Production and purification of Viral vectors for gene and cell therapy applications

Priyabrata Pattnaik, PhDDirector & Head of Biologics OperationsAsia Pacific

Production and purification of Viral vectors for gene and cell therapy applications

The cell & gene therapy market is poised for rapid growthwith projections reaching ~$10B in 5 years

Source: Seed Planning; METI; Kuick Research; Med Market Diligence;Transparency market research

Global market size & CAGRUSD B

Products in the pipeline

7474

345475

Stem celltherapy

106

17

14

Cellular therapy, other

12315

Gene therapy

14416

Phase I

Phase II

Phase III

109

1

12

8

3

2020

+36% p.a.

20182012

Kuick research (India)See Planning/METI (Japan)

Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 20162

The cell & gene therapy market can be split into 3 broad segments...

Description and examples Major players

Cell & gene therapy

Non-geneti-cally modified cell therapies

• Purified populations of allogeneic cells are expanded and injected topically

• E.g., mesenchymal stem cells

Viral gene therapies

• Drug product is virus particle, injected into patient topically (or systemically)

• E.g., Spark’s night-blindness therapy

• Autologous patient cells are extracted, genetically modified using viral transduction, then reinfused into the patient

• E.g., CAR T-cell therapy

Genetically modified cell therapies



3 broad segments

Gene Therapy Stem-cell Therapy Cell Immunotherapy

Gene Therapy

Stem-cell Therapy

Cell Immunotherapy

Nucleic acid Lenti/Adeno virus-based delivery Somatic or germline

Stem cells banking Replace or regenerate organs and tissue Broad use of bone marrow in ALL

Autogenetic CAR-T1. Most common2. Immune cells separation3. Lentivirus-based delivery4. Chimeric Antigen Receptors (CARs)

Allogeneic modified T cells1. Industrialized trend2. Genetic modification of immune cells3. Allogeneic T cell generation4. Lentivirus-based delivery or genome edit (CRISPR)

CAR-TVirusStem cells

T-cells

Virus

CAR-T4

Methods for producing genetically modified T-cells for immunotherapy

Source: Bure et al. (2016) Automation of CAR-T Cell Adoptive Immunotherapy Bioprocessing. Bioprocess International, 14(4)s: 22-31.



Overall Process Map of CAR-T

T-cell separation

Virus generationGMP-grade lentivirus Recovery rate= 70% 2.5*109 virus per trial 5*106 titer/ml 500ml bulk

T-cell modification MOI=5 1.7*109 virus per trial

Car-T Cell Amplification

Reintroduction 5*106 CAR-T/kg E.g.: 70 kg weighting 3.5*108 CAR-T per trial

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7 Production and Purification of Virus Vectors | Priyabrata Pattnaik | October 2016

Viral Vectors For Gene TherapyIntroduction

Gene Therapy – Main Applications:• Treat Heridatery Single-Gene Defects.• Cancer• Cardiovascular Diseases• Ocular Diseases• Infectious Diseases• Other Diseases Where Gene Transfer Will Have an Impact

Main Features for Virus-Based Vectors:• Lacks Viral Genes Involved in Replication.• Expression Cassette is Cloned into the Vector.• Helper Function is Provided in trans• Co-Transfection of Vector Genome and Packaging Construct Produce Recombinant Vecotor

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~60% of the vaccines undergoing clinical trials are viral based. ~ 640 viral vaccines ~ 200 viral vectors ~ 60 virus like particles

Another ~240 gene therapy products in development that utilize the same technology.

