Parvovirus Filtration Best Practices - 25 Years of Hands-On Experience

The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
PARVOVIRUS
FILTRATION BEST
PRACTICIES
Paul Genest
Applications Engineer,
BioPharm Center of Excellence - Innovation + Influence = Impact
25 YEARS OF HANDS-ON EXPERIENCE

The life science business
of Merck KGaA, Darmstadt,
Germany operates as
MilliporeSigma in the U.S.
and Canada

3
Paul Genest
Applications Engineering
(25 years in industry)
B.S. Chemical Engineering, UNH, 1994
M.S. Chemical Engineering, UNH, 1996
No. 1 Goal = Optimized Performance and
Trouble-free Operation

Agenda
1
2
3
How to measure virus filter
capacity performance?
What are the operating
parameters that impact virus
filter capacity performance?
What is the best way to size
virus filters during PD?
4
5
What is the virus removal / LRV impact
from level of filter plugging?
Scalability and the
importance of the scale down
model used for virus
validation?
6
How is validation of the virus
filter performed, what
operating parameters are
important?

How is filter capacity performance measured:
- either constant feed pressure, or
- constant feed flow, or
- combination (feed pressure and feed flow changing during processing)
- process goal is to minimize plugging / maximize L/m2 volumetric throughput, minimize filter costs ($/g)
PARVOVIRUS FILTRATION BEST PRACTICIES
5
$ $
$
high filter plugging
moderate filter plugging
no filter plugging

6
100
200
300
400
500
200 400 600 800 1000
% flow decay = 100 *
(1- 50/500) = 90%
LRV can be <or= 4
(1- 200/500) = 60%
LRV can be 5
(1- 400/500) = 20%
LRV can be 6
Flux (LMH) at
constant feed
pressure 30 psi
Volumetric Throughput (L/m2)
high filter plugging
moderate filter plugging
Filter capacity performance zone to avoid, and why?
% flow decay higher,
can drive LRV lower

7
100
200
300
400
500
200 400 600 800 1000
Flux (LMH) at
constant feed
pressure 30 psi
little to no fouling with optimal adsorptive prefilter use
(pilot FULL process simulation)
minimal filter plugging
more filter fouling during tougher validation
step with: frozen / thawed feed, decoupled
adsorptive prefilter, extra handling, virus spike,
etc. = higher % flow decay & lower L/m2
Filter capacity performance zone to target,
but (include safety factor before validation)
*** Safety Factor
before
validation
do not make clinical material to
max L/m2, BEFORE VALIDATION
after validation,
within this range
little to no fouling with optimal adsorptive prefilter use
(PD FULL process simulation)

Where is the optimal placement for the virus filter?
8
1.
BIOREACTOR
2.
CENTIRFUGE
3.
DEPTH-FILTER
4.
PROTEIN A
5.
LOW pH
INACTIVATION
9.
UF/DF
10.
0.22
FILTER
CEX AEX HIC VF
8.
Viresolve® Pro
Shield / Viresolve®
Pro parvovirus
filtration (20nm)
typical optimal position is
after the 2nd or 3rd column
6.-7.
POLISHING
X X
not
purified
enough
conc. too
high
> 25 g/L

Achieving maximum capacity / volumetric throughput (L/m2):
A. understand what is fouling the virus filter (drug product aggregates),
B. know what conditions cause the foulants / how to control them,
C. know the best way to remove foulants, if they cannot easily be prevented
9
Drug Product
Monomer ~ 4-6
nm
Virus Filter
Pore ~ 20 nm
RVLP ~
80 nm
MVM ~
20 nm
Drug Product
Di-mer
Drug Product
Tri-mer
Drug Product
Multi-mer
Virus Spike
Impurity
combined
species?
“aggregate”
Advantages of using
Adsorptive Prefilters:
2. Remove trace
aggregates: increase filter
capacity, reduce filter costs
4. Improved Robustness:
molecule 1 feed variability and
from molecule 1 to molecule 2
3. Can reverse the effects of
feed age, freeze / thaw, etc.
Factors that can impact virus
filter performance:
1. feed storage: temperature and
feed age (increase aggregate?)
2. freeze / thaw (increase
aggregate?)
5. increased concentration
(increase aggregate?)
4. pH / conductivity / additives
(change aggregate level?)
3. air/liquid interfacing,
foaming (increase aggregate?)
6. Interactions with virus spike
impurity (increase “aggregate”?)
1. Effective aggregate removal
vs. ineffective 100-200 nm
size based prefilters

