Continuous Downstream Bioprocessing By Coupling Cadence™
Single-Pass TFF With Chromatography Steps
Catherine Casey, M.Sc., Karl Rogler, Xhorxhi Gjoka, Rene Gantier, Ph.D. and Engin Ayturk, Ph. D., Pall Corporation, 20 Walkup Drive, Westborough, MA 01581 USA
RESULTS (continued)
Single-Pass Tangential Flow Filtration (SPTFF) is a revolutionary, patented technology that allows concentration
factors of 2 to 30x to be achieved in a single module pass.1-4 It provides many benefits over conventional TFF,
as highlighted below:
Benefits of SPTFF
Elimination of recirculation loop
Reduced number of pump passes
Smaller pump and tubing sizes
No mixing/foaming or minimum working volume concerns
Lower hold-up volumes for improved product recoveries
Ability to operate in-line with other unit operations
Periplasmic Extract
Figure 3
Retentate flow rate vs. feed flux for Fab in E. coli
periplasmic extract
SPTFF module could be operated at VCFs
of 4 – 11x and retentate flow rates of 8.6 to
55 mL/min (Figure 3)
Smallest SPTFF module (0.065 m2) can be
coupled with Mustang XT Acrodisc syringe
filter (0.86 mL) and XT5 capsule (5 mL)
operated at 10 MV/min
SPTFF Performance Map
g/L
E Coli
C lii Periplasmic
P i l
for 0.4 g/
Extract
L Fab in E.
60
VCF=4X
Retentate Flow Rate (mL/min)
BACKGROUND
Applications of SPTFF
Volume reduction
Concentrating to high concentrations for final formulation
Processing of fragile biomolecules
Feed
1000 L
Figure 1
Coupled SPTFF module and chromatography column
VS.
4-fold reduction in membrane resin volume
~ $0.5M savings in resin costs
Column
Feedstock
Chromatography Sorbent
Sorbent Chemistry
0.4 g/L mAb in CHO
clarified cell culture
Protein A MabSelect
SuRe (10 mL column)
Affinity
5x
0.4 g/L Fab in E. Coli
periplasmic extract
Mustang S (0.86 mL
XT Acrodisc®) syringe filter
Ion Exchange (membrane 4 - 11 x
chromatography)
1.25 g/L IgG in CHO
clarified cell culture*
Protein A MabSelect SuRe
(10 mL column)
Affinity
4x
Ion Exchange
16
2.6
50
10
7 - 60
0 - 40
34 - 230
8.6 - 55
15
4x
5
12
40
1
Column Diameter (cm)
# Cycles
Total Processing Time (hr)
Column Volume (L)
Buffer Consumption (L)
Resin Cost ($K)
125 L
Retentate
10
40
SPTFF
10
50
40
30
20
10
0
35
IgG in CHO Cell Culture (4X)
80
Feed Pressure
70
Retentate Pressure
60
50
40
30
20
10
0
0
5
Time
T
ime (min)
10
15
Pressure (psig) and Flow Rate (mL/min)
Retentate Pressure
Pressure (psig) and Flow Rate (mL/min)
Pressure (psig) and Flow Rate (mL/min)
Pressure (psig) and Flow Rate (mL/min)
70
Feed
500 L
Mustang
Capsules
Permeate
Table 3
500 L bioreactor
Membrane Volume (L)
# Cycles
Total Processing Time (hr)
Buffer Consumption (L)
Typical Capsule*
6 x 0.14
10
4.8
277
SPTFF + Mustang Capsule
2 x 0.14
27
2.2
264
* 10 MV/min flow rate, 25 g/L DBC and 0.4 g/L titer
Usable Range Of SPTFF Coupled With Mustang Capsules For Fab Capture From E. coli
CONCLUSIONS
Fab in E. Coli
olii Periplasmic Extract
E
(4X)
80
Mustang
Capsules
By placing a 3.5 m2 SPTFF module before the membrane chromatography step, significant savings can be realized:
3-fold reduction in membrane cost
Marginal reduction in buffer consumption
2-fold reduction in total processing time
Figure 2
Stability of four feedstocks when processed with an SPTFF module [a] mAb in CHO cell culture
[b] Fab in E. Coli periplasmic extract [c] IgG in CHO cell culture and [d] α-amylase in CHO cell culture
Feed Pressure
SPTFF*** + Pre-Capture Column**
30
1
3.1
14
466
170
VS.
