Strategies for Tailoring the Pore-Size Distribution of Virus Retention

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Strategies for Tailoring the Pore-Size Distribution of Virus Retention Filter Papers Simon Johan Gustafsson, and Albert Mihranyan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03093 • Publication Date (Web): 04 May 2016 Downloaded from http://pubs.acs.org on May 7, 2016

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ACS Applied Materials & Interfaces

Strategies for Tailoring the Pore-Size Distribution of Virus Retention Filter Papers Simon Gustafsson and Albert Mihranyan* Division of Nanotechnology and Functional Materials, Department of Engineering Sciences, Uppsala University, Box 534 SE-75121, Uppsala, Sweden KEYWORDS: Virus-retentive filtration, Nanocellulose, Paper making, Size-exclusion filtration, Cladophora cellulose, Hydraulic permeability

ABSTRACT

The goal of this work is to demonstrate how the pore-size distribution of the nanocellulosebased virus-retentive filter can be tailored. The filter paper was produced using cellulose nanofibres derived from Cladophora sp. green algae using the hot-press drying at varying drying temperatures. The produced filters were characterized using scanning electron microscopy, atomic force microscopy, N2 gas sorption analysis. Further, hydraulic permeability and retention efficiency toward surrogate 20 nm model particles (fluorescent carboxylate-modified polystyrene spheres) was assessed. It was shown that by controlling the rate of water evaporation during hotpress drying the pore-size distribution can be precisely tailored in the region between 10 and 25 nm. The mechanism of pore formation and critical parameters are discussed in detail. The results

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are highly valuable for development of advanced separation media, especially for virus-retentive size-exclusion filtration.

Introduction Currently, there are more than hundred different therapeutic protein products on the market, and all of these protein products possess the risk of viral contamination. The biotechnology industry struggles to find affordable, high throughput and robust virus clearance techniques.1 With respect to virus-retentive filtration, the main issue is the small size difference between the produced protein molecule and the viral particle to be removed. In particular, the smallest known viruses are around 18-22 nm in diameter,2 while the average size of human antibodies is about 12 nm.3 The few existing size-exclusion filters, which are able to remove viruses and virus-like particles, are expensive- partly, due to their complicated production methods and, partly, due to the rigorous safety validation routines implemented for quality assurance. If the intended filter further features a relatively low yield and low throughput, the overall cost-efficiency of the therapeutic protein production will become even lower.4 Therefore, at the current cost of manufacturing the virus retention filters, the industry focuses on strategies of enhancing the costefficiency of the processing in three main areas, i.e. (i) improving the selectivity of virus removal, (ii) achieving high product throughput, and (iii) attaining high yield (low fouling). The current commercially available virus-retentive filters for biotechnology use typically exhibit small-size virus removal efficiency above 4 log10, flow rates between 7 and 190 L m-2 h-1 bar-1, and protein yield above 90-95%.5 Viral contamination can occur by several means and via many contaminated sources. For instance, donor animals infected by viruses may subsequently contaminate the cell cultures derived from them and, thereby, pose a risk of infection during manufacturing of protein


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therapeutics, e.g. in ovo infection with avian leucosis virus.6 Further, many of the commonly used cell lines, such as Chinese Hamster Ovary (CHO) cells, can be infected by endogenous retroviruses, e.g. murine leukemia virus (MuLV).7 Further, serum-derived cell culture media, e.g. bovine fetal serum, may be contaminated by an array of different bovine viruses, e.g. bovine viral diarrhea virus (BVDV). Finally, errors made by the operator could expose the production chain to adventitious contamination, e.g. adenovirus. Hence, making sure there is no infection due to endogenous or adventitious viruses in the final product is crucial for production of protein therapeutics and subject to rigorous regulations by regulatory agencies, such as US Food and Drug Administration (FDA) or European Medicines Agency (EMA).8 The regulatory agencies stipulate integration of at least two orthogonal, i.e. independent, virus clearance steps during protein manufacturing to mitigate the risk of viral infection.8 Clearance of viruses (or virus-like particles) is divided into two general methods, i.e. virus inactivation and virus removal.9-12 Virus inactivation aims at reducing or eliminating virus activity by chemical or physical means. Low pH treatment, coarse solvent/detergent treatment, irradiation, and heat are commonly used as virus inactivation methods for protein solutions. Inactivation is however a limited method as there are several highly resistant virus types.2 Furthermore, there may still remain residual infectivity due to viruses in the protein solution even after inactivation as shown in several reports.13-15 The physical removal of virus particles, at least in theory, eliminates the risk of residual infection16 and is therefore preferred. Virus removal can be achieved by a variety of methods, including filtration (e.g. screening and depth filtration), partitioning and centrifugation, and chromatography (e.g. ion-exchange, hydrophobic interactions and mixed mode).17-19 Removing viruses by filtration is attractive because it is a non-destructive method, meaning that the filtration is gentle towards the biological


