Optimization of Tangential Flow Filtration Applications and Scale Up Considerations

Merck KGaA
Darmstadt, Germany
Josselyn Haas Durr
European Biomanufacturing Engineer Manager
Manufacturing Sciences and Technology, EMEA
Viral and Gene Therapy Seminar
June 25, 2019
Optimization of
Tangential Flow Filtration
(TFF) and Scale Up
Considerations

01
02
03
04
Agenda
2
Basics of Tangential Flow Filtration (TFF)
TFF AAV and Lentivirus Process Overview
Operating Parameters Optimization – Flux
controlled Microfiltration (MF)
Scale Up Considerations

3
Tangential Flow FiltrationNormal Flow Filtration
What is Filtration?
Filtration is a pressure-driven separation process that uses filters (membrane) to
separate components in a liquid (or suspension) based on their size.
There are two types of filtration:
Feed pump NFF housing
Filtrate
pump
Feed
pump
TFF
module

Tangential Flow Filtration (TFF)
Filtratefluxrate
Volume filtered
Feed Pressure
Membrane surface
Filtrate (Out)
Wall Concentration Cw
Liquid Tangential Flow
Bulk
Concentration Cb
4

No membrane
polarization
Controlled
membrane
polarization
Uncontrolled
membrane
polarization
Membrane Polarization Must be Controlled
5

Microfiltration (MF) Membrane Structure
Rated pore size
 0.10 – 0.65µm is typical in biopharm TFF
1
2
3
Typical Applications
 Concentrate cells or cell debris
 Clarify proteins or viruses from high
MW unwanted material
 Wash cells or cell debris
Typical membrane materials
 Cellulosic
 Modified PVDF
 Modified PES
0.2 µm PVDF Membrane magnified 650×
6

Ultrafiltration (UF) Membrane Structure
NMWL (Nominal Molecular Weight Limit) (also
called “cut-off”)
 1kD to 1,000kD
(~0.001 to ~0.02µm – Dextran equivalent size)
1
2
3
Typical Applications
 Concentrate proteins
 Clarify proteins
 Wash proteins from unwanted
low MW material (diafiltration)
Typical membrane materials
 Regenerated cellulose
 Polysulfones, modified Polysulfones 30 kD modified Polysulfone membrane
magnified 1,000×
7

Channel Pressures and Flows
Membrane
Membrane
Cb
Cw
k
QRQFPF PR
Pp
CpTMPΔΠ
Cb = Protein concentration in bulk solution [g L-1]
Cw = Protein concentration at membrane [g L-1]
Cp = Protein concentration in permeate [g L-1]
PF = Feed pressure [bar or psi]
PR = Retentate pressure [bar or psi]
Pp = Permeate pressure [bar or psi]
ΔΠ= Osmotic pressure [bar or psi]
QF = Feed flow rate [L h-1]
QR = Retentate flow rate [L h-1]
Qp = Permeate flow rate [L h-1]
k = Mass transfer coefficient [L/m2*h]
8
Qp

01
02
03
04
Agenda
9

10
AAV-Lentivirus Purification
Overview
purification
• Separate AAV-
Lentivirus from
impurities to meet
quality requirements
• Optimize process for
recovery and
robustness
Endonuclease
Digestion
Ultrafiltration Affinity
Chromatography
UltrafiltrationFinal filtrationFill in
assemblies
Ion Exchange
Chromatography
Endonuclease
Digestion
Ultrafiltration Anion Exchange
Chromatography
UltrafiltrationFinal filtrationFill in
assemblies
AAV DSP Process
Lentivirus DSP Process

11
AAV - Lentivirus Purification
Ultrafiltration and Diafiltration with Tangential Flow Filtration
100-300kD TFF is used at two different steps
 First TFF step, after clarification and Benzonase®
endonuclease and before chromatography
− Remove residual Benzonase® endonuclease, DNA fragments
and protein
− Concentrate virus to reduce loading time and buffer exchange
for chromatography steps
− 4-25x concentration, 5-8 diavolumes
 Second TFF step, after chromatography and before sterile
filtration of drug substance
− Achieve final concentration and exchange into formulation
buffer
− 10x concentration and ~8 diavolumes

