numbers, representing those conditions when relative diffusive transport contributions are highest, occur with the smallest viruses. With smaller viruses, transport is possible through smaller pores (lower values of r are possible for transport) and the diffusion coefficient is higher. In this study the smallest viruses used had a radius of approximately 10 nm. Recognizing that the bracketed term in Eq. (12) reaches a minimum value as the pore radius, r, approaches the virus radius, the minimum value of this term can be calculated by letting these two radii to be equal. Most of the experimental virus retention data in this paper were conducted at pressures of approximately 2.1  105 Pa with aqueous buffered solutions (μ approximately equal to 10  3 kg/m s). Assuming a diffusivity of 2  10  11 m2/s based on the Stokes–Einstein equation, the minimum value for the bracketed term is approximately 6.9. The minimum value of Ks, as calculated by Eq. (9), is approximately 38, although in contrast to the bracketed term this minimum value is approached for very large values of r. Hence, it can be shown that for the viruses and pressures used in this work, the minimum value of the Peclet number, for any given pore size for which virus may pass, is greater than 250. In actuality, the minimum value of Pem is considerably higher, since the minimum values of the two terms in Eq. (12) do not occur for the same value of r. Regardless, the Peclet number for any relevant pore sizes is much greater than unity, and relative diffusive transport through the pores under these flow conditions is negligible. This analysis and conclusion are only valid for membranes with a high degree of asymmetry, wherein the bulk of the applied pressure drop occurs across the virus retentive portion of the membrane depth. For membranes with a more symmetric morphology, the flow rate can be considerably smaller. 2.3. Model for prediction of particle retention from LLP data Virus retention is often described using a logarithmic scale, with the log reduction value (LRV) described as follows:  V f iltrate U C f iltrate LRV ¼  log ð13Þ V f eed U C f eed where Vfiltrate and Vfeed are the volumes of the filtrate and feed, respectively. For normal flow filtration, these volumes are equal and hence  C f iltrate LRV ¼  log ð14Þ C f eed Eq. (14) can be used to calculate the LRV on the basis of the entire filtrate volume for a filtration process, referred to as the pool

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LRV, or on the basis of the volume of filtrate collected during a shorter time period, referred to as the grab LRV. In order to calculate the LRV for real membranes with polydispersed pore size distributions, the particle retention described in Eq. (6) or (8) must be accounted for across the entire membrane pore size distribution. The overall LRV can be calculated by integrating to find the number of particles in the filtrate and the filtrate volume [19,20] "R 1 # 4 0 Rf n ðrÞ U r U SðrÞ Udr LRV ¼  log ð15Þ 1 4 0 f n ðrÞ Ur U dr To use Eq. (15), the expressions for S(r) from Eqs. (6)–(10) may be used. The pore size distribution can be determined from LLP testing. Eq. (15) can be rearranged and be expressed as follows: " R1 # f Q ðrÞ Udr LRV ¼ log R 1 0 ð16Þ r v f Q ðrÞ U SðrÞ U dr where fQ(r) can be determined using Eq. (5). In many cases, it is convenient to describe or smooth the measured pore size distribution using a log-normal distribution. In this work, the log-normal distribution was found to fit well with the flow-weighted distribution, fQ, as opposed to the number-weighted distribution (fn) which is usually employed. The following form of the log-normal distribution was used [16,21]:  

  ln r=μ þ b=2 1 ð17Þ f Q ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi U exp  2Ub r U 2Uπ Ub where μ is mean of the pore size distribution and the parameter b is a function of μ and of the standard deviation, σ .   2  ð18Þ b ¼ ln 1 þ σ =μ

