NATRIX SEPARATIONS INC....NATRIX SEPARATIONS INC. Introduction to High-Capacity Single Use (per...
Transcript of NATRIX SEPARATIONS INC....NATRIX SEPARATIONS INC. Introduction to High-Capacity Single Use (per...
NATRIX SEPARATIONS INC.
Introduction to High-Capacity
Single Use (per batch) Membrane Chromatography
17 November 2015
®
Agenda
• Brief introduction to Natrix Separations
• Perspectives on the evolution of upstream/downstream processing
• NatriFlo HD-Q for Flow-Through Polishing
• Impact on Process Economics
• Natrix DSP Support
• Conclusions
2
Natrix HD-Membrane ChromatographyEnabling efficient, affordable clinical DSP
Extraordinary Capacity
+ Processing SpeedConsistent, robust performance
Very small, cost-effective,
disposable columnsEnabled by productivity
Up to 90% less investment
for clinical manufacturingEliminate underutilized capital
Single-use-per-batch
DSP becoming a realityProductive columns 20x smaller
Natrix-JSR Life Sciences Partnership
• Natrix’s global distributor and strategic investor• $4B+ company with 50 year history• Polymer focus with deep development and manufacturing• Quality focused – perennial winner – Intel’s top quality
award• Global presence: EU, NA, Japan, China, Korea, India• Large and long-term commitment to bioprocess industry
• Protein A (Amsphere™, in GMP manufacturing)• Natrix, Chromacon
• Natrix a cornerstone of JSR’s life sciences initiative
Natrix Manufacturing Capabilities
• Pilot and full scale equipment installed
• Full capacity > 10 Million sq ft/yr
• Devices completed at established
• ISO 9001 Certified
PolymerizationWash
Dry
Agenda
• Brief introduction to Natrix
• Perspectives on the evolution of upstream/downstream processing
• NatriFlo HD-Q for Flow-Through Polishing
• Impact on Process Economics
• Natrix DSP Support
• Conclusions
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The Evolution of Upstream Processing
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1,000L SS Bioreactor1982
1,000L SS Bioreactor1994
2,000L Single-Use Bioreactor
TODAY
The Evolution of Downstream Processing
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PRE-PACKED COLUMN
NATRIX
SINGLE-USE PER BATCHSS COLUMN
Natrix enables robust, compact, low cost
flow-through and bind/elute operations
Agenda
• Brief introduction to Natrix
• Perspectives on the evolution of upstream/downstream processing
• NatriFlo HD-Q for Flow-Through Polishing
• Impact on Process Economics
• Natrix DSP Support
• Conclusions
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How does the Natrix technology work?
Reinforcing mesh is filled with functionalized porous hydrogel
> Hydrogel provides binding groups and final pore structure
Functionalized, durable composite membrane is created in a single step
Identical functional binding group chemistry as resins – C, Q , mixed-mode, affinity
Flexible, reinforcing fiber mesh provides strength and structure
Natrix Membranes:
Dominated by
Advective Flow
High Binding Capacity for
Proteins, Virus and DNA, High
Flow Rates
Conventional Column
Chromatography
Natrix Advective
Chromatography
Natrix Flow/Binding Dynamics vs. Other Methods
Resins