AAV Purification
Pellicon® Tangential Flow Filtration (TFF) Devices
>25 years
proven record
for virus
production
Pellicon® 2 Cassettes
Pellicon® 3 Cassettes
 Short flow path for higher
flux and higher resolution
separation capability
 Choice of flow channel
configuration providing
process optimization
capability
 Predictable, fast, true linear
scale-up from laboratory size
modules to industrial
assemblies
 Brings higher resolution,
improved yields and superior
back-pressure resistance
12
• 2 choices between Biomax® polyethersulfone (PES) and
Ultracel® regenerated cellulose (RC) membranes

13
AAV – Lentivirus Purification
TFF Membrane and Process Considerations
Schematic of a 2-pump TFF system
May need to control permeate flow
rate to reduce fouling and product
loss
100kD or 300kD MWCO is recommended for
AAV/Lentivirus TFF operation
 Potential balance between higher impurity
clearance vs. assuring higher virus recovery
Control permeate flow rate (e.g. 30LMH) is
needed to reduce fouling and product loss
 Flux may be too high due to high molecular
weight cutoff of membrane
− Increased concentration polarization of virus
at membrane surface
− Product loss and fouling
Membrane tested Recovery
Lentivirus Biomax® 300kD (Vscreen) 65%
Biomax® 500kD (Vscreen) 57%
AAV Biomax® 100kD (Cscreen)
Ultracel® 100 & 300 kD works
too
100%

01
02
03
04
Agenda
14

15
Achieving Flux Control in a TFF Operation
Permeate flow (flux) control to a fixed, robust setpoint throughout a process is
achieved through the use of a permeate pump – the flux controlled TFF
processes are also referred to as 2-pump TFF

16
With or Without Permeate Control
Contaminant removal and Antigen retention vs. VCF under different set of operating conditions
0
10
20
30
40
50
60
70
80
1 2 3 4 5 6 7 8
Volumetric Concentration Factor
Contaminantremoval(%)
Contaminant removal
Optimum operating conditions
98
98.2
98.4
98.6
98.8
99
99.2
99.4
99.6
99.8
100
Antigenretention(%)
Antigen
retention
0
2
4
6
8
10
12
14
16
Contaminant removal
Non-optimum operating conditions
Optimum operating conditions: feed flow = 6 LPM/m², TMP< 0.4 bar, permeate controlled at 30 LMH
Non-optimum operating conditions: feed flow = 6 LPM/m², TMP> 1 bar, no permeate control

17
• Selection of appropriate parameters
• Cross flow velocity
• Operating Flux
• Shearing and retention study at different
controlled flux
• Determination of critical flux
• Critical flux is the maximum permeate that
the system can sustain before the membrane
becomes polarized.
• To stabilize the system, we recommend
maintaining the system flux no higher than
75% of critical flux.
Optimization of MF TFF
Source: Seat Yee Lau; Priyabrata Pattnaik, PhD; Takao Ito, PhD; and Bala
Raghunath, PhD. “Impact of Process Loading on Optimization and Scale-Up
of TFF Microfiltration.” BPJ, Volume 13, Issue 2 (Summer 2014)

18
Typical Results from Critical Flux Experiments
Time
Flux
TMP
Retention
Flux 1
Flux 2
Flux 3
Flux 4
Flux 5
Critical flux
At a Constant Cross Flow Rate

19
First Step in Process Development
Need to stay below
the critical flux to
ensure robust
operation
TMP
Flux
MF-TFF
Critical Flux
Ti
Tf
Tf
TiTf
Ti
Note: Ti, Tf refer to TMPs at Initial & Final times
respectively (say, ti =0 & tf = 20 min)
TMP becomes unstable, i.e. does not remain
constant, at or after critical flux
Determine ‘Critical’ Flux under total recycle conditions
32 Unstable → TMP begins
to steadily rise w/ time
(at constant flux)
1 Critical flux is the flux
at which the process
becomes unstable
A high TMP will lead to
a lower flux

