# Comparative Analysis of Dry and Wet Porometry Methods for Characterization of Regular and Cross-Linked Virus Removal Filter Papers

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## Abstract

**:**

## 1. Introduction

## 2. Material and Methods

#### 2.1. Filter Preparation

^{®}, Merck Millipore, Burlington, MA, USA) using vacuum suction in a funnel. The cellulose cake on the filter was then removed and dried at 40 °C for 12 h using a hot-press (Rheinstern; Mainz, Germany).

#### 2.2. Cross-Linking of Filter

#### 2.3. Scanning Electron Microscopy

#### 2.4. Atomic Force Microscopy

#### 2.5. Nitrogen Gas Sorption Analysis

^{TM}Silica-Alumina SSA 210 m

^{2}·g

^{−1}(lot number: A-501-49) standard prior to analysis. The deviation between the pore-size mode of the calibration data from the nominal standard values was 0 nm. The sample was degassed for 6 h at 95 °C at 5 °C·min

^{−1}ramp temperature prior to analysis. The pore-size distribution was calculated according to the Barrett-Jonyer-Halenda (BJH) method [24] from the desorption branch of the isotherm. Here and throughout the text, pore-size mode refers to the position of the highest peak.

#### 2.6. Liquid-Liquid Porometry (LLP)

_{2phase}(ΔP) is the volumetric flow of intrusion liquid when the wetting fluid is present in the membrane at a set pressure, Q

_{Intrusion fluid}(ΔP) is the volumetric flow of intrusion fluid in absence of wetting fluid present in the membrane, ΔP is the pressure, F

_{Q}(r) is the flow weighted pore-size distribution, k is the shape factor which is set to 1 approximating a cylindrical pore shape, and γ is the interfacial surface tension between the two fluids, which has been reported as approximately 6.3 × 10

^{−4}N at 22 °C [27]. The measured volumetric two-phase flow data were fitted to a weighted smooth spline function using Matlab. Equation (3) was used with a step size increment of 2 nm to obtain the weighted flow fraction bar plot, a Gaussian function was then fitted to the histogram in order to visualize a probable pore-size distribution.

#### 2.7. Cryoporometry by Differential Scanning Calorimetry

^{−1}rate to −20 °C and then heated to 5 °C at a heating rate of 0.7 K·min

^{−1}.

_{on-pk}is the difference of the depressed melting point to the true melting point of bulk water, r is the radius of the pore. A

_{m}, B

_{m}and δ

_{m}are probing liquid dependent constants. Herein, we use the following values for A

_{m}= 19.082, B

_{m}= −0.1207, and δ

_{m}= 1.12 calculated by Landry [10]. The differential pore volume (dV/dr) was calculated using Equation (5):

_{on-pk})/dr is the melting point depression from Equation (4), m is the mass of the sample, ∆H

_{f}(T) is the temperature-dependent melting enthalpy, and ρ(t) is the temperature-dependent density.

_{m}

^{0}is the equilibrium melting temperature of water.

^{2}= 0.99). The estimated function is then used as dQ/dt in Equation (5) to calculate the pore-size distribution.

^{−1}heat rate) and the mean peak temperature was calculated to 0.61 ± 0.1 °C.

#### 2.8. Flux Measurement

#### 2.9. Virus Removal by EPT and LVP

_{50}) at 8-fold dilution. EPT was used for samples with high viral loads. LVP was performed for samples with expected low virus loads, provided that such indications had been demonstrated during EPT.

_{2}concentration of 5.0 ± 0.5% by volume. After the first phase of the incubation, i.e. after 6–8 days, the supernatants were transferred onto fresh cell indicator MTPs and incubated for another 5–7 days. The total duration for the incubation phase was 11–15 days, enabling a clear evaluation of CPE.

_{0}: decadic logarithm of highest dilution factor of the sample, which causes the infection of all parallel cultures (= 8)

_{10}3);

_{i}: observed reaction rate starting and including the rate at Y

_{0};

_{10}5).

_{p}: number of virus-positive wells;

_{w}: sample volume per well (0.2 mL).

