Promises and Challenges of Manufacturing and Testing Viral Producer Cell Lines
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To date, manufacturing of lentivirus (LV) vectors for gene therapy commonly relies on transient transfection of adherent HEK293 cells. This method is costly, time-consuming, difficult to scale-up and poorly reproducible, rendering large-scale applicability to fulfill increasing demand of LV in clinical pipelines cumbersome. The use of suspension-adapted transient producer cell lines for LV production has overcome some of these challenges. Furthermore, successful creation of stable producer cell lines would allow creation of master and working cell banks easily amenable to commercial production. The ideal producer cell lines should demonstrate stability in growth and gene expression, and be easily adaptable to chemically defined culture conditions and optimized for high-titer virus production. The availability of more robust producer cell lines thus represents an important scalable first step towards manufacturing processes that are conducive to large-scale production. Ultimately, these producer cell lines must be screened to satisfy various biosafety and regulatory implications. In this webinar, you will learn: • Process development for transient and stable producer cell lines • Screening of cellular gene targets via CRISPR to improve LV production from producer cell lines • cGMP and Regulatory readiness: Cell line characterization and release testing through BioReliance® global service offering
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Promises and Challenges of Manufacturing and Testing Viral Producer Cell Lines
- 1. Merck KGaA Darmstadt, Germany Henry George Elie Hanania, PhD Marian McKee, PhD Effective and high titer production of viral vectors
- 2. 2 The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada.
- 4. 4 Cell and Gene Therapy Approved Products* ANDEXXA (coagulation factor Xa (recombinant), inactivated-zhzo) ---------------------------------Portola Pharmaceuticals, Inc. ALLOCORD (HPC Cord Blood) ----------------------------------------------------SSM Cardinal Glennon Children's Medical Center LAVIV (Azficel-T) -----------------------------------------------------------------------------------------------Fibrocell Technologies MACI (Autologous Cultured Chondrocytes on a Porcine Collagen Membrane) ----------------------------------------Vericel Corp. CLEVECORD (HPC Cord Blood) ------------------------------------------------------------------------Cleveland Cord Blood Center GINTUIT (Allogeneic Cultured Keratinocytes and Fibroblasts in Bovine Collagen) ------------------Organogenesis Incorporated HEMACORD (HPC, cord blood) -------------------------------------------------------------------------------New York Blood Center Ducord, HPC Cord Blood ------------------------------------------------------------------------Duke University School of Medicine HPC, Cord Blood -------------------------------------------------------Clinimmune Labs, University of Colorado Cord Blood Bank HPC, Cord Blood -------------------------------------------------------------------------------------MD Anderson Cord Blood Bank HPC, Cord Blood --------------------------------------------------------------LifeSouth LifeSouth Community Blood Centers, Inc. HPC, Cord Blood-------------------------------------------------------------------------------------------- Bloodworks Bloodworks IMLYGIC (talimogene laherparepvec) ----------------------------------------------------BioVex, Inc., a subsidiary of Amgen Inc. KYMRIAH (tisagenlecleucel) ----------------------------------------------------Novartis Pharmaceuticals Corporation LUXTURNA -----------------------------------------------------------------------------------------Spark Therapeutics, Inc Plasma Cryoprecipitate (For Further Manufacturing Use) ----------------OCTAPHARMA Pharmazeutika Produktionsges.m.b.H. PROVENGE (sipuleucel-T)------------------------------------------------------------------------------------------- Dendreon Corp. Sterile Cord Blood Collection Unit with Anticoagulant Citrate Phosphate Dextrose Solution USP (CPD) MacoProductions, S.A.S. YESCARTA (axicabtagene ciloleucel)---------------------------------------------------------------Gilead (Kite Pharma) *Licensed products from the Office of Tissues and Advanced Therapies (OTAT) 4
- 5. 5 Gene therapy is revolutionizing medicine Drug development involves complex and costly manufacturing processes
- 6. 6
