MilliporeSigma - Viral Safety - 33
Poster Gallery
Implementation of a Virus Barrier Media
Filter into Fed-Batch Bioprocesses
Efficient Virus Clearance Across
the MilliporeSigma Downstream
Purification Portfolio
Kimberly Mann, Michael McGlothlen, Joe Orlando, Jonathan Broe, Patricia Kumpey, Kristina Cunningham, Yuanchun Zeng, David Nhiem,
Robert Smith, Christina Carbrello, Venkata Raman, Jeremy Perreault, Kevin Rautio
Ushma Mehta, Chris Gillespie, Kevin Galipeau, Michael Doty, Trish Greenhalgh, and Michael Phillips
EMD Millipore Corporation, Bedford MA, USA
EMD Millipore Corporation, Bedford MA, USA
No change in cell culture performance
MAb #1 Cell Growth in Shake Flasks
MAb #1 Viability in Shake Flasks
100
90
VB- #1
VB- #3
VB- #4
60
VB+ #5
VB+ #6
50
VB+ #7
VB+ #8
40
30
5
20
10
6
8
10
0
12
2
-
4
6
Days
8
10
12
Days
Cell performance in shake flasks
Two recombinant CHO cell lines were cultured in 125 mL shakers in fed batch culture. MAb #1 utilized Cellvento CHO-200 medium and feeds
and mAb #2 (data not shown) utilized Ex-Cell® CHO media and feeds. No change in cell growth was observed when media and feeds were
processed with (VB+) or without (VB-) Viresolve® Barrier filters. Cell viability was also unaffected. Osmolarity, pH, glucose, glutamate, lactate,
or NH3 levels were within limits (as measured by BioProfile® FLEX biotech analyzer). Titer, as measured by POROS® Protein A HPLC, was also
consistent.
®
Direct implementation of Viresolve® Barrier filter into bioreactor processes
Viresolve® Barrier filters are sterilizable and may be used in place of a 0.1 μm filter.
Model Stream B
1200
1000
800
600
400
2000
1500
1000
500
200
0
0
MilliporeSigma
Lonza
Thermo Fisher Scientific
GE Healthcare Life Sciences
VBVB-
VB-
14
VB-
2.5
VB-
VB+
VB+
8
VB+
2
Clarification
0
0
4
6
8
10
12
14
Viresolve® Barrier filter shows high capacity for efficient filtration
of most commercially available chemically defined media.
16
VB-
VB-
VB-
Joaquina X Mascarenhas 1, Nikolay Korokhov2, Ademola Kassim 1, Jason Tuter
David Onions 2, Kevin Kayser 1
1SAFC® MilliporeSigma, Saint Louis, Missouri, 63103, USA
2Bioreliance®, MilliporeSigma, Rockville, Maryland, 20850, USA
1
,
VB +
VB +
VB -
VB +
0.8
Absorbance at 230nm
Absorbance at 230nm
VB VB -
VB +
Retention time (min)
Aggregate Profile
Weak cation-exchange chromatography was employed to analyze
the Eshmuno® A purified antibody mAb#2 from bioreactor culture.
No notable differences were seen in the amount of acidic, neutral
or basic peaks between antibody purified from either culture
system with (VB+) or without (VB-) Viresolve® Barrier filtration.
The size exclusion chromatography profile for mAb#2 showed very
high amounts of monomer for antibody purified from cultures,
which was unaffected by filtration with (VB+) or without (VB-)
Viresolve® Barrier filters. Consistently, very small amounts of a
high molecular weight species were noted and no fragments were
observed.
0.6
G0F
LU
140
0.4
CHO Cells Sialic Acid Mutant Library
0.2
The introduction of animal origin free (AOF) media has significantly reduced
the incidences of adventitious virus contamination in biological production
CHO
Wild-Type
SLC35A1
Genotypes
CHOZN GS
systems. Nevertheless, contamination by the parvovirus Minute Virus of Mice
(MVM) also known as Mouse Minute Virus (MMV) remains a continuing
challenge
on average
we Fingerprinting
see one major
contamination
Nuclearand
Magnetic
Resonance
- episode of MVM
Inductively
Coupled Plasma/Optical
Emission
Structure
N-Linked
Aromatic
Region Although infrequent, infection of a Spectrometry
every
~5 years.