20
Critical Flux – Notable Points
Typically preferred
1 Critical flux can vary between 20 – 100
lmh
• Depends on the solution conditions
• Concept applies across a range of
clarification applications
‒ Mammalian /bacterial /yeast cell
clarification
‒ E.coli lysate clarification
‒ Red blood cell clarification
‒ Alum (in adjuvant processing)
concentration
2 Different methods of analysis to
determine critical flux
• TMP vs. time
• TMP vs. flux
• Permeability vs time
• All are valid methods

21
Determining the Optimum Flux
TMP
Flux
MF-TFF
Critical Flux
Ti
Tf
Tf
TiTf
Ti
Note: Ti, Tf refer to TMPs at Initial &
Final times respectively (say, ti =0 & tf
= 20 min)
TMP becomes unstable, i.e. does not
remain constant, at or after critical
flux
75%
of CF50%
of CF
Choose 2 fluxes below critical flux and determine capacity
• Select 75% and 50% of critical flux

22
TMP
L/m2
MF-TFF
TMPMax
C=60 L/m2
C=40 L/m2
Choose 2 fluxes below critical flux and determine capacity
 Select 75% and 50% of critical flux
 Capacity is determined by carrying out a volumetric concentration experiment

23
Determine area requirements in two ways:
Area based on capacity limitations
• A1 = VB/Vcapacity
1
2 Area based on flux-time
• A2 = VB/(Flux x tB)
Determine flux at which A1 = A2 → Optimum flux

24
5
10
15
20
25
30
5
10
15
20
25
30
10 15 20 25 30 35
Area,VB/(JtB),m2
Area,VB/Vcapacity,m2
Flux, lmh
Example: Optimum Flux Determination
A1
A2

25
5
10
15
20
25
30
5
10
15
20
25
30
10 15 20 25 30 35
Area,VB/(JtB),m2
Flux, lmh
A1 A2

26
5
10
15
20
25
30
5
10
15
20
25
30
10 15 20 25 30 35
Area,VB/(JtB),m2
Flux, lmh
Optimum
Flux
A1 A2

01
02
03
04
Agenda
27

Ultrafiltration / Diafiltration Scaling Rules
Scale LINEARLY whenever possible
 Don’t use predictions to estimate performance
at different scales or operating conditions
Maintain constant fluid dynamic profile
in channels for different scales of operation
 Maintains concentration polarization profile
 Affects yield, quality, consistency, product
profile and process time
Linear scaling
Non-linear scaling
28

Keep constant between scales of operation
Ultrafiltration / Diafiltration Scaling Rules
1
2
3
4
Fluid Dynamic
Parameters
Feed and filtrate rates
per membrane area
 Feed flow rate =
5L/min/m2
Feed, retentate,
and filtrate pressures
Feed and filtrate volume
per membrane area
 Loading =
100L/m2
Process design
(concentration factor,
diavolumes, fed-batch ratio)
 VCF = 20X, DV = 8N,
fed-batch ratio = 4
29

30
Scale Up: Do Not Project or Extrapolate Specific Area
Loading = 19 L/m2
Amin = 290 L/19 L/m2 = 15 m2
And NOT Amin = 290 L/(20 lmh x 6
h) = 2.4 m2
║
Loading = 290 L/2.4 m2
= 120 L/m2
√
32 Large Scale Process
• 20 X conc; 10 N
• Up to 6 hours
available for the
process
• VB = 200 L; VP =
290 L
1 What is the area
requirement for the
process?
Small Scale Test
• Ultracel® 300kD
• CF = 30 lmh;
Optimum flux
chosen = 20 lmh
(66% of CF)
• 2 L w/ 0.1 m2 @ 20
lmh to 20 X (no
diafiltration, VP =
1.9 L)

31
Scaling a MF-TFF Process
32 Must Run Process
Simulation
1 Don’t short cut scale
down process time
Projecting Capacity is
Difficult and Risky
trial time must = process time
Vprocess/Aprocess= Vtrial/A trial
Same VCF and N
 Add Capacity Safety Factors
accordingly
Feed variability
 Variable feed =Greater safety
factor
Multiple cycle variability
 CIP effectiveness
Safety factors in the range of 1.5-
2.0

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