Virus Purification

Quick Purification Strategy

H1N1 Purification

Efficient virus recovery and impurity removal are challenges in the purification of viruses suchas influenza A virus subtype H1N1. As a capture chromatography medium, Nuvia HP-Q Resin
resulted in high virus recovery and excellent impurity removal in a single purification step.
Moreover, Nuvia HP-Q Resin does not require benzonase treatment or the intensive filtration
step of H1N1 culture harvest before loading. Here, we summarize H1N1 purification onNuvia HP-Q Resin.

ProcedureH1N1 Purification on Nuvia HP-Q Resin

Optimized Protocol for Nuvia HP-Q Resin

Format: Foresight Nuvia HP-Q 1 ml Column, 5.83 x 1011 viral RNA (vRNA) copies/ml resin

Supernatant

Microfiltration (0.22 mm)

AEX using Nuvia HP-QResin

500
450
400
350
300
250200150
100500

1.5 M NaCI

250
200
150
10050240

245

250
255
Volume, mlA 230
A 254
Conductivity
vRNA recovery, %HA recovery, %260

Conductivity, mS/cm

Absorbance, mAU

Adherent Vero cell590 mM NaCI0StepMobile PhasepHCVEquilibration

20 mM Tris, 100 mM NaCI

7.55Sample loading 20 mM Tris, 100 mM NaCI

7.5–Wash

20 mM Tris, 100 mM NaCI

7.5

10–30

Elution

20 mM Tris, 590 mM NaCI

7.5

10High salt strip

20 mM Tris, 1.5 M NaCI

7.5

5

Clean in place

1 M NaOH>1210Remark
5.83 x1011 viralRNA
copies/mlof resin* Operational linear flow rate: 84 cm/hr.

HA, hemagglutinin.

Results

CI
M

059l tu

viaCu

1,5Nu

00

remsu
pernatHP
an-Qtflomw
MthNa
ro1,ugCI50helu
0mat
MeNaCIelu
atee
atelu

eatelu
CIflo

Na
M
m

059

Natou
wthranatrnpesu
reviaNul tu

HP-Qeelu
CINa00m

MCut

atan
atelu

rn

CIpe

Nasu

M
1,5rem

l tu

059

Cu

kD
250

ategh

SDS-PAGE and Western Blot Analyses

10075
5037HA
HA1NA2520

HA2

M/NP

Average size: 128 ± 4.18 nm
Reported size range: 80–180 nm
Purified H1N1 virus was highly monodispersed with 99.8%
monomer with a diameter of 128 nm.

15SDS-PAGE

Anti-HA WesternVisit bio-rad.com/NuviaHP-Q for more information.

Anti-Influenza A WesternData generated by Bioprocessing Technology Institute (BTI) Singapore.

Quick Purification Strategy: H1N1 Purification

Results
Purification Step

HA Recovery

HCP/HA, mg/mg

Host cell DNA/HA, ng/mg

Culture supernatant

100.0

13.9

442.5

Microfiltration

100.0

13.0

221.3

Nuvia HP-Q eluate

80.0

1.10.3

–

≤6.7

≤0.67

EP guideline (for 15 HA dose)**e ma.europa.eu/en/documents/scientific-guideline/guideline-influenza-vaccines-quality-module-revision-1_en.pdf
HA, hemagglutinin; HCP, host cell protein.

Highlights
Benzonase treatment not required in this workflow
High binding capacity■ Effective impurity removal■■

Reported Purification Schemes

HA Recovery, %HCP/HA, mg/mg

hcDNA/HA, ng/mg

Reference, DOI

53.0

7.7

34.5

10.1002/bit.21109

AEC, hydrophobic interaction chromatography

92.2

17.2

2.0

10.1016/j.jchromb.2019.03.037

AEC monolith

89.0NANA

10.1016/j.jchromb.2017.06.086

Pseudo-affinity (saturated membrane)

94.2

2.1

23.0

10.1002/bit.22345

Pseudo-affinity (saturated membrane)

64.0

13

3.8

10.1002/jctb.5474

Diafiltration/microfiltration/ultrafiltration size exclusion
chromatography, anion exchange chromatography (AEC)

Visit bio-rad.com/NuviaHP-Q to get technical details about the resin used in this workflow.
Visit bio-rad.com/ProcessApplications to see other applications in which these resins can be used.
Visit bio-rad.com/processlibrary to speak with a scientist or request a customized purification seminar.
BIO-RAD is a trademark of Bio-Rad Laboratories, Inc. All trademarks used herein are the property of their respective owner.

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