fermenter can be
(Asn)
®
™
NMR fingerprinting
Advanced
CHO Fed-batch
medium
The graph above
shows the concentration ratio of metals in the
catastrophic
for ofa Ex-Cell
producer,
resulting
in the
loss of product,
temporary
®
shows no change
after filtration.
shows thatclean
the
CHOwhich
media and
feed followingO-Linked
Viresolve® Barrier filtration
withdrawal
frombefore
the and
market
and NMR
extensive
downEx-Cell
costs,
can
(Ser-Thr)
component levels have not changed and no new components have
compared to Millipore Express® PLUS filtration
as measured
reach
a total in the tens of millions of dollars. In addition
to the loss of
been introduced. This same trend was observed in the aliphatic
by ICP-OES. No significant differences were observed in metal
associated
a the
contamination
event
can also haveconcentrations.
a potential impact
region - of revenue,
particular note
Pluronic® F68 peak
was unchanged
Phenotype
N-linked and
Asialylated
O-linked WT
(data not
shown). patient safety and have regulatory implications.
on drug
supply,
0.0
-
EX-CELL® CD CHO Fusion
-/EX-CELL® Advanced™ CHO Feed w/Glucose
EX-CELL® Advanced™ CHO Fed Batch
COSMC-
MGAT-/-
100
CHOZN WT
80
Slc35A1-
120%
G0
60
Man5
40
20
0
10
N-linked WT
O-linked
trunc
VB VB VB -
G1F
VB +
VB +
VB +
MVM Viral Resistance Testing
120
12
14
Glycan Analysis
N-linked
trunc
Glycan
O-linked
WT
COSMC-
Mgat1-
60%
40%
16
18
0%
20
Retention time (min)23.5%
20%
0.6%
0.0%
T=0
4.3%
T=21
analysis of the purified mAb#2 was performed using 2-AB fluorescent labeling and NP-UPLC. Consistent glycan patterns for both culture
HoursViresolve
post- infection
®
systems were observed for the antibody produced from media and feeds with (VB+) or without (VB-)
Barrier filtration.
Concentration Ratio (Viresolve® Barrier Filtered/
non-Viresolve® Barrier filtered)
®
vitamins following Viresolve® Barrier filtration compared to Millipore Express® PLUS filtration for Ex-Cell® CHO media and feed.
Knockouts:1B1, 1C3, 1C8, 1G8, 1G11, 2A6
MVM: A Threat Even in ACF Free Processes
In addition, the secreted antibodies showed no differences in the glycosylation
pattern, amount of aggregates
or charge
variants.
Preliminary
work
(not shown)
in in
Schematic
of work flow
of studying
the effect
of Slc35A1
knockout
recombinant
IgG
expressing cell line.
Mobius® 50 L bioreactorsa shows
similar
results.
assay
Wild-Type: 1A5, 1A10, 1A11, 1D8, 1E12, 2B3, 2B8, 2C1, 2C6
WCB
Bank
Filtration of media and feeds with Viresolve® Barrier filter exhibits efficient
filtration performance, high virus retention and minimal cell culture impact, and
offers a viable option to improve the overall virus risk mitigation strategy for the
manufacture of biotherapeutics.
10
MilliporeSigma, Viresolve, Millipore Express, Cellvento, Eshmuno, Mobius and the Vibrant M are trademarks of Merck KGaA, Darmstadt,
Germany. and product quality characterization
Growth, productivity
Cell
Company Co. LLC. All other trademarks
Reported By are the property of their respective owners.
Virus Ex-Celland Advanced
are trademarksYear
of Sigma-Aldrich
EHDV
CHO
1988
Bioferon GmbH
Bioferon GmbH
Lit. No. PS6649EN00, Ver. 3.0 Copyright © 2017 EMD Millipore Corporation. All Rights Reserved.
MMV
CHO
1993
Genentech
Genentech
HPLC Titers
MMV
CHO
1994
Genentech
Genentech
Reovirus
Homo 1˚Kidney
1999
Abbott Labs
FDA
Reovirus
CHO
?
?
BioReliance
Cache Valley
CHO
1999
Amgen / CMO
Amgen
Cache Valley
CHO
2000
?
BioReliance
Slc35A1 KO Clone
Vesivirus 2117
CHO
2003
Boehringer-Ingelheim
Boehringer-Ingelheim
Wild Type Clone
Cache Valley
CHO
2003
?
BioReliance
CHO-K1 producing (WCB BANK)
Cache Valley
CHO
2004
?
