A new, integrated, continuous purification
process template for monoclonal antibodies
Alex Xenopoulos*
Alison Dupont, Christopher Gillespie, Ajish Potty, Michael Phillips
Processing Technologies
Merck Millipore
Bedford, MA (USA)
Integrated Continuous Biomanufacturing
A new ECI conference
Castelldefels, Spain
October 20-24, 2013
Highlights
We developed a flow-through purification train that enables
an integrated, continuous process
We have novel solutions for continuous clarification and
capture
Bench-scale proof of principle for several mAbs shown
Breakthrough improvements not possible unless you look at
new technologies
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CONFIDENTIAL
Monoclonal antibody production
A mature, robust industry
Yet, several issues remain
Templated process
Protein A chromatography
Stability
Capital and utilities
Large footprint
Frequent bottlenecks
Sterility
Cleaning validation
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New alternative template
2° depth
filtration
Bioreactor
CEX b/e
chrom
AEX f/t
chrom
Virus
filtration
UF/DF
Centrifuge
1° depth
filtration
Bioreactor
w/ precipitation
Clarification
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Protein A
b/e chrom
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Protein A
b/e chrom
continuous
Capture
Carbon f/t
device
AEX f/t
device
CEX f/t
device
Purification/polishing
CONFIDENTIAL
Comparison of templates – icons sized by device volume
3.3 m2
14.1 L
14.1 L
19.3 L
0.6 L each
4.4 m2
Clarification
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5L
Capture
0.4 L
3L
Purification/polishing
1,000 L @ 2 g/L
CONFIDENTIAL
Comparison of templates – pool tanks
500 L
1000 L
250 L
50 L
Clarification
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Capture
Purification/polishing
CONFIDENTIAL
Clarification assisted by precipitation and using novel
Clarisolve™ filters results in post-Protein A benefits
Status
Three launched Clarisolve™ filters optimized
for particle size
Portfolio of flocculants
Continuous harvesting and loading of protein
A column successful and beneficial
Benefits
Elimination of centrifuge up to 6,000 L
1000
Increased throughput (<3x membrane area)
900
Depth Filtered
DNA removal (1-2 LRV)
Advantages persist post protein A
 Reduced turbidity
 Enhanced HCP clearance
 Reduced resin cleaning
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Turbidity (NTU)
800
700
Smart Polymer
600
500
400
300
200
100
0
4
4.5
5
5.5
pH
6
6.5
7
CONFIDENTIAL
Capture with continuous multicolumn chromatography
and incompressible Protein A resins offers savings
80
buffer/
resin
savings
70
Status
time savings
DBC @ 1% BT (g/L)
60
Two incompressible resins available
 Prosep® Ultra Plus
 Eshmuno® A
50
40
30
20
Two-column continuous
One-column batch
10
0
Continuous loading from clarified harvest
and continuous loading to purification
train successfully shown
Benefits
Higher productivity, especially at low
residence times
0
1.5
2
2.5
3
3.5
RT (min)
Effective
DBC (g/L)
Productivity
(g/L/hr)
1-column batch
4
39
1-column batch
0.22
7
7
19
3-column
continuous
0.22
37
136
Effective
DBC (g/L)
RT
(min)
Consumed
resin (L)
Consumed
buffer (L)
Batch
39
4
21
2646
Continuous
45
0.5
2.8
2009
87%
24%
Savings
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1
Residence time (min)
Resin and buffer savings
8
0.5
CONFIDENTIAL
Protein A capture cannot be beaten as part of a holistic
process evaluation
Why not CEX chromatography?
Why not precipitation?
