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William G. Whitford Integrated Continuous Biomanufacturing Castelldefels, Spain October 20-24, 2013

Single-use Technology Supporting Continuous Biomanufacturing

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True biomanufacturing origins

• Recall Roger-Marc Nicoud’s hieroglyphic claim of continuous production

• 2050 BC Sumeria Cuneiform • Customer receipt of a 4.5L batch of the “best beer” from his brewer • Documentation, metrics, chain of custody, quality assessment !

“ World's oldest beer receipt?”, The Free Library 02 Jan 2006

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Why single-use (SU) now dominate

Reduce Contamination Risks • Classical systems at 5-20%

Lower Initial Investment Costs • Up to 74% capital savings • Reduced time, facility and services

Lower Operating Costs • Up to 90% water reduction • Up to 45% faster changeover • Up to 40% energy reduction

Business Continuity • Faster speed to market • Ease of capacity expansion • Campaign change-over speed

Sustainability • Reduced environmental stress • Up to 50% reduced operator use

Many “Flexibilities”

SU bioreactor compared to stainless

Some SUBs use stainless steel bioreactor principles, providing

seamless transition to single-use

Fed-batch production of GS CHO in SFM

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SU many process flexibilities

• Open architecture of subcomponents (e.g., operating systems and monitoring probes)

• Process flow / configuration latitude (e.g., layout, porting, and connectivity changes)

• System and unit operation modularity (e.g., from skids to containers to manifolds)

• Geographical relocation ease (e.g., SUT support equipment move to new site)

• Physical location latitude (e.g., undedicated building or manufacturing suites)

• Hybrid (classical + SUT) capability (e.g., classical columns / probes in SUS)

• Product type / classification change (e.g., serum to animal-product free)

• Process platform latitude (SUT supports all animal cells / culture media)

• Process scale latitude (e.g., rapid, inexpensive scale-up and scale-out)

• Process format latitude (e.g., microcarrier/suspension culture support)

• Process mode latitude (e.g., batch, fed-batch, and perfusion culture)

• Future-proofing (e.g., change ease / economy supports updates)

• Scheduling ease (e.g., extremely short change-over times)

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SU subcomponent flexibility examples

Connectors / porting Sensors / probes

Controllers Filters

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Upstream single-use technology

• Cell culture: both seed expansion / production

• Various approaches: perfusion culture support

• Media, buffer and process liquids preparation

• Liquid pumping, filtration, collection, shipping

• On-line contents monitoring sensors/samplers

• Transport/storage of intermediate and product

• Heat exchange of media/buffer to warm or cool

• Cryopreservation of seeds and intermediates

Single-use Bioreactor

Single Use Mixer

Mixtainer

Arca

Smartainer

Smartainer II

Powdertainer

Arca

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Downstream single-use technology

Harvest • Single-Use Mixer • SU tanks, bins, drums Microcarrier Harvest • SU Harvestainer Clarification • SU depth filtration • SU centrifugation Virus Inactivation • Single-Use Mixer • SU tanks, bins, drums Filtration • SU 0.2μ capsule filters • SU TFF Virus Removal • SU 15nm capsule filters

Buffer / Fluid Preparation • Mixtainer • Single-Use Mixer Cation/Anion Exchange • SU cation packed bed columns • SU anion membrane adsorption UF/DF • SU 30kd MWCO cassettes • SU TFF Adsorption Chromatography • LU adsorption columns • LU SMB skids Waste Collection • SU tanks, bins, drums Fill/Finish • SU flow path skids

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New SU products in downstream operations

• New SU automated flow paths • Thermo Scientific SU B.P.C.s • ASI SU Heat Exchanger • Quattroflow SU pumps • Pall SU TFF Module • Acro pinch valves

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SU systems

• There are now fully single-use modular facilities

• GMP equipment / systems

• cGMP-compliant facility design

• Grade C and D processing areas

• Start-to-finish support, including IQ / OQ

• Speed to GMP production (nominally 1-2 years)

• Pre-engineered, modular, ‘off-the-shelf’ solutions

Michael Dieterle, Boehringer Ingelheim: Global Disposable Manufacturing: IBC BPI, Shanghi, 2013, with permission

