Mixed-Mode Chromatography

Virus Reduction and Impurity Removal Potentialof Nuvia aPrime 4A Resin
William H. Rushton,1 Xuemei He,1 Irene Chen,1 Sharon Bola,1 Akunna Iheanacho,2 Coral Fulton,2
Jenifer Dean,2 William Kerns,2 Kaye Peden,2 Katelyn Pritchard,2 Erin Reynolds,2 and Sreyry Schaerdel2
1
Bio-Rad Laboratories, Inc., 6000 James Watson Drive, Hercules, CA 94547
2
Texcell North America, Inc., 4991 New Design Road, Frederick, MD 21703

Abstract

We utilized a design of experiments (DOE) approach to investigate the effect of buffer pH andconductivity on the clearance of the minute virus of mice (MVM) as well as the removal of other productor process-related impurities by the mixed-mode chromatography Nuvia aPrime 4A Resin. Results fromthis study have offered insights on the interactions between the resin and MVM particles, as well as
the design space for the removal of this virus. Although there is no single solution for every product orpurification process, practical considerations for developing a mixed-mode chromatography processwith effective impurity clearance will be discussed in this application note.
Introduction
Incorporating Viral Safety During Downstream Processing

For all biotechnology products derived from cell lines of humanor animal origin, there is a risk of viral contamination from thesource material or from adventitious agents during the production
process. Adventitious agents, including viruses, can enter a
production process from different routes. Various strategies
have been employed to reduce the risk of contamination during
the purification process. Virus reduction, or viral clearance, involves
the removal or inactivation of viral contaminants and is one of
the strategies employed to enhance product safety.
The main objectives of viral clearance studies are to evaluate
process steps that can be considered effective in removing
or inactivating viruses and to quantitatively estimate theoverall level of virus reduction obtained by the manufacturing
process. Regulatory authorities require that manufacturersof biotherapeutics perform a risk assessment and evaluateorthogonal process steps dedicated to virus inactivation and/or
virus removal.

The downstream processing of a monoclonal antibody (mAb) relies
heavily on chromatographic separation. A variety of ion-exchange,
affinity, size exclusion, hydrophobic interaction, and mixed-mode
chromatography resins are available from many commercial
vendors and are designed to remove product- and process-related
impurities. Product-related impurities for mAbs include aggregatesor fragments, charge variants, and posttranslational modification
variants. Process-related impurities include host cell proteins (HCPs),
nucleic acids from host cells (hcDNA/RNA), endotoxins, viruses,
and if affinity capture is used, fragments of recombinant Protein
A. Because obtaining adequate viral clearance while expediting
process development is still a concern among process engineersand developers, Bio-Rad Laboratories, Inc., in collaboration with
Texcell North America, Inc., evaluated the virus reduction potential ofthe mixed-mode chromatography Nuvia aPrime 4A Resin.

H 3C

O
N+

H 3C

Fig. 1. Mixed-mode ligand for Nuvia aPrime 4A Resin.

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Virus Reduction and Impurity Removal Potential of Nuvia aPrime 4A Resin

Experimental Approach
A DOE approach was taken for a basic mAb (pI 9.0) to investigate
the effect of buffer pH, NaCl concentration, residence time, andfeedstream load level to remove impurities, such as MVM, HCPs,
dsDNA, endotoxin, and mAb aaggregates, using Nuvia aPrime
4A Resin. Initial experiments were performed with Mini Bio-Spin
Columns, each containing 100 μl of Nuvia aPrime 4A Resin, to
optimize process conditions. A two-level full factorial screening
design suggested by JMP Software was employed. A total of
18 different conditions with buffers in the pH range of 6.0–8.0
and NaCl concentrations up to 150 mM were evaluated (Table 2).
Before sample application, spin columns containing resin were
equilibrated with the appropriate buffer. Each feedstream was spiked
with MVM and applied to the spin column. The load level of thefeedstream ranged from 30–70 mg/ml and was incubated with resin
to mimic a residence time of either 3 or 7 minutes. At the end of the
incubation period, the flowthrough was collected, and samples wereassayed for virus titer using infectivity assays. The results are shownin the contour plot from the DOE studies (Figure 2).

