Updated Edition 2020 cGMP Cell & Gene Therapy...impurities including polyethylenimine, host cell protein, residual plasmid DNA, host cell genomic DNA, etc . Refer to AAV/Plasmid DNA

cGMP Cell &

AAV, Adenovirus, and LentivirusPD and Production Handbook

Updated Edition 2020

Gene Therapy

Vigene cGMP overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Vigene manufacturing technologies and platforms . . . . . . . . . .5

Facilities and Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Production Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Purification Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

Vigene manufacturing services . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

AAV manufacturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

Adenovirus manufacturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Lentivirus manufacturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

GMP-ReadyTM plasmid manufacturing . . . . . . . . . . . . . . . . . . . . 11

BSL-3 virus manufacturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Aseptic filling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Cell and virus banking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

AAV QC and plasmid DNA QC assays . . . . . . . . . . . . . . . . . . . . . 13

Process development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Table of Contents

www.vigenebio.com © 2018 Vigene Biosciences

3AAV, Adenovirus and Lentivirus Gene Therapy cGMP PD and Production Handbook

Vigene - the cGMP Viral Vector Experts

Vigene’s mission is to make gene therapy affordable .

We strive to achieve our vision by combining new manufacturing technologies and high yield production cell lines with a rigorous quality control system for cGMP .

Vigene Biosciences, Inc . (hereafter Vigene) is a leading gene and cell therapy contract development and manufacturing organization (CDMO) that strives to deliver the highest quality processes and products to our clients .

Vigene’s two divisions (basic research and cGMP production) work together seamlessly to offer “concept to commercial” capability to our clients .

On the basic research side, Vigene is developing, manufacturing, and distributing state-of-the-art AAV, lentivirus and adenovirus based reagents including HHMI/Janelia Research Campus AAV Biosensors .

On the cGMP clinical production side, Vigene is combining proven production technologies with rigorous regulatory compliant cGMP production to meet the needs and expectations of clinical material clients . Vigene offers FDA and EMA compliant cGMP production for lentivirus, adenovirus and AAV . Headquartered in Rockville, MD, Vigene is a leader of both research grade and cGMP grade AAV, lentivirus and adenovirus, serving over 1000 academic, biotech and pharmaceutical labs .

Vigene is constantly striving to consistently provide high-quality clinical phase biologic products and services to meet the needs and expectations of

Vigene Biosciences4 www.vigeneGMP.com

our customers in gene delivery technologies . Further, our management and employees comply with cGMP regulations including 21 CFR 11, 210, 211, and 610 and the relevant EU GMPs . By 2019, Vigene will be operating ten state-of-the-art cGMP production suites with a total of >10,000 sq ft cleanroom space, adding 5 new GMP suites to our existing 5 cGMP production suites in 2018 .

Our facilities meet all US and EU requirements for Phase I & II . The new facilities and systems will meet Phase III and commercial stage clinical material manufacturing requirements . We have been successfully audited by multiple biotech and pharma clients . All products are segregated and produced on a campaign basis and validated change-over and cleaning procedures are deployed between each project . Currently, more than 20 batches of clinical materials have been successfully produced and released .

Vigene features strong Process Development and GMP teams with accomplished virologists and technologists . In addition, we have an experienced team of Quality Assurance (QA) and Quality Control (QC) professionals who work alongside the facility team, material team, PD team

and GMP team to ensure end-to-end quality compliance .

Previous GMP grade clinical materials:1 . Adeno-Associated Virus (Lot sizes of 1E+15 – 1E +16 GC)2 . Adenovirus (Lot sizes of 1E+12 – 1E +13 viral particles or

VP)3 . Lentivirus (Lot size of 1E+10 – 1E +12 Infectious Units or

IFU)


Vigene manufacturing technologies and platforms

Facilities and Technologies

Ten carefully designed cGMP production suites allow parallel campaigns and maximal flexibility in customizable bioprocesses . We leverage proprietary production cell lines and experienced manufacturing teams, as well as partnership with leading technology innovators to provide a variety of scalable bioprocesses and superior product quality based on our clients’ material needs, budget and long-term development plans .

