Novel Membrane Processes and Devices

SPF Technologies

Gastón de los Reyes SPF Technologies

Boston MA Gaston.de.los.Reyes@SPFtechnologies.com

30Aug’21

SPF Technologies • Separation, Purification & Filtration devices and processes for

Bioprocessing and Life Science Labs.

• Founded in 2005 to develop Single-pass TFF (Leon Mir; Gastón de los Reyes)

• Business model: development and licensing.

Technology Portfolio

Ultrafiltration Devices

• Single-pass TFF (SPTFF) in Bioprocessing — Licensed to Pall Corp. in 2007

• Single-pass TFF (SPTFF) in Laboratories — VacuCon™ platform

• Ultra-thin Channel Filtration (UTF™) in Bioprocessing

Chromatography

• ChromaWeb™ Platform — under development

• Chromassette® Platform — Licensed to JSR Corp. in 2015

UF Devices for the Purification of Biomolecules in Bioprocessing and Life Science Labs.

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BIO

PR

OC

ESS

ING

LI

FE S

CIE

NC

E L

AB

S 1970

1980

1990

200

0

2010

2020

SPF 2005

TFF in Bioprocessing Pellicon® 1968

Centrifugal UF 1980s

Single-Pass TFF (SPTFF) in Bioprocessing 2007

Ultra-Thin Channel Filtration (UTF™) 2015

SPTFF in Lab (VacuCon™) 2021

1960

s Tan

gent

ial F

low

Filt

ratio

n (T

FF)

Mini TFF in Lab

Single-pass TFF (SPTFF) in Bioprocessing

4

Flux Decay in Dead-ended UF.

J = 19.377·t-0.516 R² = 0.97135

0

10

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100

0 2 4 6 8 10

J [lm

h]

t [min]

Dead-ended Flux Decay 10 g/L IgG @ 55 psi | 30K MWCO

Flux decays > 20x within 10 minutes

…continues to decay forever.

This is why TFF was invented 50 years ago.

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Concentration Polarization and the birth of Tangential Flow Filtration (“TFF”).

Membrane

Conc. Boundary

Layer

Cb

Cg

Permeation

Flux

PERMEATE Compartment

PERMEATE Compartment

Membrane

Membrane

Tangential Flow

Short Channels

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TFF • A parallel array of • Short-Channel Filtration Cassettes • At VERY HIGH feed rates

• Very LOW conversion • Effluent must be recirculated. 7

Cassette 1

Cassette 2

Cassette 3

Cassette 4

Cassette 5

Cassette 6

Cassette 7

Cassette 8

Cassette 9

Cassette 10

FEED RET PERM

Cassette 7

Cassette 8

Cassette 1

Cassette 2

Cassette 3

Cassette 4

Cassette 9

Cassette 10

Cassette 5

Cassette 6

SPTFF • A staged array of • Short-Channel Filtration Cassettes • At LOW feed rates

• Very HIGH conversion • Single-pass… continuous.

FEED

RET

PERM

Conventional TFF Process

Feed Tank

Recirc. Pump

TFF Module

Recirc. Loop

Batch Process

Processing times 4~6 hours

Limited concentration factors due to large change in tank volume 8

SPTFF Process....

Complete Elimination of Recirculation Loop Including Feed Tank (no need to accumulate batch) 10X smaller pump

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The TFF Paradigm The SPTFF Paradigm Short Flow Path Long Flow Path High Velocity Low Velocity Thin Channels Thinner Channels

The SPTFF Paradigm Shift

TMP Profile

0

20

40

60

80

0 20 40 60 80 100 120 140 160Distance in Channel

TM

P

Low Conversion —› Recirculation

High Conversion —› Single-Pass

Batch Process Continuous Process No Staging Internally staged modules

Uniform TMP Large TMP Profile Moderate Feed Pressure High Feed Pressure

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Key Benefits of SPTFF

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• Low residence time ≈ 1 minute = low exposure/degradation • Very high concentration factors • Much simpler system = simpler operation • Inline integration with other process steps to (a) reduce volume,

(b) increase productivity, etc.

1. Final Concentration of monoclonal Antibodies (mAb)

• mAb therapy requires very high dosages

• Final concentration ≥ 200 mg/mL

• Not possible with conventional TFF

2. In-process volume reduction

• Tankage reduction.