The filterability impact from the specific drug product molecule:
some drug products are well behaved molecules, easy to work with, nothing makes them form aggregates
some drug products are sensitive molecules, hard to work with, they easily form aggregates
10
Factors that can impact virus
filter performance:
1. feed storage: temperature and
feed age (increased aggregate?)
2. freeze / thaw (increased
aggregate?)
5. increased concentration
(increased aggregate?)
4. pH / conductivity / additives
(changes in aggregate?)
3. air/liquid interfacing,
foaming (increased aggregate?)
6. Interactions with virus spike
impurity (increased “aggregate”?)
store in cold room at 2-8 C
(track and be aware of feed age)
if you must freeze, thaw using method (slow
vs. fast) that generates the least amount of
aggregate
only gentle handling, filter and siphon using a
dip-tube filling from the bottom up, pour slowly
down the side wall, vacuum filter gently
ideally have drug product in buffer where
aggregation is minimized (pH, condo., additives)
operate at the lower, not higher concentrations
use lowest % virus spike possible to hit
target LRV, use purest virus spike possible
the top and bottom photos are from:
P. Genest, H. Ruppach, C. Geyer, M. Asper, J. Parrella, B. Evans
and A. Slocum, Artifacts of virus filter validation, BioProcess Int 11,
2013, 54–61.
the two middle photos, I took while in a virus spiking lab
foam

11
Impact from changing only drug product pH (no adsorptive prefilter)
decreasing pH reduced reversible drug product aggregate, increased VF capacity
blinded data from Viresolve® Vpro user PD
optimization project
less
plugging
lower pH
more
plugging
higher pH
* specific behavior can vary from molecule to molecule

Behaviors with feed pressure, feed concentration, & buffer flushing
12
0.2
50.2
100.2
150.2
200.2
250.2
300.2
0 20 40 60 80 100 120 140 160
FLUX
(LMH)
at
30
psi
Time (minutes)
2x10^6 MVM (60 min pause) 1
baseline (no virus)
~ same fouling, but SLOW
~ same filter plugging,
FASTER processing
~ same filter plugging,
FASTEST processing
more filter fouling
at highest feed g/L
feed processing buffer flushing
Viresolve® Pro Solution Performance Guide
Lit No. RF1013EN00 Rev. B 11/14 DP SBU-12-07371
Printed in the U.S.A. ©2014 EMD Millipore Corporation,
Billerica, MA 01821 U.S.A. All rights reserved.
data I generated at Millipore R&D virus spiking lab
reversible
polarization
irreversible
fouling

Adsorptive Prefilter Development (increases in virus filter capacity)
diatomaceous earth containing depth filter or membrane with added ion exchange chemistry
13
ineffective size
based prefilters
G. Bolton, S. Spector and D. Lacasse, Increasing the capacity of
parvovirus-retentive membranes: performance of the Viresolve™
Prefilter, Biotechnol Appl Biochem 43, 2006, 55–63,
https://doi.org/10.1042/BA20050108.
G. Bolton, A. Brown, C. Bechtel, J. Bill, H. Liu, J. Liu, D.
McDonald, et al., Increasing parvovirus filter throughput of
monoclonal antibodies using ion exchange membrane adsorptive
pre-filtration, Biotechnol Bioeng 106, 2010, 627–637,
https://doi.org/10.1002/bit.22729.
effective
adsorptive
prefilters

