ASGCT - 2020 Annual Meeting I May 12 – 15, 2020 I Virtual

Phase-Appropriate Viral Clearance Strategy for Sf9/Baculovirus Based Manufacturing of Gene Therapy ProductsAbhiram Arunkumar, Matthew Luther, Blake Hotz, Danielle Ladwig, Nripen SinghVoyager Therapeutics Inc., 75 Sidney Street, Cambridge, MA 02139, USA

SummaryPhase-based approach to viral clearance in rAAV processes• Developed an rAAV purification process with four robust and orthogonal viral reduction unit

operations.–Detergent Treatment, Affinity Chromatography, Anion Exchange Chromatography, and Viral Nanofiltration.

• Established panel of four model viruses that are relevant to rAAV Sf9/BACV manufacturing processes.– Baculovirus (BACV), Vesicular Stomatitis Virus (VSV), Human Adenovirus Type 5 (Ad5) & Reovirus Type 3 (Reo3).

• Demonstrated the applicability of a phase-appropriate viral clearance strategy.– Early phase strategy to support clinical studies.– Late phase strategy to support regulatory applications for commercial approval.

AbstractDue to the inherent risk of adventitious and endogenous virus contamination in the manufacturing process of a biotherapeutic modality, clearance of both classes of virus is a vital objective in the development of a robust purification process.

In this work, we evaluate the clearance of model endogenous and adventitious viruses with different physico-chemical characteristics in recombinant Adeno-associated virus (rAAV) material produced in the Sf9/Baculovirus production platform using both early and late stage purification. The ability to successfully demonstrate the clearance of potential adventitious viruses ensures the safe and continuous supply of clinical and commercial products.

Results from this study show the reduction of both model adventitious and endogenous viruses using four orthogonal purification steps. These results also provide guidance in choosing viral clearance parameters for rAAV processes and related implications for commercial manufacturing of rAAVs for gene therapy.

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IntroductionConvergence of CNS and rAAV Gene Therapy

• Genetically-validated targets with significant unmet medical need

• Targeted delivery to regions of the brain and spinal cord

• Durable transgene expression as CNS neurons are terminally differentiated

• Immune-privileged site reduces risk of immune response

• Tissue and cell-specific targeting within the CNS

• No AAV-related SAEs to date in >200 patients treated in CNS

• Does not readily integrate into the target cell genome, reducing potential for oncogenesis

• Ability to manufacture at commercial quality and scale

Severe Neurological Diseases

AAV Gene Therapy

rAAV manufacturing technologies derived from: Clément, N., and J.C. Grieger. 2016. Manufacturing of recombinant adeno-associated viral vectors for clinical trials. Mol Ther Methods Clin Dev. 3(16002). Available from:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4804725/

Adeno-Associated Virus (AAV) Adenovirus Lentivirus Herpes Simplex Virus-1

~20 nm ~100 nm ~110 nm ~200 nm

Episomal Episomal Integrative Integrative

+ mild immune response+ replication deficient + efficient transfection + persistent gene expression + high capacity (≤ 150kb)

- limited capacity (< 5 kb) - inflammatory response - risk of insertional mutagenesis - risk of insertional mutagenesis

Product Cell Line Infection /TransfectionProduction

MediaOverall

Risk Comments

rAAV Sf9 BACV Animal-Origin Free (AOF) LOW - insect cell line- non-pathogenic infectionrAAV HEK293 (adherent) CaPO4 / PEI Animal-Derived Additives MEDIUM

- human cell line- animal-derived (serum)

rAAV HEK293 (suspension) CaPO4 / PEI AOF MEDIUM- human cell line- chemical transfection

rAAV HeLa Ad5 AOF HIGH - human cell line- pathogenic infection agentrAAV sBHK (other) HSV AOF HIGH - mammalian cell line- pathogenic infection agent

Unit Operation Mechanism Test ArticleDetergent Treatment Inactivation Clarified Lysate