• 4~8X volume reduction without any hardware.

• Pressurized feed line.

3. Increase Productivity of Protein-A capture. • Increase feed concentration 5~10X to 20~80 mg/mL.

• Reduces chromatography column load time by 5~10X.

Multiple New Applications Created by SPTFF Process

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1.  SPTFF adopted rapidly and widely in Bioprocessing due to… • New capabilities —› new applications. • Same membranes • Existing TFF module could be readily adapted to build SPTFF modules.

2. As with all paradigm shifts, there will be pushback. • “Membranes are going to foul at lower velocities and higher TMP.” • “Flux will be too low.”

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Closing Remarks on SPTFF Process

UTF™ Technology Ultra-Thin Channel Filtration

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UTF Concept — The Anti-TFF

• Dead-ended ultrafiltration… minimum mixing.

• Exploits concentration polarization Intentionally builds Boundary Layer of concentrated solute

• Key technical features … Microfluidic channel array of very thin and short channels:

• Channel height is of same order as BL thickness (50~100 µm) • Channel length 2~6 cm

… Rapid cycling … Constant pressure process

A single-pass UF process… but the opposite of TFF

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UTF Concentration Process

Fig. 1A

Fig. 1B

Fig. 1C

100

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106

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107105101

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1. Load 60s

Fig. 2A

Fig. 2B

Fig. 2C

100

100

100109

113

115

111 117104109

109

2. Recover 5 s

Conc. Boundary Layer

Membrane UTF Channel

Perm Plate

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UTF Washing Process

Fig. 1A

Fig. 1B

Fig. 1C

100

100

100

106

104

110

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107105101

101

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1. Load 60s

Fig. 2A

Fig. 2B

Fig. 2C

100

100

100109

113

115

111 117104109

109

3. Recover 5 s

Conc. Boundary Layer

Membrane

UTF Channel

Perm Plate

700

703Fig. 7

702708

709

721b721a

2. Wash 100s

Conc. Boundary Layer

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0

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100

0 20 40 60 80 100 120

J [lm

h]

t [s]

Dead-ended Flux Decay 10 g/L IgG @ 55 psi | 30K MWCO

UTF Concentration Process

Semi-continuous… rapidly cycled process.

Recovery 5 seconds

Load 60 seconds

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A Prototype UTF Plate with blue-BSA

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FEED PORT

RETENTATE PORT

FLOW

3.5

cm

50 µm channels

Concentration of BSA

Conc. Boundary Layer

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• Stable flux of 65 lmh over 70 cycles (1 hour) • Steady State achieved in 5 cycles

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0 20 40 60 80 100

VC

F []

J [lm

h]

Cycle

UTF Flux Stability — IgG @ 5 g/L J VCF

Concentration of IgG

Conc. Boundary Layer

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0

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F []

J [lm

h]

Cycle

UTF Flux Stability — IgG @ 5 g/L J VCF

Reduced VCF ≈ 7.5

• Stable flux of 25 lmh over 70 cycles (2+ hours) • Steady State achieved in 5 cycles

Single-pass Buffer Exchange

• Single-pass Buffer Exchange with SPTFF possible but not yet commercialized.

… More complicated than conventional diafiltration.

… Higher buffer consumption.

• UTF process is capable of Buffer Exchange

… Removal factors of 10~40X possible with

… buffer consumption comparable to conventional diafiltration (40~80 mL/g).

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Key benefits UTF vs. SPTFF

• Simpler process.

… only one degree of freedom in Concentration process (load time)

… another degree of freedom in Wash process (wash time)

• Versatile module design.

Same module can be used for different applications.

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Closing Remarks on UTF Technology

One never knows when a new useful idea is going to spring up.

• The removal of the Boundary Layer from UF channels was a problem that I had been thinking for 30 years before UTF came to mind!

• The development of the VacuCon device paved the way for this discovery.

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SPTFF Technology in Laboratory Applications VacuCon™ Concentrator

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Lab Ultrafiltration of 50~200 mL Samples TODAY Mini-TFF Systems —

Complicated & Difficult

•  Complicated operation; training required •  Hardware intensive…

Pump, valves, gauges, reservoirs, tubing

•  Max concentration factor ≈ 20X.

