672 iCELLisTM Fixed Bed Technology for Adherent Cells Is an Efficient Scalable System for Viral Vector Production Applications Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The Ameri[.]
Trang 1Molecular Therapy Volume 23, Supplement 1, May 2015
CLINICAL TRANSLATION OF VECTOR PRODUCTION AND PROTOCOL PREPARATION II
670 Probing the Stability of rAAV Capsids and
Genomic Constructs Using Differential Scanning
Calorimetry in Concert With Mobile Phase
Modifi ers
James D Maratt,1 Samantha F Povlich,1 Christopher J Morrison,1
Gwendolyn M Wilmes,1 Samuel C Wadsworth,1 K Reed Clark.1
1 Pharmaceutical Development, Dimension Therapeutics,
Cambridge, MA.
The recent resurgence of using rAAV vectors as commercially
viable therapeutic agents has highlighted the need to identify stable
capsid / genomic construct combinations which can both endure a
typical cGMP manufacturing process and possess a long product
shelf life In this work we investigate the inherent stability of
various capsids and genomic constructs using differential scanning
calorimetry (DSC) DSC measures the amount of energy required
to increase the temperature of a sample and reference and can be
used to determine the Tm of the molecule in question Numerous
commercially relevant rAAV serotypes containing several genomic
constructs (self-complimentary and oversized genomes) were
subjected to melting curve analysis using DSC to compare stability
Additionally, various mobile phase modifi ers were added to solution
in order to probe the stability of rAAV vector, using organics and
kosmotropic / chaotropic salts These results demonstrate DSC as an
effective tool to examine inherent rAAV vector capsid stability and
support a role for the serotype and vector genome structure to affect
the observed stability
671 Development of a Successful
Lyophilization Process for Lentiviral Vector
Clinical Batches
Nicolas Delacroix,1 Gael Ouengue Mbele,1 Julien Deroyer,1
Guillaume Deplaine,1 Cecile Bauche.1
1 THERAVECTYS, Villejuif, France.
Since lentiviral vectors are incrinsingly used in various applications
(vaccination, gene therapy, T-cell therapies…) and given that so far
the best way to keep these vectors is a -80°C storage, more adapted
ways of formulation and storage must be developped
Previous attempts have been made to freeze-dry viral vectors :
adenovectors and Moloney Monkey Leukemia Virus-based vectors
(Cruz et al., 2006) and lentiviral vectors (Shin et al, 2010) None
of these attempts have proved satisfactory as the fi nal lyophilized
products were not suitable for pharmaceutical applications (mainly
due to unacceptable levels of contaminants observed in the fi nal
products)
We developped an new method to freeze-dry our clinical grade
batches of lentiviral vectors This method allows us to store the
batches at -20°C and +4°C while maintaining the characteristics
of the particules after reconstitution and the ability to use them as
intended (direct injection, T-cell modifi cation…) Our method allows
the lyophilization of lentiviral vectors either produced in adherent
cells cultures (with FBS) or in suspension cells cultures (without FBS
and with synthetic culture medium) The vectors are protected during
the freeze-drying process by the addition of a lyoprotectant (sucrose
or threalose) and stability is evaluated over time
Quality controls have been performed to evaluate the impurity profi les of the lentivector batches before the freeze-drying process and after the reconstitution of the particules (titer, total proteins, total DNA, HEK 293T proteins), and we demonstrated that the profi les were not impacted by the process and that the batches were still fi tting the international pharmacopea recomandations
We also made some immunogenicity evaluation of our vaccine candidates before and after the lyophilization process, and we were able to show that the process had no impact on the breadth and intensity of the cellular immune response we are able to elicit after a direct injection of the reconstituted particles in mice
THERAVECTYS is a Paris-based, privately-owned, fully integrated discovery & clinical development biotech company THERAVECTYS develops lentiviral vectors used for human vaccination and immunotherapy applications (CAR-T cells and TCR) An in house cGMP facility is now up and running, allowing
us to produce and charaterize our own lentiviral vector clinical batches A qualifi ed freeze-dryer has been installed in the plant, to systematically encompass this formulation and repartition step in our bioproduction processes
672 iCELLisTM Fixed-Bed Technology for Adherent Cells Is an Effi cient Scalable System for Viral Vector Production Applications
Hanna P Lesch,1,2,3 Piia Valonen,2 Kati Heikkilä,2 Eevi Lipponen,1,2 Achim Müller,2 Eva Räsänen,2 Tarja Tuunanen,2 Joonas Malinen,3 Anne Martikainen,3 Tuula Salonen,3 Elisa M Nurminen,2 Minna Hassinen,1,2 Minna Karhinen,2 Nigel Parker,1,2 Robert Shaw,1,2 Seppo Ylä-Herttuala.3,4
1 FKD Therapies, Kuopio, Finland; 2 FinVector Vision Therapies, Kuopio, Finland; 3 Department of Biotechnology and Molecular Medicine, A.I Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland; 4 Science Service Center and Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland.