BioReliance
Hu Adenovirus
HEK 293
?
Eli Lilly
Eli Lilly
MMV
CHO
2006
Amgen
Amgen
Vesivirus 2117
CHO
2008
Genzyme, Belgium
Genzyme
Vesivirus 2117
CHO
2008
Genzyme, USA
Genzyme
Vesivirus 2117
CHO
2009
Genzyme, USA
Genzyme
MMV
CHO
2009
Merrimack
Merrimack
PCV-1
Vero
2010
GlaxoSmithKline
GlaxoSmithKline
PCV-1/PCV-2
Vero
2010
Merck
Merck
The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Table 1: Known incidences
biomanufacturing.
of
viral
contamination
events
in
100
9
8
80
7
Viable Cell Density
Titer (ug/ml)
View Larger
8.51
6.92
8.41
Eluted
5.78
Hold
8.31
Eshmuno® A
resin: C2
Flow-through
8.29
Wash
6.19
Eluted
5.85
Virus Load (log TCID50)
2.5
LRV
Virus Load (log GC)
3.1
4.02
LRV
2.5
4.3
6.05
3.85
6.94
8.51
6.72
2.5
8.31
3.5
4.63
2.5
4.34
6.05
3.47
Virus
Size (nm)
Enveloped
Physicochemical
resistance
MVM
18-26
No
High
Retroviridae
X-MuLV
80-110
Yes
Low
6
60
5
4
40
Low pH Virus Inactivation
This step is dedicated to the reduction of enveloped
virus and a short hold at low pH fits well in the
process following elution at low pH from Protein A
column. Duplicate aliquots of post Protein A elution
pool were adjusted to pH 3.6 with 100 mM
Acetic Acid then challenged with XMuLV at 1E+06
TCID50/mL (~1% (v/v)), and incubated at room
temperature for 60 mins. Rapid virus inactivation
was observed and after 5 minutes, no infectious virus
was detected, resulting in LRVs of at least 4 log.
Prefiltration and Process Improvements:
Enhancing Virus Filter Performance with
the Use of Adsorptive Depth or Surface
Modified Prefilters
Eshmuno® S resin is a strong cation exchange media
with tentacle structure enabling fast, efficient purification.
Excellent purification across Eshmuno® S resin was
achieved with binding at pH 5.5 and elution with Tris pH
8.5, 10 mS/cm. These conditions avoided dilution of the
feed before AEX polishing.
Table 2 summarizes the properties of the feeds used in
each step. Media were selected with consideration to the
efficiency of purification with a view to minimizing dilution
or modification for downstream purification.
Eshmuno® S resin did not contribute to virus
safety targets as less than 1 log virus was removed
(data not shown). However, purification over CEX media
was critical to achieving acceptable viral clearance over
Eshmuno® Q AEX resin as shown in the graph.
Amanda Katz, Damon Asher, Patricia Greenhalgh, Janice Lonardo, Ben Cacace, and Jeff Hartnett
Clearance of MVM and XMuLV across Eshmuno® Q resin
TableMillipore
2. Feed Specifications
EMD
Corporation, Bedford, MA
was evaluated at pH 7.5, 8.0 and 8.5. MVM removal was
Step
insensitive to loading pH, with LRVs of at least 5 log out
Protein
Conductivity
Feed
Conc
pH
@ 25°C
Media
to the 0.25 kg/L target loading (data not shown). XMuLV
(mg/mL)
(mS/cm)
evaluated
removal was highly dependent on pH with effective
reduction at the target 0.25 kg/L loading only achieved
Clarified
Protein A
Supernatant
1.3
7.2
12-15
Eshmuno® A
at pH 8.0 and pH 8.5, the optimal conditions for HCP
resin
pool
removal.
Figure
2 Viresolve® Prefilter Conditioned Feed
Post
Protein A
23.2
5.5
3-4
resin
Improvements
in upstream process development often
generateAEX
complex, high titer process streams, placing
Q
Post
CEX
11processing
8
Eshmuno®demands
considerable
on downstream
steps. 6
resin
Protein aggregates in these feeds influence hydraulic
Nanofiltration
performance
of
virus filters resulting in over-sized platforms
Post AEX
10
5
6
Viresolve® Pro
and a significant
impact on process economics. Virus filters
Solution
from a broad range of manufactures provide robust viral
clearance but the impact of aggregates on flux is dependent on
the filter. The impact of conditioning protein solutions using
prefiltration was assessed with several monoclonal antibody
feed streams. Adsorptive depth or surface modified membrane
The purification
metfilter
mAb
yield
and to
purity
prefilters
upstreamscheme
of the virus
were
shown
remove
targets.from protein solutions, enhancing performance of the
foulants
HCPHCP
Levels
ininProcess
Intermediates
Levels
Process Intermediates
virus filter.