Example of vectors:

Adenovirus (70-100 nm)

Lentivirus (80-100 nm)

HSV (300 nm)

Viral Vectors – Introduction



Features AAV LentivirusGenome ssDNA ssRNA(+)Virus Coat Non-Enveloped EnvelopedDiameter 18 – 26nm 80 – 130nmPackaging size 4.5 kb 8 kbInfection Range Dividing & Non-Dividing Cells Mostly DividingPost Infection Mostly Non-Integrating* Integrates into Host GenomeGene Expression Long lasting (?) ProlongedMain Advantage Non-pathogenic; Non-inflamatory Persistent Gene TransferMain Disadvantage Small packaging capacity Oncogenesis may occur

Characteristics of AAV & Lentivirus

^ AAV (mainly serotype 2) may integrate into Chromosome 19


AAV Serotype Tissue Tropism

AAV1 Muscle, Heart, Ocular, CNSAAV2 CNS, Kidney, Muscle, Testes

Various in vitro applicationsAAV4 Lung, AAV5 Lung, CNS, Ocular, PancreasAAV6 Lung, HeartAAV7 Muscle, LiverAAV8 Liver, Muscle, Ocular, CNS, HeartAAV9 Lung, Liver, Muscle, Heart, CNS, Kidney, TestesAAVrh10 Pleura, CNSOther Various

AAV Serotypes and Tissue Tropism


Transient Transfection Mediated by PEI or Calcium Phosphate. Co-transfection of AAV production cells with 3 plasmids:

• Plasmid with AAV ITR and the gene of interest• Plasmid with AAV rep/cap• Plasmid providing the helper genes isolated from adenovirus.

Wild Type Adenovirus Infection Into Cell Lines with AAV rep/cap Genes and AAV Vector.

Infection using two HSV viruses harboring the gene of interest and the rep/cap genes to produce AAV.

Infecting sf9 cells with two baculoviruses harboring the gene of interest and the rep/cap genes to produce AAV.

Production Modes for Recombinant AAV

Sufficient Production of virus vector copies


Adenovirus vectors manufacturingKey requirements and needs

Antigens attributes have to meet pre-set Critical Quality Attributes

Reproducibility & consistency

Biosafety

Sufficient recovery to achieve acceptable economics

Ex: nucleic acid content, product identity & safety, etc.

Titer

Regulatory

Recovery

Purity & Quality

Process Schematics of AAV vector production and Purification


UF/DF BenzonaseTreatment

Primary Purification

Chromatography

Media and Inoculums Preparation

Cell growth in BioreactorHEK 293 Cells

Virus InoculationTransfection

VirusHarvest (Lysis)

PrimaryClarification

UF/DFSterile

Filtration

SecondaryClarification

SecondaryPurification

ChromatographyFill & Finish

Thaw and Expand

Cells and Seeds

Optional BenzonaseTreatment





Chromatography




VirusHarvest


UF/DFSterile

Filtration




Thaw and Expand

Cells and Seeds



Amplification & culture of HEK-293

Bioreactors

T-Flasks*

*Source: Wang and Rivière (2015) Manufacture of tumor-and virus-specific T lymphocytes for adoptive cell therapies, Cancer Gene Therapy (2015) 22, 85–94.

Segura et al., Biotechnology & Bioengineering, 2005, 90 : 391-404

Virus Total Protein DNA

MCBInnoculationShake/spin

Sf-9

HEK 293EB66® Cells

BHK21

Sf-9

HEK 293EB66® Cells

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Minimize Your Process Development Efforts Leverage Merck’s Experience with Various Cell Lines in Mobius® Bioreactors

CHOHybridoma

SP 2/0

Vero

h-MSCT cells

HepaRG®

Adherent

Suspension

Public references at the bench (2 L) … and production scale (50 to 2000 L)

Vero

Power/Volume

Agitation speed

MDCK

S2 Drosophilia

mAb / Rec-protein

Virus Production (Suspension)

CellTherapy

Virus production (Adherent)

h-MSC

HepaRG®

CHO

Hybridoma

*application data available

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http://www.fhcrc.org/en.html

http://www.fhcrc.org/en.html

http://bioindustryassociation.files.wordpress.com/2013/04/cell_therapy_catapult-logo-18-4-131.png





• Standard media: EX-CELLTM 293 Serum-Free Medium for HEK 293 Cells in suspension

• Custom media on demand

• Supplements

Cell culture Media preparation

Durapore® or Express® 0.22 or

0.1 µmSterilizing

Or Viresolve®

Barrier (available soon)

Lynx sterile connectors


Bioreactor Biosafety: Preventing contamination

Control

Protect

Maximum control of animal derived materials In-coming test of critical materials Move to animal free raw materials processes. Recombinant versions of Trypsin, insulin, albumin etc

(Cellprime® range).