Stable pressures successfully achieved for all four feedstocks targeting 4 – 5x VCF (Figure 2)
The volume reduction and concentration for larger scale operations will result in significant cost savings,
which could potentially be achieved through
– Reduced processing times
– Reduced buffer volumes and associated costs
– Use of smaller columns and/or resin/membrane
– Eliminating the need for break tanks between TFF and chromatography steps
mAb in CHO Cell Culture (5X)
4-fold reduction in buffer consumption
Similar total processing times
3.5 m2
Stability Of SPTFF And Chromatography Coupled Process
30
SPTFF
F
• Case Study 2: Protein Capture with Membrane Chromatography at 500 L Scale
RESULTS
25
250
** 20 cm bed height, 300 cm/h linear velocity, 30 g/L DBC and 0.4 g/L titer
*** VCF of 5x and retentate flux of 12 LMH
Feed
500 L
20
15
T
Time
ime (min)
200
Retentate
200 L
Pre-Capture Column**
60
1
3.4
57
1865
678
*Bovine IgG was added to a CHO clarified cell culture to obtain a higher IgG concentration feedstock
10
100
150
Feed Flux [LMH]
Table 2
1000 L Bioreactor
Operating Conditions of SPTFF Module
VCF
PF (psig)
PR (psig) QF (cc/min) QR (cc/min)
5
50
Pre-capture concentration of a 0.4 g/L mAb in CHO feedstock with the utilization of a 17.5 m2 SPTFF module,
could result in significant savings:
Table 1
Case Studies
0
0
SPTFF
Feedstocks concentrated 4 to 11-fold
with SPTFF module before loading onto
Permeate
chromatography media
SPTFF retentate flow rate adjusted according
to chromatography flow rates:
– Protein A MabSelect◆ SuRe◆ and HyperCel™ STAR AX sorbent: 1 column volume (CV) per minute
– Mustang® S membrane: 10 membrane volume (MV) per minute
60
10
Permeate
Feed
80
20
17.5 m2
Retentate
Operating Conditions (Table 1)
HyperCel STAR AX
(10 mL column)
30
• Case Study 1: Pre-Capture Concentration at 1000 L Scale
STRATEGY
0.5 g/L α-amylase
in CHO cell culture
VCF=8X
Case Studies: Coupling SPTFF and Chromatography
Feed
1000 L
A 4-in-series SPTFF module comprised of
seven regenerated cellulose (Delta)
10 kDa T01 TFF cassettes
Total membrane area: 0.065 m2
TFF retentate loaded directly onto
chromatography column/capsule
VCF=5X
40
0
SPTFF can be coupled with various unit operations to enable continuous bioprocessing. One placement of the
SPTFF module is immediately before loading onto a chromatography sorbent. In this study, the process stability,
volumetric concentration factor (VCF) ranges, and process economics of operating SPTFF and chromatography
steps in-line were explored in detail.
System Setup (Figure 1)
50
Feed Pressure
70
Retentate Pressure
60
50
40
30
20
10
0
0
5
10
15
T
Time
ime (min)
20
25
-Amylase in CHO Cell Culture (4X)
80
Feed Pressure
References
70
Retentate Pressure
60
Four feedstocks (mAb, Fab, IgG and α-amylase) were successfully operated under steady-state run conditions
when a SPTFF module was coupled with a chromatography column/membrane. The versatility of the SPTFF
module to be operated over a wide range of flow rates and concentration factors before loading onto the
chromatography sorbent was demonstrated.
Advantages of coupling SPTFF with chromatography:
Enables continuous bioprocessing
Reduces chromatography load times
Downsizes all future unit operations and reduces system footprints
Increases the likelihood adoption of other single-use technologies (i.e., disposable biocontainers, flow paths,
equipment, etc.) for even more cost savings
1. Mir, Leon and Gaston de los Reyes. Method and apparatus for the filtration of biological solutions. US Patents 7,384,549 B2 (June
10, 2008), 7,682,511 B2 (March 23, 2010), 7,967,987 B2 (June 28, 2011) and 8,157,999 B2 (April 17, 2012).
50
2. Casey, C., Gallos, T., Alekseev, Y., Ayturk, E., and Pearl, S. Protein concentration with Single-Pass Tangential Flow Filtration,
Journal Membrane Science, 384(1-2) (2011) 82-88.
40
30
3. Pall publication USD2789: Cadence™ Systems Employ New Single-Pass TFF Technology to Simplify Processes and Lower Costs
(Pall Life Sciences)
20
10
4. Dizon-Maspat, J., Bourret, J., D’Agostini, A. and Li, F. Single Pass Tangential Flow Filtration to Debottleneck Downstream Processing
for Therapeutic Antibody Production. Biotechnology and Bioengineering, 109(4) (2012) 962-970.
0
0
5
10
T
Time
ime (min)
15
Contact: +800.717.7255 (USA) • +41 (0)26 350 53 00 (Europe) • +65 6389 6500 (Asia/Pacific) • E-mail: biopharm@pall.com • Web: www.pall.com/biopharm
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© 2014, Pall Corporation. Pall,
, Cadence, Mustang, HyperCel and Acrodisc are trademarks of Pall Corporation. ® indicates a trademark registered in the USA.
◆MabSelect SuRe is a trademark of Sigma Aldrich. 10/14, GN14.6047