Characteristics of Nuvia aPrime 4A Resin

Nuvia aPrime 4A is a hydrophobic anion exchange resin. The resin
is designed with a positively charged hydrophobic functional ligand(Figure 1). This functional ligand can interact with biomolecules
through electrostatic and hydrophobic interactions.
The resin particles have open pores to allow efficient mass transfer
and are mechanically strong. These features make Nuvia aPrime 4A
Resin suitable for downstream processing, as the purification step
can be operated at fast flow rates. All technical properties of thisresin are listed in Table 1.
Table 1. Properties of Nuvia aPrime 4A Resin.
Property

Description

Functional group

Aromatic hydrophobic anion exchanger

Base matrix composition

Macroporous highly crosslinked polymer

Median particle size

50 ± 10 µm

Ligand density

100 ± 20 µeq/ml

Dynamic binding capacity(DBC)*≥50 mg/ml at 300 cm/hr

Recommended linear
flow rate

50–300 cm/hr

Pressure-flow performance**

Under 3 bar at flow rateof 300 cm/hr

Compression factor

1.1–1.25

Experiment
NumberpH stability***

Short-term: 2–14

Shipping solution

20% ethanol + 1 M NaCl

Regeneration

1 M NaOH

Sanitization

1 M NaOH

Storage conditions

20% ethanol

Storage temperature
Chemical stability†

Shelf life††

5 years*	10% breakthrough capacity determined with 1.0 mg/ml of an acidic mAb(pI ~6.9) in 20 mM NaPO4, pH 7.8.
** 20 x 20 cm packed bed (1.25 compression factor).
***	No significant change in ligand density after 200 hr contact at 22ºC.
†	No significant change in ligand density after 1 week contact at 22ºC.
†† Stored at room temperature in 20% ethanol + 1 M NaCl.

Table 2. DOE setup for flow-through mode.
Buffer pH

Buffer [NaCl],mM

Residence Time,
min

1

6

150

3

70

2

8

150

7

30

3

6

150

7

70

4

6

0

7

30

5

8

0

7

70

Room temperature

6

8

0

3

30

1 M NaOH, 1 M HCl, 25% acetic acid, 8 M urea,
6 M guanidine HCl, 3 M NaCl, 1% Triton X-100,
20–70% ethanol, 30% isopropanol

7

7

75

5

50

8

6

150

3

30

9

6

0

3

30

10

8

0

3

70

11

8

150

7

70

12

8

150

3

70

13

6

0

3

70

14

8

150

3

30

15

6

0

7

70

16

8

0

7

30

17

7

75

5

50

18

6

150

7

30

Nuvia aPrime 4A chromatography can be operated in both bindand elute mode and flow-through mode. Basic proteins that have ahigh pI would carry a positive charge at neutral pH, which will cause
repulsion with the resin/ligand, and these proteins will be present
in the flow-through fractions. Conversely, acidic species will havea negative charge at neutral pH and will bind to the ligand. Target
proteins with a low pI, HCPs, hcDNA/RNA, or endotoxins will bindto the resin through hydrophobic and electrostatic interactions.
Depending upon the pI and surface hydrophobicity of the target
molecule, buffer pH and conductivity can be manipulated to achieve
the desired protein selectivity and recovery.

© 2021 Bio-Rad Laboratories, Inc.

2

ColumnLoad, mg/ml

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Virus Reduction and Impurity Removal Potential of Nuvia aPrime 4A Resin

Fig. 2. Contour plot from DOE studies. MVM LRV, minute virus of micelog reduction value.

To confirm the results of the spin column DOE studies, follow-up
experiments were performed with packed Foresight Columns(inner diameter: 0.8 × 10 cm, 5 ml). pH and NaCl concentration[NaCl] (or conductivity) were evaluated. Each buffer condition
evaluated is listed in Table 3. Column loads were spiked witheither MVM (experiments 1–4) or xenotropic murine leukemia virus(XMuLV) (experiment 5). MVM is a nonenveloped, single-stranded
DNA parvovirus that ranges in size from 18 to 26 nm. XMuLV is ahighly charged, enveloped, single-stranded RNA retrovirus that
ranges in size from 80 to 120 nm.

MMV LRV

5.5

Buffer Condition

Spiking Virus
MVM

1

10 mM sodium
phosphate, 150 mM
NaCl, pH 8

MVM

2

10 mM sodium
phosphate, 200 mM
NaCl, pH 8

3

10 mM Bis-Tris, 150 mM
NaCl, pH 8

MVM

4

10 mM sodium
phosphate, pH 6

MVM
XMuLV

5

10 mM sodium
phosphate, 150 mM
NaCl, pH 8

2.5

150
Buffer [NaCl],mM

5
Residence
time, min

70

50

60

40

7
30

6

6.0

8
Buffer pH

70
Protein load
level, mg/ml

Fig. 3. Impact of pH, NaCl concentration, residence time, and load ratio on
MVM clearance. MVM LRV, minute virus of mice log reduction value.