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Agenda

1. Single−Use Systems in Biomanufacturing

2. Single−Use Systems in Continuous Biomanufacturing

3. Vaccines and Single−Use Continuous Biomanufacturing

4. Next-Gen Products and SU Continuous Biomanufacturing

5. New Continuous Biomanufacturing−Enabling Technologies

6. Continuous Biomanufacturing Applications Data / Case Studies

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Continuous bioproduction (CB) not a new idea

• Major biologicals sponsors currently use CP-type ops • Many perfusion approaches upstream

• Simulated moving bed (SMB) chromatography

• Continuous and automated fill / finish procedures

• Many existing small-molecule CP operations apply to biopharm

Product Protein Pharma Kogenate-FS rFactor VIII Bayer Xigris rActivated protein C Eli Lilly Cerezyme Beta-glucocerebrosidase Genzyme Fabrazyme Agalsidase beta Genzyme Myozyme Alglucosidase alfa Genzyme ReoPro mAb-abciximab Janssen Remicade mAb—infliximab Janssen Simponi mAb—golimumab Janssen Stelara mAb—ustekinumab Janssen Rebif Interferon beta-1a Merck-Serono Gonal-f rFSH Merck-Serono Simulect mAb-basiliximb Novartis

Biopharmaceuticals Employing Perfusion Culture

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Now contract manufacturers

• Appreciate performance and flexibility of perfusion • CMC biologics advertises 108 cells/ml by perfusion • Improved process is called High Density Perfusion • Productivity anticipated from 0.3 to 2-3 gram/L/day • Making 2000L SUB useful up to the ton/year scale

With permission

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Single-use continuous bioproduction

• Relevant applications appearing • SU in continuous bioproduction reviews

• Hybrid intensified-perfusion insect cell culture

Bögli, et al, BPI 10(5)s May, pp 40-49, 2012

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Upstream SU CB enabling products / technologies

• Commercial “perfusion” related systems • Both attached and suspended cell culture

• Reusable, limited- single-use and hybrid

• Varieties of media exchange and control

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Downstream SU CB Components

Rapidly Growing

• SU heat exchange • SU clarification

• Centrifugation, depth filtration • Suspension or adherent • Class VI, γ-irradiated

• SU pumping, valving • Quaternary diaphragm pumps

• SU capture columns • SMB continuous chromatography

• SU TFF Systems • Concentration and diafiltration

• SU surge reservoirs • Thermo Scientific BPCs • Gamma-irradiated • Extremely configurable

• SU polishing

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Developing downstream CB amenable to SU

Continuous Purification in Two-phase Systems Rosa, et al. Biotechnology Journal. Mar, 2013

• Two phase extractions • Flocculation / precipitations • Viral inactivation and/or removal • Advanced capture chromatographies • Polishing: Some now, some in development

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Single-use technologies currently in CB

• Preparation and storage of media / buffers in SU mixers

• SU liquid and gas filtration of many types, including TFF

• Storage of media and buffers for CP feeding in SU BPC

• Distribution of process fluids in metered SU manifolds

• SU storage and metered distribution of dry powders

• SU or hybrid bioreactor cell culture in seed generation

• Production in SU or hybrid-SU perfusion bioreactors

• Continual appearance of new SU probes and sensors

• SU real-time automated online multi-analysis interface

• SU flow-path on-line real-time controlled feed porting

• Clarification by SU centrifugation or filtration into BPC

• Purification in SU traditional or PCC chromatography

• Final fill in SU and/or automated and closed apparatus

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SU / hybrid perfusion culture enabling products

SU Perfusion Support Commercial Examples SU / Hybrid PC Application

Stirred tank Thermo Scientific Single-Use Bioreactor (Thermo Scientific) and PBS Air-Wheel®

(PBS Biotech)

SU PC when combined with perfusion-enabling technology. Suspension and adherent (eg, microcarrier) culture.

Roller bottle Nunc™ TufRol™ Roller Bottles (Thermo Scientific) in RollerCell 40 (CELLON S.A)

SU PC closed media exchange with some CPP control. Adherent culture.

Stacked array flask RepliCell (Aastrom) and Xpansion (Artelis) SU PC closed media exchange with some CPP control. Adherent culture.