Affinity Chromatography Selective-based Removal Clarified LysateAEX Chromatography Charge-based Removal Affinity Pool

Virus Retentive Filtration Size-based Removal TFF Pool

DETERGENTTREATMENT

CGMPTarget

Scale DownTarget

Viral ClearanceTarget Comments

Feed Volume (mL) X MIN MIN Scale down by volumeFeed pH X MATCH MATCH Match CGMP target, neutralAgitation (RPM) X MATCH MIN Worst-case for viral clearanceDetergent Conc (% w/v) X MATCH MIN Worst-case for viral clearanceInactivation Temp (oC) X MATCH MIN Worst-case for viral clearanceHold Time (min) X MATCH 0 – MIN Worst-case for viral clearance

AFFINITYCHROMATOGRAPHY

CGMPTarget

Scale DownTarget

Viral ClearanceTarget Comments

Column Bed Height (cm) X MATCH MATCH Match CGMP target, scale down by diameterColumn Diameter (cm) X MIN MIN Minimize required feed materialLoad Challenge (L/Lr) X MATCH MIN Worst-case for viral clearanceResidence Time (min) X MATCH MAX Worst-case for viral clearanceProduct Eluate Volume (CV) X MATCH MAX Worst-case for viral clearance

VIRUS RETENTIVEFILTRATION (VRF)

CGMPTarget

Scale DownTarget

Viral ClearanceTarget Comments

Membrane Area (m2) X MIN MIN Minimize required feed materialLoad Challenge (L/m2) X MATCH MAX Worst-case for viral clearanceFeed Pressure (psi) X MATCH MATCH Match CGMP targetPre-Use Flush Volume (L) X MATCH MIN Worst-case for viral clearancePre-Use Integrity Test PASS PASS PASS Match CGMP targetPost-Use Integrity Test(s) PASS PASS PASS Match CGMP target

Clinical Phase Model Virus Family Genome Enveloped Size (nm) Resistance pl Rationale

Early, Late

Baculovirus(BACV) Baculoviridae dsDNA Yes

30-100 (capsid),200-450 (length) Low

3.3 –4.3

Known processcontaminant

Early, Late

Vesicular StomatitisVirus (VSV) Rhabdoviridae ssRNA Yes 45-100 Low

5.4 –6.2

Model for knowncell line contaminant

Late Human AdenovirusType 5 (Ad5) Adenoviridae dsDNA No 70-80 Medium ~4.5Helper virus may

rescue AAV replication

Late ReovirusType 3 (Reo3) Reoviridae dsRNA No 60-80 Medium ~3.9Representative

dsRNA virus

PROCESSFRACTION

EARLY STAGE LATE STAGEBinding Non-Binding Binding Non-Binding

Load X X X XFlow Through NT X X XWash NT X X XPre-Peak NT NT X NTPeak X NT X NTPost-Peak NT NT X NTStrip NT NT X X

LRV Inactivation/Removal Capacity≤ 1 Not significant (not robust)

1 – 2 Indicative (supportive)2 – 4 Moderate (supportive)≥ 4 Significant (robust)

Step RunLRV (TCID50)

Early-Phase Study Late-Phase Study BACV VSV BACV VSV AD5 REO3

Detergent Addition1 ≥ 5.2 ≥ 4.5 4.3 ≥ 5.7 NT NT2 ≥ 5.1 ≥ 4.4 4.4 ≥ 5.8 NT NT

Affinity Chromatography1 4.1 ≥ 4.6 4.2 5.7 2.5 3.82 4.6 5.2 3.7 4.8 2.3 3.3

Used NT NT 5.1 4.6 3 3.9

AEX Chromatography1 5.7 ≥ 6.9 4.2 ≥ 6.0 ≥ 5.3 6.82 6 ≥ 6.7 4.7 ≥ 5.8 ≥ 5.5 7

Used NT NT 4.9 ≥ 6.0 ≥ 5.4 7.2

Virus Retentive Filtration1 ≥ 5.1 ≥ 4.7 ≥ 6.2 ≥ 4.0 ≥ 3.4 ≥ 7.12 ≥ 4.7 ≥ 5.0 ≥ 6.1 ≥ 3.8 ≥ 3.2 7.2

