This poster presentation outlines the different filtration strategies and performances in the upstream and downstream process to develop a scalable, cost-efficient and GMP-compliant Foot and Mouth Disease (FMD) vaccine production: • Introduction to foot and mouth disease (FMD) and background on FMD vaccines • Review of cell culture, clarification, and concentration/diafiltration steps for the production and purification of the FMD vaccine • Suggested further actions based on data outlined in this poster To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: www.emdmillipore.com/mlab

1. Filtration strategies for optimal
development and purification of a
FMD virus produced in BHK21 cells
Inoculum Propagation Bioreactor
Cell Lysis and
Virus Inactivation
Depth Filtration
Clarification
UF/DF
Final
Filtration
FormulationFill-Finish
Media Preparation
Filter
Batch
Vol
Process
Loading
Amin1 Recommended
Configuration
Resultant
Area
Final
Safety
Factor
L L/m² m² m² x
Durapore®
0.22 µm
1000
724.6 0.71 1x OptiCap XL20 1.38 1.9
Millipore Express®
SHF or PHF 0.2 µm 925.9 0.48 1x OptiCap XL20 1.08 2.2
Millipore Express®
SHC 0.5/0.2 µm 7692.3 0.08 1x OptiCap XL3 0.13 1.6
Millipore Express®
SHR 0.1 µm 1666.7 0.32 1x OptiCap XL10 0.60 1.9
Millipore Express®
SHR-P 0.5/0.1 µm 1020.4 0.38 1x OptiCap XL20 0.98 2.6
1
Amin is the minimum calculated filter area to achieve filtration (with no safety factor)
Filter Filter details
Membrane
Area (cm2
)
Catalogue
number
Millistak+®
HC C0HC 30DE + 60DE 23 MC0HC23CL3
Durapore®
0.45 µm PVDF, Bioburden Reduction 3.5 SPHLA25NB6
Milligard®
PES 1.2/0.45 µm PES, Bioburden Reduction 3.5 SMP4A25NB6
Trial flux
(LMH)
Trial loading
(L/m²)
Trial endpoint inlet
pressure (psi)
Harvest turbidity
(NTU)
Filtrate pool
turbidity (NTU)
152 177.4 20 (Pmax) 260 9.5
Membrane filter
Membrane filter
loading (L/m²)
Trial
Flux decay (%)
Sterilizing-grade
filter Vmax™ (L/m²)
Qi
(LMH)
Durapore®
0.45 µm
478.9 98 545.2 23716.7
Milligard®
PES
1.2 /0.45 µm
596.0 54 1667.2 25671.9
Table 7: Sizing recommendations for 1000 L
The Milligard®
PES 1.2/0.45 µm filter showed better performance
than the Durapore®
0.45 µm filter and would be the preferred
option for the bioburden reduction step, before concentration and
diafiltration of the product.
Concentration/Diafiltration
Following the previous clarification optimization, ME VAC was
looking at developing the concentration step of their FMD vaccine.
The Biomax®
membranes used in this study are made of
polyethersulfone and are resistant to harsh chemicals used
in cleaning, biological decontamination, and sanitization. The
polyethersulfone Biomax®
membrane has been designed to reduce
non-specific protein binding. These membranes are available in up to
4 screen formats: V screen (suspended), C screen (coarse), A screen
(fine), D screen (for high viscosity).
Table 8: Ultrafiltration membranes tested
Figure 5: Flux excursion
No pressure instability was observed with increasing permeate
fluxes. Using the 300 kDa, the trial was stopped at 67.2 LMH with
a final TMP of 5.9 psi with the permeate valve completely open.
The starting TMP was 3.96 psi meaning that the trial was stopped
with a 1.5x increase in pressure. For the 1000 kDa membrane, a
1.37x increase in pressure was noted at the end of the optimization
study, with a permeate valve completely open. It is usual to consider
pressure instability when the increase in pressure is > 1.5-2.0
and recommended to use the membranes at 75% of the maximal
working flux.