Wave-action based BIOSTAT® RM (Sartorius Stedim) and WAVE (GE Healthcare)

SU PC with perfusion-enabling technology. Suspension and/or adherent (eg, microcarrier)

Hollow Fiber LSBR (FiberCell Systems) SU fully-controlled PC. Suspension / adherent Moving packed-bed BioCell MB & MBS (Bioreactor Sciences) SU fully-controlled PC. Suspension / adherent Fixed packed-bed iCELLis™(ATMI) , Celligen FibraCel (NBS)

and TideCell (Cesco) SU or hybrid fully-controlled PC. Adherent and/or some suspension culture.

Perfusion Enabling Tech Commercial Examples

Perfusion Application

Hollow fiber media exchange ATF System (Refine Technology) and KrosFlo® (Spectrum)

Hybrid or full SU PC when combined with certain bioreactors. Suspension or adherent (eg, microcarrier) culture.

Centrifugal media exchange Centritech (Carr) and kSep®(KBI Biopharma) SU PC as combined with certain bioreactors. Suspension or adherent (eg, microcarrier)

Sonic wave media exchange CYTOPERF (APIcells), BioSep (Applikon) and (FloDesign Sonics)

SU or hybrid PC when combined with certain bioreactors. Suspension culture.

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CB modifies single-use concepts

Use just once – for 3 months?

Concept

Definition CB-specific Modification

Reusable Equipment or material intended for use for an indefinite number of times: especially in different production cycles or batches, and after salvaging or preparation by special treatment or processing.

None

Multi- or limited-use Equipment or material intended for use in a process for a limited number of times: determined by validated procedure or subsequent testing.

CB can increase the time and throughput volumes involved in each “use”, review of number of iterations is advised.

Single-use Equipment or material intended for use in a process for one time and then retired from use.

CB can increase the time and throughput volumes involved in each “use”, review of validation requirement is advised.

Hybrid Equipment or material composed of both reusable and single-use components.

None

Disposable Equipment or material intended for use either for one time or for use in a process in a limited number of times, and then retired as waste or garbage.

Same alteration as either “Single-use” or “Multi-use”, depending upon the intent.

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Agenda

1. Single−Use Systems in Biomanufacturing

2. Single−Use Systems in Continuous Biomanufacturing

3. Vaccines and Single−Use Continuous Biomanufacturing

4. Next-Gen Products and SU Continuous Biomanufacturing

5. New Continuous Biomanufacturing−Enabling Technologies

6. Continuous Biomanufacturing Applications Data / Case Studies

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Vaccine manufacturing trends

• Vaccine manufacturing technology developing • Vaccine manufacturers numbers increasing • Single-use implementation growing

A. DeArment, DSN, Sept.11, 2013

16.15 Single use technologies in bacterial vaccines production: Current state of the art and future directions This presentation will address the application of single-use disposable bioreactors in the upstream process development. Dr Tatjana Plješa, Bacterial Vaccine Production DPT, Institute of Virology, Vaccines and Sera Torlak, Serbia

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SU in vaccine manufacturing

• Eg, Novavax employing single-use technology • Uniformity from seed culture through diafiltration • T. J. Hahn, BioProcessing Journal, August, 2013

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CB in vaccine platforms

• Animal cell-based production • Cell lines from HEK 293 to CHO to Sf9 • Vectors from Lentiviruses to Baculovirus to Alphavirus • Evolving stakeholders, eg, Lentigen, Protein Sciences, Novavax

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CB vaccine product candidates

Viral • Subunit, recombinant, psudeotyped, nanoparticles (micelles) VLP, VL replicon… • CB probably only through

• Recombinant protein expression • Persistent (non-lytic) infection, lysogeny

• Already 2000L SU virus production

Jordan, et al, BioProcess International 10(8) Sep, 2012

Gene Transfer and Expression in Mammalian Cells, Makrides, Editor. Gulf Professional Publishing, 2003

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Anchorage-independent CB vaccine formats

• CB-amenable (non-lytic) anchorage-independent (suspension) cultures

• A number of platform approaches • Non-lytic MVA platforms • Persistent baculovirus infections • rAnimal cells expressing antigenic proteins

• A number of reactor styles • Classic reusable bioreactors • Single-use bioreactors (SUBs) • Including perfusion implementations