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samples to be purified. Size-exclusion filtration is preferred to depth filtration due to the risk of particle breakthrough from depth filters and overall higher sensitivity to processing parameters. Currently, size-exclusion virus-retentive filters are produced from synthetic polymers, e.g. cuprammonium regenerated cellulose, polyether sulfone (PES), and polyvinylidene difluoride (PVDF), Although ceramic virus retentive filters are also available20-21 their use is normally limited to water treatment applications because ceramic filters are heavy, brittle, and nondisposable, limiting their practical utility in biotechnology.22 The polymeric virus-retentive filters usually are asymmetric type membranes or hollow fibers produced using phase-inversion technique, which requires rigorous control of the process parameters and the use of various organic and inorganic solvents to obtain the desired pore-size distribution.23 Another drawback of phase-inversion produced asymmetric membranes is the low porosity of their functional skin layer, which ultimately determines the maximum hydraulic permeability of the filter. The latter limits the flow rate across the filter and thereby the throughput. It has been argued that nonwoven type of filters may provide faster flow rates than filters produced by phase inversion processing due to generally higher porosity but their use so far has been hampered by the choice and cost of suitable nanofibrous material.24 Recently, the world’s first size-exclusion nanocellulose-based filter paper material was reported capable of removing large-size viruses with high efficiency and produced by hotpressing technique rather than by phase inversion.25 This membrane is a non-woven µm-thick filter paper, with a well-defined pore-size distribution suitable for virus removal.25 The virus removal properties of the nanocellulose filter were validated for swine influenza virus A (SIV A) and xenotropic murine leukemia virus (xMuLV).25-26 The use of sustainable, naturally derived


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raw material and ease of manufacturing, which avoids phase inversion processing, are highly appealing for development of high performance cost-efficient industrial virus-retentive filters. It infers from above that controlling the pore-size distribution is important for size-exclusion virus filtration. However, strategies for tailoring the pore-size distribution exactly in the region, which is most beneficial for virus removal, have so far not been studied in detail. Regular nanocellulose derived from wood undergoes hornification during drying and produces a highly compact, non-porous structure.27 The hornification is the result of capillary forces exerted on cellulose nanofibers, moving the elementary fibrils to close proximity, when water is evaporated.28 Hence, by altering the surface tension and/or volatility of the matrix filling liquid, the pore-size distribution can potentially be tailored to the desired size. The latter is essentially the theoretical background for manufacturing porous nanocellulose aerogels via solventexchange processing.29-31 Unfortunately, solvent-exchange processing is not cost-efficient on the large scale and may further utilize hazardous organic solvents, having a negative environmental impact. Therefore, another strategy to tailor the pore-size distribution of nanocellulose-based filters would be to control the kinetics of water evaporation during hot-pressing. Here, we demonstrate how this simple strategy can be realized in practice for manufacturing advanced separation media. The virus removal filter paper described in this work is produced using highly crystalline Cladophora cellulose derived from filamentous green algae, rather than conventional nanocellulose from wood. Cladophora cellulose is presumably the only known nanocellulose to retain its large surface area and porosity upon conventional drying from water.32 It has been argued that this property of Cladophora cellulose is rooted in its exceptionally high degree of crystallinity, relatively thick elementary fibrils, and overall higher mechanical


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stiffness. The overall goal of this work is to demonstrate how the pore-size distribution of the nanocellulose-based virus-retentive filter can be tailored.

Materials and Methods Materials Crystalline cellulose from Cladophora sp. algae was obtained from FMC BioPolymer (G3095–10 batch; USA). Fluorosphere® (carboxylate-modified) fluorescent microspheres consisting of polystyrene (20 nm in radius; F8786) were purchased from ThermoFisher Scientific.