Both membranes can be used with approximately the same
hydraulic performances. The 300 kDa membrane tested was
completely new while the 1000 kDa had been previously used by ME
VAC for concentration without controlled monitoring of the system
pressure and flowrates (feed and permeate). Its performance during
this optimization study can therefore be affected by this previous
use and potential membrane unbalanced polarization.
Table 9: Recommended operating parameters
Further actions:
• Test a process simulation to prove feasibility at sufficiently high
loading with permeate pump control.
• Repeat multiple batches to investigate the effectiveness of the
CIP procedure.
• Assess product recovery and impurity removal by sampling the
retentate and permeate lines.
Figure 2: Throughput profile over time
Table 3: Sizing recommendations for 1000 L
Clarification
Two harvests were tested for clarification using a the Pmax/Tmax
methodology at a constant flowrate.
Table 4: Depth filter and prefilters tested
Figure 3: Millistak+®
HC C0HC pressure profile
Figure 4: Millistak+®
HC C0HC turbidity profile
Table 5: Summary of Millistak+®
HC C0HC filtration results
The results indicate that the Millistak+®
HC C0HC filter is capable of
handling the BHK21 cell culture in a single step, without requiring a
secondary depth filter.
The filterability of the filtrate was eventually assessed on bioburden
reduction membranes. Constant pressure tests were performed
at 10 psi with Durapore®
0.45 µm and Milligard®
PES 1.2/0.45 µm
small scale filters in order to determine the theoretical maximum
volume of each solution filterable on the membrane. This maximum
volume is called “Vmax™” value. The initial filtrate flux (Qi) was also
determined in order to estimate the minimum area required to filter
the batch.
Table 6: Summary of results from Vmax™ experiments
12
10
8
6
4
2
0
0 20 40 60 80 100 120 140 160 180
Turbidity(NTU)
Throughput (L/m²)
Filter Filter details
Catalogue
number
Membrane
area
(cm2
)
Durapore®
0.22 µm filter PVDF (bacterial retention) SVGLA25NB6 3.5
Millipore Express®
SHF 0.2 µm filter PES (bacterial retention) SGEPA25NB6 3.5
Millipore Express®
SHC 0.5/0.2 µm filter PES (bacterial retention) SHGEA25NB6 3.5
Millipore Express®
SHR 0.1 µm filter
PES (bacterial, mycoplasma
retention)
SVEPA25NB6 3.5
Millipore Express®
SHR-P 0.5/0.1 µm filter
PES (bacterial, mycoplasma
retention)
SHVEA25NB6 3.5
Prefilter
Diff.
Pressure
Trial
Loading
Trial Flux
Decay
Vmax™
psi L/m² % L/m²
Durapore®
0.22 µm filter
10
540.0 69.6 1782.09
Millipore Express®
SHF 0.2 µm filter 1437.1 71.6 2080.43
Millipore Express®
SHC 0.5/0.2 µm filter 1260.0 2.5 1758003.38
Millipore Express®
SHR 0.1 µm filter 888.6 36.8 N/A
Millipore Express®
SHR-P 0.5/0.1 µm filter 965.7 35.8 2685.21
Filter
Amin
(m²)
Suggested
config.