- 7. 7 1.Grow cells (i.e. HEK293T) • adherent culture in undefined media usually with serum 1.Produce up to four different expression plasmids • gag-pol/rev/env/transfer with GOI 1.Transfect at “optimized” ratio of above plasmids into cells • though efficient, most transfection reagents cost $$$$$$ 1.Harvest viral particles 48-72 hours post transfection • 2 harvests max 1.Measure viral titer, concentration and purification of r-viral particles • centrifugation, selective precipitation, ultrafiltration, chromatography, diafiltration Viral Vector Production Challenges Current LV production processes are difficult and costly to scale
- 8. From Challenges to Solutions Creation of improved platforms for viral manufacturing SimpleComplicated Critical Needs •Scalable •GMP and closed system •Economically viable processes •Clinical-grade or GMP raw materials Key Technology Gaps •Identify process influences that affect attributes •Implement standardized and controlled processes 8
- 9. Transient LV producer cell lines LV production in selected cells grown in suspension via transient transfection Optimized scaling-up of cell culture and vector harvest Optimized for effective, high-titer production of LV Improved downstream purification efficiencies and recovery 9 Lentivirus production platform Optimized scale-up and processing efficiency
- 10. 10 • Via single cell cloning and screening for improved growth and production characteristics • Suspension adaptation in Chemically Defined mediums • Via targeted cell engineering against potential cellular liability genes • E.g. apoptotic pathways- Bax, Bak, caspases, etc • Viral “defense” genes; Induction of INF, apoptosis • Reduction of cellular toxicity (VSV-G receptor) • Via KD and/or KO screens to identity novel gene targets and pathways. Improving the LV production potential of the HEK293T cell line
- 11. 11 Improving LV Production in HEK 293T LV Production Analysis
- 12. 12 • Cell lines with better production features • Suspension cell growth/bioreactor robust o Clonally derived cell lines that have been selected for improved growth characteristics, higher transfection efficiency, and high viral productivity. o Good growth rates and minimal clumping in suspension condition o Ability to maintain high LV production phenotype in bioreactor conditions Improving LV Production in HEK 293T Suspension-adapted
- 13. 13 • Support fast growth rates, minimal clumping at high cell densities, high transfection compatibility using linear PEI and high viral productivity. • Need vendors that can supply at scales required to support long term clinical and end product manufacturing. • Vendors that source from qualified raw materials with supporting regulatory documentation • Media supplements, feeds, and viral “boost” reagents/enhancers • Can reagents be added that improve viral stability, accumulation? Chemically Defined Medium (no animal components; only with known components and formulations) Improving LV Production in HEK 293T
- 14. 14 Improved viral plasmids (i.e. packaging and transfer vectors) (i.e. stronger promoters, inducible promoters, synthetic promoters that are stronger and smaller in size) High quality manufacturing and characterization of plasmids. o % supercoiling o endotoxin reduction Establishment of optimized packaging plasmids (viral gene) ratios and quantities per cell at various scales Elimination or reduction of “extraneous” vector sequences to improve transfection efficiency/viral production Improving LV Production in HEK 293T Packaging plasmids
- 15. 15 • Chemical • siRNA/shRNA/miRNA • Gene editing (CRISPR/ZFN’s) • Overexpression Improving LV Production in HEK 293T Targeted Knockdown/ Knockout
- 17. 17 LV Production Platform Further Improvements Stable LV producer cell line LV production in suspension culture without transfection (grow and go) Inducible or constitutive expression of viral components Mirror the current Mab production processes
- 18. 18 Stable Producer Cell Lines Cell Line Construction Currently still using serum and undefined medium Not suspension adapted (though grown on microcarriers) Using Tet on® /off® inducible system to control at least VSV-g env production Therefore process will still be a limited batches post induction. Some using a different env protein (i.e. RD114; less transduction efficiency) to avoid toxicity associated with VSV-G env over expression Titers typically not reaching levels similar to transient production processes. Are inclusion of selection reagents regulatory acceptable ?
- 19. 19 LV Producer Production Platform Challenges and Step-wise Solutions……….for the future.