Facility Highlights:• 10 state-of-the-art GMP production suites (>10,000 sq ft); Independent HVAC systems for all

GMP production suites • Compliant and unidirectional personnel, material and product flow• Successfully audited by EMA and FDA-compliant clients• BSL-3 Capabilities: BSL-3 containment cGMP cleanrooms for the production of organisms or

toxins featuring single pass air• 24-hour monitoring and notification service on facility and critical equipment• Vigene places a heavy reliance on disposable systems to reduce changeover and validation

costs

Suspension process Ultracentrifugation

Chromatographypurification

Adherent process

iCELLis 500 (m2)

Upstream platforms Downstream platforms

Stirred-Tank Reactor 200L-500L


Production Cell LinesSuspension HEK293 and 293T Vigene has developed proprietary suspension

HEK293 and 293T cell lines that grow under animal component-free conditions . The cell lines are optimized for rapid and scalable production of all serotypes and chimeras of adeno-associated virus (AAV) and can also be developed for adenovirus and lentivirus production . Vigene has also developed a proprietary suspension-culture-based helper-free process for AAV production using our production cell lines with typical yields of 1E+5 - 1E+6 GC/cell .

Insect Sf9 cellsVigene has entered an exclusive cGMP partnership with

Virovek to use Virovek’s advanced BAC-to-AAV production technology for cGMP production . This technology utilizes the baculovirus expression system to produce AAV vectors in sf9 (Spodoptera frugiperda) insect cells under serum-free conditions . AAV vectors generated from this scalable and efficient platform have been proven to infect and transduce a myriad of mammalian cell lines in vitro and a broad range of cell types in vivo . BAC-to-AAV production technology routinely produces 1E+15 GC with every liter of culture during the PD phase, making it possible to generate a 5E+16 - 1E+17 GC GMP batch with a 500L bioreactor .

C: 73% M:5% Y:31% K:0%

C: 49% M:41% Y:41% K:5%

Pantone 319C

Pantone Cool Gray 9C

Color Matching

CMYK

Pantone Coated

Vertical Arrangements Horizontal Arrangements

High yield proprietary production cell line

All license fees for BAC-to-AAV during phase 1, 2, 3 clinical trials and all milestones associated are waived if cGMP


Cell Culture ProcessScale-up of adherent cell culture The scale-up of adherent cell culture using HYPERstacks or roller bottles can be very labor intensive and inefficient . Vigene uses iCELLis 500 (Pall Life Sciences), the world’s first fully-integrated, single-use high-density adherent cell culture bioreactor to scale up adherent HEK293, 293T and HeLa culture processes . Central to the iCELLis bioreactor technology is the use of a compact fixed-bed, filled with custom macrocarriers . This matrix provides up to 500 m2 available area for cell growth, which is surface- equivalent of 3,000 Roller Bottles (1700 cm2) or 277 HYPERStacks (36 Layers) .

Scale-up of suspension cell cultureVigene uses 200L - 500L single-use stirred-tank bioreactors for high density culture of suspension cells (HEK293-S, 293T-S, HeLa-S and sf9 insect cells) . High titer viral vectors can be produced through either transfection or infection method in batch mode . Bioreactor systems can also be operated in fed-batch and perfusion modes to enhance the upstream process and increase volumetric yield .


Purification ProcessUltracentrifugation Vigene has extensive experience in iodixanol gradient ultracentrifugation for virus isolation and

empty/full capsid separation . However, due to the limited scalability and evolving regulatory guidance, we only recommend this method for small amounts of materials for early stage studies or when chromatography-based purification methods are not available .

Chromatography-based purificationVigene has the capability of processing 200L - 500L batches with microfluidization, depth filtration, tangential flow filtration and multi-column chromatography purification . Process optimization services can be provided to improve recovery rate and impurity removal . A comprehensive range of impurity assays has been established to monitor residual impurities including polyethylenimine, host cell protein, residual plasmid DNA, host cell genomic DNA, etc . Refer to AAV/Plasmid DNA QC assays for more information .


Vigene manufacturing services

AAV Manufacturing Upstream production platforms

• Triple transfection of adherent cells (HEK293, 293T cells)• Triple transfection of suspension cells (HEK293, 293T cells)• Baculovirus based AAV production (Sf9 cells) in partnership with Virovek• Helper virus based production of adherent cells (HEK293, 293T cells)• Helper virus based production of suspension cells (HEK293, 293T cells)

Vigene has developed scalable and proprietary upstream platforms consisting of high producing HEK293 cell lines (typically produce at 1E+5 - 1E+6 GC/cell) and optimized adherent/suspension culture transfection methods for rAAV production . We typically produce at >1E+14 GC/L with both adherent and suspension HEK293 platforms . In addition, cGMP Master Cell Banks have been created and fully tested for both adherent and suspension HEK293 cell lines, allowing streamlined manufacturing and cutting down campaign time .

