Benzonase® endonuclease is a powerful tool for degrading all forms of (deoxy)ribonucleic acids (DNA/RNA) in cell culture harvests to base pair (BP) lengths under 8 units. It is effective over a wide range of conditions including temperature, pH, and varying concentrations of Mg2+, detergents, chelating agents, and monovalent cations. It is often employed in the production of viral vaccines, completely digesting DNA and RNA to improve clearance and reduce solution viscosity. Removing Benzonase® endonuclease, a 60 kD dimer, from the process stream post-treatment may be achieved with Tangential Flow Filtration (TFF) with the appropriate molecular weight cut-off membrane (MWCO), typically 300 kD. This process has heretofore not been well described in the literature, so the present study fills this gap pairing Benzonase® endonuclease treatment of a DNA-spiked inactivated flu virus cell culture harvest with Pellicon® 2 cassettes, for clearance of the digested DNA and remaining Benzonase® endonuclease by diafiltration.

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Benzonase, Biomax, Pellicon and Ultracel are registered trademarks of Merck KGaA, Darmstadt,
Germany. All other marks are the property of their respective owners.
© 2014 EMD Millipore Corporation, Billerica, MA, USA. All rights reserved.
Benzonase®
endonuclease: use and clearance with
a TFF step in a cell culture-based vaccine process
2.2 Diafiltration
UF/DF device
Pellicon®
2 cassette 0.1 m2
V screen with Biomax®
300 kDa
membrane (cat# P2B300V01, lot # C1NA15687)
Buffer solutions
Tris EDTA and EDTA buffer
Test parameters
• Diafiltration: 30 LMH
• Feed flow rate: 5 L/min/m²
• TMP: 0.15 psig
• Number of diavolumes: N = 8
• Time: 80 min
2.3 ELISA analysis for Benzonase®
endonuclease
DF samples and standards were added to an ELISA plate and
incubated for 2 hours (capture), washed with buffer, incubated
for 1 hour with Rab-POD (linkage), washed, developed with TMB
for 15 minutes, then stopped with 0.2 N sulfuric acid. The ELISA
was read at 450 nm on a plate reader.
3. Results
3.1 The digestion of DNA by Benzonase®
endonuclease
Optimization study
During the diafiltration experiment, samples were withdrawn
from the recirculation vessel and analyzed by ELISA at 450 nm.
The A450 data was transformed into concentrations via a
standard curve. The starting concentration was 36 ng/ml, and it
underwent a 3 log reduction over the course of an 8 diavolume
exchange. The concentration is plotted in the accompanying
graph as % remaining (C/Co) vs. diavolumes and against
theoretical sieving values expected for a 60 kD protein and a
300 kD membrane. The experimental data follows the expected
trend, near a sieving value of 1 (complete passage).
A mass balance on the system was calculated at 5 diavolumes
(table below). The recovery in the permeate was 48.9%; including
the retentate yielded 49.5%. This result suggests that adsorptive
loss of Benzonase®
endonuclease to the membrane and system
could be as high as 50%.
Diafiltration plot - % remaining in retentate
• Shows clearance of Benzonase®
endonuclease versus
theoretical sieving levels
• Sieving is around 1, taking assay precision of +/- 15% into account
• 3 log reduction in Benzonase®
endonuclease by 8 DV
• LOD achieved by 8 DV (spiking to 100 U/ml enriched DF
experiment)
Adam Sokolnicki, Dana Kinzlmaier - EMD Millipore and Frédéric Sengler - Merck Millipore
Conclusion
This experiment was to examine the behavior of
Benzonase®
endonuclease during diafiltration with
Pellicon®
2 cassettes with Biomax®
300 kD membrane.
The data demonstrated 99.5% clearance at 5 diavolumes
and > 99.9% clearance after 8 diavolumes. The overall
diafiltration profile closely follows a theoretical sieving
value of 1, with some variability likely due to the ELISA
assay. Adsorption of Benzonase®
endonuclease onto
the membrane or system components was implicated
by a permeate recovery below 50%. Membrane
adsorption could be minimized through the use of a
300 kD Ultracel®
PLCMK membrane. For the clearance
of Benzonase®
endonuclease after DNA digestion of
cell cultures producing viral vaccines, 300 kD is an
acceptable MWCO.