- 21. 21 Commercial Viral Vector Manufacturing Requirements and Realities cGMP Compatible • Raw Materials and Reagents • Equipment • Consumable Sets Reproducible • Product Quality • Product Yield • Titer • Purity Scalable • Equipment • Units of Operation Robust • Mode of Operation • Stability • Tolerance • Feasible Range • Process Time Challenges for Clinical/Commercial Manufacturing •Limitation in achieving desired cell mass for production. •Mode of production is primarily based on transient transfection
- 22. 22 Eliminate the need for transient transfection Time (processing time, manipulations, multiple runs) Cost (plasmids, transfection reagents, supplies, multiple runs) Risk of contamination (manipulation, multiple runs) Variability, uncontrolled reproducibility (impact of transfection on titer and yield) scalability Suitable for scale up in order to meet demand Suspension cells in bioreactors Multiples harvests Controlled cytotoxicity Expression is controlled by inducible systems; chemical induction. Multiples harvests Commercial Viral Vector Manufacturing Requirements for Producer Cell Lines
- 23. 23 Producer cell lines need to start with a well characterized bank Consistency (lot to lot) Free from adventitious agents Documented history (including reagents and supplies used for production) Cell analysis using genotypic and phenotypic markers Testing for the presence of the vector(s) Testing for the integrity of the vector(s) Testing for vector gene expression Limits of cell expansion Sterility Commercial Viral Vector Manufacturing Raw Materials 23
- 24. 24 Producer Cell Lines Challenges for Commercial Viral Vector Manufacturing Cell Line Stability (Lentivirus) Loss of titer overtime due to epigenetic silencing of promoters Continuous use of antibiotics (selective pressure) to maintain stability of consistent productivity Cell Line Stability (AAV) Integrated rep-cap and rAAV vector sequences may lose stability over time without maintaining drug selection Interference with AAV promoters and impact during cell expansion Level of contaminants (wild type or helper viruses) Process challenges Large volume of material generated, processed and stored Multiple harvests (storage) Appropriate midstream processing and downstream purification
- 25. 25 Producer Cell Lines To be on the Right Track Cell Line Stability (lentivirus) Optimize Codon usage Optimize viral envelop glycoproteins Increase Productivity Optimize culture conditions; Media development Need stand alone producer cell lines No infection (wild type or helper viruses) No transfection (GOI part of the producer cell line) Suitable for scale up in order to meet demand Suspension cells in bioreactors Multiples harvests 25
- 26. Basic Product Safety and Characterization 26
- 27. Vector and Cell Safety and Characterization Plasmid/Virus Master Cell Bank (MCB) Working Cell Bank (WCB) Process Development (Growth/Production/Modification) Master/Working Virus Bank (MVB/WVB) Drug Substance Drug Product Cell Identity Safety Purity Identity Safety Stability Lot Release Testing Container Closure Shipping QA/QC In- Process Testing In- Process Testing In- Process Testing 27
- 29. Master/Working Cell Bank for Vector Production Cell Bank Characterization Purity Identity (cf ICH Q5D) Bacteria, fungi- sterility Mycoplasma Virus (cf ICH Q5A) Broad specificity - in vitro/in vivo assays Species specific – human/bovine/porcine Retroviruses – PCR/EM/PERT New technology – Next Generation Sequencing (NGS) 29
- 30. ELISAGOI 30 PCR Identity Assay − Gene of Interest Platform − Product Specific Development − Validation AAV Titration ELISA − AAV9 − Detects viral particles − Quantitative (Titer) LV/RV Titration ELISA − Detects p24/p30 − Titer can be calculated NGS − Virus identity test − Confirm Identity − Whole genome sequencing − Difficult to sequence genomes − Comparison to known reference Viral Vector Characterization Identity NGS ID
- 31. NGS-AAT Sterility 31 Sterility − BACT/ALERT® 3D − Detects changes in pH due to bacterial growth − Real time sample monitoring − Objective readout Adventitious Virus − Detection and identification of adventitious agents − Circumvents toxicity and neutralization issues − Can be combined with ID test Replication Competent Virus − Cellular assay − Three or more rounds of amplification − CPE, qPCR or QPERT endpoint Mycoplasma − PCR − Equivalent sensitivity and specificity to compendial method − GMP and EP 2.6.7 compliant − TAT and sample requirements better suited for cell therapies Mycoplasma rcVirus Viral Vector Characterization Purity http://www.med.upenn.edu/gtp/images/e20_ aav_med_full.jpg
- 32. AAV TCID50 32 TCID50 − Infection of HeLaRC32 cells with serial dilutions of test sample − Virus is detected using CMV promoter based PCR − Scoring of +/- wells − Spearman/Karber Calculation of TCID50 Genomic Particle Count − Droplet Digital PCR based − CMV promoter for AAV − E4 for Ad5 − 5’UTR for Lentivirus − Near 5’UTR for Retrovirus − Client Specific GOI − Absolute quantitation − No need for standard curve Particle Count Characterization of Virus Vectors Titer/Potency Ad5 FFU Ad-FFU − Infection of 293 cells with serial dilutions of test sample − Virus is detected using anti- hexon Ad5 antibodies − Foci count
- 33. ResidualsDNA Sizing 33 Residual DNA Sizing − Use DNA library approach to survey residual DNA. − Fragment size and distribution based on Bioanalyzer analysis. − Universal for all DNAs Viral Vector Testing Residuals and Additional Characterization Size Distribution Virus Size Distribution − Dynamic Light Scatter − Minimal sample manipulation − Small sample volume − Mean size and size distribution Host cell residuals − ELISA and PCR based assays to detect and quantify levels of residual DNA and proteins from host cells. Process Residuals − Benzonase − BSA − AAV Affinity
- 34. Gene Therapies Testing is a Continuous Process M/WCB Stability TestingVSS or Plasmids Final Product PD Drug Substance 34
- 35. 35 •Challenges for viral vector manufacturing remain •Producer cell lines o Current approaches o Future improvements o Commercial benefits •Cell line and viral vector testing o confirming identity, titer, and to demonstrate purity Viral vector production: Where are we? Closing GOAL Effective and high titer production of viral vectors at scale
- 36. Rockville - US 36 Gene Editing and Novel Modalities Product Development & Services Carlsbad - US Virus Manufacturing & Testing Services Cell Line & Gene Editing Development Virus and Gene Therapy Product & Process Development St. Louis - US Bedford, MA - US Glasgow – UK
- 37. The vibrant M is a trademark of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources. © 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.