variants of the membrane used commercially in Viresolves Pro devices (EMD Millipore, e.g., catalog number VPMCVALNB9), which is designated VPMEMBRANE. Membranes were cast during development specifically to create different pore size distributions. While the testing in the current study was done entirely with a single layer of membrane, commercial devices contain two layers of membrane to increase the LRV assurance since the LRV of two adjacent layers has been shown to be additive [22]. Composite, asymmetric PVDF membranes were also used in this study. These membranes feature a hydrophilic functionalized surface and were designed specifically for parvovirus retention. These membranes are designated as PPVG and are used commercially in Viresolves NFP devices (EMD Millipore, e.g., catalog number SVPVSMLNB9). Testing in the current study was done with a single PPVG layer in contrast to commercial devices, which contain three adjacent layers of membrane to increase the level of LRV assurance. Scanning electron micrographs of cross-sections of VPMEMBRANE and PPVG are shown in Fig. 1(a) and (b), respectively. A description of each of the various membrane sub-types used in this study is given in Table 1. 3.2. LLP test fluids The LLP experiments utilized CorrTest™ wetting and intrusion fluids (EMD Millipore catalog number SJ4M034E99) as the two immiscible fluids. These two fluids form upon mixing polyethylene glycol 8000 (PEG-8000), ammonium sulfate, and water in specific proportions to achieve two phases in equilibrium. The resulting Table 1 Designations and descriptions of membranes used in this study. Membrane designation

Description

VP1 VHP1 VHX1

Standard VPMEMBRANE (hydrophilized) Non-hydrophilized VPMEMBRANE Small pore size variant of non-hydrophilized of VPMEMBRANE Large pore size variant of non-hydrophilized of VPMEMBRANE Small pore size variant of VPMEMBRANE (hydrophilized) Large pore size variant of VPMEMBRANE (hydrophilized) Standard PPVG (hydrophilized)

3. Materials and methods 3.1. Membranes Many of the LLP and virus retention tests were performed on asymmetric co-cast PES membranes. The structure and pore size distribution of these membranes were designed specifically for the retention of parvoviruses, and the surface was modified using a proprietary process to render it hydrophilic. These membranes include

VHX2 VX1 VX2 NP1

Fig. 1. Cross-section of virus membranes (a) VPMEMBRANE; (b) PPVG.

S. Giglia et al. / Journal of Membrane Science 476 (2015) 399–409

PEG-rich (59% PEG, 40% ammonium sulfate, 1% water) and ammonium sulfate-rich (25% ammonium sulfate, 0.04% PEG, 75% water) phases corresponded to the membrane wetting and intrusion fluids, respectively [11]. The interfacial tension between the fluids has been reported as approximately 6.3  10  4 N/mat 22 1C and increases by about 1.2  10  5 N/m/1C over the temperature range 15–50 1C [11] 3.3. Contact angle measurements To obtain contact angle information for the VPMEMBRANE variants, the sessile drop method was used to measure the three phase contact angle of intrusion fluid, wetting fluid, and both hydrophobic and hydrophilized polyethersulfone films. PES film was submerged in intrusion fluid and the bubble was the wetting fluid. A goniometer was used to measure the contact angle. The advancing and receding contact angles were measured four times on a single sample and the average of the advancing and receding angles was taken as the Young's contact angle. Contact angle measurement for the PPVG membrane is less straightforward than for the VPMEMBRANE because unlike the VPMEMBRANE, the surface chemistry of the porous membrane cannot be readily reproduced on a non-porous film. Therefore, the contact angle for the hydrophilized PVDF membrane was assumed to be the same as for the hydrophilized PES membrane. As will be discussed in Section 4, the calculation of pore size is relatively insensitive to small changes in contact angle for contact angles less than about 301. It should also be noted that the PPVG membrane appeared to wet readily with the wetting fluid, consistent with the assumption of a low contact angle.

