Updated Edition 2019 cGMP Cell & Gene Therapy · AAV enovis n enivis ene e c n ocion nboo 3 Vigene...
Transcript of Updated Edition 2019 cGMP Cell & Gene Therapy · AAV enovis n enivis ene e c n ocion nboo 3 Vigene...
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)
5AAV, Adenovirus and Lentivirus Gene Therapy cGMP PD and Production Handbook
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
Vigene Biosciences6 www.vigeneGMP.com
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
7AAV, Adenovirus and Lentivirus Gene Therapy cGMP PD and Production Handbook
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 .
Vigene Biosciences8 www.vigeneGMP.com
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 .
9AAV, Adenovirus and Lentivirus Gene Therapy cGMP PD and Production Handbook
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
Vigene Biosciences10 www.vigeneGMP.com
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
11AAV, Adenovirus and Lentivirus Gene Therapy cGMP PD and Production Handbook
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
Vigene Biosciences12 www.vigeneGMP.com
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 .
13AAV, Adenovirus and Lentivirus Gene Therapy cGMP PD and Production Handbook
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)
Vigene Biosciences14 www.vigeneGMP.com
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