Viresolve™ Prefilter Viresolve® Pro Shield
Feed pH and conductivity impact adsorptive prefilter performance
mixed mode / hydrophobic Viresolve™ Prefilter (VPF) is less impacted,
where cation exchange Viresolve® Pro Shield (Shield) is more impacted by pH & condo
14
data generated by
Millipore R&D
same data from
previous slide
data I generated in
PD lab

The choice of best adsorptive prefilter ViresolveTM Prefilter (VPF) vs. Viresolve® Pro Shield H
pros and cons [filter suppliers have adsorptive prefilter selection guides to help:
VPF, Shield, Shield H, Millistak+® HC Pro (X0SP)]
15
(3) steps for capacity optimization:
1. run virus filter by itself
2. run with various adsorptive prefilters
3. based on results / preferences, set
process design (z preliminary L/m2 capacity
with adsorptive prefilter A, B, C or D)
membrane filter with different
surface chemistry, works at
higher pH and condo, where
regular Shield does not
clean fully synthetic, silica-
based depth filter with lower
extractables, vs. diatomaceous
earth containing VPF depth filter

Adsorptive Prefilter Watchouts
L/m2 loading / binding site dependency AND residence time dependency
16
G. Bolton, S. Spector and D. Lacasse, Increasing
the capacity of parvovirus-retentive membranes:
performance of the Viresolve™ Prefilter, Biotechnol
Appl Biochem 43, 2006, 55–63,
https://doi.org/10.1042/BA20050108.
Photos (left) are from:
P. Genest, H. Ruppach, C. Geyer, M. Asper, J. Parrella,
B. Evans and A. Slocum, Artifacts of virus filter
validation, BioProcess Int 11, 2013, 54–61.
adsorptive PF overload
binding sites saturated
adsorptive PF feed pressure / flow too
high, not enough residence time for
aggregate to bind

Flowrate
(L/m2/h)
or
Permeability
(L/m2/h/psi)
Initial flowrate or
permeability with buffer
Guidelines for virus filter sizing in PD, run full process simulation
(watchout for adsorptive binding site saturation or change in virus filter fouling profile)
17
Flowrate
(L/m2/h)
or
Permeability
(L/m2/h/psi)
Initial flowrate or
permeability with buffer
?
predicted
extrapolated
volumetric
throughput
endpoint with
short run
?
predicted
extrapolated
volumetric
throughput
endpoint with
short run
adsorptive prefilter and virus filter virus filter alone

Guidelines for virus filter sizing in PD, watchout for high filter plugging
(high filter plugging can potentially lower virus removal level [LRV, Log Reduction Value])
18
(LRV)
LOG
Reduction
Value
% flow decay = 100 * (1 - Q/Qi)
1
2
3
4
5
6
20 40 60 80 100
Viresolve® NFP
Viresolve® PRO
specific
behavior
confirmed
during
validation

No impact to capacity, but at any point, watchout for process pause
(process pause can potentially lower virus removal level [LRV, Log Reduction Value])
19
(LRV)
LOG
Reduction
Value
1
2
3
4
5
6
1. feed pressure /
strong convective
flow brings virus
to original point of
capture by a
retentive pore
2. process pause =
zero pressure and flow
3. virus diffuses to a
new location
4. virus passes through
non-retentive pore into the
filtrate, with re-start
Viresolve® NFP Viresolve®PRO
solid bar, before process pause
hollow bar, after process pause
specific behavior
confirmed during
validation