Filter preparation In order to prepare the filter, 300 mg of Cladophora cellulose was dispersed in 75 ml deionized water using high-shear ultra-sonication (750 W; 20 kHz; 13 mm probe; Vibracell; Sonics, USA) with 30 second pulsing at 70 % amplitude for 20 minutes. For the preparation of 11-µm thick membranes, 16.67 ml of the original dispersion was diluted to 50 ml with deionized water. For the 67-µm thick membranes 100 ml of the original dispersion was used without dilution. The cellulose dispersion was drained over a nylon filter membrane (Durapore®; 0.65 µm DVPP; Merck Millipore) under water suction (-0.8 bar) in a funnel. The collected wet cellulose mass was then removed and dried at different temperatures using a hot-press (Rheinstern; Germany) to produce a flat sheets. The nylon support was removed using tweezers without disrupting the integrity of the filter. The cellulose samples were dried at various temperatures, viz. 47, 80, 100, 140, 160, and 200 ˚C.


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Scanning Electron Microscopy A Zeiss Leo 1550 FEG SEM system was used. The images were taken at 1.4 kV acceleration voltage, using 30 mm aperture and an in-lens detector. The samples were sputtered with Au/Pd prior to the analysis to avoid charging effects.

Membrane Porosity The total porosity of the membrane was calculated from the ratio between the bulk and true density as follows:

ఘ

ߝ% = ቀ1 − ఘ್ೠ೗ೖ ቁ

(1)

೟ೝೠ೐

where ε% is the total porosity, ρbulk is the membrane bulk density calculated from membrane dimensions using the mass-to-volume ratio, and ρtrue = 1.64 g cm-3 is the true density of Cladophora cellulose.32 The thickness of the produced membrane was measured using a digital 10-3 mm precision calliper (Mitutoyo Absolute, Japan). The thickness of each membrane was measured at 10 different positions, and in total 8 membranes were evaluated.

Atomic Force Microscopy A Bruker Dimension Icon AFM system using a Bruker silicon nitride SCANASYST-AIR probe was used to obtain the images. The probe had a symmetric pyramid geometry with a nominal tip radius of 2 nm. The sample was mounted on a magnetic holder using a double adhesive tape. The images were acquired in the peak-force tapping mode, using the


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manufacturer’s ScanAsyst automatic optimization algorithm. Post processing of the pictures was done in Bruker NanoScope Analysis v. 1.6 software.

Pore-size distribution analysis To analyse the pore structure of the membranes, the nitrogen gas adsorption isotherms were obtained with ASAP 2020 instrument (Micromeritics, USA). The samples were outgassed for 6 hours in vacuum at 95 C. The specific surface area of the produced samples was assessed according to the Brunauer-Emmet-Teller (BET) method using a multipoint analysis for P/P0 between 0.06 and 0.29 33. The pore-size distribution was assessed according to the Barret-JonyerHalenda (BJH) method

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from the desorption branch of the nitrogen gas sorption isotherm. The

instrument was calibrated using Micrometrics™ Silica-Alumina SSA 210 m2/g (lot number: A501-49). The deviation between the pore-size mode of the calibration data from the nominal standard values was 0 nm. Here and throughout the text, pore-size mode refers to the position of the highest peak.

Hydraulic permeability A Millipore UF Stirred Cell (47 mm) fitted with the Cladophora cellulose filter and a postfilter support membrane (Munktell; General Purpose Filter Paper) was used to calculate the rate of water flow. The filter was pre-wetted for 15 min prior to the filtration. Five different overhead pressures were applied per sample, viz. 2, 3, 4, 5, and 6 bar. The outflowing water was collected at 15-minute intervals at each pressure to determinate the flow. Deionized water was used throughout experiments.


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Removal of surrogate nanoparticles The same setup as described above was used to assess the efficiency of removal of the surrogate Fluorosphere® particles (23±3nm). A volume of 25 ml of deionized water was spiked with 10 µl of Fluorosphere® dispersion. An aliquot of 5 ml was left as the feed control. The remaining volume of 20 ml of the spiked dispersion was added to the stirred cell and filtrated through 11-µm thick membranes dried at 47 and 200 ˚C, respectively. Five samples were tested for each set of membranes. The fluorescence intensity in the emission range of 590-630 nm with 2 nm increment was tested prior to and post filtration using a TECAN Infinite M200 spectrophotometer. The excitation wavelength was set at 554 nm. The Origin™ statistical software was used to integrate the area under the curve (AUC) for fluorescent emission. The significance of difference statistical analysis was further performed in Excel Windows by a twosided t-test, assuming unequal variance (p=0.01). The log10 reduction value (LRV) was calculated using equation 2 as follows ஺௎஼೛ೝ೐

‫݃݋݈ = ܸܴܮ‬ଵ଴ ൬஺௎஼

೛೚ೞ೟

൰

(2).

