Process Chromatography

Single-Step Influenza and Dengue Virus Purification withMixed-Mode CHT Ceramic Hydroxyapatite XT MediaYae Kurosawa,1 Payal Khandelwal,2 Daniel Yoshikawa,2 and Mark Snyder 2
1HOYA Technosurgical Corporation, Tokyo, Japan
2Bio-Rad Laboratories, Inc., 6000 James Watson Drive, Hercules, CAAbstract

CHT CERAMIC
HYDROXYAPATITE

Past studies have shown that CHT™ Ceramic Hydroxyapatite Media can be used for virus
purification. We purified influenza and dengue viruses on the newest member of the CHT
family, CHT XT Media. We recovered over 75% of each virus in a single step of purification.
In addition, >90% reduction of contaminating proteins and >60% removal of dsDNA
were seen in both samples. This positions CHT XT as an additional media for simple,
fast, and reproducible viral purification and overcomes the shortcomings of conventional
purification methods.

Introduction
Virus preparations have been used as vaccines for over two
hundred years, and more recently for gene therapy platforms.
For gene delivery, conventional viral purification methods suchas density gradient ultracentrifugation and precipitation can bedifficult to scale and may not meet the purity standards required
for a therapeutic. Consequently, chromatography has emerged
as an advanced tool to meet large-scale therapeutic virus
purification requirements. Viruses bind to chromatographic resins
by electrostatic interactions.
We have previously demonstrated mammalian virus purification
with the mixed-mode CHT Ceramic Hydroxyapatite Media(Kurosawa et al. 2014). CHT Media are a group of mixed-mode
calcium affinity/cation exchange supports. The high negative
surface charge on many viruses allows them to bind tightly to CHT
calcium sites, permitting significant purification with high (>80%)
yields. Such tight binding has also been utilized to provide robust
viral clearance, which can be used to ensure viral safety duringbiomanufacturing (ICH Expert Working Group 1999, Moritz 2005).
CHT XT is the newest addition to the CHT family of media. It has
been designed for superior physical robustness to ensure it canbe used repeatedly over a large number of cycles. It maintains
the unique separation properties of CHT for aggregate removal
and monoclonal antibody purification. In order to test its virus
purification capabilities, we performed single-step purificationsof influenza and dengue viruses using CHT XT Media.

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Materials and Methods
The viruses used in this study are shown in Table 1.
Table 1. Virus type and size.
Virus

GenomeEnvelope

Size, nm

Influenza

ssRNA

+

80–120DenguessRNA+

50

Virus Production

Influenza virus: Strain A/California/07/2009 was cultured on MDCKcells in minimal essential medium (MEM; Gibco) containing 10%
fetal bovine serum (FBS) and L-glutamine in 225 cm2 flasks. Whenthe cells were almost confluent, influenza virus was inoculated onto
the cell monolayer at a 1:3,000 dilution in 75 ml of D-MEM/F-12
with trypsin, penicillin, and streptomycin (flu culture medium). Thesupernatant was harvested on days 3, 4, and 11 and clarified
through 0.45 µm filters.
Dengue virus: Type 2 strain ThNH7/93 was cultured on C6/36 cells.
Cells were grown in 225 cm2 flasks that were precoated with
poly-L-lysine at 100 µg/ml in phosphate buffered saline (PBS).
Cells were cultured in Minimum Essential Medium Eagle (modified)(modified EMEM, MP Biomedicals) containing 10% FBS at 28°C for1 week. After the cells reached confluence, dengue virus type 2 wasinoculated at a 1:1,000 dilution onto the cell monolayer in 75 ml ofmodified EMEM containing 0.5% FBS and MEM Vitamin Solution(Thermo Fisher Scientific) and cultured at 28°C. The medium waschanged at day 3 and culture fluid (about 75 ml) was collected atday 7. The culture fluid was filtered through a 0.45 µm filter to remove
cells and large cell debris.