CultiBag RM 2-L perfusion

FiberCell Systems LS-HFBR bioreactor

Thermo Scientific S.U.B. with Refine Technology ATF

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Agenda

1. Single−Use Systems in Biomanufacturing

2. Single−Use Systems in Continuous Biomanufacturing

3. Vaccines and Single−Use Continuous Biomanufacturing

4. Next-Gen Products and SU Continuous Biomanufacturing

5. New Continuous Biomanufacturing−Enabling Technologies

6. Continuous Biomanufacturing Applications Data / Case Studies

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Hard to organize “next-gen” system / modes

• Many developing • Cell culture formats • Expression systems • Entity and vaccine types

• Two examples • Constitutive mammalian virus protein

expression by a viral promoter in insect cells using a plasmid vector

• Constitutive mammalian cell expression of a jellyfish protein by viral promoter using an insect virus vector

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CB in next-gen biopharmaceuticals

• Antibody conjugates (~20 in development) • Most pre-conjugates in CHO / NSO suspension transfectoma

• NSO: Wyeth-Ayerst Mylotarg (withdrawn) • CHO:

• Adcetris from Seattle Genetics • Kadcyla from Roche/Genentech

• Amenable to perfusion / CB • Linker / pro-drug conjugation by CP?

2010

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SU continuous fermentation in biopharma

• Borrowing from other biotechnology • Increasing publications appearing • Even commercialized products

High Cell Density E. coli Cultivation in Different Single-Use Bioreactor

Systems, Dreher, et al, Chem Ing Tech

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SU CB in next-gen products

Next Generation Technology SU Potential CB Potential Biosimilars, biobetters Yes – CB Yes Cocktails / polyclonals Yes – CB Yes Next Generation antibodies Yes – CB Yes o Bi- multi-specific antibodies (bisAbs, BiTEs, DARTs) Yes – CB Yes o New CD20, HER2 and EGFR Ab’s Yes – CB Yes o ADCC – enhanced MAb oncotherapeutics Yes – CB Yes o Recombinant antibody mixtures Yes – CB Yes o Glycoengineering, Fc AA mutations Yes – CB Yes o Engineered Ab (eg, scFv, VH/VL, mini-Abs) Yes – CB/CF Yes o Fragments and Ab–like proteins (FAb, Fcab, nanobody) Yes – CB/CF Yes o Immunoconjugates, eg, ADCs Yes – CB/CF Pre-conjugate New protein biologics for cancer, osteoporosis, ophthalmic… yes – CB/CF Yes Designed Ankyrin Repeat Proteins (DARPins) Soon – CF Yes Domain antibodies, dAbs, other next–generation fragments Soon – CF Yes Anticalins, Adnectins and other alternative scaffolds Soon – CF Yes Dual ligand peptides and assemblies Soon – CF Yes Viral vectors and vaccines Yes – CB Non-lytic

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Agenda

1. Single−Use Systems in Biomanufacturing

2. Single−Use Systems in Continuous Biomanufacturing

3. Vaccine and Single−Use Continuous Biomanufacturing

4. Next-Gen Products and SU Continuous Biomanufacturing

5. New Continuous Biomanufacturing−Enabling Technologies

6. Continuous Biomanufacturing Applications Data / Case Studies

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CDI Bioscience: RP Shift®

• Recall Mike Flickinger on non-dividing cell productivity

• RP Shift enables • Increased recombinant protein production • By “shifting” cells out of proliferation cycle • Cells produce in reduced growth / division • Paused cells secrete product /do not grow

• RP Shift technology operates by • Anti-apoptotic factor conditional activation • RP-1 repressor elements (RP-1) • Two cyclin dependent kinase inhibitors

• RP Shift inducible technology details • Contains anti-apoptotic factor cDNA • Genes under lac-inducible promoter • Expressed factors pause cells in G1 / G0

rChOk1 A

rChOk1 b

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CDI Bioscience: PACE™ CHO Cells

• Engineered with RP Shift technology • PACE CHO-DG44X • PACE CHO-KX

• Robust cell lines with high “unshifted” growth rate • Easy to maintain, transfect, select, expand • Easy to optimize process • Stable producers • Easy to scale-up

• Improves overall production • Function in batch and fed-batch culture • Demonstrated performance in perfusion culture • Large-scale, multiple products, single-use systems