_{0}: log

_{10}total virus load spiked start material

_{n}: log

_{10}total virus load related filtrate

## 3. Results and Discussion

^{3}for the cross-linked compared to 0.43 g/cm

^{3}for the regular filter. Based on the SEM images of cross-sections, we hypothesize that cross-linking reduces the space between individual nanocellulose sheets. Figure 5B shows the PSD from LLP data. The analysis of the LLP data produces a multimodal size distribution, which highlights the difficulty of measuring very low flows with high accuracy. However, there is no statistical difference between the pore modes, i.e. 24.3 ± 2.5 nm for the regular and 22 ± 1.4 nm for the cross-linked sample. Even though the flow commenced at a lower pressure for the cross-linked sample, the model reveals that the higher increase of flux at ~1.2 bars compared to the regular filter shifts the pore mode towards a smaller diameter. Figure 5C shows the PSD obtained from CP-DSC analysis. The pore mode of 24.0 ± 0.8 nm is assessed for both the regular and 24.0 ± 0.6 nm for the cross-linked filters. Similar to NGSP analysis, the pore volume is slightly lower for the cross-linked sample.

## 4. Conclusions

## Author Contributions

## Funding

## Acknowledgments

## Conflicts of Interest

## References

- Van Reis, R.; Zydney, A. Bioprocess membrane technology. J. Membr. Sci.
**2007**, 297, 16–50. [Google Scholar] [CrossRef] - Elford, W.J. Principles governing the preparation of membranes having graded porosities. The properties of "gradocol" membranes as ultrafilters. Trans. Faraday Soc.
**1937**, 33, 1094–1104. [Google Scholar] [CrossRef] - Bauer, J.H.; Hughes, T.P. The preparation of the graded collodion membranes of Elford and their use in the study of filterable viruses. J. Gen. Physiol.
**1934**, 18, 143–162. [Google Scholar] [CrossRef] [PubMed] - Barnard, J.E.; Elford, W.J. The causative organism in infectious ectromelia. Proc. Royal Soc. Lond. B-Conta
**1931**, 109, 360–380. [Google Scholar] [CrossRef] - Elford, W.J. A new series of graded collodion membranes suitable for general bacteriological use, especially tint filterable virus studies. J. Pathol. Bacteriol.
**1931**, 34, 505–521. [Google Scholar] [CrossRef] - Galloway, I.A.; Elford, W.J. Filtration of the virus of foot-and-mouth disease through a new series of graded collodion membranes. Br. J. Exp. Pathol.
**1931**, 12, 407–425. [Google Scholar] - Ferry, J.D. Ultrafilter membranes and ultrafiltration. Chem. Rev.
**1936**, 18, 373–455. [Google Scholar] [CrossRef] - Morison, K.R. A comparison of liquid-liquid porosimetry equations for evaluation of pore size distribution. J. Membr. Sci.
**2008**, 325, 301–310. [Google Scholar] [CrossRef] - Giglia, S.; Bohonak, D.; Greenhalgh, P.; Leahy, A. Measurement of pore size distribution and prediction of membrane filter virus retention using liquid-liquid porometry. J. Membr. Sci.
**2015**, 476, 399–409. [Google Scholar] [CrossRef] - Landry, M.R. Thermoporometry by differential scanning calorimetry: Experimental considerations and applications. Thermochim. Acta
**2005**, 433, 27–50. [Google Scholar] [CrossRef] - Asper, M. Virus Breakthrough after Pressure Release During Virus Retentive Filtration; PDA: Barcelona, Spain, 2011. [Google Scholar]
- Dishari, S.K.; Venkiteshwaran, A.; Zydney, A.L. Probing effects of pressure release on virus capture during virus filtration using confocal microscopy. Biotechnol. Bioeng.