403

was measured by collecting permeated fluid onto 5 kg load cells (Tadea-Huntleigh Model 1042) for a sufficient length of time for reliable flow rate determination. Flow rates as low as 0.1 mL/min could be measured, using a criteria of the r2 value 40.999 on straight line fit of mass (translated to volume) versus time measurements. The temperature of the fluid entering the membrane was measured using a k-type thermocouple (Omega). All porometry testing was carried out at 2272 1C. To determine the flow rate through all the continuous pores in the membrane, the permeability of the intrusion fluid was first measured between 345 and 69 kPa in 69 kPa increments. Membrane permeability with water was measured similarly. Next, wetting fluid was introduced to the membrane, at a pressure of 138 kPa, for 10 min to ensure that all the pores were filled with the wetting fluid. The membranes were then intruded with the intrusion fluid at an initial pressure of about 30 kPa. At this pressure, pores larger than about 60 nm will be intruded in accordance with Eq. (2a). After equilibrating for one minute, flow through the membrane was recorded by collecting the permeate into beakers positioned onto load cells as described earlier. If there was no measurable flow after 5 min, pressure was increased stepwise to the next pressure level in 7 kPa increments. After the intrusion pressure reached 240 kPa (corresponding to the pressure to intrude pores of about 10 nm in diameter or larger), pressure was increased in increments of 35 kPa until a final intrusion pressure of about 345 kPa was reached. For each membrane in this study, four membrane samples were tested as replicates. The 90-mm discs were cut from across a 30 cm wide membrane roll to represent the width of the roll. 3.5. Viruses and assays

3.4. LLP testing A schematic of the experimental setup is shown in Fig. 2. Stainless steel holders (EMD Millipore) were used to contain 90 mm discs with an effective surface area of 48 cm2. The tight side of the membrane was oriented such that it faced the fluid entrance. A 0–345 kPa pressure controller (SMC ITV2031) was used to establish the system pressure and each device was fitted with a 0–345 kPa pressure transducer (Cole-Parmer 68075-46) to measure pressure at the inlet of each device. Flow through of the membranes

In contrast to conventional virus clearance testing, where viruses of different physicochemical characteristics are evaluated, virus selection for this study was driven by size. The parvoviruses Minute virus of Mice (MVM) and Porcine Parvovirus (PPV) were the smallest viruses used and are routinely used in validation studies of biopharmaceutical manufacturing processes. Bacteriophage PR772 is the suggested model phage for characterization of larger-pore-size virus filters [23] and bacteriophage Phi X-174 is commonly used as a model for parvovirus for studying small-virus retentive filters.

Fig. 2. Schematic of LLP experimental setup.

S. Giglia et al. / Journal of Membrane Science 476 (2015) 399–409

Reports of the sizes of these model viruses in the literature depend on the virus preparation and the measurement method [24,25]. As this is a study of virus retention in aqueous solution, the mean size for each virus determined by dynamic light scattering (DLS) was used for analysis. The size range reported for each virus represents reported values using a variety of sizing methods, Table 2. Stocks of bacteriophage Phi X-174 were purified by CsCl gradient ultracentifugation and purchased from Promega Corp (Madison WI, catalog number I-1041). PR772 was obtained from ATCCs (Manasas, VA; accession number BAA-769) and purified stocks were prepared using the methods described in PDA [3]. Source stocks of MVM and PPV were obtained from ATCCs (Manasas, VA; accession numbers VR1346 and VR742). High titer stocks of MVM were generated as previously described [31]. High titer stocks of PPV were generated by infecting confluent ST cells (ATCCs CRL-1746) with PPV in Advanced/F12 Dulbecco's Modified Eagle's Medium (DMEM), Invitrogen (Catalog no. 12634-028), using the same propagation and purification procedure as described for MVM, however the culture media was changed two days post-infection and the crude lysate was harvested after a total of four days incubation. All purified bacteriophage and virus stocks were aliquoted and stored in buffer at  80 1C for use in spiking studies. Bacteriophage titers were determined using standard plaque assay methods. MVM titers were determined using a tissue culture infectious dose 50% (TCID50) assay [31] PPV titers were also determined using TCID50 assays and using ST indicator cells and infectivity was determined after an incubation of seven days. The retentive capability of the various filter devices is expressed as LRVs which were calculated from the concentrations using Eq. (14). 3.6. Virus retention testing Samples for virus retention testing, in the form of 25-mm discs, were cut from the same section of the membrane roll used for LLP testing and installed with the tight side of the membrane downstream into over-molded plastic devices in a format similar to Viresolves Pro Micro Devices (EMD Millipore). In single layer format, these devices contain about 2.8 cm2 of effective filtration area. Before retention testing, device integrity was confirmed using a binary gas test, the details of which have been described elsewhere [32]. This test is capable of detecting defects of about 1 μm in diameter or greater in this device format. Each selected membrane roll was tested in a minimum of three devices. Buffer was prepared for each day of testing by adding 50 mM sodium acetate and 100 mM sodium chloride to deionized water from a Milli-Qs laboratory water purification system (EMD Millipore); pH was adjusted to 5.0 using glacial acetic acid and filtered using 0.22 mm Stericups filters. Buffer without protein was used as the challenge solution to eliminate any impact of protein fouling on virus retention. All challenge solutions were spiked to a target concentration of 5  106 pfu/mL bacteriophage or 5  106 TCID50/ mL PPV or MVM, and processed through buffer-flushed 0.22 mm (MVM, PPV and Phi X-174) or 0.45 mm (PR772) Stericups filters (EMD Millipore). Prechallenge load samples were collected before and after processing over the Stericups filters and assayed for titer. An additional ‘hold’ sample was held at room temperature for