Diffusional limitations
High Binding Capacity for
Proteins. Limited capacity for
large molecules (virus, DNA),
Low Flow Rates
Conventional Membranes
Limited surface area
Low Binding Capacity,
High Flow Rates
Conventional Membrane
Adsorber Chromatography
• Disposable products – use for a single batch
• Intended for flow-through applications in single-cycle mode
• Pilot, Process are pleated construction with Q membrane chemistry
NXF-01Recon Mini
0.2mL
NXF-02Recon0.8mL
NXF-10Pilot15mL
NXF-20 - 60Process 150- 600115mL- 460 ML
NatriFlo™ HD-Q membrane adsorbers
HD-Q DBC @ 10% BT 5 MV/min
NatriFlo™ HD-QBinding vs Conductivity
25mM Tris, pH 8.1
High dynamic binding capacity
Very fast kinetics
6 seconds residence time
200 mg/ml BSA
NatriFlo™ HD-QBSA Capacity VS Flow
Increasing ionic strength decreases binding capacity
Natrix™ HD-Q membrane’s high binding capacity provides wide margin for salt tolerance
0
50
100
150
200
250
300
2 7 12 14
Dyn
amic
BSA
bin
din
g ca
pac
ity
(mg/
mL)
Conductivity (mS/cm)
NatriFlo HD-Q Capto Q
Q FF Mustang Q
Sartobind Q Sartobind STIC
Chromasorb Capto Adhere
Resin: 1 CV/minMembrane: 10 MV/min
NatriFlo™ HD-QBinding vs Conductivity in Tris
HD-Q Phosphate Tolerance (BSA, 10%BT)
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50
100
150
200
25 50 75
Dyn
amic
BSA
bin
din
g ca
pac
ity
(mg/
mL)
Phosphate concentration (mM)
NatriFlo HD-Q Capto Q
Q FF Mustang Q
Sartobind Q Sartobind STIC
Chromasorb Capto Adhere
Resin: 1 CV/min
Membrane: 10 MV/min
Process design and operational flexibility
• Equilibration: Phosphate buffer, pH 8.0
• Sample: 1 g/L BSA in equilibration buffer
Process RobustnessGood HCP Reduction at High Protein Loads
HCP breakthrough for a comparative load up-to 4 kg/L
Feed HCP: 104 ppm; DNA: 1.7 ppmpH: 7.5; Conductivity: 5 mS/cm.Flow rate: 10 MV/min
HCP reduction performance for a load @ 10kg/L
8 g mAb/L purified using protein A20 mM phosphate + 100 mM NaCl, pH 7Flow rate = 10MV/min (Residence time 6 s)
No end in sightMembrane 1
Membrane 2
NatriFlo HD-Q
Collaborator II Patheon
Collaborator IV Study.NatriFlo™ HD-Q showed good scalability from lab to pilot and process scale at 1kg/L load.
Buffer = 25 mM Tris-HClpH = 8.0Flow rate = 10 MV/min.
Collaborator V Study.NatriFlo™ HD-Q demonstrated very efficient clearance at all scales.
Buffer: 20mM PO4 with 100mM NaClpH = 7.0Flow rate 10 MV/min
Collaborator IV Gallus
NatriFlo™ HD-Q Scale-UpLab Performance Predicts Larger Scales for HCP Reduction
HD-Q Shows High DNA Binding Capacity
AEX Media0
5
10
15
20
25
30
Dyn
amic
DN
A b
ind
ing
cap
acit
y (m
g/m
L)
NatriFlo HD-Q
Capto Q
Q FF
Mustang Q
Sartobind Q
Sartobind STIC
Chromasorb
Capto Adhere
Resin: 1 CV/min
Membrane: 10 MV/min
Effective clearance of protein AND DNA
• Equilibration: 25 mM Tris, pH 8.0
• Sample: 0.1 g/L Herring testes DNA in equilibration buffer
NatriFlo HD-Q Endotoxin Clearance
• Endotoxin clearance (>4 LRV) from buffer (25 mM Tris + NaCl, pH 8) spiked with endotoxin (> 1000 EU).
• >4 LRV for endotoxin as high as 9 million EU/mL of membrane at both 5 and 15 mS/cm
• Protein free buffer only experiment since endotoxin can interact with proteins in a process specific manner.