Results and Discussion Cellulose paper sheets were produced at different drying temperatures using the hot-press method as described in the experimental section. As it will be evident from the discussion below, slower kinetics of drying favored smaller pore-size mode as compared to drying at higher temperatures. For clarity we discuss the results of two sets of samples having varying thickness and dried at different temperatures. First, membranes having 67 µm thickness were dried at 80 ˚C and 200 ˚C, herein denoted as Mem-67-80 and Mem-67-200. Then, another set of membranes of 11 µm thickness was dried at 47, 80, and 200 ˚C, herein denoted as Mem-11-47, Mem-11-80,


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and Mem-11-200 to further elucidate the effect of drying kinetics on the filter structure. More extensive data on 67 µm membranes dried at variable temperatures are presented in Supporting Information. S1, S2 and S3 SEM and AFM imaging Figure 1 shows the top-view SEM images of the filter membranes. All filters feature an open structure of intertwined nanofibers as previously described.25 Figure 2 shows the typical crosssection of the 67-µm filter as presented in Figure 1. The thickness of the filter estimated from the SEM image correlated well with the caliper measurements summarized in Table 1. It is seen in Figure 2 that the filter consists of a layered structure of regularly arranged individual skin sheets. Each skin sheet has an estimated thickness of 50-100 nm composed of numerous entangled Cladophora cellulose nanofibres. The observed stratified structure, which is similar to the famous French mille-feuille (or “a thousand leaves”) puff pastry, contrasts strongly to all currently known advanced virus-retentive polymer filter architectures produced by phase-inversion processing. Figure 3 shows AFM images of the studied membranes. The AFM images support the conclusions from the SEM images in Figure 1, concerning the open porous structure of filters. It is observed in Figure 3 that the nanofibers of cellulose are slightly wrinkled for Mem-67-200 sample, as opposed to smooth surface of nanofibers of Mem-67-80, which could be due to different thermal treatment regimes. Both SEM and AFM images suggest that the pores in the filter paper are generated by the voids between nanofibers. The AFM images for 11-µm membranes also show open porous structure similar to that seen for 67-µm membranes. However, it can be observed in Figure 3 that, compared to Mem-11-80 sample, a higher degree


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of fibre-fibre interaction exists for Mem-11-47, as verified by the presence of fiber aggregates forming high-density nods. Pore-size distribution analysis Figure 4 and Table 1 summarize the results of the nitrogen sorption analysis for all samples dried at varying temperatures. One of the most interesting results of the gas adsorption analysis is related to the demonstrated ability to control the pore-size distribution of the membranes by altering the water evaporation rate during drying. For Mem-67-200 and Mem-67-80 the pore-size distribution mode shifts from 24 to 18 nm as seen in Figure 4 A. A similar trend can be observed in Figure 4 B for Mem-11-200, Mem-11-80, and Mem-11-47 samples, wherein a shift from 25 nm to 11 nm, respectively. Thus, it can be concluded that higher drying temperature generally implied larger pore-size mode, whose position is further affected by the filter thickness. As it is seen in Table 1, the total porosity and total pore volume of 67 µm filters were largely unaltered by drying conditions and varied only slightly around 35% and 0.36 cm3 g-1 for the thicker membranes. The total porosity of Mem-11-47 sample was 38% and for Mem-11-80 and Mem-11-200 sample 42%, respectively. Also, the total pore volume, calculated from the isotherms in Figure 4 C and presented in Table 1, for Mem-11-80 and Mem-11-200 samples was larger than that of all other studied samples, i.e. 0.52 cm3 g-1. These differences could be attributed to varying heat distribution patterns in thin membranes as compared to more robust thicker filters. The nitrogen sorption isotherms reveal further information about the pore structure of the membranes. The nitrogen sorption isotherms presented in Figure 4 C clearly feature a hysteresis loop between the adsorption and desorption branches of the isotherms. The existence of a hysteresis in all isotherms suggests an interconnected pore structure in accordance to the


