Mammalian Virus Purification Using Ceramic Hydroxyapatite

Yae Kurosawa,1 Maiko Saito,1 Daniel Yoshikawa,2 and Mark Snyder2 Tech 1HOYA Technosurgical Corporation, Tokyo, Japan Note 2Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA, USA

Process Separations Bulletin 6549

Abstract Conventional techniques for mammalian virus purification produce variable quality, quantity, and significant loss of particle infectivity. Here, we propose the chromatographic separation of viral particles of diverse sizes and from different families, such as dengue, Japanese encephalitis, influenza, mouse hepatitis, adenovirus, poliovirus, and feline calicivirus, using CHT™ Ceramic Hydroxyapatite Media (Resin). The separation of viral particles from impurities in at least one case was best observed on CHT Ceramic Hydroxyapatite Type II Media when compared to three other apatite media, suggesting the importance of determining which media works best for a specific virus. Additionally, factors such as adjusting the flow rate and gradient slope showed variable differences in the separation of viral particles on ceramic hydroxyapatite media.

Introduction In order to study a virus, a pure, high-quality, infectious Viruses can infect mammalian cells and cause diseases such population is required. Conventional techniques for as influenza, hepatitis, yellow fever, smallpox, and AIDS. Since mammalian virus purification, for uses such as vaccine some biotherapeutic products are produced using mammalian production or biological studies, can produce material cell lines or plasma, the risk of viral contamination in these of variable quality and quantity, often with significant loss products is a concern and guidelines have been enforced to of particle infectivity. alleviate this risk. Chromatographic separation of viral particles In this paper, we report the use of ceramic hydroxyapatite from process intermediates is a key part of ensuring viral media for purification of a wide variety of mammalian viruses. safety in biotherapeutics (ICH Expert Working Group 1999, Chromatography using ceramic hydroxyapatite media Möritz 2005). Additionally, purification of viral particles is used is simple, easily scalable, and results in a concentrated extensively in the study and characterization of these infectious preparation of highly active virus. agents. Understanding aspects of a virus, such as how it infects host cells, uses the host cells for reproduction, and Materials and Methods evades the host immune system, aids scientists in determining The viral particles used in this study are shown in Table 1. how to use viruses for research and therapy.

Table 1. Viral type and size. Virus Family Genus Genome Envelope Size, nm Dengue Flaviviridae Flavivirus ssRNA + 50 Japanese encephalitis Flaviviridae Flavivirus ssRNA + 50 Influenza Orthomyxoviridae Influenzavirus ssRNA + 80–120 Mouse hepatitis Coronaviridae Coronavirus ssRNA + 100–150 Adenovirus Adenoviridae Mastadenovirus dsDNA – 90 Poliovirus Picornaviridae Enterovirus ssRNA – 30 Feline calicivirus Caliciviridae Vesivirus ssRNA – 30–38

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Bulletin 6549 Rev A US/EG 14-0393 0914 Sig 1213 Virus Purification

Viral Activity Assays A Viral activity was determined using the assays shown in Table 2. 50 0.06 1,000

Protein contaminants were detected by UV absorbance at 0.05 280 nm and SDS-PAGE analysis. DNA derived from host cells 40 800 was quantified using the Quant-iT PicoGreen dsDNA Assay Kit 0.04 30 600 (Life Technologies Corporation, USA). 0.03

20 0.02 400

Table 2. Detection methods used for viral activity. Absorbance HA titer, HAU/ml

Detection Method Virus mS/cm Conductivity, 0.01 10 200 Hemagglutination (HA) test Dengue, influenza, adenovirus 0 Plaque assay Japanese encephalitis 0 0 50% tissue culture infective dose Poliovirus, feline calicivirus, 0 5 10 15 20 25 30 35 40 (TCID50) mouse hepatitis Elution volume, ml

B Standard Chromatography Protocol 50 0.06 1,000

Chromatography was performed using Bio-Rad’s BioLogic 0.05 DuoFlow™ System. Columns (4.6 x 35 mm, Sugiyama Shoji 40 800 Co., Ltd., Japan) with a 10-μm frit were packed with 40-µm 0.04 30 600 CHT Ceramic Hydroxyapatite Type II Media. Frit pore size was 0.03 important, as smaller porosities significantly reduced virus 20 0.02 400 recovery (Y. Kurosawa, unpublished data). The flow rate was Absorbance HA titer, HAU/ml 1 ml/min. The purification protocol is outlined in Table 3, unless mS/cm Conductivity, 0.01 10 200 otherwise noted. 0