To date many early phase gene therapy trials have been successful However, phase III and commercial phase have brought further challenges in producing viral vectors in suffi cient large quantities Upscaling the process on suspension cells is feasible, but many viral production applications are still applicable only in adherent settings Scaling up the adherent system has proven to be troublesome and costly The PALL iCELLisTM disposable fi xed-bed bioreactors offer
an effi cient option for viral vector manufacturing in large quantities
in an adherent environment In iCELLisTM Nano, the cultivation area 0.53-4 m2 for smaller batches, is ideal for process development purposes In iCELLIs500 the cultivation area varies between 66 and 500 m2 and is ideal to satisfy demand for phase3/ commercial requirements
We have optimized adenovirus type 5 manufacturing using iCELLisTM Nano HEK293 cell cultivation, infection and harvest
of the virus by lysing the cells inside the bioreactor were effi cient,
Trang 2Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy S268
CliniCal TranslaTion of VeCTor ProduCTion and ProToCol PreParaTion ii
reaching total yield of 3.4 x 10^14 vp/batch When upscaling the
process into 100 m2 cultivation area with iCELLis500, 1.2 x 10^16
vp/batch were produced
iCELLisTM technology is applicable also to other vector types
which require for example plasmid transfection Virus can also be
harvested by perfusion from the medium Lentiviral vector production
in 293T cells was tested in iCELLisTM Nano and we achieved high
plasmid transfection efficiency, leading to the comparable titers and
productivity as in flasks To conclude, iCELLisTM equipment has
provided us an efficient way to manufacture large batches of different
kinds of gene therapy products suitable for large preclinical animal
models and up to phase III trial and beyond
673 Establishment of a National Reference
Standard for Reverse Transcriptase Activity Assay
and Its Applicability in Quality Control of Gene
therapy Products
Yonghong Li,1 Chen Wang,2 Lei Yu,3 Chunming Rao,1 Junzhi
Wang.1
1 National Institutes for Food and Drug Control, Beijing, China;
2 Beijing Tiantan Biological Products Corporation Limited, Beijing,
China; 3 Beijing Institute for Drug Control, Beijing, China.
The reverse transcriptase (RT) activity assay is widely used in
the quality control of biological products including gene therapy
products for it can detect all infectious retroviruses in biological
starting materials, cell banks and final products But the lack of
reference standard for RT activity results in significant systemic
error and incomparability between laboratories To standardize RT
activity assay, this study established a sensitive real time PCR and
developed the first national reference standard, which was prepared
from a raw material of recombinant moloney murine leukemia virus
reverse transcriptase Our tests indicated that the candidate reference
standard met relevant requirements of the national reference standard
The activity value of candidate RT standard was determined as 26.4 U
per ampoule by a collaborative calibration among four laboratories,
with the within-laboratory geometric coefficients of variation (GCVs)