• Arrow indicates limit of detection
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pac
ity
at 4
LR
V (
mill
ion
EU
/mL
of
me
mb
ran
e)
Conductivity (mS/cm)
Viral clearance for HD-QExperimental (also see Poster)
• NatriFlo HD-Q Recon Mini (0.2 mL)
• Flow rate: 10 MV/min
• Sample volume: 200 – 430 mL (titer
dependent)
• Virus spike: 1% (v/v)
• Sample: Biosimilar mAb
– HCP: 15 – 45 ppm
– Aggregates: 1.5 – 3.0 %
Protein A
(Amsphere JWT)
Low pH Virus Inactivation
Cation-exchange
(POROS XS)
UF/DF
HD-Q Hydrogel Membrane Adsorber
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Robust, Load Independent MVM Clearance
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9
0 5 10 15 20 25
MV
M C
lear
ance
(LR
V)
Load (kg/L)
10 mM Phosphate, pH 7.5
25 mM Phosphate, pH 7.5
25 mM Tris, pH 7.5
25 mM Tris, pH 8.0
Load independent MVM clearance over wide design space
Load: 0.25 – 20 kg/L membrane
(0.25 kg/L typical resin load)
Phosphate vs. Tris
Sample: 10 g/L mAb, 10 mS/cmResidence time: 6 seconds
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MVM Clearance:Effect of pH & Conductivity at 10 kg/L Load
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2
4
6
8
6.5 7.0 7.5
MV
M C
lear
ance
(LR
V)
pH
2.5 mS/cm 5 mS/cm 10 mS/cm7 LRV at pH 6.5-7.5
High LRV clearance at lower pH balanced by lower conductivity
Load: 10 kg/LSample: 10 g/L mAb in 10 mM Phosphate + NaClResidence time: 6 seconds
Note: Up arrow (↑) indicates limit of detection
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Higher MVM Clearance with HD-Q
40x smaller disposable device with similar processing time
LoadHD-Q: 10 kg/LCapto Q: 0.25 kg/L
Residence time:HD-Q: 0.1 minuteCapto Q: 3 minute
Sample: 10 g/L mAb in 10 mM Phosphate + NaClNote: Up arrow (↑) indicates limit of detection
MVM Clearance: HD-Q vs. Capto Q
7.45.8
4.4 4.2
0
2
4
6
8
2.5 5.0
MV
M (
LRV
)
Conductivity (mS/cm)pH 6.5
HD-Q (10 kg/L) Capto Q (0.25 kg/L)
6.9 7.16.8 6.7
0
2
4
6
8
2.5 5.0
MV
M (
LRV
)
Conductivity (mS/cm)pH 7.0
HD-Q (10 kg/L) Capto Q (0.25 kg/L)
Similar and better LRV at pH 6.5-7.0
HD-Q productivity/L much higher
(>1000x kg/L-min.)
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Clearance of MuLV, PRV & Reo-3
HD-Q gave high LRV at 10 kg/L load and 10 mS/cm conductivity for three other model viruses
Load: 10 kg/L membraneSample: 9.8 g/L mAb in 20 mM phosphate + NaCl, pH 7.5 & 10 ms/cmResidence time: 6 seconds
Note: No virus breakthroughUp arrow (↑) indicates limit of detection
4.95.9 6.3
0
2
4
6
MuLV PRV Reo-3V
iru
s C
lear
ance
(LR
V)
Agenda
• Brief introduction to Natrix
• Perspectives on the evolution of upstream/downstream processing
• NatriFlo HD-Q for Flow-Through Polishing
• Impact on Process Economics
• Natrix DSP Support
• Conclusions
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Flow-Through Polishing of 4000 g of mAbNatriFlo HD-Q vs. Q Resin Column (10 g/L concentration)
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Q Resin Column
≤150 g/L
32 L Column Volume
(45 cm ID x 20 cm H)
300 cm/h (7.95 L/min)