0 0 Table 3. Standard purification protocol. 0 5 10 15 20 25 30 35 40 Elution volume, ml Step Mobile Phase pH Volume, ml 600 mM sodium Fig. 1. Chromatograms of the separation of dengue virus type 2 by Wash 7.2 5 phosphate CHT Type II Media. A, flow rate at 1.0 ml/min;B , flow rate at 0.1 ml/min. UV 10 mM sodium absorbance at 260 nm (—); UV absorbance at 280 nm (—); conductivity (—); Equilibration 7.2 10 phosphate viral activity in HA test (—). 10 mM sodium Sample loading 7.2 10 phosphate Other serotypes of dengue virus also bound to and eluted 10 mM sodium Wash 7.2 10 phosphate from CHT Type II Media. The approximate elution points in the Gradient elution from sodium phosphate gradient for each serotype are shown in Elution 10–600 mM sodium 7.2 15 Table 4. Types 2 and 4 eluted at roughly the same position. phosphate 600 mM sodium Wash 7.2 5 phosphate Table 4. Elution points of dengue serotypes in a sodium phosphate gradient. Virus Serotype Approximate Elution Point, mM Results 1 250 Dengue Virus 2 450 Figure 1A shows the recovery of dengue virus type 2 from 4 425 cell culture fluid. HA activity was recovered near the end of the gradient, separated from the bulk of A280-absorbing material and from dsDNA (Kurosawa et al. 2012b). Figure 1B demonstrates that decreasing the flow rate by tenfold improves the sharpness of the elution peaks and, hence, separation. In both cases, recovery of HA activity was greater than 95%. Recent studies have indicated that adsorption of dengue virus particles to the surface of CHT Type II Media is similar to their adsorption to cells (Saito et al. 2013).

Influenza Virus A Chromatography of influenza virus A/Beijing/262/95 and 50 1.2 1.2 x 106 2,000 A/Panama/2007/99 (Schickli et al. 2001) cultured in the 1.0 1.0 x 106 40 presence of 0.02% and 0.20% BSA, respectively, is shown 1,500 in Figure 2. 0.8 8.0 x 105 /ml 50 30 0.6 6.0 x 105 1,000 A 20

50 0.8 3,000 Absorbance 0.4 4.0x 105 ng/ml dsDNA, Infectivity, TCID Infectivity, Conductivity, mS/cm Conductivity, 500 2,500 10 40 0.2 2.0 x 105 0.6 2,000 0 0 0 0 30 0 5 10 15 20 25 30 35 40 Elution volume, ml 0.4 1,500

20 B Absorbance

1,000 HA titer, HAU/ml 50 1.2 1.2 x 104 2,000 Conductivity, mS/cm Conductivity, 0.2 10 1.0 1.0 x 104 500 40 1,500

0 0.8 8.0 x 103 /ml 0 0 50 30 0 5 10 15 20 25 30 35 40 Elution volume, ml 0.6 6.0 x 103 1,000 20 Absorbance 0.4 3 ng/ml dsDNA, B 4.0 x 10 Infectivity, TCID Infectivity, Conductivity, mS/cm Conductivity, 50 500 0.8 700 10 0.2 2.0 x 103 600 40 0 0.6 0 0 0 500 0 5 10 15 20 25 30 35 40 Elution volume, ml 30 400 0.4 Fig. 3. Chromatography of two strains of MHV (A, MHV-NuU; B, MHV-S). UV absorbance at 260 nm (—); UV absorbance at 280 nm (—); conductivity (—); 20 300 Absorbance viral infectivity in TCID50 (—); dsDNA (—). Culture fluid contained 10% fetal bovine HA titer, HAU/ml serum (FBS). 0.2 200 Conductivity, mS/cm Conductivity, 10 100 Nonenveloped Viral Particles 0 0 0 Nonenveloped viral particles can be purified by ceramic 0 5 10 15 20 25 30 35 40 Elution volume, ml hydroxyapatite chromatography in the same way as enveloped

Fig. 2. Chromatography of influenza virus. A, A/Beijing/262/95; viruses. Adenovirus (AdV) type 27, feline calicivirus (FCV) B, A/Panama/2007/99. UV absorbance at 260 nm (—); UV absorbance A391 (Hirano et al. 1986), and poliovirus (PV) Sabin type 2 at 280 nm (—); conductivity (—); viral activity in HA test (—). all adsorbed to CHT Type II Media (Figure 4), although they showed different elution times. HA activity is separated from a small BSA peak and a significant amount of material that did not bind to the column. Recovery, as measured by the HA assay, was 98% for the A/Beijing/262/95 virus. Higher concentrations of sodium phosphate are required to elute the A/Panama/2007/99 virus. In addition, the retention time was not affected by the source (allantoic fluid vs. cell culture; data not shown). Mouse Hepatitis Virus Mouse hepatitis virus (MHV) is a coronavirus (CoV), a genus that includes SARS-CoV. Two strains of MHV (MHV-NuU and MHV-S) (Hirano et al. 1981) were applied and bound to CHT Type II Media. Both were eluted at 26–28 minutes in the gradient (Figure 3).