ranging from 2.7% to 9.6% and the between-laboratory GCV values
being 9.8% The thermal accelerated stability test showed that no loss
of activity was observed when the candidate RT standard was stored
at 4°C , 25°C and 37 °C compared with -20°C even after 9 months
The applicability study showed that the cell bank and viral bank of an
adenovirus gene therapy product were positive and the final products
were negative As a conclusion, the candidate preparation was suitable
to serve as a Chinese national standard for RT activity assay and can
be used in the quality control of gene therapy products
674 Development of the Retroviral Vector
Manufacturing Process for MM-TK Therapy
Simona La Seta Catamancio,1 Manuela Cota,1 Patrizia Mangia,1
Ralitsa Arnaudova,1 Paolo Morandi,1 Marisa Marzocca,1 Michele
Manfredini,1 Francesca Rossetti,1 Elena Spoldi,1 Giuliana Vallanti,1
Gian-Paolo Rizzardi,1 Catia Traversari.1
1 MolMed S.p.a, Milan, Italy.
The MM-TK Drug Product (DP) is a medicinal product used
as adjunctive therapy in leukaemia patients undergoing stem cell
transplantation It is constituted of T lymphocytes genetically modified
ex vivo with the γ retroviral vector SFCMM-3 Mut2 #48 encoding the
HSV-TK Mut2 suicide gene and the cell surface marker ∆LNGFR
During clinical development, different improvements were introduced
in retroviral vector (RVV) manufacturing in order to obtain simple
and robust processes producing RVV with low level of impurities and
high performance in lymphocyte transduction Single-use bioreactors
are currently replacing conventional bioreactors also for mammalian
cell culture They allow to increase biological and process safety by reducing cleaning and sterilization procedures and the risk of cross contaminations In this context, we are currently evaluating different single-use, closed-system bioreactors for the manipulation of adherent packaging cell lines A feasibility study is on-going, using a single-use bioreactor based on the 2D multi-tray stacks technology Xpantion system (Pall LifeSciences) This study explores the role of gas control
on cell growth and its impact on the RVV quality attributes evaluating key metabolites, as glucose and lactate, and viral titer during cell expansion In parallel a manufacturing process in 10-layers cell factories (CFs) has been developed and validated The results obtained with the Am12/ SFCMM-3 Mut2 #48 producer cell line grown up to
10 days in XP10 and in XP50 bioreactors showed an infectious viral titer on the reference cell line CEM A3.01, a transduction efficiency
on T cells and an impurities level close to those obtained with the CFs Process development and comparability data will be presented in more detail
675 High Quality Plasmid DNA Manufacturing for Ex-Vivo Protein Synthesis and Viral Vector Production for Gene Therapy
Neha Tiwari, Jill Beilowitz, Carlos Sampson, Dorothy Peterson
1 Process Development, VGXI, Inc, The Woodlands, TX.
High quality plasmid DNA is required for a variety of therapeutic purposes Although use as a DNA vaccine is the most popular, a high quality plasmid DNA product is also required as the raw material for processes like viral vector production for gene therapy, ex-vivo protein synthesis, etc Contrary to a DNA vaccine, plasmid products used as raw materials have varied requirements in terms of purity and presence of contaminant levels in the final product It is essential to have a large scale plasmid production method which can be tailored for the specific plasmid product based on its potential use Our company has designed a plasmid production strategy, consisting of
5 purification steps, for removal of specific contaminants It can be tailored to manufacture plasmid for specific uses at both research and large cGMP scale
Plasmid pVGXIa, a raw material for Ex-vivo protein synthesis, was produced using a modified purification approach Since the plasmid was the raw material for protein synthesis and not a drug product, removal of only host cell protein (HCP) as a contaminant was critical during purification Plasmid paste from the fermentation process was purified using a modified 3 step method designed for the removal
of HCP The 3 step method consisted of our company’s patented Airmix® technology, Ion Exchange separation and Buffer-exchange The final purified plasmid contained £0.2% of HCP Total circular form of plasmid DNA was 100% A 25% reduction in production cost and 10% increase in yield at the 10L scale were achieved by customizing the purification strategy This same approach can be utilized for cost effective production of high quality plasmid DNA for viral vector generation for gene therapy at large scale