Area
(m²)
Safety
Factor
Process
loading
(L/m²)
C0HC 5.3 7x 1.1 m² 7.7 1.5 130.0
Durapore®
0.45 µm filter 1.84 3x Opticap®
XL20 filter 3.7 2.0 268.8
Or Milligard®
PES 1.2/0.45 µm filter 0.61 1x Opticap®
XL20 filter 1.2 2.0 833.3
Device Membrane Screen Catalogue # Area (m2)
Pellicon®
2 cassette
Biomax®
300 kDa membrane
C
P2B300C01
0.1
Biomax®
1000 kDa membrane P2B01MC01
Membrane
Pump
Flowrate
TMP
(psi)
Set permeate Flux
(LMH)
300 kDa
4 LMM
5 50
1000 kDa 6.5 51
0
100
200
300
400
0 2 4 6 8 10 12 14 16
Throughput(L/m²)
Time (min)
Durapore®
0.22 m filter Millipore Express®
SHF 0.2 m filter Millipore Express®
SHC 0.5/0.2 m filter
Millipore Express®
SHR 0.1 m filter Millipore Express®
SHR-P 0.5/0.1 m filter
0
5
10
15
20
25
0 20 40 60 80 100 120 140 160 180
Pressure(psi)
Throughput (L/m²)
3.90
10.24 18.00 25.80
43.20 51.60
64.80 67.20
0.80 7.50 8.40
27.60
44.40
61.20 66.00 67.80
8
7
6
5
4
3
2
1
0
0 20 40 60 80 100 120
TMP(psi)
Time (min)
300 kDa – Flux (LMH)
1000 kDa – Flux (LMH)
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Lit. No. MS_PS7158EN
Summary
The present work shows MilliporeSigma portfolio capabilities for both the upstream (cells, cell culture media, filtration) and
downstream (Clarification and TFF) steps allowing the production and purification of the FMD vaccine:
• Cellvento®
BHK-200 serum-free medium for FMDV production in MilliporeSigma BHK21 cells can easily be filtered
using Millipore Express®
filters (for bacteria or and/or mycoplasma clearance)
• High capacity and low turbidity achieved on the clarification step allowing a very
cost-efficient and low foot print scale-up with the Millistak+®
HC C0HC filter
• Pellicon®
2 300 kDa or 1000 kDa cassette can be implemented for the
concentration and diafiltration of the FMD vaccine
Abstract
ME VAC is working on the development of a Foot and Mouth
Disease (FMD) vaccine and has entered a collaboration
with our company, starting with the use of Cellvento®
BHK-200 cell culture media. This media was developed for
the growth of specifically adapted BHK21 cells and allows
the replication of the FMD virus in a completely serum-
free environment. The present work outlines the different
filtration strategies and performances in the upstream and
downstream process to develop a scalable, cost-efficient
and GMP compliant FMD vaccine production.
MilliporeSigma: Y. Cherradi, N. El Hajjami, Y. Gaabouri, A. Boumlic
ME VAC: L. Dudnikov
Introduction
A ME VAC - MilliporeSigma collaboration
Foot and mouth disease (FMD) is a severe viral disease capable of
infecting and sometime killing cattle, swine, sheep, goats and other
cloven-hoofed ruminants, thereby strongly affecting the production
of livestock. Massive vaccination with at least 80% coverage is one
of the control strategies implemented to prevent virus introduction
and development. Typical FMD vaccines are inactivated viruses
initially grown in mammalian cell culture. In this study ME VAC and
MilliporeSigma collaborated to explore optimal filtration strategies to
build cost-efficient upstream and downstream operations.
Figure 1: FMD Vaccine Process
Cell Culture Media
Different cell culture media can be used for BHK21 cell propagation.
Most propagation media contain serum, as high as 10%. In order
to increase biosafety by preventing risks of bovine spongiform
encephalopathy (BSE), transmissible spongiform encephalopathy
(TSE) agents and mycoplasma contaminations, use of a serum-
free medium was preferred. The Cellvento®
BHK-200 medium is a
serum-free cell culture medium, formulated without animal derived
component and optimized for the culture of suspension BHK21 cells
at high-density and viability with efficient propagation of viruses.
During preparation of the media, a sterilizing-grade filtration is
performed and different filters were screened in order to define the
most efficient option.
Table 1: Sterilizing-grade filters tested
The medium was challenged against different sterilizing-grade filters
(for bacterial and/or mycoplasma retention). The best capacity was
observed with the Millipore Express®
SHF 0.2 µm and SHC 0.5/0.2
µm filters for the sterilizing-grade options. The latter is however
showing no signs of plugging in comparison with the single layer
Millipore Express®
filter. Very similar performances were obtained
between the two mycoplasma-retentive Millipore Express®
filters.
Table 2: Filtration trial results