Steps of virus filter development, relative level of difficulty, relationships
20
process development
(proof of concept, capacity
optimization, sets initial L/m2 design
expectation)
level of difficulty = 3-6
pilot scale up
(proof of scalability, generation of
clinical material?)
virus validation,
virus spiking study
(proof of virus removal capability,
sets maximum L/m2 throughput)
large scale implementation
(sets SOP based on
all best practices,
max L/m2 set by validation)
LOD 1 = easiest
LOD 10 = hardest
lod based on
sensitivity of the feed
lod easier with fresh
feed & in-line
adsorptive prefilter
use appropriate safety
factor to cover L/m2 in
tougher validation
environment
test the frozen / thawed
and adsorptive prefilter
decoupling impacts prior
to validation (required for
validation)
feed & in-line
lod harder with frozen /
thawed feed & de-
coupled adsorptive
prefilter
lod harder with extra
handling (multiple transfers,
vacuum filtration, etc.)
lod harder with virus
spike addition
feed & in-line
watchouts
do not UF concentrate the feed
material prior to validation (not
in the process, not
representative)
do not run max L/m2 seen
in PD for clinical material
prior to tougher validation
(may not achieve this in
tougher validation
environment)

Virus filter scalability (linked to the scale down model for validation)
21
Volumes at
300 and 900 L/m2
90 – 300 ml
5 – 15 L
20 – 60 L
70 – 200 L
150 – 450 L
450 – 1400 L
@ 5g/L
2300 – 7000 g
Viresolve® Pro Solution Performance Guide
Lit No. RF1013EN00 Rev. B 11/14 DP SBU-12-07371
Printed in the U.S.A. ©2014 EMD Millipore Corporation,
Billerica, MA 01821 U.S.A. All rights reserved.

The virus filter validation process (for virus filter alone)
22
Feed Sample
(Cvf)
30 psi
2. Filter ‘Spiked’ Solution
3.1 cm2
* ~ 107 – 108 XMuLV or MMV
~ 225 ml Feed 726 L/m2
~ 0.23 ml of *virus prep
(0.1 vol % spike)
1. Spike Virus
0.22um
or
0.45um
Goal = demonstrate target virus LRV at target filter throughput (L/m2)
3. Assay & Calc. LRV








=
p
vp
f
vf
V
C
V
C
LOG
LRV
Filtrate Sample
(Cvp)
viruses too large to pass
through parvovirus filter
pores
small MVM virus can pass
through parvovirus filter
pores

23
• Run at low pressure /
flow (controlled to same
flow as would occur with
the adsorptive prefilter
coupled to the parvovirus
filter) = proper residence
time for effective
aggregate removal
• Dip tube used to gently
place the adsorptive
prefilter filtrate in
collection vessel =
minimal air liquid
interfacing / minimal new
aggregate formation
before the parvovirus
filter
• Note that adsorptive
prefilter filtrate sits for
process time (30-120
minutes), new aggregate
does not typically, but can
reform with time before
the parvovirus filter
• Pour virus
spiked
adsorptive
prefiltered /
sterile filtered
filtrate slowly /
gently down side
wall of the feed
vessel =
minimal air
liquid interfacing
/ minimal new
aggregate
formation before
the parvovirus
filter
• Low vacuum and filtrate
down side wall = minimal
air liquid interfacing /
minimal new aggregate
formation before the
parvovirus filter
The virus validation process (with adsorptive prefilter)
decoupled, 2X process time (1X for adsorptive prefilter, 1X for virus filtration)
VIRUS
MONODISPERSITY
MICROFILTRATION
(NO GROSS VIRUS
AGGREGATION)

24
6. a type of parvovirus filter
(removes virus, shows maximum
capacity / minimal fouling with
effective in-line removal of aggregates
[both short residence time between
filters and no harsh handling])
3. virus feed injection syringe
(pushes an amount of virus into the
drug product feed, post adsorptive
prefilter, pre parvovirus filter)
1. a drug product feed
(either pumped direct from a bind
and elute chromatography column or
supplied to filters from pressurized
feed vessel via pressurized gas at
constant 30 psi, as shown, or from a
collected chromatography pool [flow
through or bind and elute step])
4. an in-line
static mixer
(mixes
injected virus
with drug
product feed)
5. feed sample syringe
(pulls at the same rate as the virus feed
injection, provides a measure of total virus
added in the drug product feed, post
adsorptive prefilter, pre parvovirus filter)
syringe pump
(push and pull)
filtrate
collection
(sample
assayed to
show how
much virus
got removed
by the virus
filter)?
Virus filter validation
with in-line virus injection
(coupled, direct in-line, minimal time in between the two filters, minimal
handling in between the two filters,
more representative of manufacturing process)
2. an adsorptive prefilter
(used for aggregate removal to protect
the parvovirus filter from fouling)

