1. Integrated Continuous Biomanufacturing A new ECI conference
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

2. 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
AX | ECI Castelldefels | 21Oct2013

3. 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
AX | ECI Castelldefels | 21Oct2013

4. New alternative template
2° depth filtration
Protein A
b/e chrom
CEX b/e chrom
AEX f/t chrom
Virus filtration
Current template
UF/DF
Clarification
Capture
Purification/polishing
Bioreactor
Centrifuge
Protein A
b/e chrom
continuous
1° depth filtration
Carbon f/t device
AEX f/t device
CEX f/t device
Bioreactor
w/ precipitation
Alternative template
AX | ECI Castelldefels | 21Oct2013

5. Comparison of templates – icons sized by device volume
Current template
Clarification
Capture
Purification/polishing
3.3 m2
14.1 L
14.1 L
19.3 L
0.6 L each
4.4 m2
5 L
0.4 L
3 L
Alternative template
1,000 2 g/L
AX | ECI Castelldefels | 21Oct2013

6. Comparison of templates – pool tanks
Current template
1000 L
500 L
Clarification
Capture
Purification/polishing
250 L
Alternative template
50 L
AX | ECI Castelldefels | 21Oct2013

7. 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
Increased throughput (<3x membrane area)
DNA removal (1-2 LRV)
Advantages persist post protein A
Reduced turbidity
Enhanced HCP clearance
Reduced resin cleaning
AX | ECI Castelldefels | 21Oct2013

8. Productivity (g/L/hr)
Capture with continuous multicolumn chromatography and incompressible Protein A resins offers savings
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
RT (min)
Effective DBC (g/L)
Productivity (g/L/hr)
1-column batch 4 39 7
0.22 19
3-column continuous 37
136
1000L feedstock, 1 g/L
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%
AX | ECI Castelldefels | 21Oct2013

9. 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

10. Purification in flow-through mode using novel adsorbers, minimum interventions, fewer pool tanks and one skid
Low pH VI Pool
CEX Pool
AEX Pool
VF Pool
Traditional
Process
CEX b/e
AEX f/t
VF with prefiltration
Proposed
Process
Low pH VI Pool
VF Pool
Carbon + AEX f/t
CEX f/t + VF
In-line pH
AX | ECI Castelldefels | 21Oct2013

11. (leached Protein A, HCP, fragments)
Novel flow-through adsorber functionalities work synergistically to remove several classes of impurities
Larger acidic HCP,
DNA, viruses
AEX
mAb Aggregates
CEX
Cell culture components
Insulin, methotrexate, Pluronic F68®, hygromycin, antifoam C
Process-related impurities
DNA, HCP, leached Protein A, viruses
Product-related impurities
Aggregates, fragments
MAb
Low MW high
Low MW impurities
(leached Protein A, HCP, fragments)
Carbon
acidic pI basic
AX | ECI Castelldefels | 21Oct2013

12. 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
AX | ECI Castelldefels | 21Oct2013

13. Internal bench-scale experimental case studies: Robustness of flow-through purification train (3 mAbs)
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
AX | ECI Castelldefels | 21Oct2013

14. Loadings of activated carbon and f/t CEX devices were 0.5 – 1.0 kg/L
External trials: Robustness of flow-through purification train (7 mAbs) #
Monomer yield (%)
Aggregates (%)
Fragments (%)
HCP (ppm) 1 91
5.1  0.8
1.2 à 0.1
688 à 4 2 83
1.0 à <0.1
0.3 à 0
64 à <1 3 87
1.6 à 0.6
n/a
80 à 3 4 86
2.0 à 0.8
0.2 à 0
350 à 7 5 84
0.13 à 0
155 à <1 6 85
9.2 à 2.7
600 à 6 7
3.0 à 0.8
1468 à 7
Loadings of activated carbon and f/t CEX devices were 0.5 – 1.0 kg/L
AX | ECI Castelldefels | 21Oct2013

15. Internal case studies: Product quality
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
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
AX | ECI Castelldefels | 21Oct2013

16. Cost of Goods: where is the advantage?
% cost savings for DSP process
5 5,000 L
commercial
1 1,000 L
clinical
Old batch  New continuous
24%
35%
AX | ECI Castelldefels | 21Oct2013

17. Process modeling: advantages of proposed template
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)
2.4
1.0
 58%
Processing time
hrs 55 30
 45%
Cost
$/g of mAb
219
109
 50%
1,000 2 g/L | 2 kg batch | ~70% yield
AX | ECI Castelldefels | 21Oct2013

18. 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
AX | ECI Castelldefels | 21Oct2013

19. 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
AX | ECI Castelldefels | 21Oct2013

21. Abstract
We have developed a new monoclonal antibody purification template comprised of precipitation-based clarification, protein A capture chromatography and flow- through polishing that offers a robust, single-use manufacturing solution while significantly reducing overall cost of goods (COGs). Modeling studies verify that the individual clarification, capture and flow-through polishing solutions offer significant advantages as stand-alone unit operations. Additionally, these technologies were designed to be integrated in a continuous purification process template. Proof of principle for single-batch, continuous operation was obtained at bench scale. Performance of the integrated process matched or surpassed that of the traditional batch process. Advantages of pairing unit operations will be presented and discussed. Extensive process modeling both supported and guided the experimental work and documented cost and operational advantages of the new process template.
AX | ECI Castelldefels | 21Oct2013