This document presents a new continuous diafiltration (CDF) system as an efficient solution for intensified or continuous bioprocessing. The CDF system uses tank cycling to keep the membrane in use for diafiltration for most of the time. A 24-hour demonstration of the CDF system showed stable and consistent performance over 24 cycles with less than 6% variation in key parameters like cycle time, flux, conductivity, yield and product concentration. The CDF solution provides 6-8 fold higher membrane utilization compared to batch diafiltration with comparable buffer usage but significantly reduced footprint and linear scalability. It is a robust solution for continuous processing that does not require process re-development.

1. Merck KGaA
Darmstadt, Germany
Akshat Gupta and Elizabeth Goodrich
October 10, 2019
An Efficient and cGMP-
Friendly Solution to
Diafiltration for
Intensified or Continuous
BioProcessing

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

3. Agenda
01 The Case for Continuous Diafiltration
02 An Efficient Solution to Continuous Diafiltration
03 24-Hour Process Demonstration
04 Conclusions

4. The Case for
Continuous
Diafiltration
01

5.  Deliver target molecule in final formulation buffer and at specified concentration
 Removes low MW solutes
 Typical mass loadings: 150 g mAb/m2-hr or ~ 0.5 kg/m2 for typical 3-hr processing time
 Largest systems of 6 high holder with 120m2 limited to 60 kg batches
 Large tank & footprint, high capital and long lead times
 High working volume & potential product recovery dilution
 Long setup (install, flush, IT) and turnaround (flush, clean, NWP, sanitize, store)
General mAb process:
Bioreactor Clarification Protein A
Capture
Cation
Exchange
Purification
Anion
Exchange
Polishing
Viral
Inactivation
Virus
Filtration
Concentration
Diafiltration
Formulation
Sterile Filtration
Final Fill
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 2019
Background: Batch Formulation Step
5

6. Evolution to Continuous Processing
Intensified Upstream
& harvest
Intensified
Capture
In-Line VI
In-Line Buffer
Flow Through
Polishing
SPTFF &
Continuous
DF
• Manufacturers pursuing continuous processes are starting to address this step
• Existing solutions present implementation challenges
• Can we do better than batch UF/DF?
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 20196

7.  Typically comprised of 3 steps (UF, DF, UF) plus Product Recovery
 Diafiltration occurs at constant volume and constant (specified) concentration
DF consumes the majority of process time (membrane area) of the overall operation
Final Formulation Batch UF/DF Operation
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 20197
Initial concentration
Diafiltration
Final concentration
0
20
40
60
80
100
120
140
160
0% 20% 40% 60% 80% 100%g/Lconcentration
process time
UF UFDF
~10 to 70g/L ~8 to 10 Diavolumes @
Copt (70g/L)
~70 to
150g/L

8. Standard batch DF optimizes membrane area and buffer usage
• Diafiltration at constant volume (i.e level control) results in
more efficient buffer exchange versus a series of UF
concentrations followed by buffer dilution
• Diafiltration at the optimal protein concentration provides the
best balance between flux during DF and volume needed to
be diafiltered
• Results in lowest required membrane area
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 20198
DF Optimization Parameter = CbDF * JDF
Optimum Cb
for DF

9. Process Objectives
How can we make
DiaFiltration
Continuous
And
more efficient
>3.5 log clearance +
safety factor
~8 DV buffer exchange
Improve membrane
utilization
Minimum process
re-development
Comparable buffer
Usage
Flexibility to
accommodate
challenging molecules
Reduce system
footprint
Straightforward,
Robust performance
Scalable
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 20199

10. An Efficient
Solution to
Continuous
Diafiltration
02
10 An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 2019

11.  Sequence of inline concentration and
inline dilution steps
 In many industries 2-3 stages are
sufficient but biotech needs about 6
such steps to get 8 diavolumes
equivalent clearance
 For same process time, more area
than batch DF
 For same g protein, more buffer than
batch DF
 Not easy-to-use: Difficult to balance 6
DF flows and 6 permeate flows over
extended operation
Continuous Option 1: Inline UF/DF
Protein solution
Water
Equilibration
buffer
Sanitization
buffer
Permeate
Waste
Diafiltration
buffer
Concentrated,
purified
protein
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 201911

12.  Have two systems
cycling back and forth
 Fill holding tank
 System1 DF
 System 2 recovery
& turnaround
 Ease-of-use & efficiency
of batch
 Membranes only work
50% of the time, but
better than 12%
Permeate
Waste
Diafiltration buffer
Protein solution
Water
Equilibration buffer
Sanitization buffer
Permeate
Waste
purified protein
System 1 Diafiltering System 2 Recovering, Flushing, Filling
Continuous Option 2: Cycle batch DF systems
Diafiltration buffer
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 201912