**2015**, 112, 2115–2122. [Google Scholar] [CrossRef] [PubMed] - Woods, M.A.; Zydney, A.L. Effects of a pressure release on virus retention with the Ultipor DV20 membrane. Biotechnol. Bioeng.
**2014**, 111, 545–551. [Google Scholar] [CrossRef] [PubMed] - Jackson, N.B.; Bakhshayeshi, M.; Zydney, A.L.; Mehta, A.; van Reis, R.; Kuriyel, R. Internal virus polarization model for virus retention by the Ultipor
^{®}VF Grade DV20 membrane. Biotechnol. Progr.**2014**, 30, 856–863. [Google Scholar] [CrossRef] [PubMed] - Asper, M.; Hanrieder, T.; Quellmalz, A.; Mihranyan, A. Removal of xenotropic murine leukemia virus by nanocellulose based filter paper. Biologicals
**2015**, 43, 452–456. [Google Scholar] [CrossRef] [PubMed] - Gustafsson, S.; Lordat, P.; Hanrieder, T.; Asper, M.; Schaefer, O.; Mihranyan, A. Mille-feuille paper: A novel type of filter architecture for advanced virus separation applications. Mater. Horiz.
**2016**, 3, 320–327. [Google Scholar] [CrossRef] - Gustafsson, S.; Mihranyan, A. Strategies for Tailoring the Pore-Size Distribution of Virus Retention Filter Papers. ACS Appl. Mater. Interfaces
**2016**, 8, 13759–13767. [Google Scholar] [CrossRef] [PubMed] - Metreveli, G.; Wågberg, L.; Emmoth, E.; Belák, S.; Strømme, M.; Mihranyan, A. A Size-Exclusion Nanocellulose Filter Paper for Virus Removal. Adv. Healthc. Mater.
**2014**, 3, 1546–1550. [Google Scholar] [CrossRef] [Green Version] - Quellmalz, A.; Mihranyan, A. Citric Acid Cross-Linked Nanocellulose-Based Paper for Size-Exclusion Nanofiltration. ACS Biomater. Sci. Eng.
**2015**, 1, 271–276. [Google Scholar] [CrossRef] - Gustafsson, S.; Manukyan, L.; Mihranyan, A. Protein–Nanocellulose Interactions in Paper Filters for Advanced Separation Applications. Langmuir
**2017**, 33, 4729–4736. [Google Scholar] [CrossRef] - Liu, J.; Willför, S.; Mihranyan, A. On importance of impurities, potential leachables and extractables in algal nanocellulose for biomedical use. Carbohydr. Polym.
**2017**, 172, 11–19. [Google Scholar] [CrossRef] - Ibrahim, N.A.; Abo-Shosha, M.H.; Elnagdy, E.I.; Gaffar, M.A. Eco-friendly durable press finishing of cellulose-containing fabrics. J. Appl. Polym. Sci.
**2002**, 84, 2243–2253. [Google Scholar] [CrossRef] - Harifi, T.; Montazer, M. Past, present and future prospects of cotton cross-linking: New insight into nano particles. Carbohydr. Polym.
**2012**, 88, 1125–1140. [Google Scholar] [CrossRef] - Barrett, E.P.; Joyner, L.G. Determination of Nitrogen Adsorption-Desorption Isotherms—Estimation of Total Pore Volumes of Porous Solids. Anal. Chem.
**1951**, 23, 791–792. [Google Scholar] [CrossRef] - Erbe, F. The determination of pore distribution according to their sizes in filters and ultra-filters. Kolloid Z.
**1933**, 63, 277–285. [Google Scholar] [CrossRef] - Bechhold, H.M. Schlesinger, and K. Silbereisen, Pore size of ultra filters. Kolloid Z.
**1931**, 60, 172–198. [Google Scholar] [CrossRef] - Phillips, M.W.; DiLeo, A.J. A Validatible Porosimetric Technique for Verifying the Integrity of Virus-Retentive Membranes. Biologicals
**1996**, 24, 243–253. [Google Scholar] [CrossRef] - Kärber, G. Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Naunyn Schmiedebergs Arch. Exp. Pathol. Pharmakol.
**1931**, 162, 480–483. [Google Scholar] [CrossRef] - Spearman, C. Review of The Method of ‘Right and Wrong Cases’ (‘Constant Stimuli’) without Gauss’s Formula. Psychol. Bull.
**1909**, 6, 27–28. [Google Scholar] [CrossRef] - Chen, D. Viral clearance using traditional, well-understood unit operations (session I): Virus-retentive filtration. PDA J. Pharm. Sci. Technol.
**2014**, 68, 38–50. [Google Scholar] [CrossRef]

