Updated Edition 2019 cGMP Cell & Gene Therapy · Adenovirus (Lot sizes of 1E+12 – 1E +13 viral particles or VP) 3 . Lentivirus (Lot size of 1E+10 – 1E +12 Infectious Units or

cGMP Cell &

AAV, Adenovirus, and LentivirusPD and Production Handbook

Updated Edition 2019

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-source 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 our

customers in gene delivery technologies . Further, our management and employees

Vigene Biosciences4 www.vigeneGMP.com

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)

4 . Other viruses (HIV, dengue virus, HPV etc)


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

Chromatography

purificationAdherent process

iCELLis 500 (m2)

Upstream platforms Downstream platforms

Stirred-Tank Reactor 200L-500L


Production Cell Lines

Suspension 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 cells

Vigene 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

manufactured

with Vigene


Cell Culture Process

Scale-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 culture

Vigene 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 Process

Ultracentrifugation

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 purification

Vigene 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 platforms

We 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 plasmid

transfectionAdherent HEK293/293T

Adherent HEK293/293T

Adherent HeLa

Suspension HEK293/293T-s

Suspension HEK293/293T-s

Suspension HeLa-s

Suspension sƒ

Helper virus

co-infection

Baculovirus-

insect cells

GMP-source plasmid

manufacturing

Proprietary

high-yield

suspension cell lines

Exclusive license for

BAC-to-AAV

technology

Virus packaging method


Adenovirus Manufacturing

Adenovirus 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 .

Adenovirus

Vigene 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 Manufacturing

Lentivirus 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 produc-tion 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-source plasmid manufacturing

Vigene manufactures GMP-Source 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-Source 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 Filling

200-2000 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 Banking

MCB & 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 General 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 13 years and a viralvector technology powerhouse.

■ AAV GMP

■ Adenovirus GMP

■ Lentivirus GMP

■ Retrovirus GMP

■ Plasmid GMP-Source and GMP

Vigene Bioproduction

Ordering

[email protected] Free (USA): 1-800-485-5808

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

Technical Support

Email: [email protected] Free (USA): 1-800-485-5808

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

Updated Edition 2019 cGMP Cell & Gene Therapy · Adenovirus (Lot sizes of 1E+12 – 1E +13 viral particles or VP) 3 . Lentivirus (Lot size of 1E+10 – 1E +12 Infectious Units or

Documents