Purification Summary
* Pores
of virus filters are sized to pass most proteins and
10000
exclude
viruses
1000
323
186
173
* Therapeutic
protein feed streams may contain low levels of
100
10
aggregates
or other foulants that gradually plug the virus
10
filter. Virus
1 filter fouling may become more pronounced as
A
Q
S
d
the feed stream
ed titer increases.
ed
o
oa
o
o
ifi
ifi
r
cla
Replicate # 2
XMuLV LRV
3.5
5
≥4.2
≥4.2
10
≥4.7
≥4.7
30
≥4.9
≥4.9
60
≥4.9
≥4.9
(15 g/L)
6
5
4
3
2
1
0
0.1
0.25
r
Cla
un
®
un
®
EX
L
un
®
m
hm
A
halso
hm
* AdsorptionUnof monomer variants
or
fragments
may
Es
Es
Es
contribute to membrane plugging
No CEX : D1: pH 7.5
Post CEX : pH 8.0
Post CEX: pH 8.5
No CEX : D2: pH 7.5
Viresolve® Pro Shield H
Arrow denotes complete XMuLV retention
Surface modified membrane prefilter
500
0.9
450
0.8
Virus Filtration
400
0.7
In
most biologic processes, virus filtration is the
350
0.6
dedicated step for reduction of both enveloped and
non300
0.5
enveloped virus by size exclusion. Post AEX pool was
250
®
Viresolve Pro Device
pH
adjusted then processed over
a pre-filter Viresolve
0.4
Viresolve Prefilter with
200
Pro Device
Pro
Shield, spiked with MVM atViresolve
2E+06
TCID50/mL
0.3
150
(~0.2%
(v/v), then processed over Viresolve® Pro filter
0.2
100
device
at constant pressure of 30 psi.
®
®
®
*downstream
Support membrane:
0.2 µm PES
of the filtration
devices, even following
recovery,
resulting
in reduction
of greater
than
*product
PES membrane
surface
modified
via cross-linked
mixed5.9
log.chemistry
mode
* Surface chemistry binds to aggregates and other foulants
Device
Viresolve Pro # 1
Figure
5 Viresolve®
Viresolve® Pro # 2
0.1
50
Sample description
LRV
Pool+ wash
≥5.93
* Effective operational range: pH > 5.5 or conductivity
≥5.93
> 25 mS/cm
(see Figure 7)Pool at 5.0 Kg/m2
®
At the 5.0 kg/m2 target loading, processing was 0.0
0
stopped,
and
product
recovered.
No virus
0
100
200
300
400
500 was detected
600
2
Pool
at 5.0 Kg/m
Pro
Shield
H Test
MilliporeSigma, Eshmuno, Viresolve, Clarisolve and Mobius are
virus
protein aggregate
trademarks of Merck KGaA, Darmstadt,
Germany. All other
trademarks are the property of their respective owners. Lit. No.
PS1245EN00 Ver. 2.0 07/2017
©2017 EMD Millipore Corporation. All rights reserved.
Pool+ wash
0.9
0.4
200
Protein A (Eshmuno® A resin)
40 mg/mL
Low pH hold at pH 3.6
NA
100
* Surface chemistry binds to aggregates and other foulants
0
* Effective operational range: pH < 6 and conductivity
®
resin)
80 mg/mL
CEX(see
(Eshmuno
< 30 mS/cm
FigureS7)
AEX ( Eshmuno ® Q resin)
250 mg/mL
Virus Filtration (Viresolve® Pro Solution)
5 kg/m2
Cumulative mean LRV
0
MVM LRV
XMuLV LRV
2.5
2.5
100
n/a
0
200
Throughput (L/m2)
300
≥4.9
20
1
0
0
2
4
Days in culture
6
8
10
nanoporous
membrane
0
≥5.3
≥4.9
13.7#
18.2#
In the absence of a prefilter, average mass capacity =
≥5.9
≥5.9*
0.7 kg/m2.
