Diafiltration (DF) is at the heart of the final downstream process step for a majority of mAb-based and other therapeutic molecules. However, as process templates have adapted to be more flexible, handle larger batch sizes, require lower plant footprint, and run in an integrated or continuous mode, DF has been one of the last unit operations to change. This poster describes a solution for continuous diafiltration that includes the following characteristics: • Requires only a small modification to standard operating strategies • Delivers a continuous process • Yields significant reductions in membrane area and system size 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. Continuous DF via Tank Cycling Dramatically Reduces
Membrane Area and System Size
• 350 g IgG diafiltered using 0.11 m2 of membrane
• Membrane actively in use for diafiltration ~84% of total time
• 3.2 kg/m2 processed in ~19 hours (= 4 kg/m2/day)
• 15 m2 for 60 kg batch in 1 day (12k L bioreactor @ 5 g/L titer)
• 0.11 m2 for 200 L perfusion bioreactor (@ 1.2 g/L and 2 VVD)
Summary
• We have developed a solution for Continuous Diafiltration
that utilizes two recirculation tanks to cycle between
filling/emptying and diafiltering which enables continuous
flow in and out of the system
• Significantly reduced membrane area due to higher
utilization of filter and extended process time
• Smaller volume sub-batch cycles reduce tank footprints
• Loading filter modules in series to increase conversion per
pass offers opportunity to reduce pump passes
• Smaller system size facilitates use of sanitized/sterilized
closed single-use flowpaths
• Smaller piping simplifies recovery for high product yield
• Leverages known batch DF operations  insignificant
process changes or risks during development or scaleup
• Can be used to de-bottleneck existing facilities (process
larger batches on existing equipment) and enable fully
continuous DSP for perfusion processes
Bench-scale demonstrations are available
Results
Concept Testing:
• Polyclonal hIgG (Seracare Life Sciences, Inc.)
• Pellicon® 3 cassette with Ultracel® 30kD membrane
2 design cases evaluated:
• Low area: standard crossflow,
standard membrane loading
• Low pump pass: low crossflow,
series membrane loading
New Approach to
Continuous Diafiltration
Other industry options to replace batch DF leave room for
additional efficiency gains:
• Single-Pass Inline Diafiltration (ILDF)
• Cycling of two separate systems
MilliporeSigma’s approach to Continuous Diafiltration (CDF)
maintains well-established, robust batch DF operation
using constant-volume DF for the most efficient use of
membrane area and buffer. However, it utilizes two cycling
recirculation tanks to process aliquots of protein
sequentially over an extended duration, so the membrane
is nearly always in-use for active protein processing.
Introduction
Diafiltration (DF) is at the heart of the final downstream
process step for a majority of mAb-based and other
therapeutic biomolecules. For many years, it has provided
a cost-effective, efficient, and robust method for achieving
> 3 logs of buffer exchange in a unit operation that is also
able to manipulate the final product concentration to a
desired target. But as process templates have adapted to
be more flexible, handle larger batch sizes, require lower
plant footprint, and run in an integrated or continuous
mode, DF has been one of the last unit operations to
change. Practices from other industries have been adapted
to provide alternatives to traditional batch-based DF.
However, it has been challenging to exceed established
expectations around unit operation productivity and
maintain a process that is easily implementable and GMP-
friendly.
Here, we describe a solution for continuous diafiltration
that requires only a small modification to standard
operating strategies, while delivering not only a continuous
process, but also significant reductions in membrane area
and system size. The process has been run for 24 hours
with no cleaning, and there was no noticeable degradation
in pressure, flux, buffer exchange, or yield. The solution
allows for flexibility in process design to accommodate
periodic cleaning, adjustable aliquot volumes, a variety of
product recovery techniques, and methods to reduce pump
passages for shear-sensitive molecules.
24-Cycle Process Performance Consistency
www.emdmillipore.com
A Novel Approach to Diafiltration for
Intensified or Continuous Processing
MilliporeSigma, the vibrant M, Pellicon, and Ultracel 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.
