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

6

The Evolution of Upstream Processing

7

1,000L SS Bioreactor1982

1,000L SS Bioreactor1994

2,000L Single-Use Bioreactor

TODAY

The Evolution of Downstream Processing

8

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

9

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)

0

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

0

3

6

9

12

5 15

Bin

din

g ca

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

22

Robust, Load Independent MVM Clearance

0

1

2

3

4

5

6

7

8

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

23

MVM Clearance:Effect of pH & Conductivity at 10 kg/L Load

0

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

24

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.)

25

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

26

Flow-Through Polishing of 4000 g of mAbNatriFlo HD-Q vs. Q Resin Column (10 g/L concentration)

27

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

28

• 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

29

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

30

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

32

• On-site support for

• Process development

• Process optimization

• Technology integration

• 100% dedicated to DSP/Chromatography

• Significant DSP experience

Thank you!

• Peter Tunon

Tunon@natrixseparations.com

• Geert Lissens

Geert.lissens@jsrmicro.be

34

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

35

• 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

37

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

20

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).