To verify the results of the DOE, further studies were executedusing packed scale-up columns. A summary of the viral clearance
results for column loads spiked with MVM is included in Table 4.
In brief, all buffer conditions resulted in greater than 4.2 logs ofclearance with the exception of one buffer condition. This buffercondition (10 mM sodium phosphate, pH 6.0) achieved only
2.23 logs of clearance and was included as a negative control
to demonstrate confirmation of a predicted trend. A summary of
the viral clearance results for the column load spiked with XMuLV
is included in Table 5. For XMuLV, only one buffer condition wastested (10 mM sodium phosphate, 150 mM NaCl, pH 8.0) andgreater than 5.07 logs of clearance was achieved. The results of
the runs with Foresight Columns are in good agreement with thespin column DOE predictions. The mixed-mode chromatography
step with Nuvia aPrime 4A Resin was effective in removing both
MVM and XMuLV.

Test Article

mAbX

mAbX, monoclonal antibody X; MVM, minute virus of mice; XMuLV, xenotropic
murine leukemia virus.

Buffer pH and NaCl concentration also effect the elimination ofother impurities during Nuvia aPrime 4A chromatography. Running aflow-through purification step at higher pH and NaCl concentrations
may enhance the clearance of dsDNA and mAb aggregates whilecausing a minor compromise in endotoxin removal (Figure 4). These
results demonstrate the overall impurity clearance efficiency of Nuvia
aPrime 4A Resin as an intermediate polishing step for purificationof a mAb. Investigational viral clearance studies can assist in
defining the design space of a process step that targets productrecovery and impurity removal.

Before sample application, Foresight Columns were equilibrated withthe appropriate buffer. A residence time of 5 min was utilized witha load ratio of 50–55 mg/ml of resin. Each run was performed inflow-through mode and all chromatography was performed using an
AKTA Pure Fast Protein Liquid Chromatography (FPLC) System.

© 2021 Bio-Rad Laboratories, Inc.

4.0

1.0

Table 3. Experimental conditions: scale-up studies with Bio-Rad
Foresight Columns.
Experiment
Number

4.0
3.0
5.0

5

8.0

4

7.5

150
3

7.0
Buffer pH

100

6.5

50

6.0

8.0
0

0

7.0

50

7.5

100

Results and Discussion
A DOE approach was used to establish effective clearance
conditions for MVM and product- and process-related impurity
clearance with the mixed-mode chromatography resin, Nuvia
aPrime 4A Resin. As MVM contains capsid proteins with a pI of
6.1–6.2, there is a net negative charge on the surface of the protein
at near neutral pH (6–8). The contour plot illustrates that the upper
right quadrant with high pH and high NaCl concentration leads togreater MVM log reduction value (LRV). These results demonstratethat MVM is more effectively retained by Nuvia aPrime 4A Resinat higher pH and NaCl concentrations. Buffer pH and conductivity
have the potential to impact electrostatic as well as hydrophobic
interactions. At pH 8, the virus particles interact with thequaternary amines of the Nuvia aPrime 4A ligand electrostatically.
The hydrophobic interaction is enhanced by the higher NaCl
concentration in the buffer. Residence time did not seem to havemuch effect on MVM removal while the load ratio did show a slight
positive effect on MVM clearance (Figure 3).

MVM LRV
<1.5
<1.6
<1.7
<1.8
<1.9
<2.0
<2.1
<2.2
<2.3
<2.4
<2.5
<2.6
<2.7
<2.8
<2.9
<3.0
≥3.0

6.5

Buffer [NaCl], mM

150

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Virus Reduction and Impurity Removal Potential of Nuvia aPrime 4A Resin

Conclusions
Regulatory authorities require viral clearance evaluations for
manufacturing unit operations. Therefore, the virus removal or virus
inactivation capacity of these operations should be determined
during process development.

Table 4. Removal of MVM by mixed-mode chromatography.
Calculations for log Reduction Value
Mixed-Mode
Chromatography(Nuvia aPrime
4A Resin)Experiment 1 Experiment 2 Experiment 3 Experiment 4

log10 total virusof hold control

7.25

7.53

7.67

7.43

Minus the log10
total virusof product

<3.04

<3.00

<2.97

5.20

log reduction
value

>4.21

>4.53

>4.70

2.23

Studies using a DOE approach were performed to establish
effective viral clearance conditions for a mAb using Nuvia aPrime
4A Resin. The viral clearance studies, as well as the overall impurity
clearance achieved, demonstrate the efficiency of Nuvia aPrime 4A
Resin as an intermediate polishing step.
Bibliography
Viral assay procedures
Blair GE (1985). Virology: A Practical Approach, Mahy BWJ, ed. (Oxford: IRL Press, Ltd.).

Table 5. Removal of XMuLV by mixed-mode chromatography.
Calculations for log Reduction Value
Mixed-Mode Chromatography(Nuvia aPrime 4A Resin)

Experiment 5
7.22

Minus the log10 total virus of product<2.15

log reduction value>5.07

European guidance for design of viral clearance studies
Committee for Proprietary Medicinal Products (1996). The European Agency for the
Evaluation of Medicinal Products. Note for Guidance on Virus Validation Studies:
The Design, Contribution and Interpretation of Studies Validating the Inactivation
and Removal of Viruses. CPMP/BWP/268/95. https://www.ema.europa.eu/en/virusvalidation-studies-design-contribution-interpretation-studies-validating-inactivationremoval, accessed August 31, 2021.