<1.5 Hours
(1 cycle)
NatriFlo HD-Q
≤10,000 g/L
0.46 L Membrane
Volume
4.6 L/min
<2 Hours
(1 cycle)
Clinical Phase 1: Options for a 1000 gram batch
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• Prime
• Sanitize
• Equilibrate
• Load
• Discard
• Pack
• Validate
• Document
• Sanitize
• Equilibrate
• Load
• Strip
• Regenerate
• Store1000 g mAb
80 L (12.5g/L)
Q PolishingNatriFlo P150
0.115 L MV 1.15 L/min
10,000 mg mAb/mL
<2 hours
1 cycle
Column 20 cm ID x 20 cm H
6.3 L BV
300 cm/hr (1.6 L/min)
150 mg mAb/ml
<3 hours/cycle,
2 cycles
DSP Platform Productivity Factors Key Cost Factors
Economic ImpactPhase 1: 500 L reactor, 2.5 g/L (Model)Column 20 cm ID/6.3 L vs. NatriFlo HD-Q P150
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Summary Results Column Natrix HD-Q Savings
Media/Device Expense/Campaign $19,635 $4,785 $14,850 Column Capital Expense/Campaign $50,000 NA $50,000 Column Labor Expense/Campaign(200 hours vs. 1 hour)
$40,600 $200 $40,600
Buffer Cost per Campaign $200 $63 $137
Cost per Campaign $110,435 $5,048 $105,387
Other Operational Factors
Buffer Consumption (Liters) per Campaign
400 L 126 L 274 L
Total Processing Time per Batch 5.3 hours 2.8 hours 2.5 hours
1 campaigns
3 batches
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Conclusions for HD-Q
• Great LRV clearance on four model viruses (MVM, MuLV, PRV & Reo-3) at 10 kg/L load; MVM up to 20 kg/L load
• > 6 LRV MVM clearance at 10 mS/cm at pH 7.5 or 8.0 in both Tris and Phosphate buffers
• No breakthrough for MuLV, PRV & Reo-3
• Superior MVM clearance & productivity compared to Capto Q resin
• 0.115 L NatriFlo HD-Q P150 = 5 L Capto Q column
• 0.460 L NatriFlo HD-Q P600 = 20 L Capto Q column
Dedicated Process Science and Integration support
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• On-site support for
• Process development
• Process optimization
• Technology integration
• 100% dedicated to DSP/Chromatography
• Significant DSP experience
Thank you!
• Peter Tunon
• Geert Lissens
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www.jsrlifesciences.com
www.natrixseparations.com
Appendix
• Brief introduction to Natrix
• Perspectives on the evolution of upstream/downstream processing
• NatriFlo HD-Q for Flow-Through Polishing
• Natrix HD-Sb for Bind-Elute Capture and Polish
• Impact on Process Development Productivity
• Natrix DSP Support
• Conclusions
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• CEX group – sulfonic acid• HIC group – t-butyl• High binding capacity and salt
tolerant• Target application
• Capture or polishing of mAbs• Removal of mAb aggregates and HCP• Bind/elute or flowthrough• Flexible binding & elution conditions• Base stable
HD-Sb Salt Tolerant CEX w HIC modality
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First in the Natrix HD-Sb line
• HD-Sb Recon
• 0.87 mL nominal membrane volume
• Two layer flat sheet membrane configuration
• Female Luer inlet and outlet connections
• Larger devices under development
4.0
4.5
5.0
5.5
pH
70
8080
90
90
5 8 10 13 15 18 20