Table 2 Virus size determinations (nm) by DLS.

the duration of the test. Details of tests that contained protein solutions have been described elsewhere [33]. Virus filtration devices were flushed with deionized water for 10 min followed by buffer for 10 min at a constant pressure of 207 kPa. Following wetting out of the devices, the buffer was replaced by spiked challenge solution and the devices run at a constant pressure of 207 kPa. Filtrate from each run was collected on a balance and cumulative volume was tracked with process time. Filtrate grab samples were collected at points corresponding to approximately 100 L/m2 and assayed for titer using the virus appropriate indicator test systems. There was no measurable flux decline at 100 L/m2 compared to the clean water flux.

4. Results and discussion 4.1. Pore size distributions LLP data were used to generate flow rate vs. pressure porometry curves, and a typical result is shown in Fig. 3. The raw data were smoothed by using a moving 3-point third degree polynomial fit of the flow rate vs. pressure data. Pore size was calculated from the intrusion curve fitted data per Eq. (2a), with the use of contact angle information, listed in Table 3. As discussed earlier, the contact angle for the hydrophilized PVDF membrane (PPVG) was assumed to be the same as for the hydrophilized PES membrane (VPMEMBRANE). Provided that the actual contact angle for the PPVG membrane is within the range of 0–301, the maximum error in the calculation of pore size resulting from the contact angle assumption is about 8%. The surface tension values employed in Eq. (2a) were as described in Section 3.2, including the adjustment of surface tension with temperature. The smoothed data of Fig. 3 was transformed into flow fractions through 1 nm pore size increments from 1 to 40 nm. These values were fitted to a log-normal probability density function, Eq. (17), to arrive at m and σ values as shown in Fig. 4. The coefficients of variation (COV) of the m and σ values of the four replicates of this 2.5 Intrusion fluid only data Intrusion fluid only linear fit Intrusion fluid through wetting fluid data Intrusion curve polynomial fit

2.0

Flow Rate (ml/s)

404

1.5

1.0

0.5

0.0 0

10

20

30

40

50

Pressure (PSIG) Fig. 3. LLP intrusion curve for hydrophobic VPMEMBRANE VP1.

Table 3 Contact angle (θ) measurements on hydrophilic and hydrophobic VPMEMBRANE.

Virus

Mean DLS Size (nm)

Size range (nm)

Source

Hydrophilic PES film

Hydrophobic PES film

PR772 Phi X-174 (φX) MVM/PPV

82 27 22

53–88 26–32 18–26

[26,24,27] [28,24,29,26,23] [30,24,25]

Advancing

Receding

Mean

Cos (θ)

Advancing

Receding

Mean

Cos (θ)

33

8

20

0.94

24

8

16

0.96

S. Giglia et al. / Journal of Membrane Science 476 (2015) 399–409

membrane that were tested were less than 3%. These low COV values were typical of all the membranes tested in this study.

