a new, integrated, continuous purification process template for monoclonal antibodies alex...
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A new, integrated, continuous purification process template for monoclonal antibodies
Alex Xenopoulos*Alison Dupont, Christopher Gillespie, Ajish Potty, Michael PhillipsProcessing TechnologiesMerck MilliporeBedford, MA (USA)
Integrated Continuous BiomanufacturingA new ECI conference
Castelldefels, SpainOctober 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 processProtein A chromatography
Yet, several issues remain
StabilityCapital and utilitiesLarge footprintFrequent bottlenecksSterilityCleaning validation
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New alternative template
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Clarification Capture Purification/polishing
Current template
Alternative template
Bioreactor Centrifuge
2° depth filtration
Protein Ab/e chrom
CEX b/e chrom
AEX f/t chrom
Bioreactorw/ precipitation
1° depth filtration
Protein Ab/e chromcontinuous Carbon f/t
deviceAEX 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
Current template
Alternative template
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
Current template
Alternative template
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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0
100
200
300
400
500
600
700
800
900
1000
4 4.5 5 5.5 6 6.5 7
Tu
rbid
ity
(NT
U)
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 71-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
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50
60
70
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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 evaluationWhy not CEX chromatography? Cheaper resin Cheaper unit operation
Two dilution steps – volume increaseLonger processing timeHigher water/buffer useLower selectivityLess virus removalLower yieldIncreased process developmentLess templatable
More expensive
Why not precipitation? Single-use Buffer consumption Processing time
More materialsAdditional unit operationsPrecipitant removalNo product concentrationDilution stepsNo purificationIncreased process development
More expensive at commercial scale
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CONFIDENTIAL
Purification in flow-through mode using novel adsorbers, minimum interventions, fewer pool tanks and one skid
Prop
osed
Proc
ess
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
alPr
oces
s
CEX b/e AEX f/t VF withprefiltration
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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 AggregatesCEX
Low MW impurities(leached Protein A, HCP, fragments)
Carbon
Cell culture components Insulin, methotrexate, Pluronic
F68®, hygromycin, antifoam CProcess-related impurities DNA, HCP, leached Protein A,
virusesProduct-related impurities Aggregates, fragments
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CONFIDENTIAL
Benefits of flow-through purification
Disposable chromatography devices connected without pool tanksNo bind/elute chromatographic stepsMinimal interventionsOrthogonal mechanisms for impurity removalNeeded pH adjustments incorporated in skidOne skid (protein A elution TFF) is possibleEnables 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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mAbMonomer
Yield(%)
AggregatesProtA VF pool
(%)
HCPProA 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 à 0.1 688 à 42 83 1.0 à <0.1 0.3 à 0 64 à <13 87 1.6 à 0.6 n/a 80 à 34 86 2.0 à 0.8 0.2 à 0 350 à 75 84 1.6 à 0.6 0.13 à 0 155 à <16 85 9.2 à 2.7 n/a 600 à 67 91 3.0 à 0.8 n/a 1468 à 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 processYield 92% 87%
Process-related impuritiesHCP: 11 ppm
Leached ProtA: 10 ppmDNA: < 10 ppb
HCP: 2 ppmLeached ProtA : 4 ppm
DNA: < 10 ppbProduct-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 Lcommercial
1 g/L @ 1,000 Lclinical
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
($/g
)
5 kL @ 5 g/L commercial laborconsumablesmaterialsfacility
6537
4142
155
91
62
39
0
100
200
300
400
Old batch New continuous
DSP
cos
t ($/
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 proposedIt 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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