Pro

duct

ivity

, P

CD

rCHOk1 B

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PACE™ rCHO Cell Producers

Non-fed Batch Performance

Recombinant producer PACE cells

• Halted proliferation • Cell volume increased • Cell lifespan increased • A flattened morphology • Viability 0 – 15% higher • Increased mitochondria • More endoplasmic reticulum • Environmental stress resistant • Hydrodynamic force / shear resistant • Similar results in all major cell platforms • Specific productivity (PCD) 2-6 fold higher

0

1

2

3

4

0 1 2 3 4 5 6 7Day

VCD

(106 /m

l)

Parental No Shift RP Shift

0

1

2

3

4

0 1 2 3 4 5 6 7Day

VCD

(106 /m

l)

Parental No Shift RP Shift

0

1

2

3

4

0 1 2 3 4 5 6 7Day

VCD

(106 /m

l)

Parental No Shift RP Shift

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Agenda

1. Single−Use Systems in Biomanufacturing

2. Single−Use Systems in Continuous Biomanufacturing

3. Vaccines and Single−Use Continuous Biomanufacturing

4. Next-Gen Products and SU Continuous Biomanufacturing

5. New Continuous Biomanufacturing−Enabling Technologies

6. Continuous Biomanufacturing Applications Data / Case Studies

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Single-Use 50L S.U.B and KrosFlo System

Runs 1-3

• Equipment • HyClone HyPerforma TK S.U.B. (50L) • Spectrum Labs KrosFlo® Perfusion System • mPES, 0.5 mm ID, 0.54 m2, 30 kDa pore size

• Materials • CHO cell line • HyCell CHO medium

• Analytics • ViCell XR (Beckman Coulter) • Octet 384 (ForteBio)

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Results S.U.B. / Khameleon System

• Runs 1 and 2 • Frit and open pipe

• Run 3 • Prototype drilled-hole sparger

2

1

3

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Single-Use 50L S.U.B and ATF 6

Run 4

• Equipment • HyClone HyPerforma TK S.U.B. (50L) • Refine Technology ATF 6 • PES, 1.0 mm ID, 2.5 m2, 0.2 µm pore size • Frit and open pipe

• Materials • CHO cell line • HyCell CHO medium

• Analytics • ViCell XR (Beckman Coulter) • Octet 384 (ForteBio)

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Hexameric IgG: 1.15 Md Original failed polymerization vs

optimized hexameric product

Bennett, A., et al, JBC Sep, 2007

SU HFB continuous culture

SU Hollow Fiber Bioreactor (HFB) culture

• Proprietary IgG1 Hexamer • Productive perfusion culture maintained over 2 months • 40% improper monomeric form in serum-supplemented T-150s • 95% hexameric form in optimized CDM-HD perfusion culture SFM

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SU HFB continuous production of MSCs

Biomemetic conditions

• 3-D matrix culture • 5 kd MWCO fiber barrier • Continuous gradient of renewed media (plasma-like) • Generating / maintaining a required autocrine "cocktail”

Red masses of cells generated on fibers post day 10

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SU HFB continuous production of MSCs

Materials and Methods • Placental mesenchymal stem cells (PMSC) • From full term human placenta digest • Raw harvest of mixed cell types

• Hollow fiber perfusion bioreactor (HFB) culture • 5kd MWCO polysulfone cartridges • FiberCell Systems Duet®

• 3,000 cm2; 20 ml ECS • Perfusion rate: 60 ml/minute • Harvest each 2 – 7 days into T-flasks • >2 month continuous productive culture

Cartridge harvest T-flask culture (D4)

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SU HFB continuous production of MSCs

Results

• Initial placenta harvest marker phenotype distribution w/controls (Table 1) • Continuous 3 mo culture ECS yields enhanced MSC precursors (Table 2)

Table 2. Direct cartridge-harvest vs post 3-5 day flask culture

Table 1. Phenotype pre-loading and from ECS every 3-7 days. Last two rows are the % of indicated phenotype

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Alliances and partnerships

• SUS providers aligning with perfusion technology • Reduced facility classification • Continuous • Disposable • Closed • Coming: Integrated?

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Conclusion

1. Many SUS exist to support biomanufacturing

2. Developing SUS support continuous biomanufacturing

3. SUS support most next-gen products and platforms in a continuous format

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