MONOLITHIC SUPPORTS FOR THE DOWNSTREAM
PROCESSING OF INACTIVATED INFLUENZA VIRUS PARTICLES
M. Leskovec1, L. Urbas1, M. W. Wolff2,3, C. Ziemann2, A. Štrancar1, U. Reichl2,3, P. Kramberger1
1
BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia
2 Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, Magdeburg, Germany
3 Bioprocess Engineering, Otto-von-Guericke-University, Magdeburg, Germany
INTRODUCTION
Monolithic supports represent a new generation of chromatographic media. Due to
their large inner channel diameters and enhanced mass transfer characteristics,
methacrylate monoliths (CIM® monolithic columns) offer efficient and fast
separation of large biomolecules like pDNA, viruses and monoclonal antibodies.
High binding capacity for viral particles, good product recovery and resolution are
also benefits of monoliths. During loading of MDCK cell-derived H1N1 inactivated
influenza virus particles onto monolithic columns, increased back pressure is
sometimes observed. This is especially an issue if a large amount of virus needs to
be purified since the back pressure depends on the loading volume. The goal of this
work was to determine the factors contributing to this effect. We tried to prevent
the increased back pressure by treating virus harvests with different precolumn
UV signal (280nm)
Buffer B (%)
4500
120
4000
3000
80
2500
60
2000
1500
40
1000
1×1013
The capacity was approximately
virus
particles per ml, and did not depend on the
pretreatment.
Buffer B [%]
100
3500
A [mAU]
RESULTS
Virus material, filtered (cellulose acetat filter; pore
size 0.45 μm) or pre-treated with precolumn phase
(Lipid removal agent (LRATM), Amberlite® XAD 7HP
and epoxy precolumn) was loaded on a CIMac QA
column until the dynamic binding capacity (DBC)
for the virus was reached. DBC was calculated at
10%
of
the
breakthrough,
using
the
hemagglutination assay.
phases (LRA™ - Lipid removal agent, Amberlite® XAD 7HP, epoxy monolithic
column) and by filtering the virus material before loading it onto the column. To
compare different pre-treatment strategies of the virus material the dynamic
binding capacity of CIMac QA for virus was first determined, resulting in
approximately 1×1013 virus particles per ml. Than loadings of the pre-treated virus
material at 75% of the column capacity were performed and mass balances for the
virus, DNA and proteins were investigated. Another goal of this work was to find a
good regeneration strategy for the columns where increased back pressure
occurred. For this reason different regeneration procedures using lipase,
benzonase, 2-propanol and NaOH treatment were tested on the columns with
increased back pressure.
Differently pre-treated virus samples were
loaded on the column. Loadings corresponded to
approximately 75% of total column capacity.
Mass
balances
of
virus
particles
(hemagglutination assay), proteins (Bradford
Ultra method) and DNA (PicoGreen assay) were
evaluated.
20
500
0
0
0
10
20
30
40
50
60
t [min]
Sample pretreatment
filtration
Amberlite® XAD 7HP
LRATM
Epoxy
Virus capacity at 10%
breakthrough [HA
VP/ml]
1.59E+13
6.58E+12
1.68E+13
9.62E+12
Flowthrough (0.1 M NaCl)
0,8
Elution (2 M NaCl)
100
100
80
80
80
60
Filtration
Amberlite
LRA
Epoxy
40
20
0
60
Filtration
Amberlite
LRA
Epoxy
40
20
0
Virus Protein
DNA
content content content
(%)
(%)
(%)
60
Filtration
Amberlite
LRA
Epoxy
40
20
0
Virus Protein
DNA
content content content
(%)
(%)
(%)
Virus Protein
DNA
content content content
(%)
(%)
(%)
Back pressure increase during 75% DBC loadings of H1N1 inactivated influenza
virus particles on CIMac QA was monitored. Measurements before and after
loading, after elution and after regeneration with 1 M NaOH are depicted in the
chart below.
60
NaOH
2-propanol
Lipase
Benzonase
55
Pressure [bar]
Pressure [MPa]
1,0
Elution (0.6M NaCl)
100
After loading non-treated virus material on CIM QA 1 ml columns, four different
regeneration procedures were tested. Back pressure was monitored at 3 ml/min
flowrate before and after loadings, after cleaning with 20 ml of the selected
procedure and after 2 hour regeneration with 1 M NaOH.
1,2
Conditions:
• Column: CIMac QA
• Buffer A: 50 mM MES + 0.1 M NaCl, pH 6.7
• Buffer B: 50 mM MES + 2 M NaCl, pH 6.7
• Flow: 1 ml/min
• UV detection: 280 nm
0,6
0,4
before load
after load
after elution
after regeneration
50
45
40
35
0,2
before load
after load
after cleaning (20cV)
after 2h NaOH
CONCLUSIONS
• Dynamic binding capacity of CIMac QA column for H1N1 inactivated influenza
was approximately 1×1013 virus particles per ml (haemagglutination test) and
it was not dependent on sample pre-tratment strategy.
• Virus recovery on CIMac QA was in the range from 80 to 100%, where 73-90%
was eluted in fraction E1 (0.6 M NaCl) with very good purity (protein and DNA
concentrations were low).
30
Filtration
Amberlite
LRA
Epoxy
• The monolithic epoxy precolumn was the most appropriate pre-phase to avoid
increasing back pressure when loading large amounts of sample on the
monolithic column.
• The optimal regeneration procedure to decrease back pressure was sanitization
with 1 M NaOH.