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Test the Process! After choosing the membrane,module, and all operating parameters,run the entire process to ensure thatperformance meets all criteria foracceptability. During the process,monitor flows and pressures. Collectsamples of all initial and final streams.Calculate process time to ensure that itis within the expected range. Test thequality of the final product withreliable assays, ideally the assays thatwill be used during actual processingfor qualifying product release.

In order to understand where theproduct is going during a process, it isimportant to calculate not only yield,but also mass balance. Determine thetotal protein in each of the retentate,the filtrate, and the unrecoverableholdup volume. Ideally, these amountssum to the total amount that was putinto the unit operation. If they fallshort, there were likely someadsorption and/or solubility lossesduring the process. However, if theamount of protein unaccounted for is alarge percent of the total, either theprocess is not operating correctly orsome operating parameters need tobe changed to reduce the losses. Theyield and mass balance follow thelaw of conservation of mass where:

The subscripts o, r, f, and h refer tooriginal, retentate, filtrate, and holdup,respectively. The percent yield in anyone of the streams can be calculatedby dividing the amount protein in thatstream by the total amount in thefeedstock. For instance, the yield inthe retentate is calculated as:

Finally, to understand how robust aprocess is to feedstock variability andmultiple cycles, it is very helpful to runthe process several times. Althoughthis is not always possible, especiallywhen feedstock is extremely limited, itcan help guard against unexpectedperformance degradation once the

process is in place. In addition, it willhelp to ensure that the processparameters were not determinedbased on a best-case run that is notreproducible.

Putting the Process Together Once a protein processing procedurehas been developed, it must beintegrated into a complete process.The typical sequence of steps in anultrafiltration/diafiltration process areoutlined in figure 16.

Set Up and Pre-Use Cleaning Before installation of membranes intoa new TFF holder, thoroughly cleanand flush all components of the holderand system to remove potentialcontaminants that were introducedduring manufacture and assembly.Scrubbing exposed surfaces with asoap solution and recirculating thesolution through all piping with the useof special cleaning gaskets, followedby extensive flushing with high qualitywater removes residual dirt and oils.

After new membranes have beeninstalled, and before their first use onproduct, clean, sanitize,depyrogenate, and flush the assemblyto remove membrane preservativesand any contaminants introducedduring installation. Please refer to the

appropriate Millipore MaintenanceProcedures for recommendedcleaning, sanitization, and depyro-genation solutions, recirculation times,and temperatures.

Integrity and PermeabilityTesting In order to ensure that the installedmembranes have not sustained anydamage during storage and handling,Millipore recommends integrity testingall TFF assemblies prior to startup andafter each post-use cleaning. An airdiffusion test identifies problems suchas macroscopic holes in the membrane,cracks in the seals, or improperlyseated modules.

Labscale™ Benchtop TFF System withPellicon XL module. Complete, linear scalable solution for small-volume processing.

Vo * Co = Vr * Cr + Vf * Cf + Vh* Ch

Yield [%] = 100* Vr * Cr / Vo* Co

Store

Set up SystemInstall Membranes

Clean Membranesand System

Test Integrityand Permeability

Equilibrate withProcess Buffer

ConcentrateDiafilter

Remove Productfrom System

Clean Membranesand System

Test Integrityand Permeability

Figure 16. Typical sequence of steps in aTFF process

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2 - 15 ml samplesThe Vivaspin 15 concentrator is adisposable ultrafiltration device foruse in swing bucket centrifugesaccommodating 50 ml tubes.Vivaspin 15 is used for theconcentration of biological samples inthe 2 - 15 ml range. The innovativedesign (US Patent no. 5,647,990,second patent pending), simplicity,speed and exceptional concentraterecoveries are the main features ofthe concentrator.

In a single spin, 15 ml solutions canbe concentrated up to 300x. Samplescan be typically concentrated in 10-30 minutes with macromolecularrecoveries in excess of 95%. Thelongitudinal membrane location andadjacent thin channel, provideoptimum cross flow conditions evenfor particle laden solutions, thecentrifugal force pulling particles andsolids away from the membrane tothe bottom of the device.Macromolecules collect in animpermeable 50 µl concentratepocket integrally moulded below themembrane surface, therebyeliminating the risk of filtration to dryness.