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. Lit. No. MS_PS2889EN Ver 1.0 10/18
Elizabeth Goodrich, Akshat Gupta, Herb Lutz
MilliporeSigma, Burlington, MA, USA
Corresponding Author: Elizabeth.Goodrich@emdmillipore.com
Discussion
Comparison of CDF to Batch and ILDF
In the chart above, various performance metrics for two configurations of
Continuous DF and for Inline DF are normalized to typical Batch DF values.
• Inline DF has benefits of a single pump pass, no recirculation tank, and a
low feed flow rate. However, because SPTFF has much lower flux than
standard TFF, ILDF requires 75% of the area as batch DF even though
the process is stretched to 19 hours. This is also seen in the productivity
(g/m2hr), where ILDF is 22% as efficient as batch. And, since ILDF
relies on sequential concentration and dilution for buffer exchange,
significantly more buffer volume is needed, requiring a large tank.
• Continuous DF uses constant-volume batch DF operating mode, so its
productivity, pump passes, and buffer consumption are equivalent to
batch. However, since the process time is increased to 19 vs 3 hours, the
required membrane area is >6-fold lower (loading in g/m2 is >6-fold
higher). This has a direct impact on feed flowrate (>6-fold lower) and
piping size. In addition, since only ~1/20th of the batch volume is in the
recirculation tank at any given time, the tank size is significantly smaller.
• Low pump pass Continuous DF is achieved by running 2 filters in series.
This allows the feed flowrate to be dropped ~3X while still achieving the
required cycle time, meaning that pump passes are 1/3 of the batch
case. While the membrane area reduction is not quite as large as the
CDF low area case, it is still >3-fold lower than batch DF. This could be
an important consideration for highly shear-sensitive molecules.
The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada
Manual Bench-top CDF System
Pellicon® Mini Cassette holder; peristaltic feed and DF
pumps; two 500 mL recirculation tanks with stir bar
mixing; level switch; load cells for buffer, permeate, and
recovery vessels; pressure, conductivity, and UV sensors
Typical mass loading for Ultrafiltration (UF):
• 150 g mAb/m2-hr or 450 g/m2 for 3 hour process time
Largest systems of 6-high holder accommodate 120m2
• Maximum batch limit approximately 54 kg mAb
• Large tank & footprint, high capital cost, long lead times
• High working volume, potential product recovery dilution
• Long setup (install, flush, IT) and long turnaround (flush,
clean, NWP, sanitize, store)
How can DF be made more efficient?
Opportunities
• Increase utilization from 12% (3 hr process / 24 hr) to
reduce membrane area
– Extra time spent with set up, turnaround, idle time
– Single-use Pellicon® Capsule reduces set up and
turnaround (flush, IT, sanitization)
– Extending process time would directly reduce
membrane area
• Reduce tankage
– Change batch to continuous operation
– Balance continuous production line
Address Risks/Concerns
• Reduce protein degradation by reducing pump passes
– Could add modules in series to increase
conversion per pass
• Reduce bioburden
– Sanitized/sterilized closed system
– Closed, single-use Pellicon® Capsule will aid
bioburden control
• Mitigate membrane fouling
– Include opportunity for periodic cleaning flush
Tank
I
Tank
II
Recovery
Tank To next
process
step
From
previous
process
step
Step Tank Function
Previous
Process
Step
Tank I Feeds Tank I
with Protein