Single-Step Influenza and Dengue Virus Purification with Mixed-Mode CHT Ceramic Hydroxyapatite XT MediaChromatography

TCID50 Influenza Virus Infectivity Assay

CHT XT Media was packed into 4.6 x 35 mm columns (0.58 ml
bed volume) and viral purification was performed at a flow rateof 360 cm/hr as shown in Table 2 for influenza and Table 3for dengue. All experiments were carried out in triplicate andrepresentative data are shown.

A confluent monolayer of MDCK cells in 96-well microplates wasused for this test. MDCK cells (2 x 104 cells/well) were cultured influ culture medium for 1 day prior to the testing. Each virus fraction
underwent tenfold serial dilution with flu culture medium. Diluted
viral fractions (50 µl) were added to each well (n = 3) and cultured
for one week. Cytopathic effects were studied under a microscope.
Titers were calculated using the Reed-Muench method.

Table 2. Influenza purification protocol.
StepMobile Phase

Pre-wash

1 M NaOH

pHVolume, ml

Wash

5

Viral Purity Analysis

600 mM sodium phosphate

7.2

10Equilibration

10 mM sodium phosphate

7.2

10Wash

24% of 600 mM sodium phosphate

7.2

10Sample loadingand washing

24% of 600 mM sodium phosphate

7.2

24Elution

Gradient elution from 150–600 mM(24% to 100%) sodium phosphate

7.2

11Column wash

600 mM sodium phosphate

7.25pHVolume, ml

7.2

10Viral purity was assessed by electrophoresis on a 12.5%
SDS‑PAGE gel and by immunoblotting. The protocols used forthese analyses were as described previously (Kurosawa et al.
2012). Cell culture fluid was concentrated tenfold and all othersamples were concentrated 30-fold by ultrafiltration with a 10 kD
MWCO filter before use. Bio-Rad’s Gel Doc EZ Imaging System
was used to quantitate band density. For immunoblotting,primary antibody anti-HA mAb (Abnova) was used at a 1:200dilution with the influenza virus samples and mouse anti-D2serum (made in house by HOYA Technosurgical Corporation forthese experiments) was used at a 1:500 dilution with the dengue
virus samples. Secondary antibody anti-mouse IgG (Fc)-POD(American Qualex) was used at a 1:1,000 dilution. The bands
were visualized with EzWestBlue (ATTO Corporation).

Table 3. Dengue purification protocol.
StepMobile Phase

Pre-wash

1 M NaOH

Wash

600 mM sodium phosphate

5Equilibration10 mM sodium phosphate

7.2

20

Sample loading andwashing

10 mM sodium phosphate

7.2

20

ElutionGradient elution from 10–600 mM
sodium phosphate

7.2

15Column wash

600 mM sodium phosphate

7.2

5Results
Influenza Virus
Viral Activity

The chromatographic results from the influenza virus purification
are shown in Figure 1. Influenza virus elutes at approximately
250 mM phosphate, with good separation from the bulk ofUV-absorbing material and partial resolution from dsDNA.
Hemagglutination activity (red bars on the chromatograms)
measures the titer of virus that can bind to molecules present on
the surface of red blood cells and cause agglutination. As seenin Figures 1A and 1B, HA activity is predominantly seen between
retention volumes of 26 and 31 ml. The total viral activity recoveredfrom these fractions was calculated to be about 75% of the starting
activity. This activity was well separated from the bulk of the DNAand A280-absorbing impurities. The samples showing bulk viral
activity were pooled for further analytical assays.

Viral Activity Assay

Influenza viral activity in the recovered fractions was determinedvia hemagglutination analysis (HA test) with chicken red bloodcells. Activity of the dengue virus in the recovered fractions wasdetermined via HA test with goose red blood cells as described
previously (Kurosawa et al. 2012).
Estimating Double-Stranded DNA (dsDNA)

The concentrations of dsDNA in fractions were determinedusing the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher)according to the manufacturer’s instructions.
Protein Analysis in Virus Fractions

Viral fractions were analyzed for total protein concentrations withthe Micro BCA Protein Assay Kit. Host cell protein (HCP) analysis
for the influenza viral fractions was performed with the MDCK HCP
ELISA Kit (Cygnus Technologies).