Publication that describes many of the challenges that exist in validation
Feed sample collection, feed shipping, frozen / thawed feed, decoupled adsorptive prefilter, virus spike addition,
extra handling from microfiltration for virus monodispersity, etc.
25
Figure 2 and Photo 1 are from:
P. Genest, H. Ruppach, C. Geyer, M. Asper, J. Parrella, B. Evans and A. Slocum,
Artifacts of virus filter validation, BioProcess Int 11, 2013, 54–61.
other photos, I took while in a virus spiking lab

Publication that describes many of the challenges that exist in validation
Feed sample collection, feed shipping, frozen / thawed feed, decoupled adsorptive prefilter, virus spike addition,
extra handling microfiltration for virus monodispersity, etc. the need for a dip-tube
26
Figure 3, Figure 6, and Figure 5 are all from:
P. Genest, H. Ruppach, C. Geyer, M. Asper, J. Parrella, B. Evans and A.
Slocum, Artifacts of virus filter validation, BioProcess Int 11, 2013, 54–61.

The importance of virus prep purity, the path to more pure virus preps
27
CRUDE
ULTRA 1: lot 1
ULTRA 1: lot 2
ULTRA 2: lot 1
ULTRA 2: lot 2
ULTRA 3: lot 1, lot 2 and lot 3
RUNspike
work around method 1
1012 titer bacteriophage in place
of 107 titer mammalian viruses
work around method 2
PDA Journal of Pharmaceutical Science and
Technology, Technical report, No. 47, Preparation of
virus spikes used for virus clearance studies, 2010,
University of Iowa, Iowa City (Iowa).
D.R. Asher, A. Slocum, K.F. Bergmann, P. Genest, A.B.
Katz, J.J. Morais, et al., Predicting virus filtration
performance with virus spike characterization,
BioProcess Int 9, 2011, 26–36.
Cabatingan, M. Impact of virus stock quality
on virus filter validation. Bioprocess
International 2005, 3 Supplement 7), 39-43
A. Slocum, M. Burnham, P. Genest, A.
Venkiteshwaran, D. Chen and J. Hughes, Impact of
virus preparation quality on parvovirus filter
performance, Biotechnol Bioeng 2013, 110,

28
Even with purified virus preps, higher % virus spikes can still
reduce filter capacity (use only enough virus to hit target LRV)
Can do virus scoping runs with various % spikes to see the
capacity impact (and not assay, until final % spike is decided)
P. de Vilmorin, A. Slocum, T. Jaber, O. Schaefer, H. Ruppach
and P. Genest, Achieving a successful scale-down model and
optimized economics through parvovirus filter validation using
purified TrueSpikeTM viruses, PDA J Pharm Sci Technol 69,
2015, 440–449, https://doi.org/10.5731/pdajpst.2015.01054.
low
purity
virus
spikes
high
purity
virus
spikes
high
purity
virus
spikes
high
purity
virus
spikes
high
purity
virus
spikes
More pure prep collaborations (using Millipore generated pure viruses)