TOTAL Log Reduction ≥ 20 ≥ 20 ≥ 18 ≥ 20 ≥ 11 ≥ 17

ANION EXCHANGECHROMATOGRAPHY

CGMPTarget

Scale DownTarget

Viral ClearanceTarget Comments

Column Bed Height (cm) X MATCH MATCH Match CGMP target, scale down by diameterColumn Diameter (cm) X MIN MIN Minimize required feed materialLoad Challenge (vg/mL*r) X MATCH MAX Worst-case for viral clearanceResidence Time (min) X MATCH MIN Worst-case for viral clearanceProduct FT/Wash Volume (CV) X MATCH MAX Worst-case for viral clearance

Insect Cell Line (Sf9) With Non-pathogenic Infection Agent (BACV) Minimizes Risk During Large-scale CGMP Manufacturing of rAAV

Principal Viral Vectors Used in Gene Therapy

- Barrier to Entry (MCB/WCB Characterization)- Raw Material Sourcing (Qualification Program, Supply Chain)- Facility Design (Modular Suites)- In-Process Testing (Bulk Harvest/Drug Substance Sampling)(Indicator Cell Line - Vero, MRC-5, BHK)

- Viral Clearance/Inactivation (Process Steps - Chromatography) (Dedicated Steps - VRF)

Risk Management Incorporates a Multifaceted Approach

rAAV manufacturing technologies derived from: Clément, N., and J.C. Grieger. 2016. Manufacturing of recombinant adeno-associated viral vectors for clinical trials. Mol Ther Methods Clin Dev. 3(16002). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4804725/

1. Demonstrate ability of the manufacturing process to clear specific relevant viruses2. Estimate process robustness by characterizing ability to clear nonspecific model viruses

Viral Clearance Studies are Mandated Prior to Entering Clinical Trials and for Commercial Launch Of Biologics

VIRAL

SAFETY A SSU R A N C E

REMOVE

PREVENT

DETECT

Implement Robust Clearance Technologies

OptimizeSampling

and TestMethodologies

EnsureSafety of Raw

Materials andProcesses

“The risk of viral contamination is a feature common to all biotechnology products derived from cell lines.” – ICH Q5A (R1)• Viral contamination events in biomanufacturing are rare

but can have serious consequences– Potential impact on patient safety and drug supply– Legal, regulatory, and financial implications– “In use” and manufacturing hold time stability studies– Clinical device compatibility

• Reported major viral contamination events in biopharmaceutical manufacturing:

Wisher, M. 2018. Viral Risk Mitigation: A Global Regulatory Perspective. Bioprocess Int, 16(10). Available from: https://bioprocessintl.comsponsored-content/viral-risk-mitigation-a-global-regulatory-perspective/

MethodsUnit Operations• Selection based on prior knowledge, scale-down model, and step reproducibility• Presence of detergent in clarified lysate test article impacts infectivity

Detergent Treatment• Scale-down qualification not required → well-characterized model, approved study protocol• Demonstrate kinetics of inactivation → faster loss of infectivity = greater virucidal effect

Affinity (Binding) Chromatography• Scale-down qualification required → UV, cond, and pH profiles comparable to large scale

Anion Exchange (Non-Binding) Chromatography• Scale-down qualification required → UV, cond, and pH profiles comparable to large scale

Virus Retentive Filtration (VRF)• Scale-down qualification required → pressure and flux profiles comparable to large scale