As determined by agarose gel electrophoresis, the 10 U/ml
dose of Benzonase®
endonuclease digests any DNA from
a high BP number streak to a small BP number band.
The small DNA strands pass completely through the
300 kD membrane and into the permeate.
x-axis: base pair length (50 – 17,000 bp)
y-axis: relative intensity of signal
Reference peaks at 50 and 17,000 bp bracket sample
Control shows range of DNA present
10 U/ml digestion shows degradation of DNA over time until only small BP number remain
Flu vaccine culture supernatant
Merck Millipore is a division of
Viresolve, Millistak+, Pellicon, Millipore Express and ProSep are registered trademarks
of Millipore Corporation. Fractogel and Eshmuno are registered trademarks of Merck
KGaA, Darmstadt, Germany. The Merck Millipore logo is a trademark of Merck KGaA,
Darmstadt, Germany. © 2011 Merck Millipore
Virus Removal by Tentacle Ion Exchange
Media Eshmuno® Q and Eshmuno® S
INTRODUCTION
The risk of viral contamination is a feature common to all
biological compounds derived from animal or human origin.
Besides chemical inactivation and specific filtration units
to reduce the risk of viral contaminations, in many process
validation chromatographic steps are considered to contribute to
the overall virus reduction.
In contrast to the well known virus clearance feature of anion
exchangers there is an increasing interest in the virus removal
capacity of cation exchange materials. Here, the new tentacle
resin Eshmuno S has been evaluated for its capabilities to
remove viruses.
Additionally, the anion exchange material Fractogel was re-
evaluated with mAb containing feed stream and compared to
the new material Eshmuno Q. Here, the binding and nonbinding
mode, as well as fresh and used gels were investigated using a
scaled down chromatography unit.
Appropriate model viruses as MuLV, an enveloped RNA retrovirus,
PRV, an enveloped DNA herpesvirus, and PPV, a non-enveloped
small DNA parvovirus were selected according to ICH Topic 5A.
These viruses vary in their biophysical and structural features and
they display a variation in resistance to physical and chemical
agents or treatments.
Experimental setup
Equipment: Äkta basic 100 and/or Äkta purifier 100,
Unicorn Software 5.11
Column : 100*10mmID, 7.85 mL
Fresh material - maximum 10 cycles
Used material – more than 100 cycles
Linear Flow rate: 300 cm/h or 150 cm/h
Cation exchange Chromatography - Eshmuno S - binding mode
Buffer A: 30mM Na-acetate, pH 5.0 (± 0.02)
Buffer B: 30mM Na-acetate, 1M NaCl, pH 5.0 (± 0.02) .
The conductivity was 2.5 mS/cm. The buffer was filtrated through
a 0.2 µm filter.
Load: 68 mL of 5 mg/mL IgG (MW: approx. 150 kDa; pI Mab: 7.5 – 8.6)
in 30mM Na-acetate, pH 5.0
Virus Spike: 1 % v/v spike ratio
Anion exchange chromatography - Eshmuno Q - non binding mode
Buffer A: 40 mM Tris/HCl, pH 7.5 (± 0.02)
Buffer B: 40 mM Tris/HCl ,1 M NaCl, pH 7.5 (± 0.02)
The conductivity was 2.9 mS/cm. The buffer was filtrated through
a 0.2 µm filter.
Load: 91 mL of 5 mg/mL IgG (MW: approx. 150 kDa; pI Mab: 7.5 – 8.6)
in 40 mM Tris/HCl, pH 7.5
Packing of the columns, sanitisation, leakage testing as well as HETP
determination was performed before release of the column. All the
experimental work was performed in a GLP environment.
Common mAb purification scheme
Bettina Katterle2, Ellen Reuter2, Andreas Stein1 and Lothar R. Jacob1
1 Merck KGaA, Frankfurter Str. 62, 64293 Darmstadt
2 Charles River Biopharmaceutical Services, Gottfried-Hagen-Str. 20, 51105 Köln
Table 1: Different effective methods to reduce viral contaminations in the
processing of blood plasma derived and recombinant products
Method Mode Example Remark
Chromatography Adsorption
Fractogel® TMAE
Eshmuno® Q
Eshmuno® S
5-6 log10, Non-robust
Filtration Size exclusion Viresolve® Pro device > 4 log10, robust
Solvent/detergent Chemical inactivation Tributyl Phosphate Robust
Physical treatment Inactivation Heat, UV irradiation Robust
CONCLUSIONS
• The effectiveness of virus removal by ion exchange media was
evaluated. For this purpose cell culture supernatant taken from a
production process was spiked with the viruses, MuLV and PRV.
