Post on 18-Jun-2018
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
CONFIDENTIAL
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 Templated process Protein A chromatography
Yet, several issues remain Stability Capital and utilities Large footprint Frequent bottlenecks Sterility Cleaning validation
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CONFIDENTIAL
New alternative template
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Clarification Capture Purification/polishing
Bioreactor Centrifuge
2° depth filtration
Protein A b/e chrom
CEX b/e chrom
AEX f/t chrom
Bioreactor w/ precipitation
1° depth filtration
Protein A b/e chrom continuous Carbon f/t
device AEX f/t device
CEX f/t device
Virus filtration UF/DF
CONFIDENTIAL
Comparison of templates – icons sized by device volume
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Clarification Capture Purification/polishing
3.3 m2
4.4 m2
14.1 L 14.1 L 19.3 L
0.6 L each
5 L 0.4 L 3 L
1,000 L @ 2 g/L
CONFIDENTIAL
Comparison of templates – pool tanks
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Clarification Capture Purification/polishing
1000 L 500 L
50 L
250 L
CONFIDENTIAL
Clarification assisted by precipitation and using novel Clarisolve™ filters results in post-Protein A benefits
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0100200300400500600700800900
1000
4 4.5 5 5.5 6 6.5 7
Turb
idity
(NTU
)
pH
Depth Filtered
Smart Polymer
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 Increased throughput (<3x membrane area) DNA removal (1-2 LRV) Advantages persist post protein A Reduced turbidity Enhanced HCP clearance Reduced resin cleaning
CONFIDENTIAL
Capture with continuous multicolumn chromatography and incompressible Protein A resins offers savings
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RT (min) Effective DBC (g/L)
Productivity (g/L/hr)
1-column batch 4 39 7 1-column batch 0.22 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
Savings 87% 24%
Status Two incompressible resins available Prosep® Ultra Plus Eshmuno® A
Continuous loading from clarified harvest and continuous loading to purification train successfully shown Benefits Higher productivity, especially at low residence times Resin and buffer savings
0
10
20
30
40
50
60
70
80
0 0.5 1 1.5 2 2.5 3 3.5
DBC
@ 1
% B
T (g
/L)
Residence time (min)
Two-column continuousOne-column batch
time savings
buffer/resinsavings
CONFIDENTIAL
Protein A capture cannot be beaten as part of a holistic process evaluation Why not CEX chromatography? Cheaper resin Cheaper unit operation
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 expensive
Why not precipitation? Single-use Buffer consumption Processing time
More materials Additional unit operations Precipitant removal No product concentration Dilution steps No purification Increased process development
More expensive at commercial scale AX | ECI Castelldefels | 21Oct2013 9
CONFIDENTIAL
Purification in flow-through mode using novel adsorbers, minimum interventions, fewer pool tanks and one skid
Prop
osed
Pr
oces
s
Low pH VI Pool
VF Pool
Carbon + AEX f/t
CEX f/t + VF
In-line pH
Low pH VI Pool
CEX Pool
AEX Pool
VF Pool
Trad
ition
al
Proc
ess
CEX b/e AEX f/t VF with prefiltration
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CONFIDENTIAL
Novel flow-through adsorber functionalities work synergistically to remove several classes of impurities
MAb
acidic pI basic
Low
M
W
high
Larger acidic HCP, DNA, viruses
AEX
mAb Aggregates CEX
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
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CONFIDENTIAL
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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CONFIDENTIAL
Internal bench-scale experimental case studies: Robustness of flow-through purification train (3 mAbs)
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mAb
Monomer Yield (%)
Aggregates 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
CONFIDENTIAL
External trials: Robustness of flow-through purification train (7 mAbs)
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# 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
CONFIDENTIAL
Internal case studies: Product quality
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Current process Alternative process Yield 92% 87%
Process-related impurities HCP: 11 ppm
Leached ProtA: 10 ppm DNA: < 10 ppb
HCP: 2 ppm Leached ProtA : 4 ppm
DNA: < 10 ppb Product-related impurities
(% HMW/Main/LMW) 1/98/1 0.5/99/0.5
Charge variants (% Acidic/Main/Basic) 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
CONFIDENTIAL
Cost of Goods: where is the advantage?
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% 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%
12
5
3
3
15
16
4
2
0
10
20
30
40
Old batch New continuous
DSP
cost
($/g
)
5 kL @ 5 g/L commercial laborconsumablesmaterialsfacility
6537
4142
155
91
62
39
0
100
200
300
400
Old batch New continuous
DSP
cost
($/g
)
1 kL @ 1 g/L clinical laborconsumablesmaterialsfacility
CONFIDENTIAL
Process modeling: advantages of proposed template
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Parameter for DSP portion 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%
Processing time hrs 55 30 45%
Cost $/g of mAb 219 109 50%
1,000 L @ 2 g/L | 2 kg batch | ~70% yield
CONFIDENTIAL
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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CONFIDENTIAL
Acknowledgments
Downstream Technologies, MM • Kevin Galipeau • Meghan Higson • Jad Jaber • Mikhail Kozlov • Matthew Stone • William Cataldo • Romas Skudas • Jeff Caron • Jonathan Steen • Scott Bliss • Dennis Aquino • Wilson Moya
Analytical Technologies, MM • Rong-Rong Zhu • Michael Bruce Team Supply, MM • Michael McGlothlen • Patricia Kumpey • Paul Hatch Business Development, MM • Fred Mann BioPharm Services, Inc • Andrew Brown
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