• As a minimum: sterile filtration of cell culture media (Express SHC for example)

• Implementation of a Mycoplasma or a Virusbarrier: filtration of cell culture media for example using Viresolve® barrier

• Sterile Sampling with NovaSeptum• Aervent filters for the aeration (0.22µ)• Sterile connectors (Lynx)





Chromatography




VirusHarvest (Lysis AAV)


UF/DFSterile

Filtration




Thaw and Expand

Cells and Seeds



Adherent cells may be lysed in situ or detached from the growth substrates

Lysis can happen by freeze-thaw lysis, mechanical homogenization or chemically via the use of surfactants.

Large volume suspension cultures may be treated with surfactants, e.g. Triton X-100, or homogenized with a mechanical device.

Nuclease treatment is incorporated following lysis to reduce viscosity and facilitate subsequent filtration and chromatography steps.

Cell lysis to release virus particles

Purification


Clarification Centrifugation

Centrifugation allows effective removal of microcarriers, but not so much for cell/debris separation; the viral yield recovery is typically low (≤30-50%)

Post-centrifugation clarification using double layer PES filter offer low throughput due to insufficient clarity of the post-centrifuge viral supernatant and cause additional losses of viral yield


Clarification strategiesCell density & size matters

Low Concentration

High Concentration

Small particlesColloids

Charged Depth Filters

TFF Filters

Surface Filters

Non-charged Depth Filters

Filter capacities depend on cell density and degree of lysis and particle size distribution

Large/hard particles

Milligard, Polysep, Polygard CN

Polygard CR, Clarisolve 60HX

Prostak, Pellicon 2

Millistak+

Clarisolve 20/40 MS


Case study: Clarification of AAV8 • Adeno-associated virus harvest clarification. Previous process

using a competitive depth filter.

• Clarisolve 20 MS was selected for primary clarification

• No pre-treatment required

• Adeno-associated virus harvest clarification. Previous processusing a competitive depth filter.

• Clarisolve 60 HX was tested for primary clarification.

Increase of 4 x throughput vs previous depth filtration filter = reduction of footprint

• No pre-treatment required

Depth filtration based clarification – primaryAAV case studies

20MS: Polypropylene & Millistak® media 60HX: Polypropylene media construction with no (+)-charged resin binder and no DE offer inert surface, hence no virus binding issues


Millistak+ D0HC can be also used for the clarification of virus lysates despite of (+) charges (cellulose fibers with Diatomaceous earths).

Can be an option to test side by side with Clarisolve(similar format, same holder for large scale) & NFF filters

Example: Capacity range from 30 – 300 L/m2 for Adenovirus from Per.C6 cell harvest

Depth filtration based clarification Millistak+ for primary clarification


NFF based clarification of AAVFiltration train example

Cell/Virus

Harvest

Polysep II

1.2/0.5 µm

Durapore0.45µm

Pellicon 2 Biomax 100-300kD Polygard CR

5µm

OR

Clarigard3 µm

Clarification Bioburdenreduction

Concentration


Clarification systems with Single Use flow pathsSemi-automated systems

For POD formats (Millistak+ & Clarisolve®

For NFF filter capsules





Chromatography




VirusHarvest


UF/DFSterile

Filtration




Thaw and Expand

Cells and Seeds


Solution: Genetically engineered endonuclease that cleaves all forms of DNA and RNA.