4.2. Virus retention A range of VPMEMBRANE samples was manufactured spanning a range of pore size distributions. As-cast membrane samples in hydrophobic form were evaluated for pore size distribution using LLP, and hydrophilized membrane samples were tested for virus retention with bacteriophage Phi X-174. Fig. 5 shows the fitted pore size distribution curves for three different VPMEMBRANE samples representing low, medium, and high pore diameters (m) and distribution widths (σ), including the sample of Fig. 4. As described in Section 2.3, particle passage through these pore size distributions can be calculated using Eq. (16). Predicted LRV as a function of particle size for each of the three pore size distributions of Fig. 5 is shown in Fig. 6, along with measured LRV from retention testing with Phi X174. The good agreement between the model predictions and measured values supports the understanding that sieving is the primary mechanism for virus retention in these membranes.

In addition to the hydrophobic membrane samples described above, two hydrophilized PES membrane samples representing medium and high pore size ranges were characterized using LLP and tested against a panel of viruses (described in Section 3.5). The pore size distributions, as fitted to a log-normal probability density function, of these membrane samples are shown in Fig. 7. The predicted and measured LRV values are shown in Fig. 8. As with the Phi X-174 results shown previously, the measured data with two species of parvovirus as well as Phi X-174 and the larger PR772, were in good agreement with the model predictions. An important difference in performance between these two membranes is not only that the membrane with the largest pores exhibited lower LRV values, but the larger mean pore size membrane, and with the wider pore size distribution, also showed a lower sensitivity of LRV to particle diameter. Consistent with the model prediction, the difference in LRV between Phi X-174 and the parvoviruses, MVM and PPV, was less for the membrane with the larger mean pore size and broader pore size distribution than it was for the membrane with the smaller mean pore size and narrower pore size distribution. In line with the model predictions,

6

μ = 15.1 σ = 2.8

0.14

VHP1 μ = 15.1 σ = 2.8

VHX1 μ = 10.2 σ = 2.6

7 0.16

405

5

LRV

Flow Fraction

0.12 0.10

4 VHX2 μ = 22.6 σ = 4.9

3

0.08

2

0.06 0.04

1

0.02

0 0

10

0.00 0

5

10

15

20

25

30

20

30

40

Particle Diameter (nm)

35

Pore Diameter (nm)

Fig. 6. Predicted and measured Phi X-174 LRV. Text adjacent to curves indicate membrane sample identifiers and m and σ values in nm. Symbols indicate measured LRV and lines indicate model predictions.

Fig. 4. Data shown in Fig. 3 transformed into a pore size distribution.

VP1 μ = 17.4 σ = 2.7

VHX1 μ = 10.2 σ = 2.6

0.15

0.15

VX1 μ = 20.8 σ = 3.5

Frequency

Frequency

VHP1 μ = 15.1 σ = 2.8 0.10

VHX2 μ = 22.6 σ = 4.9 0.05

0.10

0.05

0.00

0

10

20

30

40

Pore Diameter (nm) Fig. 5. Pore size distributions of three PES membrane samples. Text adjacent to curves indicate membrane sample identifiers and the fit values of m and σ in nm, according to Eq. (17).

0.00

0

10

20

30

40

Pore Diameter (nm) Fig. 7. Pore size distributions of two hydrophilized PES membrane samples. Text adjacent to curves indicate membrane sample identifiers and m and σ values in nm.

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there was no detectable passage of the relatively large (82 nm) bacteriophage PR772 with either membrane. Although the differences are small, the results in Fig. 8 show a slightly higher measured LRV for PPV compared to MVM for the larger pore size membrane, which is surprising given the comparable size of these two species of parvovirus. To probe this further, an additional VPMEMBRANE variant sample of comparable relatively large pore size (m ¼19.3 nm, σ ¼ 3.3 nm) was characterized using LLP and challenged with PPV and MVM, Fig. 9. In this case, the LRV for MVM was slightly higher than for PPV, again with close agreement between predicted and measured LRVs. No consistent trend in relative LRV differences between MVM and PPV was identified and differences in mean LRV between these two viruses, which were approximated 0.5 logs, is within experimental uncertainty. Although the preceding results demonstrate that virus retention by VPMEMBRANE and variants of it can be predicted from the pore size distribution as measured using LLP, in principle these techniques could be applied to different virus filters for which the same model assumptions hold. To demonstrate this point, testing was performed on PPVG membrane. Differences between PPVG and VPMEMBRANE include the base polymer material (PVDF instead of PES), the applied surface chemistry (proprietary), and the membrane morphology (sharper transition between substrate and retentive 7