Vivaspin 15

30,000MWCO

1ml

0.75

0.50

025

0.10

31

25.5

76

Technical specifications Vivaspin 15

Concentrator capacity

Swing bucket rotor 15 ml

Fixed angle rotor 8 ml

Dimensions

Total length 76 mm

Width 25.5 mm

Active membrane area 4 cm2

Hold up volume of membrane <20 µl

Dead stop volume 50 µl

Materials of construction

Body Polycarbonate

Filtrate vessel Polypropylene

Concentrator cap Polycarbonate

Membrane Polyethersulfone

Centrifugal UF — Slow & Cumbersome • Multiple 15·mL devices • Slow permeation rate • Large centrifuge required

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VacuCon™ Concentration Process Enabled by SPTFF Module Operated in Dead-ended

Mode

The Process

1. Add sample to Feed Reservoir Connect Vacuum Port to vacuum source.

2. Vacuum induces permeation within SPTFF Separation Module concentrating feed sample in retentate compartment.

3. Disconnect vacuum when sample is consumed… and recover retentate fraction into Syringe.

The performance of SPTFF with the simplicity and reliability of vacuum filtration devices.

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FEED RESERVOIR

PERMEATE RESERVOIR

SPTFF MODULE

RETENTATE COLLECTOR

Retentate Plate in VacuCon SPTFF Module

Feed Port

RetentatePort

Single-sided converging channels

8 Channels — 4 | 2 | 1 staging

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Illustration of Single-pass TFF Nature • 0.25 mg/mL Cyto C • 100 mL in 30 min. • 80% recovery at 80X concentration factor

Feed Port

RetentatePort

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Important Additional Features To Make Device Simple & Reliable…

1. Philic gate integrated into bottom of Feed Reservoir. Hydrophilic membrane stops permeation when feed reservoir empties.

2. Phobic vent bonded into UF membrane. Hydrophobic membrane effects reliable venting.

No operator intervention.

Washing in situ.

Simply add wash buffer to Feed Reservoir Retentate fraction washed inside Retentate channels. • Simple

• Reliable • No operator intervention

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Typical Permeation with IgG

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0

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60

80

100

120

0 5 10 15 20 25 30 35 40 45

VPE

RM

[mL]

t [min]

IgG Concentration in VacuCon

110 mL 0.1 mg/mL

100 mL 1 mg/mL

Typical Purification Performance with BSA & IgG

Excellent yield: > 80%

Very high concentration factors: 30~95 X

Fast Permeation: 7~45 min.

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Examples of Concentration and Recovery of Various Proteins

Protein Protein Conc.

Sample Vol.

Perm. Time

RetentateVolume Yield Vol. Conc.

Factor[mg/mL] [mL] [min] [mL] [%] []

BSA 1 34 7 1.12 86 30

BSA 1 50 15 1.14 88 44

IgG 0.1 107 20 1.12 94 95

IgG 1 53 20 1.10 85 48

IgG 1 98 45 1.14 80 86

1. First target application for SPTFF: replace of small centrifugal devices (analytical sample prep)… but never got traction!

• Suppliers of centrifugal devices engaged in a price war. “Can you make the SPTFF sample-prep device at a low cost?”

• Applied later to Bioprocessing… where it got immediate traction!

• Today targeting a badly served application: Preparative-scale samples in laboratories.

2. Features not part of the core technology — e.g., philic gate; phobic vent —invariably play an important part in creating new useful product concepts

• Focus on the application

• Think beyond the core technology.

Closing Remarks on SPTFF in Laboratory Applications

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1. Many opportunities to innovate created by Convergence in Biopharm. • Process intensification —› High productivity • Single-use • Smaller production batches • Flexible, multi-product plants • Short exposure times

2. Key factors to drive adoption in Biopharm Manuf.:

• New capabilities Final Concentration of mAb.

• Pain reduction In-process Volume Reduction; Easy-to-use & Reliable Protein Concentration in Laboratories.

Process economics not a factor.

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Summary

Acknowledgements

The SPF Team

Erik Blomquist René de los Reyes Ana Guevara Andy Proulx Ted McCabe Barry Gaiman Rob Zeller Doug Jacoby Mike Form

Andrew Zydney

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This Presentation is Dedicated to Dr. Leon Mir 1938 ~ 2013

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Manager at Millipore 1989~1992

My good friend 1989~2013

Co-founder of SPF 2005

Partner at SPF 2005~2013

SPF Technologies