Vigene also holds an exclusive license for BAC-to-AAV technology from Virovek . The suspension baculovirus-sf9 system generates 1E+17 GC of AAV vectors with a single production run (100L scale), which is recognized as the one of the most powerful and efficient AAV production platforms available .

Downstream purification platformsWe offer iodixanol gradient ultracentrifugation and large-scale chromatography purification services (IEX, AEX, affinity, SEC, etc .) based on our clients’ preferences, material needs, timelines and long-term development plans . With our experienced production, QA and QC teams, Vigene has the expertise and confidence in operating controlled processes to deliver products with optimal yield and purity to our clients .

Transient plas-mid

Adherent HEK293/293T

Adherent HEK293/293TAdherent HeLa

Suspension HEK293/293T-s

Suspension HEK293/293T-sSuspension HeLa-s

Suspension sƒ

Helper virusco-infectionBaculovirus-insect cells

GMP-ReadyTM plasmidmanufacturing

Proprietaryhigh-yield

suspension cell lines

Exclusive license forBAC-to-AAVtechnology

Virus packaging method


Adenovirus ManufacturingAdenovirus Production The cGMP production of clinical grade adenovirus vectors requires knowledge of the complex methods to generate, purify and characterize adenovirus vectors in a well established proven cGMP quality system . As a leader of adenovirus vector production, Vigene combines the leading adenovirus production technology with comprehensive cGMP procedural controls that have been developed to ensure clinical product quality, safety and consistency .

AdenovirusVigene has developed proprietary, highly efficient, and reproducible systems for high titer adenovirus production .

Production yields purified adenovirus for clinical trials in batch sizes of 1 x 1015 viral particles .

Process Specifications• Robust TFF concentration and ion exchange purification process, capable of handling culture

volumes in excess of 30 liters .• Final formulation based on client requirements

Lentivirus ManufacturingLentivirus Production The cGMP production of clinical grade lentivirus vectors for CAR-T and gene therapies requires knowledge of the complex methods to generate, purify and characterize lentivirus vectors in a well established proven cGMP quality system . As a leader of lentivirus vector production, Vigene combines the leading lentivirus production technology with comprehensive cGMP procedural controls that have been developed to ensure clinical product quality, safety and consistency for CAR-T and gene therapy clinical trials .

Vigene has developed a proprietary and highly efficient system for ultra high titer lentivirus pro-duction at clinical scale . The scalable system process for lentivirus production utilizes adherent 293T with titers of 109- 1010 IFU/ml and a total batch size up to 5x1012 IFU .

Process Specifcations• Optimized calcium phosphate transduction protocol• Closed system purification/concentration processing (TFF/ultracentrifugation)• In-house p24 ELISA and RT-PCR assays• Final product yields typically 1E+9 IFU/ml range


GMP-ReadyTM plasmid manufacturing Vigene manufactures GMP-ReadyTM plasmid DNA to support plasmid transfection-based viral packaging campaigns . In 2019, fully GMP-grade plasmid production will be possible in our new facility . With material segregation, full traceability, document control and a comprehensive range of plasmid DNA quality control assays, GMP-ReadyTM plasmid DNA can be used as critical raw material for clinical manufacturing campaigns . Refer to AAV/plasmid DNA QC assays for more information .

• Plasmid productions are conducted in segregated and dedicated production suites . Each plasmid is segregated from other plasmids and plasmid-bearing materials using physical barriers to maintain isolated manufacturing conditions .

• Full room changeover occurs prior to initiation of new projects including cleaning of surfaces and non-disposable equipment .

• The final fill and finish can either be in the GMP facility (Grade-A BSC in Grade B room) or in a Biosafety Cabinet (BSC) in a non-GMP facility with a cleaning procedure between different plasmids .

BSL-3 virus manufacturing Vigene can operate under BSL-3 for the production of organisms or toxins requiring such containment . Unlike other facilities that claim “BSL-2 plus”, Vigene can truly operate under BSL-3 . The BSL-3 suite is on a separate HVAC system with single pass air and is segregated from other suites . Only single-use technology is used in the suite and complete validated change-over is enforced after each project . Below is a list of viruses that we have manufactured in the BSL-3 cGMP suite .

• HIV• Dengue virus• HPV• Adenovirus


Aseptic Filling200-2000 Vials per day Small-scale aseptic filling of viral and non-viral products are available on site . Vialing capacity of 200 - 2000 vials per day is available with fill volumes ranging from 10 μl to 100 ml .