Single Layer LRV

6 5 4 3 2 1 0 10

100

Particle Diameter (nm) Fig. 8. Predicted and measured LRV for four viruses of different sizes on two hydrophylized membranes.

layer instead of gradual decrease in average pore diameter throughout membrane depth). PPVG membrane, (used in triple layer in NFP virus filter devices) was characterized using LLP. Fig. 10 shows the LLP data transformed into a pore size distribution. While the lognormal probability density function appears to provide a good fit to the data, a closer examination at the upper end of the distribution (Fig. 11) shows a significant population of pores that deviate from the log-normal fit. This is in contrast to the data for the VPMEMBRANE (Fig. 11(b)), where the log-normal curve fits the data well over the entire measured range. The PPVG membrane was tested in single layer against the panel of viruses described in Section 3.5 and the predicted and measured LRV values are plotted in Fig. 12. Two model predictions are shown. Model 1 is based on the log-normal distribution fit and model 2 is based on the incremental pore sizes determined from the transformed LLP data. Because the log-normal fit poorly describes the upper end of the pore size distribution, LRV predictions based on this fit differ significantly from measured values, particularly with larger virus sizes. The predicted LRV using model 2 much more closely agrees with the measured values, although even this model tended to slightly over predict LRV. While fitting to a log-normal distribution is a convenient method to describe the pore size distribution and simplifies LRV predictions, this example shows that the upper tail of the distribution may not be well described by this type of fit and caution must be exercised in using the lognormal fitted data to make LRV predictions. It should also be noted that uncertainty in the contact angle, particularly for the PPVG membrane (see Section 3.3), results in corresponding uncertainty in the calculation of pore size via Eq. (2a). Another factor to consider is that a small number of “oversized” pores that are not part of the continuous pore size distribution may have flow rates through them that are below the detection limit of the LLP test, but high enough to have a measurable effect on LRV. For example, using Eq. (1) the water flow rate through a 5 mm pore of 140 mm in length at an applied pressure differential of 345 kPa is calculated to be only about 0.002 ml/min, which would not be detectable by the LLP test of this study. For a membrane sample where the total integral flow rate is 200 ml/min, the flow rate through this pore would prevent the virus LRV from exceeding 5. As noted earlier, a separate test (i.e., an integrity test) is required for detecting defects. Previous investigators have demonstrated that virus membrane LRV can be correlated to virus size [5,13], and that membrane pore

0.18

μ = 15.4 σ = 2.6

0.16

6

0.14

5

0.12

Flow Fraction

Single Layer LRV

7

4 3

0.10 0.08 0.06

2

0.04 1

0.02 0 10

100

Particle Diameter (nm)

0.00 0

5

10

15

20

25

30

35

40

45

50

55

Pore Diameter (nm) Fig. 9. Predicted and measured LRV for PPV and MVM for a VPMEMBRANE variant, membrane VX2.

Fig. 10. LLP data for PPVG membrane NP1 transformed into a pore size distribution.

S. Giglia et al. / Journal of Membrane Science 476 (2015) 399–409

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0.010

0.010

μ = 17.4

μ = 15.4 σ = 2.6

0.006

0.004

σ = 2.7

0.008

Flow Fraction

Flow Fraction

0.008

0.006

0.004

0.002

0.002

0.000

0.000 0

5

10

15

20

25

30

35

40

45

50

55

Pore Diameter (nm)

0

5

10

15

20

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30

35

40

45

50

55

Pore Diameter (nm)

Fig. 11. Upper end of pore size distributions (a) PPVG (membrane NP1) and (b) VPMEMBRANE (membrane VP1).