 Cheaper resin
 Cheaper unit operation
 Single-use
 Buffer consumption
 Processing time
 Two dilution steps – volume increase
 Longer processing time
 Higher water/buffer use
 Lower selectivity
 Less virus removal
 Lower yield
 Increased process development
 Less templatable
 More materials
 Additional unit operations
 Precipitant removal
 No product concentration
 Dilution steps
 No purification
 Increased process development
More expensive
More expensive at commercial scale
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Purification in flow-through mode using novel adsorbers,
minimum interventions, fewer pool tanks and one skid
Traditional
Process
CEX b/e
Low
pH VI
Pool
CEX
Pool
Proposed
Process
Carbon +
AEX f/t
10
Low
pH VI
Pool
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VF with
prefiltration
AEX f/t
AEX
Pool
VF
Pool
CEX f/t +
VF
VF
Pool
In-line pH
CONFIDENTIAL
Low MW high
Novel flow-through adsorber functionalities work
synergistically to remove several classes of impurities
Larger acidic HCP,
DNA, viruses
mAb Aggregates
CEX
MAb
AEX
Low MW impurities
(leached Protein A, HCP, fragments)
Carbon
Cell culture components
 Insulin, methotrexate, Pluronic
F68®, hygromycin, antifoam C
Process-related impurities
 DNA, HCP, leached Protein A,
viruses
Product-related impurities
 Aggregates, fragments
acidic pI basic
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Benefits of flow-through purification
Disposable chromatography devices connected without pool tanks
No bind/elute chromatographic steps
Minimal interventions
Orthogonal mechanisms for impurity removal
Needed pH adjustments incorporated in skid
One skid (protein A elution  TFF) is possible
Enables integrated, continuous process template
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Internal bench-scale experimental case studies:
Robustness of flow-through purification train (3 mAbs)
mAb
Monomer
Aggregates
Yield
ProtA  VF pool
(%)
(%)
HCP
ProA VF pool
(ppm)
VF
Capacity
(kg/m2)
mAb04
88
N/A
250  2
> 3.5
mAb05
92
5.0  1.0
591  1
>3.6
mAb07
91
1.4  ~0
82  1
>3.7
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External trials:
Robustness of flow-through purification train (7 mAbs)
#
Monomer yield
(%)
Aggregates (%)
Fragments (%)
HCP (ppm)
1
91
5.1  0.8
1.2  0.1
688  4
2
83
1.0  <0.1
0.3  0
64  <1
3
87
1.6  0.6
n/a
80  3
4
86
2.0  0.8
0.2  0
350  7
5
84
1.6  0.6
0.13  0
155  <1
6
85
9.2  2.7
n/a
600  6
7
91
3.0  0.8
n/a
1468  7
Loadings of activated carbon and f/t CEX devices were 0.5 – 1.0 kg/L
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Internal case studies:
Product quality
Current process
92%
HCP: 11 ppm
Leached ProtA: 10 ppm
DNA: < 10 ppb
Alternative process
87%
HCP: 2 ppm
Leached ProtA : 4 ppm
DNA: < 10 ppb
1/98/1
0.5/99/0.5
15/71/13
13/72/15
Glycan profile
(% Gal: 0/1/2)
79/19/2
79/20/2
Higher order structure
(CD)
No change
No change
Yield
Process-related impurities
Product-related impurities
(% HMW/Main/LMW)
Charge variants
(% Acidic/Main/Basic)
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Cost of Goods: where is the advantage?
labor
5 kL @ 5 g/L commercial
consumables
40
labor
consumables
materials
facility
1 kL @ 1 g/L clinical
400
materials
facility
4
300
2
15
20
16
DSP cost ($/g)
DSP cost ($/g)
30
200
62
39
155
3
91
100
10
3
12
42
65
5
16
37
0
0
Old batch
41
Old batch
New continuous
New continuous
% cost savings for DSP process
5 g/L @ 5,000 L
commercial
1 g/L @ 1,000 L
clinical
Old batch  New continuous
24%
35%
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CONFIDENTIAL
Process modeling: advantages of proposed template
Units
Current
process
Alternative
process
% change
Equipment cost
$M
6.9
3.1
 55%
Footprint
m2
87
59
 32%
Water use (incl cleaning)
L/g of mAb
24.2
1.4
 94%
Buffer use (excl WFI)
L/g of mAb
2.4
1.0
 58%
hrs
55
30
 45%
$/g of mAb
219
109
 50%
Parameter for DSP portion
Processing time
Cost
1,000 L @ 2 g/L | 2 kg batch | ~70% yield
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Key features of the alternative template
An alternative templated process for downstream purification of mAbs is proposed
It matches performance of current templates, provides operational advantages
Features:
• Novel downstream purification process for mAbs – from bioreactor through formulation
• Connected unit operations – continuous operation, minimal interventions
• Novel unit operations developed – leverage continuous nature
• Clarification toolbox – novel depth filters, precipitating agents
• Product capture with continuous multicolumn protein A affinity chromatography –
efficient use of resin and buffer
• Flow-through polishing – no bind/elute steps, improved simplicity and economics
• Virus filtration and ultrafiltration/diafiltration – no changes
• Proof of concept and feasibility data generated – performance equivalent to current,
advantages in overall operational flexibility
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Acknowledgments
Downstream Technologies, MM
Analytical Technologies, MM
• Kevin Galipeau
• Rong-Rong Zhu
• Meghan Higson
• Michael Bruce
• Jad Jaber
• Mikhail Kozlov
Team Supply, MM
• Matthew Stone
• Michael McGlothlen
• William Cataldo
• Patricia Kumpey
• Romas Skudas
• Paul Hatch
• Jeff Caron
• Jonathan Steen
Business Development, MM
• Scott Bliss
• Fred Mann
• Dennis Aquino
• Wilson Moya
BioPharm Services, Inc
• Andrew Brown
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CONFIDENTIAL