Experiments have been performed at defined virus titer and run on
an scaled down model of the chromatographic column.
• The use of fresh and close to the end of life time chromatographic
material did not affect the viral clearance capabilities.
• The cation exchange material Eshmuno S run at pH 5.0 in the
binding mode, showed a good viral clearance. In contrast to pH
5.5 (data not shown), the removal capacity at pH 5.0 was detected
to be at least 2 log10 for MuLV and 4 log10 for PRV. Hence, cation
exchange steps contribute to the overall removal capacity of an
overall manufacturing process. However, the parameters as pH and
salt concentration must be well understood with respect to process
safety.
• Anion exchange chromatography operated in the non-binding and
binding mode is capable of effectively removing potential viral
contaminants and can be considered as a rather robust step.
• The tentacle resins Eshmuno Q and Fractogel exhibited an excellent
virus clearance with a reduction of at least 5.5 log10 for MuLV.
Acknowledgements
Our special thanks to Nina Riebesehl; Nadja Giesbrecht, André
Kiesewetter for the technical performance of the chromatography.
Table 2: Results of virus removal on different chromatographic ion exchange media
Chromatography
Media
Type of
Chromatography
and Mode
Virus
Virus (log10)
product eluate
(run 1)
Virus (log10)
product eluate
(run 2)
Eshmuno Q
AEX, binding MuLV ≥ 5.47 ≥ 5.36
AEX, nonbinding
MuLV ≥ 5.02 ≥ 4.73
PPV 6.56 ≥ 5.73
Fractogel TMAE
AEX, nonbinding
MuLV ≥ 5.66 ≥ 5.56
PPV ≥ 6.79 ≥ 7.24
Fractogel TMAE
Hicap
MuLV ≥ 5.75 ≥ 5.57
PPV ≥ 7.12 ≥ 6.89
Eshmuno S CEX, binding, pH 5.0
MuLV 2.41 2.52
PRV ≥ 5.95 3.96
Virus with a high titer, e.g. 107 to 109, is spiked into the load sample
and subsequently the chromatography is performed under the same
conditions as the manufacturing process. The corresponding fractions
are analysed for virus infection (Tissue Cytopathic Infective Dose =
TCID50 titration and large volume plating).
The log10 reduction factor (LRF) of a designated process step is
calculated by the difference of the spiked load sample and the
collected process fractions.
Figure 2. Eshmuno Q Chromatography in non-binding mode, pH 7.5 – 1% Virus
spike (v/v)
MuLV and PRV clearance under non-binding conditions on Eshmuno Q. As shown in
Table 2, Eshmuno Q anion exchange chromatography resulted in excellent log reduction
capabilities, and, thus, contribute significantly to the total LRV of the process. The
different phases of the chromatography process are indicated by the arrows and include
equilibration (F1), product flow through/wash (F2), and CIP (F3).
RESULTS
Figure 1. Eshmuno® S Chromatography in binding mode, pH 5.0 – 1% Virus spike (v/v)
MuLV and PRV clearance in product binding mode on Eshmuno S cation exchanger.
Depending on the virus, the log10 reduction during cation exchange column run can
significantly contribute to the total LRV of the process. Detailed results are summarized in
Table 2. The different phases of the chromatography process are indicated by the arrows
and include flow through/wash (F2), pre-eluate (F3), product eluate (F4), and CIP (F5).
PRINCIPLE OF A VIRAL CLEARANCE STUDY
The viral clearance studies are essential for process validation and
product safety. The virus reduction should be shown on different,
orthogonal steps.
Usually the data is obtained on scaled down and validated process
operation units. The total reduction factor for virus removal is the sum
of every single evaluated step.