7

Single Layer LRV

6

PR772

5 4 3 φX

2 PPV MVM

1

Data Model 1 Model 2

0 10

100

Particle Diameter (nm) Fig. 12. Predicted and measured LRV values for the PPVG membrane. Model 1 and model 2 are described in the text.

size as determined from LLP can be correlated to virus retention. This work shows that a mechanistic model based solely on a sieving mechanism can quantifiably predict these relationships. The model predictions were confirmed on two different types of membranes with different pore size distributions and a panel of viruses of different sizes, LLP testing can therefore provide for a rapid and an efficient way of predicting retention of different viruses by membranes, at least as a tool in membrane development 4.3. Accelerated at-line LLP test For manufacturers of parvovirus retentive filters, at-line process control can enable improved product consistency and yield. Although virus retention is a key characteristic of the membranes, viral retention testing is not rapid enough to enable real-time process control and is better suited for quality control purposes. The membrane pore size distribution, which determines the level of virus retention, is set during the membrane casting, but additional processing such as membrane hydrophilization and device assembly is required before the membrane LRV can be measured. Furthermore, common viral assays can take days or weeks to perform. Fig. 13 depicts an example of this process. To provide rapid feedback to enable at-line process control, LLP can be well suited because it can be performed quickly on hydrophobic membrane samples and can provide data which are predictive

of virus retention. The LLP test used to collect the data for Sections 4.1 and 4.2 typically takes two to three hours to execute. Because more rapid testing is desired to enable control of the membrane casting process, an accelerated LLP test was developed which can be performed in approximately 40 min. Rather than measuring the full porometry curve as in Fig. 3, this accelerated test focuses only on the critical portion of the curve which is related to pore sizes in the range of approximately 20–26 nm and allows at-line adjustments during casting. Fig. 14 shows a typical porometry curve and highlights those pressures which were tested as part of the accelerated test. This test has similarities to the previously described single point versions of the CorrTest™ procedure [11], but one key difference is that the new test incorporates testing at multiple pressures. Having data for a range of pressures allows more precise correction of temperature effects related to the surface tension and decreases test error associated with control of the pressure. To establish the utility of the accelerated LLP test, membrane samples with a range of pore size distributions were tested with the accelerated LLP test and for virus retention. VPMEMBRANE was deliberately cast under a series of different conditions to develop a family of filters of similar morphology, materials, and surface chemistry, but differing in pore size distribution. The hydrophobic membrane samples were evaluated using the accelerated LLP test to determine the flow ratio (Eqs. (3) and (4)) at approximately 23 nm. For virus retention testing, the membrane was hydrophilized and installed into small scale devices, and challenged with a Phi X-174 in the presence of IgG, with samples collected when the flux had decayed by 75%. These fouling conditions were selected because they are more representative of the industrial applications of the filters. Since the effective pore size distribution in a fouled membrane can be different from that in a clean membrane due to preferential plugging of some pore sizes over others, the pore size distribution data determined from the LLP measurements of the clean membrane cannot be used to directly predict LRV in a fouled membrane. However, a correlation was established between the LRV and the intrusion fluid flow ratio. This correlation was used to predict LRV, and Fig. 15 shows the agreement between the predicted value of the LRV (from LLP) and the measured LRV. The predictions have approximately 0.5 log accuracy, which is on the order of the common test error associated with viral assays. An example of the utility of the accelerated test is illustrated in Fig. 16 for two different membrane casting runs. For casting run 2, the LLP test at the start of the cast run indicated an LRV of the membrane higher than the target range. Although a high LRV is in itself not problematic, a membrane with an excessively tight pore

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Fig. 13. Simplified process flow diagram for virus filter manufacture.

2.5

7

Flow Rate (ml/s)

2.0

Measured single layer LRV

Intrusion fluid only Not measured Intrusion fluid through wetting fluid

1.5

1.0

0.5

Variability after process adjustments informed by at-line LLP test

6 5 Variability before process adjustments

4 3 2 1 0

0

10

20

30

40

3

2

3

50

Fig. 16. Effect of process control guided by LLP.

Pressure (PSIG) Fig. 14. Abridged LLP test focused on critical portion of the porometry curve. Center point measures fraction of flow through pores 4  23 nm.