Using the Viresolve® Pro Shield H prefilter, average mass
Figure 6 Viresolve® Pro Shield H Conditioned Feed
(13 g/L)
Cross-linked polymeric
surface coating
Figure 3: SLC35A1 knockout in an expressing production line: Growth and Productivity
Both wildtype and SLC35A1 knockout clones were isolated from the transfected population. Viability, viable cell density and
productivity were measured. A range of titer and VCD were obtained in both knockout and wildtype clones. It was determined that the
genetic modification did not impact these parameters.
Figure 3 Viresolve® Pro Shield Test Summary
16
14
Viresolve® Prefilter
12
10
Viresolve Pro Device
Viresolve® Pro Shield
with Viresolve® Pro Device
®
0.1
2 detected downstream
#conservativecapacity
estimate when
no virus
= 5 kg/m
14
protein
0.2
0.0
400
0.9
3
2
0.7
0.3
* PES membrane surface modified via cross-linked polymeric
Unit Operation
Target Loading
sulfonic acid cation exchange chemistry
View Larger
0.8
confirmed our downstream purification technologies0.6can
be300run in a connected process to deliver viral clearance
0.5
performance required by customers.
* Support membrane: 0.2 µm PES
0.2 µm
PES
≥5.93
Viresolve® Pro Device
Viresolve® Pro Shield H
with Viresolve® Pro Device
400
Surface
modified
membrane
prefilter
The purification
scheme
achieved
virus clearance
targets with a margin for safety. Importantly, the project
*Not tested, LRV from MVM used as surrogate for XMuLV
≥5.93
Summary
500
®
Viresolve
Pro Shield
Virus Clearance
Summary
The life science business of Merck KGaA, Darmstadt Germany
operates as MilliporeSigma in the U.S. and Canada.
0.5
Mass Loading ( kg/L)
Post CEX : pH 7.5
769,231
Why
Do Virus Filters Plug?
100000
1000000
3.2
XMuLV
removalusing
usingEshmuno
Eshmuno®® Q
Q with Post CEX
XMuLV
removal
CEX pool
pool
Throughput (L/m2)
100,0000
Replicate # 1
XMuLV LRV
0
Cation (CEX) and Anion (AEX) Exchange Chromatography
Methods
10000000
Time of incubation
at pH 3.6 (min)
View Larger
% Viability
Single-Cell Cloning, Screening &
reversed-phase
C-18 chromatography.
amino acids and soluble
delivered
cell derived
products.The graph above shows no significant difference in the media concentration ofSequencing
Viresolve® Pro
filter device
Parvoviridae
®
80%
Summary
®
Eshmuno® Q
resin
Virus Family
CEX
Abstract
Eshmuno S
G2F
100%
In this work, we evaluated engineering a CHO parental cell line to create a
new host
1.0 cell line that would be resistant to MVM infection by eliminating the
Figure 1: Schematic of major glycosylation structures resulting from specific gene knockouts and the correlation of cell
major receptors used by the virus to enter cells. The goal was to engineer a
surface sialic acid and MVM binding and internalization.
host cell
line resistant to MVM infection, while maintaining desired
0.8
Fluorescent microscopy panel shows the staining of fluorescent labeled Mal II Lectin. The Mal II lectin binds exclusively to sialic acid
productivity and product quality profiles. While the exact functional receptor
bound in an α-2,3 linkage conformation. CHO cell lines engineered to express lower α-2,3 surface sialic acid demonstrate increased
for MVM
binding to CHO cell surface is unknown, sialic acid on the cell
0.6
resistance to MVM infection compared to the wild-type controls.
surface has been implicated. The CMP-sialic acid transporter, solute carrier
family 35A1
(Slc35A1) is responsible for transporting sialic acid into the
The risk of virus contamination of the bioreactor remains a concern for
0.4
Golgi. Knocking out function of this gene in a cell results in asialylated
biotherapeutic
manufacturers,
thereLine
is no universal technology that provides a
Slc35A1 KO in an recombinant
IgG
ProducingasCell
glycan structures, thus eliminating the ability of MVM to bind to and enter
0.2
reliable, cost effective solution for virus removal of all components of cell culture
the cell. While the wildtype cells displayed immediate loss of viability and
media. This study evaluated the Viresolve® Barrier filter, which provides an efficient
inhibition in cell growth upon MVM infection, the absence of sialic acid on the
Thawed & Passaged
Recombinant
Working Cell
2x
IgG
0.0
Bank (10
and easy way to protect bioprocesses from adventitious virus contamination.