© 2024 Bio-Rad Laboratories, Inc.

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Single-Step Influenza and Dengue Virus Purification with Mixed-Mode CHT Ceramic Hydroxyapatite XT MediaA

A
50

4,000

0.04

2,500

50

4,000

7.0x105

3,500

6.0x105

0.14
3,500

30

20

0.04

10000

51015202530
Elution volume, ml

35

10500

500

0

4.0x105
3.0x105

2.0x105

1,000

0.02

500

1.0x105000.144,000

1.2x1050.12

3,50000400510152025 30
Elution volume, ml

3540

B

0.12

3,500

40

5,000

40

2,0000.06
1,500
0.04

3,000

Conductivity, mS/cm

2,5000.08

2,00030

2,5000.08

2,0000.06

20

1,500
0.04

1,000
100.02500

0000

510152025 30
Elution volume, ml

35

1.0x105

3,000

0.1
4,000
dsDNA, ng/ml

Absorbance

2050

3,0000.1
30

6,000

8.0x104

6.0x104

4.0x104

Infectivity, TCID50 /ml

4,000

Absorbance

0.14

HA titer, HAU/ml

50

HA titer, HAU/ml

B

Conductivity, mS/cm

2,0000.06
1,5001,00002,5000.08

Infectivity, TCID50 /ml

0.01

1,0005.0x105
HA titer, HAU/ml

1,500

3,0000.1
Absorbance

2,000

1,500

Conductivity, mS/cm

2,5000.02

dsDNA, ng/ml

20

3,0000.1240

2,000

HA titer, HAU/ml

30

0.03

Absorbance

Conductivity, mS/cm

40

1,000
10

1,0000.02

2.0x104
500004000051015202530
Elution volume, ml

35040

Fig. 1. Replicate elution of influenza virus from CHT XT. HA titer ( ); DNA ( );A280 ( ); A260 ( ); conductivity ( ).

Fig. 2. Infectivity of the influenza virus purified on CHT XT. Infectivity ( ); HA
titer ( ); A280 ( ); A260 ( ); conductivity ( ).

Viral Infectivity

Analytical Assays

In order to determine if the purified influenza virus maintained its
infectivity, a TCID50 test was performed (Figure 2) to assay forcytopathic effects (CPE). This test determines the quantity ofactive, infectious particles. A mean recovery of ~68% infectivity
was calculated for the replicates. As seen in Figures 2A and 2B,
the bulk of the infectivity is seen at retention volumes that also
showed the highest HA activity. This confirmed that fractions 26
to 32 contained active, purified virus.

Three analytical assays were performed on the pooled samples fromthe influenza virus run on CHT XT Media.The results of the analyticalassays are shown in Table 4. Single-step influenza virus purification
on CHT XT led to significant (>99%) clearance of total and host cell
proteins as well as removal of over 60% of dsDNA.
Table 4. Results from the analytical assays on the pooled influenza
samples from the HA assay.
Log Reduction
in dsDNA
0.42© 2024 Bio-Rad Laboratories, Inc.

3

Log Reductionin Total Protein

Log Reductionin HCP Content

2.82

2.54

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Single-Step Influenza and Dengue Virus Purification with Mixed-Mode CHT Ceramic Hydroxyapatite XT Media

Viral Purity

Dengue Virus
Viral Activity

SDS-PAGE analysis of the active pooled fractions from Figure 1
confirmed the significant purification of species migrating at theexpected molecular weight for the two forms of the HA protein(Figure 3A). Immunoblotting of the same samples confirmed thatthe concentrated bands are HA (Figure 3B).

The chromatographic results from the dengue virus purification are
shown in Figure 4. Dengue virus elutes at approximately 400 mM
phosphate, with good separation from the bulk of UV-absorbing
material and dsDNA. HA activity was predominantly seen between
retention volumes of 28 to 38 ml; fractions 30–35 were pooledfor further analysis. The total viral activity recovered from thesefractions was calculated to be about 87%. Viral activity was notdetected in the flow-through fractions in any of the replicates.