6 Standard Deviation of residuals = 0.46 logs

Measured single layer gLRV75

2

Casting run:

0.0

5

virus retention testing) and therefore the casting conditions were adjusted to shift the pore size smaller. For both casting runs 2 and 3, the adjusted casting conditions informed by the LLP test resulted in a shift in the pore size distribution from outside the target range to within the target range. The accelerated test is used as part of the VPMEMBRANE manufacturing process as an at-line test at membrane casting to ensure proper pore size distribution.

4

5. Conclusions

3

2

1 Dotted lines represent +/-0.5 logs

0

0

1

2

3

4

5

6

Predicted single layer LRV from LLP Fig. 15. Predicted Phi X-174 LRV from correlation between flow fraction through intruded pores at 16 psig and LRV of Phi X-174.

structure will tend to have a higher likelihood of fouling and therefore may not be able to process the expected volume of material in the expected time. Based on the LLP value, the casting conditions for this run were adjusted to shift the pore size distribution (and therefore LRV) into the target range. In another example, the LLP results at the start of the casting run 3 predicted a lower LRV than the target range (subsequently corroborated with

LLP is a highly useful and practical method for characterizing the pore size distribution of virus filtration membranes. Because the pore size measured by LLP represents the narrowest throat in a continuous pore traversing the membrane, it is particularly relevant for relating to the particle retention characteristics of the membrane. The pore size distribution can be fitted to a log-normal probability density function, which is mathematically convenient for characterizing the distribution and also for calculating virus retention. However, the upper end of the pore size distribution is not always well described by the log-normal function for all virus filters, and can lead to significant over-prediction of LRV, especially for relatively large viruses. Previous investigators have demonstrated that virus membrane LRV can be correlated to virus size [5,11], and that membrane pore size as determined from LLP can be correlated to virus retention. This work, based on two different types of membranes with different pore size distributions and a panel of viruses of different sizes, shows that a mechanistic model based solely on a sieving mechanism can quantifiably predict these relationships. The close agreement between predicted and observed

S. Giglia et al. / Journal of Membrane Science 476 (2015) 399–409

retention levels provides convincing support for size based retention being the primary mechanism of virus removal by virus filters. For a given virus size, there is a critical portion of the pore size distribution curve (where the pore size is close to the virus size) that can be correlated with virus retention. Collection of LLP data in only this critical region, rather than for the entire distribution, allows for rapid characterization of the membrane pores and enables at-line testing for near real-time feedback on membrane pore size (correlated to virus LRV) as the membrane is being cast. This quick feedback can be used to make adjustments in the membrane casting process to continuously control membrane pore size and therefore assure proper and consistent levels of virus retention.

Nomenclature an b bn Cfeed Cfiltrate D1 fn fQ k Kc Kd Ks Kt Pem Q QIntrusion

Q2 phases R r rvirus S v Vfeed Vfiltrate

coefficient in Eqs. (8) and (9) parameter defined in Eq. (18) coefficient in Eqs. (8) and (9) particle concentration in the feed (m  3) particle concentration in the filtrate (m  3) solute diffusion coefficient (m2/s) pore probability density function (m  1) flow-weighted pore probability density function (m  1) shape factor convective transport hindrance factor diffusive transport hindrance factor hydrodynamic function (see Eq. (9)) hydrodynamic function (see Eq. (8)) membrane pore Peclet number volumetric flow rate (m3/s) fluid total flow rate through all of the membrane pores when only the intrusion fluid is present (m3/ s) total flow rate when both phases are present (m3/s) ratio of two flows radius of pore (m) radius of the particle or virus (m) sieving coefficient fluid velocity (m/s) volume of the feed (m3) volume of the filtrate (m3)

Greek letters

ΔP δ γ θ λ η μ σ ϕ

transmembrane pressure (Pa) pore length (m) surface tension (N/m) contact angle between the interface and the pore wall (1) ratio of the particle size to the pore size dynamic viscosity (Pa s) mean pore diameter (m) pore diameter standard deviation (m) partition coefficient between the pore and the surrounding solution

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