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interpretation of the SEM and AFM images. According to IUPAC classification, there are two types of hysteresis, i.e. H1 and H2 types. An H1 type of hysteresis suggests a relatively homogeneous pore-size distribution throughout the sample. On the other hand, an H2 hysteresis is usually related to materials with a complex pore structure, in which pore blocking effects are of importance.35 An H2 hysteresis occurs typically for materials with ink-bottle pores, wherein the neck-size distribution is much narrower than the size of the cavities, and therefore the porefilling and emptying events occur in different regimes.35 The Mem-67-80 and Mem-67-200 samples show the characteristics of an H1 hysteresis36, where the curvature of adsorption and desorption branches are parallel to each other. This is not the case for the Mem-11-47 membrane, wherein a relatively sharp step in the desorption branch of the isotherm is clearly visible, indicating a H2 hysteresis. The observed results are in line with the interpretation of the AFM images for Mem-11-47 sample where higher degree of fiber aggregation is visible. Normally, an H2-type of hysteresis would imply a broad pore-size distribution. However, by studying closely the BJH pore-size distributions in Figure 4, it is observed that Mem-11-47 sample features an exceptionally narrow and well-defined pore-size mode centered at 11 nm. In order to explain this paradox, it should be noted that SEM and AFM techniques depict only the surface topography of the samples, whereas gas sorption analysis probes the entire volume of the sample. Considering the stratified structure of the membrane, it can be speculated that the origin of the H2-hysteresis in Mem-11-47 sample is an artifact due to its hierarchical structure. In particular, although each membrane consists of a single sheet of paper, two types of pores can be distinguished, i.e. (i) pores, which are formed by the interfibrilar space in individual sheets, and (ii) pores, which arise due to the distances between nano-sheets. Then, when the size of the pores due to the interfibrilar


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distances is substantially smaller than the average distance between individual sheets, an H2-type hysteresis arises as one seen in Mem-11-47. Hydraulic permeability A successful filter should not only show high retention selectivity based on size but also feature rapid flow. Figure 5 shows the flow properties of the membrane filters (Mem-11-47, Mem-11-80, Mem-67-80 and Mem-67-200). It is seen from this graph that the filtration rate scales linearly with the applied overhead pressure. It is further seen that the flow rate increases dramatically for the thinner filters from around 10 L m-2 h-1 bar-1 for 67-µm membranes to 80 L m-2 h-1 bar-1 for 11-µm membranes. The linearity of the plot at increasing overhead pressure in Figure 5 confirms the ability of Cladophora cellulose membrane to withstand pressures up to 6 bars without any chemical modification. For comparison, chemical cross-linking was necessary to enhance the wet-strength properties in the past to avoid micro-cracking of the nanocellulose filter 37. Here, an underlying support post-filter (general purpose filter paper) was used to provide the necessary mechanical support, thus dramatically improving the process resilience of the filter. It should be noted that the most advanced available filters, e.g. Millipore Viresolve Pro or Virosart MF, have flow rates on the order of 190 L m-2 h-1 bar-1. The reported hydraulic permeability values of the filter reported here are in the range of 80 L m-2 h-1 bar-1. Removal of surrogate nanoparticles At last, in order to verify whether the reduction in pore-size mode for membranes produced at lower temperature has practical importance for the selectivity of removal, the LRV was estimated for a set of 11-µm thick membranes dried at 47 and 200 ˚C (Mem-11-47 and Mem-11200) using surrogate 20-nm Fluorospheres. Figure 6 shows the AFM images of the filters


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following the filtration. Both images confirm the retention of 20 nm Fluorosphere particles retained in the pore space between the nanofibers. To quantify the efficiency of removal, the emission spectra of the permeate fraction are compared to the feed solution in Figure 7. It is seen that the fluorescence intensity is substantially reduced in both samples. However, Mem-11-200 shows a clearly detectable peak at 607 nm in contrast to Mem-11-47 that shows only negligible permeate fluorescence in the same region. It is estimated that Mem-11-47 exhibits LRV of 3.8, whereas Mem-11-200 shows LRV of only 2, see Table 2. The statistical analysis confirmed that the observed difference between the samples is significant (p=0.01).