at Copt
Batch DF Tank II
(previously
filled)
Diafiltration
Subsequent
Process
Step
Recovery
Tank
(Previously
diafiltered)
Holds
diafiltered
Protein for
concentration
to CFinal
0
20
40
60
80
2.0 3.0 4.0 5.0 6.0
Flux[LMH]
ln CB
Flux versus hIgG Concentration
Ln Cb hIgG in 50 mM Acetate pH 5.5
Ln Cb hIgG in PBS pH 7.2
Buffer ln Cgel
Cgel
[g/L]
k
[LMH]
Copt
[g/L]
PBS pH
7.2
5.2 183 30.12 67.33
50mM
Acetate
pH 5.5
5.4 211 33.23 77.56
Step End point Step duration
Diafilter one tank while
other tank is filling
DF buffer = 1800 mL
(9 DV)
~ 39 minutes
Recover product
from tank
Tank is fully drained ~ 3 minutes
Recover product from
lines and membrane
Displaced with 1 holdup
volume of buffer (~56 mL)
~ 4 minutes
Total time per cycle ~ 46 minutes
MilliporeSigma Cycling Tank
Continuous DF Solution
• Utilize existing PD data
• Optimal buffer and membrane use
• Standard membranes and holders
• Proven hardware and controls
• Significant reduction in size & area
Continuous DF Piping Diagram
Components shown in black are equivalent to standard
batch DF system, while components in blue are added to
enable continuous operation via tank cycling
BUFFER
FEED PUMP
DIAFILTRATION
PUMP
TANK I
TANK II
RECOVERY
TANK
To Next Unit
Operation
Continuous DF
TFF
Permeate
From Previous
Unit Operation
Bioreactor Clarification Protein A
Capture
Cation
Exchange
Purification
Anion
Exchange
Polishing
Viral
Inactivation
Virus
Filtration
Concentration
Diafiltration
Formulation
Sterile Filtration
Final Fill
Generic mAb Process Template
Use Existing PD Data from Batch Development
Determine optimum protein concentration for DF (CbDF).
Use flux at CbDF to determine aliquot volume for desired
DV’s, membrane area and cycle time.
24-Cycle Process Run on Manual System:
• 73 g/L hIgG, 200 mL/cycle = 14.6 g per cycle
• 0.11 m2 Pellicon® 3 cassette with Ultracel® 30kD membrane
• 5 L/min/m2 feed flowrate, 1 bar TMP
• 9 diavolumes per cycle
30
35
40
45
0 2 4 6 8 10 12 14 16 18 20 22 24
Cycle Number
Control Chart: DF Cycle Time
Average = 36.9 min, σ = 1.8 min
20
22
24
26
28
0 2 4 6 8 10 12 14 16 18 20 22 24
Cycle Number
Control Chart: Permeate Flux
Average = 24.6 LMH, σ = 1.0 LMH
2.90
2.95
3.00
3.05
3.10
0 2 4 6 8 10 12 14 16 18 20 22 24
Cycle Number
Control Chart: Final Conductivity
Average = 3.00 mS/cm, σ = 0.02
96.0
98.0
100.0
102.0
0 2 4 6 8 10 12 14 16 18 20 22 24
Cycle Number
Control Chart: Product Yield
Average = 98.9%, σ = 0.7 %
50
60
70
0 2 4 6 8 10 12 14 16 18 20 22 24
Cycle Number
Control Chart: Product Concentration
Average = 59.4 g/L, σ = 2.8 g/L
Control graphs: Circles show cycle data; lines indicate 24-cycle average and ±1, ±2, ±3 σ
Manual system performance. Variations are expected to be lower for an automated system.
1 1 1 1 1 1 1
0.16
6.39
1.06
1 1
0.05
0.160.32
3.13
0.52
0.36
1
0.05
0.06
0.75
1.33
0.22
0.01
2.69
0.00 0.01
0
1
2
3
4
5
6
7
Area required
(m2)
Loading per
batch (g/m2)
Productivity
(g/m2hr)
Pump Passes Buffer
Consumption
(L)
Recirculation
Tank (L)
Feed Flowrate
(L/min)
NormalizedtoBatch
All cases: no UF, 9 diavolumes
■ Batch: 3 h process, 6 LMM crossflow
■ CDF low area: 19 h process, 6 LMM crossflow
■ CDF low pump passes: 19h process, 1 LMM crossflow
■ ILDF: 19 h process
10
11
12
0 2 4 6 8 10 12 14 16 18 20 22 24
Cycle Number
Control Chart: Delta Pressure
Average = 11.2 psid, σ= 0.3 psid
100.0
90.3
0
20
40
60
80
100
Pre-Use Post-24 Cycles
%ofOriginalPermeability
Membrane Permeability before and after 24
cycles with no cleaning