Platform downstream processes in the age of
continuous chromatography: A case study
Mark Brower
BioProcess Technology & Expression
Bioprocess Development
Kenilworth, NJ
Integrated Continuous Biomanufacturing
Castelldefels, Spain
20-24 October 2013
Transition to Future Concepts
To meet increasing global demands requires…
PROCESS INTENSIFICATION
Batch Stainless
Batch Stainless /
Single Use
Continuous
Single Use Enabled
Next Generation
Bulk
mAb Downstream Purification
Primary Recovery
(Centrifugation / MF + DF)
Bulk Purification
Protein A Chromatography
6H
12H
18H
Viral Inactivation
(Low pH Hold)
24H
Fine
DNA / HCP / Viral Adsorption
Anion Exchange Chromatography
Variant and Aggregate Clearance
Cation Exchange Chromatography
Formulation
Viral Filtration
Nanofiltration
Concentration / Buffer Exchange
Microfiltration / Diafiltration
30H
• Increased flexibility
36H
• Reduced footprint
42H
• Reduced capital spend
48H
• Better resource utilization
54H
Bioburden Reduction
Sterile Filtration
60H
Continuous Processing Vision - 2,000L SUB*
Formulation:
BRF/DiaF
Anion
Exchange
Membrane
Continuous UF
A
E
X
M
Surge
Bag
S
U
B*
BRF
p
Viral Filtration
Continuous
Viral Inactivation
Single-Use
Centrifugation
p
Surge
Bag
Surge
Bag
Surge
Bag
p
*Single-Use Bioreactor
Depth
/BRF
Filtration
p
Surge
Bag
p
p
p
BRF
BioSMB
Protein A
Overall DSP Time Cycle is Dictated by the Longest Step
Other Steps are Lengthened to Compensate
p
BRF
Polishing
Step
Continuous Processing Case Study mAb 1
- Non-platform
Hour
0
kSep
9:00
DF/SF
9:30
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
7:15
12:00
SMB
11:00
VI
11:40
12:15
1:30
AEX
6:00
4:00
MMC
7:30
UF
4:15
9:00
5:00
AEX Membrane
SMB Protein A
SU Centrifuge
Harvest
Bag
SPTFF
SUB
DF / BRF
Viral Inactivation
Mixed Mode
MCC for Bind & Elute Applications
C1
• Methods based on
batch process
C7
C5
• Flexibility in loading
zone
C3
• Loading, washing,
elution, CIP carried
out simultaneously
Switch
Time
CEX CMCC Load Zone Design
Feed
nd
2
Pass
W1
Longer
residence time
in the elution
zone
Similar column
cycling
compared with
protein A
Productivity
3.7X batch
process

Feed
2 methods designed to maximize time in the elution zone
 Wash 1 in parallel 8 columns (shorter / continuous feed)
 Wash 1 in series 6 columns (longer / discontinuous feed)
W1
nd
2
Pass
SMB Transformation of Platform CEX Step
3.7 x Specific Productivity
Design Equations*
 