13. Cycle
#
Tank
I
Tank
II
Recovery
Tank
0 Feed Fill Idle Idle
1a Constant
Volume DF
Feed Fill Idle
1b Recovery
Drain
Continue Feed
Fill
Recovery Fill
2a Feed Fill Constant
Volume DF
Transfer
2b Continue
Feed Fill
Recovery Drain Recovery Fill
and Transfer
Etc.
Continuous Option 3: pre & post SPTFF, Cycle just the tanks in DF
Tank
I
Tank
II
Recovery
Tank
To next
process step
(SPTFF)
Continuous Feed
From previous
process step
(SPTFF) at Copt
 Use membrane for diafiltering most of the time
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 201913

14. Conventional batch
Added for cycling DF
- Tank II
- 3 Switching valves
BUFFER
FEED PUMP
DIAFILTRATION
PUMP
TANK I
TANK II
RECOVERY
TANK
To Next Unit
Operation
Continuous DF
TFF
Permeate
From Previous
Unit Operation
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 201914
Continuous DF via Tank Cycling

15. 24-Hour Process
Demonstration
03
15 An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 2019

16. Standard TFF Process Development strategy:
 Select feed flow rate setpoint
 Optimize TMP setpoint
 Determine optimum protein concentration for DF
 Determine required number of diavolumes
See TB032 and AN2700 for detailed information
Process parameters are the same as for Batch DF
Buffer
Cgel
[g/L]
k [LMH] Copt [g/L]
PBS pH 7.2 183 30.12 67.33
50mM Acetate
pH 5.5
211 33.23 77.56
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 201916

17. Breaking a batch DF process into cycles
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 201917
Scenario 2: CDF System needs to be designed for a given mass flowrate coming from previous unit
operation
Scenario 1: CDF System set up for evaluation with defined membrane area and defined cycle time
or cycle volume for given protein
Cycle Volume = (Flux* Area)*(Cycle time – recovery time – safety factor time) / (60*DV’s)
Cycle Time = {[(Cycle volume*DV’s*60) / (Flux*Area)] + Recovery time} / (1 – safety factor %)

18. Bench-Scale Manual CDF Setup
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 201918
Recirculation tank I and tank II
Feed pump
Membrane
Permeate
collection
Back pressure
controller
Inline
conductivity
Feed,
retentate and
permeate
pressure
Diafiltration
buffer delivery
Recovery tank

19. An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 201919
Close-up of Recycle Tanks
Recirculation tank I and tank II
Switching valves

20. CDF Performance
 Manual CDF operation over 24 cycles with sensors to monitor
− Easy to control- constant DF addition, switch cycles & recovery steps
− Pellicon 3 0.1 m2 minicassette at 5LMM crossflow
− Small tank (2L/m2) with 0.2L IgG feed/cycle at 70 g/L, 14 g/cycle
− High loading (~ 4 kg/m2/day)
− ~46 min cycle= 39 min DF (9 DV‘s, ~4 min per DV), 3 min tank drain, 4 min system flush
 Stable 24 cycle-to-cycle manual operation
− Cycle DF time 37min ±5%, Average flux/cycle 24.6LMH ±4%, DP 11.2psi ±3%
− Cycle Pool Conductivity 3mS/cm ±1%, Yield 98.9%±1%
− Pool product concentration 59g/L ±5%
− No fouling- stable NWP between cycles
− Consistent with other mAb feed runs
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 201920

21. Cycle Time, Flux and Buffer Exchange are Consistent over 24 Cycles
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
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 201921

22. Yield and Final Recovered Product Concentration and Pressure drop
are Consistent over 24 Cycles
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
55
60
65
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
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 201922
10
10,5
11
11,5
12
12,5
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

23. Membrane Buffer Permeability Maintained without Cleaning
100,0
90,3
0
10
20
30
40
50
60
70
80
90
100
110
Pre-Use Post-24 Cycles
%ofInitialBufferPermeability
Buffer permeability pre and post 24 cycles
with no intermediate cleaning
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 201923

24. 1 1 1 1 1 1 1
0,16
6,39
1,06 1 1
0,05 0,16
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
(gm/m2)
Productivity
(gm/m2.hr)
Pump Passes Buffer
Consumption (L)
Tank (L) Feed Flowrate
(ml/min)
NormalizedtoBatch
CDF Testing
Performance Comparison
All cases: no UF, 9 diavolumes
 Batch: 3h process, 6LMM crossflow
 CDF low area: 19h process, 6LMM crossflow
 ILDF: 19h process
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 201924

25. Conclusions
04
25 An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 2019

26. ▪ Successfully developed a continuous diafiltration process
▪ >3.5 log clearance + safety factor
▪ Same buffer consumption as batch process
▪ No redevelopment required batch data can be used directly
▪ 6-8 fold improvement in membrane utilization
▪ Significant reduction in system foot print
▪ Linearly scalable
▪ Flexibility to accommodate challenging molecules
▪ Robust performance demonstrated over 24 hours operation
▪ Patent application filed November 2018
Summary
An Efficient and cGMP-Friendly Solution to Diafiltration for Intensified or Continuous BioProcessing | 10 October 201926

27. Director of MSAT Applications
Engineering
Email:
Elizabeth.Goodrich@emdmillipore.com
Senior BioProcess Engineer,
MSAT Applications Engineering
Email:
Akshat.Gupta@emdmillipore.com
Elizabeth GoodrichAkshat Gupta
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