Practical Guide: Selecting the Optimal Resins for
Mammalian Virus Process Purification and Clearance
Payal Khandelwal, PhD
Bio-Rad Laboratories, Inc., 6000 James Watson Drive, Hercules, CA 94547

Purification Solutions

Bulletin 6790

Your One-Stop Shop for Mammalian Virus Purification Resins

Over the past 35 years or so, biologics such as monoclonal antibodies (mAbs), recombinant proteins, and,more recently, biosimilars and biobetters have become powerful therapeutics and been shown to be effectivein treating various human diseases. However, as all biological products run the inherent risk of possibly carrying
and/or amplifying extrinsic viral material, their successful implementation for use in humans depends on theability to effectively remove any viral contaminants. Viruses can directly infect mammalian cells and cause
diseases such as AIDS, mumps, measles, herpes, hepatitis, meningitis, and shingles. Therefore, viral clearance
from biologics is critical to their performance. On the other hand, the purification and study of these diseasecausing viruses can aid in the development of preventive and curative therapeutics. One of the most common
process steps included in downstream virus purification and clearance schemes is the use of chromatography
resins. Resins, unlike other conventional techniques, retain viral infectivity along with the quality and quantityof the virus purified and hence are the purification agents of choice.
Bio-Rad has provided a progressive selection of chromatography resins for the process-scale purification ofviruses for over 50 years. Here, we provide a brief snapshot of the different apatite-based media that can beeffectively utilized for the process-scale purification of small to mid-sized viruses. Their use is simple, easily
scalable, and results in a concentrated preparation of highly active virus. For purification resins appropriate forlarge and highly complex viruses such as adenoviruses, refer to bulletin 6807 – Selecting the Optimal Resins
for Adenovirus Process Purification.
CHT ™ Ceramic Hydroxyapatite Media Can Be Used with Diverse Virus Types
CHT Ceramic Hydroxyapatite can be used for the chromatographic separation of enveloped and nonenveloped virusesof diverse sizes from different sources and different families, such as dengue, Japanese encephalitis, influenza, mousehepatitis, adenovirus, poliovirus, and feline calicivirus (Table 1).
Table 1. Wide variety of viruses that can be purified using CHT.
Virus

FamilyGenus

GenomeEnvelope

Size, nmDengueFlaviviridae

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

ssRNA–

90

Poliovirus

Picornaviridae

Enterovirus

ssRNA–30

Feline calicivirus

Caliciviridae

Vesivirus

ssRNA–30–38

Standard Virus Purification Protocol with CHT
The general workflow of virus process purification with CHT involves six steps as shown below with specific buffer requirements ateach step (Table 2). Chromatography was performed using Bio-Rad’s BioLogic DuoFlow™ System. Columns (4.6 x 35 mm,Sugiyama Shoji Co., Ltd., Japan) with a 10 µm frit were packed with 40 µm CHT Media. Crude extract was run on the columns.

Wash

Equilibration

Sample loading

Wash

ElutionWashPractical Guide: Selecting the Optimal Resins for Mammalian Virus Process Purification

10 mM sodium phosphate

7.210ElutionGradient elution from
10–600 mM sodium phosphate

7.215

Wash

600 mM sodium phosphate

7.25

Viral Activity Is Retained with CHT Chromatographic
Purification
Viruses purified with CHT were tested using multiple methods(Table 3) for retention of viral activity. The results show that CHT
is able to purify highly active virus. Purity analysis for protein
contaminants was carried out by UV absorbance at 280 nm
and SDS-PAGE analysis and host cell DNA contamination
was monitored using the Quant-iT PicoGreen dsDNA Assay Kit.
Table 3. Detection methods used to check viral activity after
CHT chromatography.
Detection Method

Virus

Hemagglutination (HA) test

Dengue, influenza, adenovirus

Plaque assay

Japanese encephalitis

50% tissue culture infective dose
(TCID50 )Poliovirus, feline calicivirus,
mouse hepatitis

Choosing the Right Apatite-Based Media
Bio-Rad offers two kinds of CHT Media, Type I and Type II, inmultiple sizes. Type I has higher protein binding capacity andbetter capacity for acidic proteins while Type II has a lower
protein binding capacity but offers better resolution of nucleic
acids and certain proteins. We also offer MPC™ Ceramic
Hydroxyfluoroapatite and CFT™ Ceramic Fluoroapatite, whichhave slightly different properties relative to CHT. Each of theseresins behaves differently with different viruses under different
conditions. Hence the choice of resin and the conditions usedhave to be optimized for the virus being purified.
For example, dengue virus type 2 was purified using all four
apatites. The yield with MPC was only 50% versus ~80%
yields from the other three apatites. Additionally, the best
separation of virus from impurities was seen with CHT Type IIMedia due to its larger pore size (Figure 1). The importance ofchoosing the appropriate media for a specific virus was also
seen when CHT Type II and CFT Type II Media were used inthe purification of poliovirus. CHT gave a recovery of 88% while
CFT recovered 102% (Figure 2) of the virus. For details about
these purifications, refer to bulletin 6549.