**Figure 1.**Cytopathogenic effect (CPE) illustrated for reference A9 cells incubated with or without minute virus of mice (MVM): (

**A**) healthy cells and (

**B**) infected cells. CPE: cytopathogenic effect; MVM: minute virus of mice.

**Figure 2.**Atomic force microscopy (AFM) images of as-produced regular (

**A**) and cross-linked (

**B**) mille-feuille filter.

**Figure 3.**Cross-section scanning electron microscope (SEM) images of as-produced regular (

**A**) and cross-linked (

**B**) mille-feuille filter.

**Figure 4.**(

**A**) Isotherm from nitrogen sorption measurement for both regular and cross-linked filter; (

**B**) LLP isotherm for regular and for cross-linked filter; (

**C**) Cryoporometry isobar for regular and cross-linked filter. The data within the PSD was fitted to a Gaussian distribution to obtain good peak separation. LLP: liquid-liquid porometry; PSD: pore-size distribution.

**Figure 5.**(

**A**) Calculated pore-size distribution for regular and cross-linked filters from nitrogen desorption isotherm using the BJH method. (

**B**) Calculated pore-size distribution for regular and cross-linked filters from LLP measurement data. The dotted line is a visual PSD guide for the eye obtained by fitting a lognormal distribution to the data. (

**C**) Calculated pore-size distribution for regular and cross-linked filters using cryoporometry data.

**Figure 6.**Normalized flow rate values measured for regular and cross-linked samples (n = 3). The delta values indicate the reduced flow rates per bar between 1 and 5 bar.

**Figure 7.**(

**A**) MVM removal capability by mille-feuille filter at varying TM pressure, (

**B**) MVM removal capability by cross-linked mille-feuille filter at varying TM pressure, (*) no infective viral particles detected in large-volume plating. TM: transmembrane.

**Table 1.**Summary of measured pore mode for Barrett-Jonyer-Halenda (BJH), LLP, and differential scanning calorimetry (DSC), the results are the average with ±SD.

Method | Pore Mode Regular (nm) | Pore Mode Cross-Linked (nm) |
---|---|---|

BJH | 18.9 ± 1.4 ^{a} | 20.0 ± 2.3 ^{a} |

LLP | 24.3 ± 2.5 ^{b} | 22.0 ± 1.4 ^{b} |

DSC | 24.0 ± 0.8 ^{c} | 24.1 ± 0.6 ^{c} |

^{a}n = 4,

^{b}n = 3,

^{c}n = 3.

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**MDPI and ACS Style**

Gustafsson, S.; Westermann, F.; Hanrieder, T.; Jung, L.; Ruppach, H.; Mihranyan, A.
Comparative Analysis of Dry and Wet Porometry Methods for Characterization of Regular and Cross-Linked Virus Removal Filter Papers. *Membranes* **2019**, *9*, 1.
https://doi.org/10.3390/membranes9010001

**AMA Style**

Gustafsson S, Westermann F, Hanrieder T, Jung L, Ruppach H, Mihranyan A.
Comparative Analysis of Dry and Wet Porometry Methods for Characterization of Regular and Cross-Linked Virus Removal Filter Papers. *Membranes*. 2019; 9(1):1.
https://doi.org/10.3390/membranes9010001

**Chicago/Turabian Style**

Gustafsson, Simon, Frank Westermann, Tobias Hanrieder, Laura Jung, Horst Ruppach, and Albert Mihranyan.
2019. "Comparative Analysis of Dry and Wet Porometry Methods for Characterization of Regular and Cross-Linked Virus Removal Filter Papers" *Membranes* 9, no. 1: 1.
https://doi.org/10.3390/membranes9010001