A. Adenovirus A 4 50 0.25 6.0 x 10 250 A. 2,000 40 0.2 B.

5.0 x 104 40 0.2 200 35 4.0 x 104 1,500 30 0.15 30 0.15 150 3.0 x 104 25 0.1 20 100 Fl dsDNA, Absorbance 2.0 x 104 1,000 20 0.1 280 Conductivity, mS/cm Conductivity, Virus activity, HAU/ml A 0.05 10 50 1.0 x 104 15 Conductivity, mS/cm Conductivity, 0 0 0 0 500 0 5 10 15 20 25 30 35 40 10 0.05 Plaque forming activity, PFUx1000 Elution volume, ml 5

B. Feline calicivirus 0 0 0 50 0.3 7.0 x 104 3,000 1 2 3 4 5 6 7 8 9 10 11 12 Fraction number 4 6.0 x 10 2,500 40 4 B 5.0 x 10 /ml 0.2 50 2,000 25 40 0.2 30 A. B. 4.0 x 104 1,500 35 3.0 x 104 20

Absorbance 20 0.1 1,000 ng/ml dsDNA, 4 30 0.15

Conductivity, mS/cm Conductivity, 2.0 x 10 Virus activity, TCID activity, Virus 10 1.0 x 104 500 25 15

0 0 0 0 20 0.1 280 0 5 10 15 20 25 30 35 40 A Elution volume, ml 10 15 Conductivity, mS/cm Conductivity,

C. Poliovirus 10 0.05 50 0.4 4.0 x 107 Plaque forming activity, PFUx1000 5 5 40 7 0.3 3.0 x 10 0 0 0 /ml

50 1 2 3 4 5 6 7 8 9 10 11 12 30 Fraction number 0.2 2.0 x 107 Fig. 5. JEV chromatography at pH 7.0. A, mouse brain homogenate 20 infected with JEV JaGAr01; B, cell culture fluid of JEV Beijing. UV absorbance Absorbance at 280 nm (—); conductivity (—); infectious activity in plaque assay (—).

Conductivity, mS/cm Conductivity, 0.1 7

1.0 x 10 TCID activity, Virus 10 Note: Figure 5A is modified from Kurosawa et al. 2012a.

0 0 0.0 Effect of Hydroxyapatite Type on Separation 0 5 10 15 20 25 30 35 40 Elution volume, ml Figure 6 shows the separation of dengue virus type 2 from cell

Fig. 4. Chromatograms of the separation of cell lysate (A) or culture culture contaminants on four apatites: CHT Type I, CHT Type fluid (B, C) containing nonenveloped viral particles by CHT Ceramic II, CFT™ Ceramic Fluorapatite Type II, and MPC™ Ceramic Hydroxyapatite Type II Media. A, AdV type 27; B, FCV A391; C, PV Sabin Hydroxyfluoroapatite Media. Yields were 80% or higher for type 2. UV absorbance at 260 nm (—); UV absorbance at 280 nm (—); each media type except for MPC, where the yield was 50%. conductivity (—); viral activity (AdV in HA test, FCV and PV in TCID50) (—); dsDNA (—). Cell culture fluid contained 10% FBS. FI, fluorescence intensity. Although binding and elution was achieved on all four media, the separation of virus from impurities was best on CHT Type Japanese Encephalitis Virus II Media. Figure 7 shows a similar study using CHT Type II Japanese encephalitis virus (JEV) chromatography is shown and CFT Type II Media for the purification of poliovirus, with in Figure 5 (Kurosawa et al. 2009, 2012a). Irrespective of the recoveries of 88% and 102%, respectively. These results source or strain, the virus elutes at approximately 350 mM illustrate the importance of choosing the appropriate media for sodium phosphate (note that the gradient in these two cases is the separation in question. 10–400 mM and the column size is 6.8 x 20 mm). Again, there is good separation between protein contamination and the virus.

A. CHT Type I A 50 0.16 1,000 40 0.06 1.2 x 107

40 7 0.12 800 0.05 1.0 x 10 30

30 600 0.04 6 /ml

8.0 x 10 50 0.08 20 0.03 400 20 6.0 x 106 Absorbance 0.04 10 HA titer, HAU/ml 0.02 Absorbance Conductivity, mS/cm Conductivity, 200 4.0 x 106 Conductivity, mS/cm Conductivity, 0 10 TCID Infectivity, 0 0 0.01 2.0 x 106 0 5 10 15 20 25 30 35 40 Elution volume, ml 0 0 0 0 5 10 15 20 25 30 35 40 45 50 B. CHT Type II Elution volume, ml 50 0.16 1,000 B 40 0.12 800 40 0.06 1.2 x 107 30 600 0.08 0.05 1.0 x 107 20 400 30 /ml Absorbance 0.04 6