Presence of Mg2+ (1-2 mM) is required for enzyme activity. One unit of Benzonase® degrades approximately 37µg DNA in 30 min to as

low as 3-8 base pairs (<6 kDa). Benzonase® can be detected with dedicated ELISA kit. Sensitivity 0.2 ng/ml

Nucleic Acid Digestion Benzonase® Endonuclease


Regulatory: <10 ng nucleic

acid/dose

CharacteristicsOrigin Serratia marcescens

Expression E. Coli K-12 mutant

Molecular mass ~ 30 KD

Isoelectric point 6.85

Functional in pH range 6-10

Temperature 0-42°C

Bioprocess International, February 2014

Parameters Biomax 300kD

Feed flow (l/min/m²) 6

TMP (bar) <0.3

Initial flux (LMH) 30

Final flux (LMH) 30

Average flux (LMH) 30

Volumetric Concentration Factor 10

Diafiltration volume 2

Purification: First StepTarget: 25-fold conc + DF, vector recovery of 90%

Success Criteria Good yield & Retention Higher purity and

Contaminant Removal- hcDNA- HCP- Spent Benzonase

SolutionPermeate controlPellicon 2 Biomax 300 kD, V Screen for Lentivirus100 kD for AAV

Lentivirus & AAV: Typical TFF process parameters

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TFF-MF (0.1-0.65 µm) and Open UF (300-1000 kDa) optimization






Chromatography




VirusHarvest


UF/DFSterile

Filtration




Thaw and Expand

Cells and Seeds



Purification of AAV Using cation exchange Fractogel EMD SO3- as primary step

“The recovery from the Fractogel SO3 − column was almost 100% based on the infective viral particle recovery. In our laboratory the Fractogel SO3 − has consistently shown a recovery between 80 and 100%, which is dependent upon the extraction procedures and the variability in the infective viral particle assay”

+ Benzonase®

Fractogel® EMD


Purification of AAV Ion exchange schemes

(A) Two-step purification protocol involving a strong cation exchange chromatography resin (Fractogel SO3-) followed by a strong anion exchange resin (Fractogel TMAE)

(B) Capture of the AAV vector by anion exchange chromatography using a strong anion exchange resin (Fractogel TMAE) with subsequent polishing by gel filtration chromatography.


Best method of separation is ultra centrifugation, but has challenges of scalabaility.

Ion exchange chromatography with dual shallow gradient (pH & conductivity) can be used.

pI of empty particles could be higher than that of packaged virions.

AAV empty capsid separation

SOURCE: Okada et al., (Sep. 2009) Scalable Purification of Adeno-Associated Virus Serotype 1 (AAV1) and AAV8 Vectors, Using Dual Ion-Exchange Adsorptive Membranes. Human Gene Therapy, 20:1013–1021


TFF & chrom systems with Single Use flow pathsFully automated, recipe driven

Chromatography TFF (UF/DF)Controls Automated with recipe control CCP6

Operating pressure

Up to 60 psi

Operable fluid temp

4oC-30oC 4oC-45oC

Area Column diameter: 60 – 350 mm

System 1: 0.5-2.5 m2

System 2: 2.5-5 m2

SensorsPre & post columnpH, conductivity, UV, temperature, pressure

Added sensors for conductivity, UV

Flow Rate System 1: 0.1 – 2.2 L/minSystem 2: 1.6 - 8L/min

System 1: up to 18 L/minSystem 2: up to 28 L/min

OtherLinear & step gradient mix: Mixing range 10-90%Accuracy :+/-2 %

Mixing tank 50L to 100/200LOptional jacketed





Chromatography




VirusHarvest


UF/DFSterile

Filtration




Thaw and Expand

Cells and Seeds



Screening for excipients that prevent AAV2 vector aggregation

SOURCE: Wright et al., 2005. Identification of Factors that Contribute to Recombinant AAV2 Particle Aggregation and Methods to Prevent Its Occurrence during Vector Purification and Formulation. Mol. Ther., 12(1): 171-178.