1

 1   1 
 1   
CE  1 - exp - NT U 1 -    1   1    exp - NT U 1 -   
 S   S 
 S   

S
 
S
QBed  qBed
Q feed  c0
NTU  koLa 
N V

Ni V
N2 V
 koLa  1 

Qi
 Q feed Q feed  QW 1 
• 1.2cm x 3cm pre-packed columns
• Poros HS Adsorbent
• qbatch=50mg/mL
• Feed = 11-13g/L
• 2 different load zone configurations
• Good agreement between experimental and
theoretical capture efficiency
• CMCC loading was 60-73mg/mL at high yield
*Miyauchi and Vermeulen (1963)
>95%
Aggregate Clearance
– Wash in Series Configuration
• Effect of column height investigated
• 1.2 x 3.4cm, 1.2 x 6.8cm, 0.5 x 20cm
• Feed aggregation varied (low and high)
• Six 1.2 x 3.4cm columns for MCC
• 4th cycle fractionation (20 fractions per column pooled)
10%
20cm High Agg
20cm Low Agg
6.8cm Low Agg
CMCC 3.4cm Low Agg
% Aggregates
8%
• Similar pre-peak
observed in batch
and MCC Process
• Similar pool
aggregate levels
observed
6%
4%
• Little difference
observed at different
column heights
2%
0%
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Normalized Elution Volume [-]
0.8
0.9
1.0
Integration of MCC CEX into Continuous DSP
- 100L platform harvest
Formulation:
BRF/DiaF
pH UF
Continuous
A
E
X
M
100%
S
U
B*
pH
Concentration Factor [-]
80%
60%
40%
20%
BRF
0%
-20%
-40%
-60%
-80%
VI Feed
-100%
0
200
0
400
20
AEX Feed
VI
CEX Feed
40600 60 800 80 1000100 1200
120 1400
140
Time [Min]
2
Membrane Loading [L/m ]
Surge
Bag
Surge
Bag
p
Depth
/BRF
Filtration
p
Surge
Bag
p
Continuous UF
Surge
Bag
p
Viral Filtration
p
Surge
Bag
p
p
BRF
C
R
I
T
I
C
A
L
I
T
Y
BioSMB
Protein A
p
BRF
BioSMB
CEX
1.5
1.5
1.25
1.25
1
1
0.75
0.75
0.5
0.5
0.25
0.25
0
0
0
1
2
3
4
Column
• 16 Overlaid CEX Elution Profiles
• AEXM Effluent Feed
50
6
0.5
Absorbance [mAU]
Absorbance [mAU]
Continuous CEX Performance
0.4
0.3
0.2
0.1
STDEV(%) Between Columns =1.01%
0
1
Continuous Processing Case Study mAb 2
- Platform
Centrifugation
DF/BRF
Protein A SMB
Viral Inactivation
Anion Exchange
Membrane
Cation Exchange
Chromatography
SPTFF
Overall
Mass Balance = 93%
Average
Yield
97.3%
98.6%
98.1%
100%
DNA*
[ppm]
N/S
30,515
N/S
2
HCP
[ppm]
N/S
383,300
N/S
1,063
Res. ProA
[ppm]
N/S
N/S
N/S
2.1
98.8%
<LOQ
82
1.5
99.0%
84.2%
<LOQ
605
<LOQ
99.2%
99.5%
0.001
35
<LOQ
99.0%
77.9%
0.001
8.7
<LOQ
99.0%
% Monomer
N/S
N/S
N/S
89.8%
DSP Productivity Enhancement
Step
Continuous
Protein A Chromatography
[g/(L·h)]
3.1
Cation Exchange Chromatography
[g/(L·h)]
3.7
Overall
[g/day]
~3x
• MCC steps enjoy a modest specific productivity increase
• Other steps suffer from lower specific productivities because they are slowed to
accommodate the incoming flow rate
• The overall DSP will be 2-4x more productive (g/day) by operating in parallel
(dependent on Protein A column sizing)
Conclusions and Future Work
• A platform cation exchange step was transformed into a MCC
process
– 3.6X specific productivity increase
– Maintained consistent aggregate separation performance
compared to the batch process
– Integrated into continuous DSP top reflect platform operation
with 84% yield at the 100L scale
– Matched cycles with protein A step
• Interface CEX step with continuous viral filtration
• Scale up process to 2000L in 24hours
Acknowledgements
•
•
•
•
BTE
– Marc Bisschops
– Ying Hou
– Steve Allen
– David Pollard
Analytical Support
– Joe Fantuzzo
– John Troisi
– Jun Heo
Fermentation Support
– Patty Rose
– Chris Kistler
– Rachel Bareither
Protein Purification Process Development
– Nihal Tugcu
– Thomas Linden
Questions?