Model Virus Panel• Represents known contaminants and range of physico-chemical properties• High titer stocks available, including quantitative/sensitive/reproducible detection assay• Dependent on regulatory submission phase (e.g., IND/IMP, BLA/MAA)• No ssDNA model virus (e.g., PPV, MMV, BPV) to be tested as AAV is in Parvoviridae family

Early-Phase Study Design• Test partial panel of model viruses (n = 2) in duplicate (n = 2)• Test only on new resin (chromatography steps)• Small test article volume required

Late-Phase Study Design• Test full panel of model viruses (n = 4) in duplicate• Test new and used resin in duplicate for cycled steps (chromatography steps)• Model viral filter process pauses and flushes• Large test article volume required

Future DirectionsLate-Phase Study Design• Continue to develop scientific understanding of critical/key parameters– Test multiple unit operations, under a range of conditions with multiple rAAV serotypes

• Employ bracketing approach where appropriate– Validate at high and low parameters to allow operation within an acceptable range

• Build towards platform and/or modular validation strategy– Viral inactivation/removal for individual (modular) or several (platform) unit operations– Data extrapolated to biochemically similar rAAV products purified by a platform process

NT = Not tested for model virus

Unit Operation Test Article Scale Down(# runs)Viral Clearance

(# runs)Detergent Treatment Clarified Lysate 0 4

Affinity Chromatography Clarified Lysate 3 4AEX Chromatography Affinity Pool 3 4

Virus Retentive Filtration TFF Pool 3 4TOTAL -- 9 16

Unit Operation Test Article Scale Down(# runs)Viral Clearance

(# runs)Detergent Treatment Clarified Lysate 0 4

Affinity Chromatography Clarified Lysate 3 + lifetime 16 (new, used)AEX Chromatography Affinity Pool 3 + lifetime 16 (new, used)

Virus Retentive Filtration TFF Pool 3 8TOTAL -- SEVERAL 48

Sample Plan• Demonstrate process understanding by increasing process fractions during late-stage studies• Show variations in peak collection do not impact viral clearance

X = Tested for model virus, NT = Not tested for model virus

Sample Analysis• Pre-testing cytotoxicity and interference assays are required to determine sample dilution• Infectivity assays (TCID50) are the preferred method to determine viral clearance/removal• Large sample volume testing available to improve assay sensitivity

AEX Chromatography Provides Robust Viral Clearance

Platform Sf9/BACV Process Demonstrates High Overall Reduction Factor

LARGE SCALE

VIRAL CLEARANCE

BACV Removal

+DETERGENT

Crude Lysate

ClarifiedLysate

Bulk Harvest

Brx

BACV Inactiv.Cell Lysis

BrxDepth

Filtration0.2 m

Filtration

+TIME

Crude Lysate

ClarifiedLysate

Bulk Harvest

BrxCell Lysis

BrxDepth

Filtration0.1 m +35 nm

Filtration

=

where:V1 = volume of starting materialC1 = virus concentration in starting materialV2 = volume of final materialC2 = virus concentration in final material

2

4

6

8

10 15 20 25 30

VSV

Cle

aran

ceLR

V ±

95%

Con

f Lim

it

Conductivity (mS/cm, 20°C)

2

4

6

8

10 15 20 25 30

BAC

V C

lear

ance

LRV

±95

% C

onf L

imit

Conductivity (mS/cm, 20°C)

2

4

6

8

10 15 20 25 30

ADS

Cle

aran

ceLR

V ±

95%

Con

f Lim

it

Conductivity (mS/cm, 20°C)

2

4

6

8

10 15 20 25 30

REO

-3 C

lear

ance

LRV

±95

% C

onf L

imit

Conductivity (mS/cm, 20°C)

Resin A – Low pHResin A – High pHResin B – Low pHResin B – High pH

1999

1993MVM

1988EHDV

2006MVM

2008Vesivirus

1994MVM

2004Cache Valley 2010

PCV-12000

Cache Valley

2009MVM

Vesivirus2003Cache Valley

Vesivirus

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4804725/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4804725/