A123

4kD
200

A116
9750

0.3

1,000

100

8008066HA
4540

1420600

4000.1

10B
123440200

20001,000

100

800

800kD

02000510116
97
66

60dsDNA, ng/ml

Conductivity, mS/cm

2130

HA titer, HAU/ml

0.2

HA1

Absorbance

31

15202530
Elution volume, ml

3540

B
HA50

0.3

4540
31

HA1Fig. 3. SDS-PAGE (A) and immunoblotting analysis (B) of fractions. Protein
bands on the gels were visualized by silver staining and the HA proteins in active
samples were detected using an HA-specific monoclonal antibody. Lane 1: 3 μlprotein MW marker; lane 2: 10 μl cell culture fluid; lane 3: 10 μl flow-throughfractions; lane 4: 10 μl pooled fractions from HA assay.

20600

4000.1106040200

2000dsDNA, ng/ml

1430

HA titer, HAU/ml

21Absorbance

Conductivity, mS/cm

0.2

0005

1015202530
Elution volume, ml

3540Fig. 4. Replicate elution of dengue virus from CHT XT. HA titer ( ); DNA ( );A280 ( ); A260 ( ); conductivity ( ).

© 2024 Bio-Rad Laboratories, Inc.

4Bulletin 7115

Single-Step Influenza and Dengue Virus Purification with Mixed-Mode CHT Ceramic Hydroxyapatite XT MediaConclusions
Chromatography media are continuously evolving to meet theincreasing demands of the bioprocessing industry. We developed
the new CHT XT Media to meet the demand for a robust media withexcellent life time and single-step purification functionality for healthy
process economics. With both high purity and high yield for bothinfluenza and dengue viruses, we show that CHT XT can be used forsingle-step impurity clearance. This positions CHT XT as a media forsimple, fast, scalable, and reproducible viral purification.

Analytical Assays

Two analytical assays were performed on the pooled samples
from the dengue virus run on CHT XT. The results of the analyticalassays are shown in Table 5. Single-step dengue virus purification
on CHT XT led to significant clearance of protein (>93%) anddsDNA (>91%) contaminants.
Table 5. Results from the analytical assays on the pooled dengue samples
from the HA assay.
Log Reduction in dsDNA

Log Reduction in Total Protein

1.07

1.15

References
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.
51,074–51,105.

Viral Purity

SDS-PAGE analysis of the pooled active retention fractions fromFigure 4 showed that the pool contains proteins that migrate at
the expected position for dengue E and C proteins as shown on
the immunoblot (Figure 5). Note that a contaminant can be seen
migrating immediately above the E protein on the SDS-PAGE gel.

Kurosawa Y et al. (2012). Purification of dengue virus particles by one-step ceramichydroxyapatite chromatography. World J Vaccines 2, 155.
Kurosawa Y et al. (2014). Mammalian virus purification using ceramic hydroxyapatite.
Bio-Rad Bulletin 6549.
Moritz A (2005). Virus safety evaluation of biotechnology products in development.
BioProcess Int Suppl. 3, 15–16.

A
123456Visit bio-rad.com/CHTXTVirusPurification for more informationand to request samples.

kD2001169766

E protein
NS145312114B
1 23467kD
2001169766
45

E protein
NS1312114C protein

Fig. 5. SDS-PAGE (A) and immunoblotting analysis (B) of fractions. Protein bandson the gels were visualized by silver staining and the viral proteins in active samples
were detected by antiserum against dengue virus type 2. Lane 1: 3 μl protein MWmarker; lane 2: 5 μl dengue positive control; lane 3: cell culture fluid; lane 4: 10 μlflow‑through fractions; lane 5: 5 μl UV-rich fractions; lane 6: 10 μl pooled fractions
from HA assay.

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5

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Single-Step Influenza and Dengue Virus Purification with Mixed-Mode CHT Ceramic Hydroxyapatite XT MediaBIO-RAD and CHT are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions.
All trademarks used herein are the property of their respective owner. © 2024 Bio-Rad Laboratories, Inc.

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