Item Treatment Virus titer (log10
TCID50
) LRV
1 Pre-treatment 5.75
2 Hold 5.50
3 60 °C, 60 min – Tube 1 ND (≤-0.87) 4.63
4 60 °C, 60 min – Tube 2 ND (≤-0.87)
4.63
Heat inactivation of influenza A stock
ND: no infectious virus was detected
Introduction
Benzonase®
endonuclease is a powerful tool for degrading all
forms of (deoxy)ribonucleic acids (DNA/RNA) in cell culture
harvests to base pair (BP) lengths under 8 units. It is effective
over a wide range of conditions including temperature, pH, and
varying concentrations of Mg2+
, detergents, chelating agents,
and monovalent cations. It is often employed in the production
of viral vaccines, completely digesting DNA and RNA to improve
clearance and reduce solution viscosity.
Removing Benzonase®
endonuclease, a 60 kD dimer, from the
process stream post-treatment may be achieved with Tangential
Flow Filtration (TFF) with the appropriate molecular weight
cut-off membrane (MWCO), typically 300 kD. This process has
heretofore not been well described in the literature, so the
present study fills this gap pairing Benzonase®
endonuclease
treatment of a DNA-spiked inactivated flu virus cell culture
harvest with Pellicon®
2 cassettes, for clearance of the digested
DNA and remaining Benzonase®
endonuclease by diafiltration.
1. Preparation of flu cell culture
500 ml frozen, harvested cell culture (MDCK cells) infected with
influenza strain A/WS was provided by the Virology Lab Group
(A. Miura). MDCK cells were grown to confluence in DMEM
media which was then decanted, replaced with infected media,
and incubated for 3 days. The infected cell culture was decanted,
clarified by centrifugation at 2500 rpm for 10 minutes, and
passed through a 0.45 mm filter. It was then inactivated at 60 °C
for 60 minutes (titer ≤ -0.51), and frozen for storage.
2. Test methods and materials
2.1 Feed solution
Heat treatment
Live virus; 1.2 ug/ml DNA
60 °C for 60 min
Additional DNA spike 1 mg/ml Herring sperm DNA
Benzonase®
endonuclease digestion 100, 10, 1.0, 0.1 U/ml; up to 2 hrs
EDTA quench Min 10 mM EDTA target
Microchip DNA analysis Agilent Bioanalyzer microfluidic
electrophoresis 2100; sizing/quant
Electropherogram (digital)
Alternative analysis for DNA content
Electrophoresis
Electrophoresis
1.2 or 2.2 % agarose gel
Electropherogram
Control w/o Benzonase®
endonuclease 10 U/ml digestion after 15 min 10 U/ml digestion after 60 min
3.2 Clearance of DNA and Benzonase®
endonuclease by
diafiltration – Feasibility study
1. 100 bp marker
2. Undigested flu cell culture
3. 10 U/ml Benzonase®
endonuclease digest
4. 100 U/ml post retentate recirc
5. Retentate @ 1 DV
6. Retentate @ 3 DV
7. Retentate @ 5 DV
8. Retentate @ 8 DV
9. Permeate pool @ 5 DV
Samples run in duplicate (9 x 2)
Large DNA molecules present in spiked cell culture (2) are
digested by Benzonase®
endonuclease (3) and cleared by
diafiltration (5-8). Dilute, digested DNA is detected in the
permeate (9).
The DNA band strength fades with each progressive DV sample until
it is no longer visible by 8 DV. Lane 9 contains permeate at 5 DV.
100.00 %
10.00 %
1.00 %
0.10 %
0.01 %
Diavolumes (N)
%remaining
Experiment
for S = 1
for S = 0.90
for S = 0.85
Benzonase endonuclease diafiltration®
Volume (ml) Concentration (ng/ml) Mass (ng) % Recovery
Start 502 36.426 18286 -
5 DV Perm 2500 3.580 8950 48.9%
5 DV Ret 500 0.190 95.02 0.52%
References
• Benzonase®
endonuclease ELISA Kit II Instruction Leaflet and Product Guide
• Jonges, Marcel, et. al.: Influenza Virus Inactivation for Studies of Antigenicity and
Phenotypic Neuraminidase Inhibitor Resistance Profiling, J. Clin. Microbio., 2010,
48(3):928
• Protein Concentration and Diafiltration by Tangential Flow Filtration. Millipore Lit.
No. TB032 Rev. C 06/03 03-117 (Application note)
• Maintenance Procedures Pellicon®
2 Cassettes (Lit. No. P17512 Rev C 7/98) (Guide)
• Pellicon®
2 Cassette Filters (P17512) or Pellicon®
3 Filters Installation (AN1065EN00)
(User Guide)