20000HA titer, HAU/ml

4000.04
051015 20 25 30
Elution volume, ml

35400B. CHT Type II500.16

1,000400.12

8003020106000.08

4000.04

20000HA titer, HAU/mlWash

10051015 20 25 30 35
Elution volume, ml

400C. CFT Type II50403020100.16

1,6000.12

1,2000.08

8000.04

40000HA titer, HAU/ml

100051015 20 25 30
Elution volume, ml

3540D. MPC
50403020100.16

1,0000.12

8000.08600
4000.04

20000HA titer, HAU/ml

7.2

6000.0805101520253035400

Elution volume, ml

Fig. 1. Comparison of four apatites in the purification of dengue virus type 2.
UV absorbance at 260 nm (—); absorbance at 280 nm (—); conductivity (--);
viral activity in HA test (—).
A. CHT Type II Media
403020100.06

1.2 x 107

0.05

1.0 x 107

0.04

8.0 x 10 60.03

6.0 x 10 60.02

4.0 x 10 60.01

2.0 x 10 600Infectivity, TCID50 /ml

Sample loading 10 mM sodium phosphate2000

5 10 15 20 25 30 35 40 45 50Elution volume, ml

B. CFT Type II Media
403020100

1.2 x 107

0.06
0.05

1.0 x 107

0.04

8.0 x 10 60.03

6.0 x 10 60.02

4.0 x 10 60.01

2.0 x 10 600

5 10 15 20 25 30 35 40 45 50
Elution volume, ml

Infectivity, TCID50 /ml

10Absorbance

7.2

Absorbance

10 mM sodium phosphate

80030Absorbance

Equilibration

1,0000.12

Absorbance

50.16

Absorbance

7.25040

Absorbance

600 mM sodium phosphate

Conductivity, mS/cm

Wash

Conductivity, mS/cm

Volume, ml

Conductivity, mS/cm

pHConductivity, mS/cm

Mobile Phase

Conductivity, mS/cm

StepConductivity, mS/cm

A. CHT Type I

Table 2. Standard virus process purification protocol.

0Fig. 2. Comparison of two apatites in the purification of poliovirus.
UV absorbance at 260 nm (—); absorbance at 280 nm (—); conductivity (--);
infectious activity in TCID50 (—).
© 2016 Bio-Rad Laboratories, Inc.

Bulletin 6790

Practical Guide: Selecting the Optimal Resins for Mammalian Virus Process Purification

Selecting the Best Conditions for Virus Purification
with Apatite-Based Media
Efficiency in viral purification in terms of time and quantity
depends on multiple factors including the flow rate and thesalt gradient slope. We show that decreasing the flow rate bytenfold (1.0 ml/min to 0.1 ml/min) improves the sharpness ofelution peaks and hence the separation of dengue virus type 2(Figure 3). For more details on the purification of the influenza,
mouse hepatitis, and Japanese encephalitis viruses in a similarmanner on various apatite media, refer to bulletin 6549.
A. 1.0 ml/min flow rate

2010

800

0.04
0.03

600

0.02

400

0.01

200

00051015 20 25 30
Elution volume, ml

3540

HA titer, HAU/ml

30

Absorbance

Conductivity, mS/cm0.0540

References
Dove GB et al. (1990). Purification alternatives for IgM (human) monoclonal
antibodies. In ACS Symposium Series 427, M.R. Ladisch et al., eds.
(Washington, DC: American Chemical Society), pp. 194–209.
Grun JB et al. (1992). Viral removal/inactivation by purification ofbiopharmaceuticals. Biopharm 5, 22–30.
Snyder MA et al. (2009). PEG enhances viral clearance on ceramichydroxyapatite. J Sep Sci 32, 4048–4051.

1,0000.06

50

The details provided here can help you design a process
purification strategy for your virus of choice. For technical/product support or to request a quote, email your regional
Bio-Rad representative at process@bio-rad.com or contact
our customer service at 1-800-4-BIORAD (1-800-424-6723).

0Explore our extensive selection of process-scale chromatography
resins and their performance characteristics and applications(bulletin 6713). For process optimization of your virus purification,
request a sample.

B. 0.1 ml/min flow rate
0.06

302010

800

0.04
0.03

6000.02

4000.01

2000HA titer, HAU/ml

40

1,0000.05
Absorbance

Conductivity, mS/cm

5000051015 20 25 30Elution volume, ml

3540Fig. 3. Separation of dengue virus type 2 at various flow rates.
UV absorbance at 260 nm (—); absorbance at 280 nm (—); conductivity (--);
viral activity in HA test (—).

Hydroxyapatite media also support viral clearanceof 0.6–3.5 logs (Dove et al. 1990, Grun et al. 1992). At
times, they have also been shown to outperform size
exclusion chromatography by a factor of 15, ion exchange
chromatography by a factor of 75, and precipitation bya factor >150 in viral clearance (Dove et al. 1990). CHT,
specifically, has been shown to clear up to 4 logs ofxenotropic murine leukemia virus (x-MuLV) and 1–2 logsof minute virus of mice (MVM) from an antibody sample(Snyder et al. 2009). Therefore, they can be trusted as
effective media for the purification and clearance of small
to mid-sized mammalian viruses. As mentioned earlier,adenoviruses warrant the use of other mixed-mode andion exchange resins such as Nuvia™ cPrime™, Nuvia™ S(cation exchange, CEX), Nuvia™ Q (anion exchange, AEX),
UNOsphere™ S (CEX), and UNOsphere™ Q (AEX). The use of
these resins for adenovirus purification is discussed in depthin bulletin 6807.

© 2016 Bio-Rad Laboratories, Inc.

Bulletin 6790

Practical Guide: Selecting the Optimal Resins for Mammalian Virus Process Purification

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