SLC35A1 knock-out cell line lead to complete
resistance to MVM infection.
producing
Media components: Amino acids and soluble vitamins
vials)
clone
7/14/15
This approach could be EX-CELL
applied
to different
CHO host cell lines,
as well as
CD CHO Fusion
EX-CELL Advanced™ CHO Fed Batch
EX-CELL Advanced™ CHO Feed w/Glucose
Equivalency/Preliminary Studies
Study results demonstrated that media and feed compositions were unaffected by
therapeutic protein producing clonal cell lines. Incorporation of viral
®
ZFN
resistance to the MVM virus in the CHO host and subsequent production cell
One through
WCB Vial thawed the Viresolve
filtration
Barrier filter. Cultures, both in shake flasks and
Transfection
on 12/7/15 and
Figure 2: Case Study SLC35A1 Knockout in an Expressing
lines
adds
yet another
of "defense" to the current risk mitigation
passaged for a week
High
Performance
Liquidlevel
Chromatography
scaled prior
uptoto
stirred
tank Production
bioreactors,
showed no differences in cell growth or titer.
beginning
of
Line
growth & productivity
strategies
adding by
even
greater
assurance
production
of safely
Amino acidused,
analysis conducted
ion-exchange
chromatography
postof
column
Ninhydrin method
and water-soluble vitamin analysis by
®
Eshmuno® S
resin
6.94
8.54
5.7
LRV
XMuLV qPCR
8.31
Concentration
All aspects of clone development, selection, cell culture
media selection, scalable production in 3-200L Mobius®
bioreactors, and clarification over Clarisolve® 20MS
depth filters were optimized as part of the benchmark
process development.
Retention time (min)
Viral genome copies % of wild type
Vitamins: Low concentration
components are detectable
VB +
VB +
VB -
Charge heterogeneity
Fluorescence Emission (420nm)
Normalized Intesity
Abstract
Concentration Ratio (Viresolve® Barrier Filtered/
non-Viresolve® Barrier filtered)
1.0
Tryptophan:
photo unstable;
good indicator
of media
stability
VB VB -
Trissa Borgschulte 1, Henry George 1, Audrey Chang 2,
AEX
Table 1. Virus Properties
Product quality of recombinant antibody was unaffected
Media composition was not affected
Viresolve® Barrier +
Millipore Express® PLUS filters
VB+
Cell performance in 3L bioreactors
Genetic Engineering of CHO cells for
Viral Resistance to MVM
Millipore Express® PLUS filter
VB+
Recombinant CHO cell mAb #2 was expanded for fed batch production in 3L bioreactors utilizing Ex-Cell® CHO media and feeds. As in the
small-scale shake flasks, no change in cell growth was observed when media and feeds were processed with (VB+) or without (VB-) Viresolve®
Barrier filters. Titer, as measured by POROS® Protein A HPLC, was also consistent.
Given the small pore size of virus retentive filters, implementing virus filtration upstream
of the bioreactor raises the question of whether critical cell culture media components
might be removed. Therefore, it is important to evaluate the cell culture performance
using filtered media to ensure that there has not been any negative impact to the
process. Ex-Cell® CHO media, Cellvento® CHO-200 medium and corresponding feeds
were processed through Viresolve® Barrier filters, and media composition was compared
to 0.2 µm Millipore Express® PLUS filtered controls. Fed batch cultures were performed
in both shake flask and Mobius® 3L stirred tank bioreactors to verify that surfactants,
such as poloxamer (which are essential for shear protection in stirred tank bioreactors
and can be difficult to filter), had not been removed during filtration. Cell culture
performance and protein quality were evaluated.
Unfiltered
VB+
Days
View Larger
Methods
CEX
Virus
Filtration
Each unit operation was optimized to maximize step
purity and yield. Following optimization, virus clearance
was assessed using two viruses: Minute Virus of
Mice (MVM), and Xenotropic Murine Leukemia Virus
(X-MuLV), Table 1.
0.5
2
2
Virus
Inactivation
Eshmuno® A
resin
1
4
0
Protein A
Capture
XMuLV Infectivity
Virus Load (log TCID50)
Hold
1.5
6
Days
More than twice the volume of media can be filtered using
Viresolve® Barrier filter in 4 hours as compared to existing
downstream virus filtration technologies.