Final remarks on the proposed mechanism of pore formation Below we discuss the proposed mechanism of the formation of stratified structure and respective pore-size mode control. In our opinion, two critical steps in the production process of nanocellulose-based virus-retentive filter deserve special attention, i.e. flocculation/coagulation of cellulose nanofibers during wet cake formation and shrinking due to capillary forces during water evaporation. The theoretical background of flocculation/coagulation during the wet cake formation is described by the Derjaguin-Landau-Vermey-Overbeek (DLVO) theory. Thus, the wet cake formation will be affected by the charge of cellulose nanofibers and composition of the surrounding liquid, such as its pH or buffer strength. The effects of the surface charge on the pore-size distribution of Cladophora cellulose paper sheets have been discussed elsewhere38 and, therefore, will not be covered in this work. Here, we will instead focus on the effect of the drying kinetics on the pore size distribution. Once the wet cake is formed and the nanofibers are interlocked in a loose flocculated structure, the effect of contractive capillary forces during drying becomes prevalent. Prior to the hot-press drying, the relative water content and the


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interfibrilar distances in the matrix are identical for samples of similar basis weight. As the heat is applied, water is evaporated and the capillary contraction forces pull the nanofibers closer to each other. However, the rate of water evaporation is dependent on the applied temperature and the thickness of the membrane, which may affect the heat distribution. Thus, Mem-11-200 is exposed to the contracting capillary forces for a shorter period than e.g. Mem-11-47 membrane due to faster evaporation of water, which results in larger pores. There is further a detectable difference between the membranes of varying thickness, which are dried at identical temperature, which can also be correlated to the drying time. In all, being able to control the pore-size distribution of nanocellulose-based filter paper opens new possibilities for designing affordable virus-retentive size-exclusion filters for production of therapeutic proteins.

Conclusion In this work, we experimentally demonstrate the strategies for precisely controlling the poresize distribution of the non-woven nanocellulose-based virus-retentive-filter paper. By controlling the rate of water evaporation during hot-press drying the pore-size distribution can be tailored in the region between 10 and 25 nm. Rapid rate of evaporation favors broader size distribution and larger pore-size mode. The results are highly valuable for developing advanced and cheap separation media that can be tailored for specific needs.


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Figure 1. SEM images of membranes produced with different thicknesses and drying temperature. Mem-67-200, Mem-67-80, Mem-11-80 and Mem-11-47.


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Figure 2. SEM images of same cross-section of Mem-67-80. Left images is taken with 4.2 K X magnification, right images with 22 K X magnification in the same area.


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Figure 3. AFM pictures of membranes produced with different thicknesses and drying temperature. Mem-67-200, Mem-67-80, Mem-11-80 and Mem-11-47.


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Figure 4. N2 adsorption analysis of Mem-67-200, Mem-67-80, Mem-11-200, Mem-11-80 and Mem-11-47: A) BJH desorption pore-size distribution of 67 µm membranes. B) BJH desorption pore-size distribution of 11 µm membranes. C) Isotherm of 67 µm membranes. D) Isotherm of 11 µm membranes.

Figure 5. Flow at different pressure measured for membranes with different thickness. Mem-11200, Mem-11-80 and Mem-11-47 are 11 µm thick, Mem-67-200 and Mem-67-80 are 67 µm thick.


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Figure 6. AFM images of Mem-11-200 and Mem-11-47 following Fluorosphere® filtration. The spheres can be observed on the surface of the filter as both aggregates and individuals.


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Figure 7. Fluorescence intensities of feed and permeate solutions following filtration through A) Mem-11-200 and B) Mem-11-47. TABLES. Table 1 Summary of properties for membranes dried at different temperatures and thicknesses.


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11 ± 0.8 μm

67 ± 0.5 μm

Drying Temperature (˚C)

47

80

200

80

200

Pore mode (nm)

10

22

25

18

25

Drying time (min)

180

60

15

360

20

Total pore volume (cm3/g)

0.27

0.52

0.52

0.38

0.37

Basis weight (g/cm2)

29

29

29

173

173

Specific surface area (m2/g)

82

104

95

90

69

Flow rate, LMH at 1 bar

80

102

116

10

14

Total porosity (%)a

38

42

42

35

35

a

Calculated using eq. 1.

Table 2 LRV measurements and statistical analysis done with a two-sample t-test assuming unequal variances. 11 µm Membranes Drying temperature:

47 ˚C

200 ˚C

LRV (mean)a

3.82

1.97

Variance

0.14

0.95

Df*

5

t-value

3.97

P(T

Strategies for Tailoring the Pore-Size Distribution of Virus Retention

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