Conductivity
dBC Butyl IgG
<= 10
<= 20
<= 30
<= 40
<= 50
<= 60
<= 70
<= 80
<= 90
> 90
4.0
4.5
5.0
5.5
pH
5 10 15 20 25
Conductivity
dBC mAb2 Butyl
<= 10
<= 20
<= 30
<= 40
<= 50
<= 60
<= 70
<= 80
> 80
Human IgG mAb
Design Space for Binding Conditions
• DBC at 10% BT: 80 – 90 mg/mL
• Salt tolerance around pH 4.5
Model protein sep Sb pH5 4p5gL 100 MV 10 MVpmin Feb 19 2015002:10_UV1_280nm Model protein sep Sb pH5 4p5gL 100 MV 10 MVpmin Feb 19 2015002:10_Cond Model protein sep Sb pH5 4p5gL 100 MV 10 MVpmin Feb 19 2015002:10_pH Model protein sep Sb pH5 4p5gL 100 MV 10 MVpmin Feb 19 2015002:10_Logbook
0.0
10.0
20.0
30.0
40.0
50.0
mAU
20.0
40.0
60.0
80.0
mS/cm
4.0 6.0 8.0 10.0 min
pH
5 5
0 m
M N
a A
ce
tate
1 M
Na
Cl 1
00
MV
gra
die
nt
CIP
50
0 m
M N
aO
H
Model Protein Separation on the HD-Sb Recon
pH5 and 100 MV gradient: 12-min elution
Lyso
RNase Aα-CTG A
Model protein sep Sb pH5 4p5gL 10MVpmin 100 MV 9 and 13p5 mgpml load Feb 19 2015001:10_UV1_280nm Model protein sep Sb pH5 4p5gL 10MVpmin 100 MV 9 and 13p5 mgpml load Feb 19 2015001:10_Cond Model protein sep Sb pH5 4p5gL 10MVpmin 100 MV 9 and 13p5 mgpml load Feb 19 2015001:10_pH Model protein sep Sb pH5 4p5gL 100 MV 10 MVpmin Feb 19 2015001:10_UV1_280nm@11,SHFT Model protein sep Sb pH5 4p5gL 10MVpmin 100 MV 9 and 13p5 mgpml load Feb 19 2015002:10_UV1_280nm@14,SHFT Model protein sep Sb pH5 4p5gL 10MVpmin 100 MV 9 and 13p5 mgpml load Feb 19 2015001:10_Logbook
0
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40
60
80
100
120
mAU
10.0
20.0
30.0
40.0
50.0
60.0
70.0
mS/cm
30.0 40.0 50.0 60.0 70.0 80.0 90.0 ml
4.5 mg/mL
9.0 mg/mL
13.5 mg/mL Lyso
RNase Aα-CTG A
Load amount test
Binding condition: 50 mM NaOAc, pH 5.0 (Buffer A)Elution condition: 50mM NaOAc, pH 5.0, 1M NaCl (Buffer B)Gradient: 0-100% B over 100 MV, flow = 10 MV/minute
4.5mg/mL Total Protein Load
HD-Sb Performance SummaryBind & Elute Mode
Mode LoadAggregates Monom
er YieldFeed Elution
Bind & Elute
50 mg/mL
1.80% Undetectable >90%
Bind & Elute
50 mg/mL
3.30% Undetectable >90%
Bind & Elute
50 mg/mL
8.10% Undetectable >90%
-0,005
0,005
0,015
0,025
0,035
0,045
0 5 10 15 20
FeedEluate
7 8 9 10 11 12 13 14
Figure 1: Overlaid SEC chromatogram of Feed and Eluate peaks from HD-Sb in Bind & Elude Mode.
High Aggregate Removal in Bind & Elute Mode. Overlaid SEC chromatograms of Elution, Feed/flowthrough and Strip
peaks from Bind & Elude [experiments show effective aggregate removal. Load 50 mg/mL, flow rate 10 membrane
volumes/minute, sample concentration 1 g/L in equilibration buffer (45 mM Na-acetate, 130 mM NaCl, pH4.5, 16.25
mS/cm). Elution with wash buffer (20 mM phosphate, pH6.3, 6 mS/cm).
HD-Sb Performance SummaryFlowthrough Mode
Aggregates Monomer YieldFeed Flowthrough
12.80% 0.60% >90%
0
0,1
0,2
0,3
0 5 10 15
FeedFlowthrough
7 9 11 13
Figure 2: Overlaid SEC chromatogram of Flowthrough and Feed peaks from HD-Sb in Flowthrough Mode.
Load 300mg/mL, flow rate 10 membrane volumes/minute, sample concentration 9.5g/L in equilibration buffer (50 mM Na-acetate, 130 mM
NaCl, pH5.5, 10 mS/cm).