0.04 8.0 x 10 50 10 HA titer, HAU/ml

Conductivity, mS/cm Conductivity, 200 0.03 20 6.0 x 106 0 0 0 0.02 0 5 10 15 20 25 30 35 40 Absorbance 4.0 x 106 Elution volume, ml Conductivity, mS/cm Conductivity, 10 0.01 TCID Infectivity, 2.0 x 106 C. CFT Type II 0 0 0 50 0.16 0 5 10 15 20 25 30 35 40 45 50 1,600 Elution volume, ml 40 0.12 Fig. 7. Chromatograms of culture fluid of polio virus at pH 6.4. A, CHT Type 1,200 30 II Media; B, CFT Type II Media. UV absorbance at 260 nm (—); UV absorbance 0.08 at 280 nm (—); conductivity of elution buffer (—); infectious activity in TCID (—). 800 50 20 Note: the gradient in these two cases is 150–450 mM at pH 6.4 for 20 ml. 0.04 10 400 HA titer, HAU/ml Conductivity, mS/cm Conductivity,

0 0 0 Conclusion 0 5 10 15 20 25 30 35 40 Using ceramic hydroxyapatite media provided high purity, Elution volume, ml recovery, and viral activity for seven mammalian viruses of varying size and belonging to different families. We have D. MPC shown that, in at least one case, slowing the flow rate and 50 0.16 1,000 decreasing the gradient slope allowed better purification of

40 0.12 800 viral particles on CHT Type II Media, signifying the importance of determining the best settings for such factors when using 30 600 0.08 apatite media. Testing different apatites is significant for 20 400 determining which media type will work best for a specific Absorbance 0.04 HA titer, HAU/ml virus. A larger pore size, as provided by the CHT Type II

Conductivity, mS/cm Conductivity, 10 200 0 Media, allowed better separation of the dengue virus from 0 0 contaminants, compared to other apatite media. 0 5 10 15 20 25 30 35 40 Elution volume, ml Of equal significance, the use of ceramic hydroxyapatite Fig. 6. Chromatograms of dengue virus type 2. A, CHT Type I Media; media is simple and provides reproducible results, allowing B, CHT Type II Media; C, CFT Type II Media; D, MPC Media. UV absorbance an alternative to the conventional methods of viral purification. at 260 nm (—); UV absorbance at 280 nm (—); conductivity (—); viral activity in HA test (—).

© 2014 Bio-Rad Laboratories, Inc. Bulletin 6549 Acknowledgements ■■ Dengue viral particles were supplied by Professor Koichi Morita of the Department of Virology, Institute of Tropical Medicine, Nagasaki University, Japan.

■■ Experiments using influenza virus A/Beijing/262/95 and A/Panama/2007/99 were conducted in collaboration with Dr. Shota Nakamura and Dr. Takaaki Nakaya of Osaka University, Japan.

■■ The mouse hepatitis virus and feline calicivirus were supplied by Dr. Norio Hirano and Dr. Shigehiro Sato, and the poliovirus by Dr. Shigehiro Sato, of the Department of Microbiology, Iwate Medical University, Japan.

References Hirano N et al. (1981). Comparison of mouse hepatitis virus strains for pathogenicity in weanling mice infected by various routes. Arch Virol 70, 69–73. Hirano N et al. (1986). A survey of feline respiratory infections. Jpn J Vet Sci 48, 423–427. ICH Expert Working Group (1999). Viral safety evaluation of biotechnology products derived from cell lines of human and animal origin. In Federal Register 63(185), pp. 51074–51105. Kurosawa Y et al. (2009). Observation of Japanese encephalitis virus particles on ceramic hydroxyapatite by scanning electron microscopy. Med Biol 153, 607–610. Kurosawa Y et al. (2012a). Development of a purification method for Japanese encephalitis virus particles using ceramic hydroxyapatite chromatography. Med Biol 156, 410–416. Kurosawa Y et al. (2012b). Purification of dengue virus particles by one- step ceramic hydroxyapatite chromatography. World Journal of Vaccines 2, 155–160. Möritz A (2005). Virus safety evaluation of biotechnology products in development. BioProcess Int Suppl. 3, 15–16. Saito M et al. (2013). Scanning electron microscopy-based approach to understand the mechanism underlying the adhesion of dengue viruses on ceramic hydroxyapatite columns. PLoS ONE 8, e53893. Schickli JH et al. (2001). Plasmid-only rescue of influenza A virus vaccine candidates. Philos Trans R Soc Lond B Biol Sci 356, 1965–1973.

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Bulletin 6549 Rev A US/EG 14-0393 0914 Sig 1213