60
50
45
40
30
25
20
12
10
6.1
8
6
4.9
4
7.3
2
0
250
115.8 200
105.4 150
126.3 100
50
10
39.2
36.9
41.5

4.8
4.4
5.2

European guidance for virus safety testing and viral clearanceCommittee for Proprietary Medicinal Products (2008). European Medicines
Agency. Guideline on Virus Safety Evaluation of Biotechnological Investigational
Medicinal Products. EMEA/CHMP/BWP/398498/2005. https://www.ema.europa.
eu/en/virus-safety-evaluation-biotechnological-investigational-medicinal-products,
accessed August 31, 2021.
Calculation of virus titer based on plaque assay data
Darling AJ (2000). Design and interpretation of viral clearance studies forbiopharmaceutical products. Methods in Biotechnology 9, 195–209.
Virus safety of plasma-derived medicinal products
European Medicines Agency (2011). Guideline on Plasma-Derived Medicinal
Products. EMA/CHMP/BWP/706271/2010. https://www.ema.europa.eu/en/plasmaderived-medicinal-products, accessed August 31, 2021

7
4

1
1.0
0.75
0.47 0.5
0.25
075
Buffer[NaCl], mM

Statistical analysis of viral assays for viral clearance
Li N and Yang H (2012). Statistical evaluations of viral clearance studies for biologicalproducts. Biologicals 40, 439–444.

70
0
0.25
0.5
0.75
1.0

50

60

6

5
Residence
time, min

7
30
40

5

150
3
4

50

100

7.5

7
Buffer pH

8.0
0

6.0

TCID50 assay calculation by Spearman-Karber method
Hubert JJ (1984). Spearman-Karber method. In Bioassay, 2nd edition (Dubuque,Iowa: Hunt Publishing Company), 65–66.
6.5
7.0

Aggregatecontent, %Endotoxin,
EU/mg

dsDNA,
ppm

HCP, ppm

log10 total virus of hold control

Desirability

Guidance for testing worst-case conditions for virus inactivation steps
Chinniah S et al. (2016). Characterization of operating parameters for XMuLVinactivation by low pH treatment. Biotechnol Prog 32, 89–97.

50
Protein load
level, mg/ml

Desirability

Retrospective evaluation of viral inactivation and filtration data frommultiple collaborators
Mattila J et al. (2016). Retrospective evaluation of low-pH viral inactivation and viral
filtration data from a multiple company collaboration. PDA J Pharm Sci Technol 70,
293–299.

Fig. 4. Effects of buffer composition and operational parameters on impurity
removal. HCP, host cell protein.

Validation of analytical methods
United States Pharmacopeia (1995). Validation of Compendial Methods. USP 23,
item 1225, 1,982–1,994. Rockville, MD: United States Pharmacopeial Convention, Inc.
Guidance for viral clearance
United States Pharmacopeia (2016). Design, Evaluation, and Characterization of Viral
Clearance Procedures. USP 39-NF 34, item 1059.1. Rockville, MD: United States
Pharmacopeial Convention, Inc.
Q5A U.S. guidance for virus safety testing and viral clearance
U.S. Department of Health and Human Services, Food and Drug Administration,
Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation
and Research (CBER) (1998). Guidance for Industry: Q5A Viral Safety Evaluation of
Biotechnology Products Derived from Cell Lines of Human or Animal Origin. https://www.fda.gov/media/71394/download, accessed August 31, 2021.

© 2021 Bio-Rad Laboratories, Inc.

4

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Virus Reduction and Impurity Removal Potential of Nuvia aPrime 4A Resin

21 CFR Part 58, GLP
U.S. Food and Drug Administration, Department of Health and Human Services(2011). Good Laboratory Practice for Nonclinical Laboratory Studies. Title 21 Code ofFederal Register Part 58. govinfo.gov/app/details/CFR-2011-title21-vol1/CFR-2011title21-vol1-part58/summary, accessed August 31, 2021.
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World Health Organization (2004). WHO Guidelines on Viral Inactivation and Removal
Procedures Intended to Assure the Viral Safety of Human Blood Plasma Products.
WHO Technical Report, Series No. 924. who.int/publications/m/item/WHO-TRS924Annex4, accessed August 31, 2021.

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BIO-RAD and BIO-SPIN are trademarks of Bio-Rad Laboratories, Inc. in certain
jurisdictions. All trademarks used herein are the property of their respective owner.
Nuvia aPrime 4A Resin is covered by U.S. Patent Number 9,669,402 and foreigncounterparts.

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