Figure 1. Monoclonal Antibody Process Operations and
Purification Media
MVM Infectivity
Sample
Flow-through
Wash
VB+
VB+
10
Eshmuno® A
Column
Eshmuno® A
resin: C1
VB+
VB-
12
Duplicate columns were challenged with clarified harvest
HCP PPM (ng/mg)
1400
MAb #2 Titer
3
16
2500
Titer (g/L)
Model Stream A
Viable Cell Density (cells x 106/ml)
1600
Liters filterin in 4 hours/m2
Liters filtered in 4 hours/m2
MAb #2 Cell Growth in Mobius® 3L bioreactors
3000
1800
The purpose of this project was to establish
capabilities for producing a monoclonal antibody
feed enabling the development of new filtration and
chromatography products. The purified antibody
should have industry-representative titer, yield and
purity, achieved using the Eshmuno® chromatography
resin platform in combination with Viresolve®
Pro filtration devices as seen in Figure 1.
Relative Flux (J/J0)
4
XMuLV Log Reduction Value ( LRV)
2
pool co-spiked with MVM at 1E+06 TCID50/mL (0.1%
(v/v)) and XMuLV at 5E+05 TCID50/mL (~1% (v/v)).
Columns were washed in 100 mM citric acid pH 5.6,
then protein was eluted in 100 mM acetic acid pH 3.0.
Samples were assessed for titer by infectivity assays
(MVM and XMuLV) or qPCR (XMuLV only). Protein A
viral clearance results are summarized below and
show moderate removal (~2.5 log) of both MVM and
XMuLV, and approximately 0.5-1 log inactivation of
XMuLV, attributed to the low pH elution conditions.
Flux (LMH)
-
Eshmuno® A resin is a rigid, high capacity, acid and
alkaline resistant Protein A affinity chromatography
media for the purification of Fc-containing proteins
such as monoclonal antibodies. Following preliminary
optimization of load, wash and elution conditions,
Eshmuno® A resin delivered robust performance
under a variety of conditions, consistently removed
high levels of HCP, and could be loaded to 40 g/L
with protein recoveries of at least 95%.
12
Capacity (kg/m2)
0
Protein A Affinity Chromatography
The downstream purification process of any biologic has
several objectives: purity, yield, and safety for humans
or animals. A critical component of safety assurance is
reducing virus to levels that meet stringent regulatory
requirements. Virus reduction can be achieved
through multiple complementary approaches and most
processes rely on a combination of technologies that
are designed primarily to achieve purification targets,
but may also offer opportunities for virus reduction.
Relative Flux (J/J0)
VB+ #5
VB+ #6
VB+ #7
VB+ #8
10
VB- #2
70
VB- #4
15
VB- #1
80
VB- #2
VB- #3
Introduction
Flux (LMH)
20
% viable
Viable Cell Density (cells x 106/ml)
25
)
Upstream bioprocesses are at particular risk of contamination from adventitious
agents. Typical 0.1 µm filters used at this step protect bioreactors from
bacteria and mycoplasma, but offer no protection from viral contaminations.
A new polyethersulfone (PES) virus barrier filter, Viresolve® Barrier filter, has
demonstrated high levels of microorganism retention - full retention for bacteria
and mycoplasma (>8.0 LRV - Log Reduction Value) and ~ 5 LRV for small viruses,
such as parvoviruses. This filter is optimized for use with upstream components,
and therefore exhibits higher flow and capacity than downstream virus filters.
In this study, we evaluate the effects of implementing a Viresolve® Barrier filter
into upstream processing.
2
Introduction
9
7
5
Viresolve® Pro Device
Viresolve® Pro Shield H
with Viresolve® Pro Device
Table of Contents for the Digital Edition of MilliporeSigma - Viral Safety
Contents
MilliporeSigma - Viral Safety - 1
MilliporeSigma - Viral Safety - 2
MilliporeSigma - Viral Safety - Contents
MilliporeSigma - Viral Safety - 4
MilliporeSigma - Viral Safety - 5
MilliporeSigma - Viral Safety - 6
MilliporeSigma - Viral Safety - 7
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MilliporeSigma - Viral Safety - 27
MilliporeSigma - Viral Safety - 28
MilliporeSigma - Viral Safety - 29
MilliporeSigma - Viral Safety - 30
MilliporeSigma - Viral Safety - 31
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MilliporeSigma - Viral Safety - 33
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MilliporeSigma - Viral Safety - 35
MilliporeSigma - Viral Safety - 36
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