Formulation Storage & Transport

Range of formulation buffers and excipients to ensure long-term stability

• Buffers (ex: Tris, HEPES, PBS)

• Salts (freeze-dry)

• Stabilizers (ex: Polysorbate)

• Polyols: Manitol, sorbitol, PEG

• Sucrose, Trehalose (coming soon)

Emprove® dossier

Single Use Mixing & transport technologies


Bioburden monitoring is essential (<10 cfu/ml) Durapore 0.22µm filter can be used as final filter

and had to undergo retrospective validation Process need to operate at low pressure to avoid

product loss during filtration Lot release will depend on sterility testing of final

dosage form & filter IT test

Sterile filtration and storage


Wide range of buffers in solid or liquid forms• Biological buffers (organic, ex amino-acids)

• Purfication & formulation buffers (ex: NaOH, NaCl, PBS, etc..)

• Cleaning in place (ex: NaOH, HCl)

• Emprove® dossier c

Buffers

Liquid buffers: dual sourcing strategy • Berlin, Germany (batch size: up to 2000 L)

• Irvine, Scotland (batch size: up to 10000 L)


Final FillingComponent choice

CriteriaGamma compatibility

>40kGy

Statement of animal origin

USP<88> Class VIpost-gamma >40kGy

USP<85> Endotoxin,post-gamma >40kGy

USP<788> Particulates,post-gamma >40kGy

USP<661> Physicochemical,post-gamma >40kGy

Shelf life >2.5 years, post-gamma>40kGy

Total Bioburdenpre gamma

Bacteriastatis/Fungistasis,Post-gamma >40kGy

Configurable Assembly

Component Library

All availableComponents

Solutions from Merck for AAV vector production and Purification


Pellicon 2Biomax 100 kD

BenzonaseELISA Kit IIMobius Mix

Fractogel TMAE

Express HPF > SHR Mobius BioreactorNovAseptum SamplingAervent (vent filter)

Mobius Mix

Polygard CR 5µmClarisolve 60HX

Pellicon 2Biomax 300 kD

Durapore0.22µm

Polysep II 2.0/1.2

Fractogel DMAE

Mobius IntegratedFill FinishSolutions

Thaw and Expand

Cells and Seeds



Benzonase® Treatment Clarification Intermediate TFF

Tangential Flow Filtration

Last StepDrug

Substance

First stepCell thawing

Cell culture (perfusion) – 5 weeks Virus infection – 2 days

3-6

weeks

USPweek 1 to 5

DSPweek 5

DSPweek 6

Final Filtration

2 stage Ion Exchange Chromatography

Virus vector– Single Use Process manufacturing overview


Quality-control assays for clinical-grade AAV vectors

SOURCE: Arie van Oorschot (UniQure) Setting up a market scale manufacturing platform from scratch. Cell Culture World Conference. 23 February 2016


Schematic of the manufacturing steps associated testing regimes for a cell therapy production process

Source: Alison Armstrong (2016) Advances in Assay Technologies for CAR T-Cell Therapies. BioPahrm International, 28(2): 32-37.

Quality, Science and Services You Can Trust

Merck Life Science (SAFC and BioReliance) provide the greatest array of process development, manufacturing and testing support services for our clients.

Viral Based Gene Therapy products (Adenovirus, AAV, Retrovirus, Lentivirus, others)

Cell Banking Viral Banking Bulk Drug Substance Bulk Drug Product Custom Assay Development BioSafety Testing

Viral Vector Production at SAFC Carlsbad Facility



Merck offers wide range of technology, tools

and services for Production and purification of

Viral vectors for gene and cell therapy

Thank You

Priyabrata Pattnaik, [email protected]

@pattnaik_p

https://sg.linkedin.com/in/priyabratapattnaik

https://plus.google.com/109816383630328905377

Production and purification of Viral vectors for gene and cell therapy applications

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