Key Benefits of Vigene’s Aseptic Filling Services• GMP aseptic filling of viral and infectious materials• Documented to meet FDA requirements for Phase I/II and Phase III/ Commercial clinical

manufacture• Pricing is based on runs for a small, not large, facility

Please contact us to discuss your filling needs . If we are unable to assist you, we will guide you to others who might .

Cell & Virus BankingMCB & WCB; MVB & WVB The establishment of compliant, robust, and traceable cell and viral banks is critical for GMP production . Banking of master and working cell banks (MCB, WCB) for mammalian cells is performed in a separate area designed for these activities . Viral banking services are completely

segregated from non-viral activities to ensure no cross-contamination of products .

Mammalian Cell Banks• Base master & working cell lines (eg, HEK-293, Vero)• Stable recombinant protein/antibody expressing lines (CHO, NS/0, BHK-21)• Human and non-human stem cells

Viral Banks• Viral Seed Stocks• Viral vectors for gene delivery (adenovirus, retrovirus, lentivirus, AAV, others)• Viral vectors for vaccines (live, attenuated, and whole-killed)• Banking/vialing of purified phage

Quality assays for release of all banks are performed by outside testing firms to ensure safety and stability of these products .


AAV QC and plasmid DNA QC assays We offer a comprehensive range of assay development and in-process testing services to ensure the identity, quality and purity of your product and to reduce the overall costs and time it takes to get your product to the clinic .

Our current assay capabilities are:• Purity and Impurities Viral vector purity (SDS-PAGE, silver stain) Residual polyethylenimine (SDS-PAGE) Residual iodixanol (HPLC) Residual Host Cell Protein (ELISA) Residual Host Cell DNA (qPCR) Residual Plasmid DNA (qPCR) Residual benzonase (ELISA)

• Strength Vector genome titer (qPCR) Total viral particle titer (ELISA)

• Microbiological Safety and Quality Bioburden (Direct inoculation) Endotoxin (LAL/Chromogenic method)


Process development Critical to ensuring successful and economical production is having a robust and optimized manufacturing process . The goals of process development (PD) include:

- Maximizing the capacity of a production platform- Establishing a scalable, reproducible and robust manufacturing process- Reducing manufacturing costs- Extending intellectual property protection or proprietary know-how through innovation- Satisfying the regulatory requirements necessary for FDA approval

The development and optimization of biologics manufacturing processes is a difficult and complex task . For small firms, acquiring PD capabilities is expensive and often off-focus . Contracting your PD needs to Vigene allows our clients to focus on discovery and scientific research while we take on the industrial development . Vigene’s PD team works hard to ensure that you have a process that meets FDA regulatory requirements, reduces your costs going forward and maintains high quality standards .

Vigene provides the following process development services: • Process optimization using scale-down models (iCELLis® Nano, T-flask, shake flask, 1L STR

bioreactors)• Cell line engineering and adaptation• Media/feed screening and optimization• Transfection/infection optimization • High cell density culture and transfection/infection• Harvest, lysis, and clarification• Column and membrane chromatography (affinity, ion exchange, size exclusion, mixed-

mode)• Bioprocess filtration (tangential flow, hollow fiber, depth filtration)• Formulation study• Viral clearance study• Long-term stability study

2018 Vigene Biosciences . All rights reserved . All trademarks are the property of Vigene Biosciences including Vigene Biosciences, Vigene, Vigene logo, Make Gene Therapy Affordable . BAC-to-AAV is the trademark of Virovek . iCELLis is the trademark of PALL life sciences . Images are used with permission from Beckman Coulter and Gener-al Electric Company .

C: 73% M:5% Y:31% K:0%

C: 49% M:41% Y:41% K:5%

Pantone 319C

Pantone Cool Gray 9C

Color Matching

CMYK

Pantone Coated

Vertical Arrangements Horizontal Arrangements

is a power combination between a leadingmanufacturer producing preclinical and GMPPhase I/II and Phase III/commercial clinicalmaterials used in vaccine, cell & genetherapy applications for 17 years and a viralvector technology powerhouse.

� AAV GMP

� Adenovirus GMP

� Lentivirus GMP

� Retrovirus GMP

� Plasmid GMP-ReadyTM and GMP

Vigene Bioproduction

Ordering

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Telephone: 301-251-6638 Fax: 301-251-6110

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Documents

Updated Edition 2020 cGMP Cell & Gene Therapy...impurities including polyethylenimine, host cell protein, residual plasmid DNA, host cell genomic DNA, etc . Refer to AAV/Plasmid DNA