29
5 g/L Mab
Weight (g) / Volume (ml) vs. Time Data
Pressure Regulator
(15 psi)
~200 ml
4.5 g/L
Mab D
Vpro lot no
M1200607
AVP-4
Vpro lot no
M1200607
AVP-4
Valves
~200 ml
4.5 g/L
Mab D
Pressure Regulator
(30 psi)
Constant Pressure
Pump
(125 LMH)
~200 ml
4.5 g/L
Mab D
Vpro lot no
M1200607
AVP-4
Vpro lot no
M1200607
AVP-4
Valves
Pump
(250 LMH) ~200 ml
4.5 g/L
Mab D
vs. Constant Flow
Pressure Regulator
(15 psi)
~200 ml
4.5 g/L
Mab D
Vpro lot no
M1200607
AVP-4
Vpro lot no
M1200607
AVP-4
Valves
~200 ml
4.5 g/L
Mab D
Pressure Regulator
(30 psi)
Pressure Regulator
(15 psi)
~200 ml
4.5 g/L
Mab D
Vpro lot no
M1200607
AVP-4
Vpro lot no
M1200607
AVP-4
Valves
~200 ml
4.5 g/L
Mab D
Pressure Regulator
(30 psi)
Constant Pressure
Pump
(125 LMH)
~200 ml
4.5 g/L
Mab D
Vpro lot no
M1200607
AVP-4
Vpro lot no
M1200607
AVP-4
Valves
Pump
(250 LMH) ~200 ml
4.5 g/L
Mab D
vs. Constant Flow
Pump
(125 LMH)
~200 ml
4.5 g/L
Mab D
Vpro lot no
M1200607
AVP-4
Vpro lot no
M1200607
AVP-4
Valves
Pump
(250 LMH) ~200 ml
4.5 g/L
Mab D
Pump
(125 LMH)
~200 ml
4.5 g/L
Mab D
Vpro lot no
M1200607
AVP-4
Vpro lot no
M1200607
AVP-4
Valves
Pump
(250 LMH) ~200 ml
4.5 g/L
Mab D
vs. Constant Flow
P. Genest, H. Ruppach, C. Geyer, M. Asper, J. Parrella, B. Evans and A.
Slocum, Artifacts of virus filter validation, BioProcess Int 11, 2013, 54–61.
Capacity and LRV Impacts from mode of operation
constant pressure vs. constant flow

30
Virus capacity (L/m2) optimization, example case study
Jaime De Souza, Ken Scott, and Paul Genest. Virus-Filtration Process Development
Optimization. BioProcess International, (14) 4, April 2016 pp. 62-74.

1. How is virus filter capacity performance measured?, slide 4
2. How is the final design L/m2 throughput determined (PD, to pilot, to validation, to manufacturing scale)?, slide 5-6
3. Where is the best placement, within the overall process, for the virus filter (VF)?, slide 7
4. How to deal with virus filter plugging / non-optimal filter capacity?, slides 8-10 and 12-16, 15 is poll question 1
5. What are the performance behaviors from feed pressure, feed concentration, and buffer flushing?, slide 11
6. What is the best way to size virus filters during PD (process simulation)?, slide 17
7. What is the virus removal / LRV impact from level of filter plugging?, slide 18
8. What is the virus removal / LRV impact from process pause?, slide 19
9. The steps for virus filter development, their relative levels of difficulty, and how they are related?, slide 20
10. Scalability and the importance of the scale down model used for virus validation?, slide 21
11. What is the validation process (with no adsorptive prefilter)?, slide 22
12. What is the validation process (with an adsorptive prefilter decoupled)?, slide 23
13. What is in-line virus injection (with an adsorptive prefilter still coupled) and where it is helpful to use?, slide 24
14. What are some additional validation challenges / behaviors, and best practices?, slides 25-27, 27 is poll question 2
15. What is the impact from an impure virus spike and or from too high a % virus spike, what is the history regarding the
pursuit of pure virus preps?, slides 28-30, 30 is poll question 3
16. How does constant pressure vs. constant flow operation affect capacity and LRV?, slide 31
31
Table of contents, detailed (filter capacity performance)

29
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filter, Biotechnol Prog 18, 2002, 782–795, https://doi.org/10.1021/bp010193.
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Applications Engineer,
BioPharm Center of Excellence - Innovation + Influence = Impact
Paul Genest
The vibrant M, Millipore®, Viresolve® Millistak+® are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All
other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly
accessible resources.
© 2019 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

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