Editor's Notes
- In recent years, the interest in gene therapy and gene correction applications and efforts has seen a tremendous increase. . With the recent successes and approvals of treatments for what was once “incurable” or untreatable diseases, the gene therapy industry has surged in the last few years in respective research efforts and clinical activities. While a wide variety of delivery technologies of the genetic payload into the cells exists, the current methodologies in using viruses to vector, or shuttle into the target cell type, have advanced all the way from research application, through the clinic and into current approved therpies. The current formats for viral delivery focus on the use of two viral systems, AAV (or adeno associated virus) and LV (or lentiviral) particle. As seen in this slide, AAV systems are mainly being developed for use in direct injection and delivery into the patient (either tissue specific) or systemic (I,.e. whole body) applications. Ex vivo therapies, where a specific cell type such as T cells or stem cells. is first extracted from the patient , are modified with a retrovirus system, or lentiviral vector…….with the modified cells then being placed back into the patient where the modified cells will now express the protein of interest changing it into a cellular therapy. CAR-T (or chimeric antigen receptor T ) cells……is a type cell therapy being of most well know for this type of application.
- As the increase in gene therapy applications has risen, and with the successes that have come with them, so has the generation of approved products and therapies. This slide highlights some of the recent FDA approvals being granted to various pharmaceutical and biotechnology companies. A few highlights include: Kymriah which is a CAR-T therapy using a retrovirus vector to deliver a anti-CD19 CAR to treat blood based cancers. Similarly, YESCARTA is also a T cell immunotherapy indicated for the treatment of adults with relapsed or refractory large B-cell lymphoma Luxturna is an AAV based treatment for retinal dystrophy (a form of blindness due to a defective protein). All of these have seen great clinical success and have been heralded as ushering in a new generation of gene therapy applications…with as many as 400+ new gene therapies proceeding through various pre-clinical and clinical stages in the US alone.
- While gene therapy is revolutionizing medicine by either the treatment of previously incurable conditions……or by drastically improving the effectiveness over conventional small molecule based therapies, the costs of such therapies has become a major concern throughout the industry. While clinically successful, ex-vivo therapies such as those used for Kymriah are patient specific and processing intensive…….which adds to the large overall costs of such a treatment. Much of the cost is due to the complex nature for the manufacturing processes currently being used for the production of viral vectors which are being used to deliver the gene of interest into the target cell type, and the subsequent treatment and patient therapy. T Therefore, improving the efficiency of this manufacturing process ………and therefore reducing the associated costs will be the focus of my presentation.
- As I mentioned previously, the common viral vectors used in current gene therapy applications are either lentiviral or AAV derived systems. This slide highlights the features of both systems. While the genome of LV systems is a ssRNA molecule, the AAV systems are ssDNA LV systems are an enveloped virus which buds from the surface of either infected cells, or cells that have been transfected with the various viral packaging genes. Therefore, LV particles will have a heterogeneous outer surface makeup of the various host cell proteins that come about during the secretion and budding process. For gene therapy, typically LV is pseudotyped with a heterologous envelope protein VSV-G envelope which expands the overall tropism (i.e. infectivity range) of recombinant virion particle. Due to this expanded tropism……One key feature of LV is it’s ability to transduce or infect……a wide variety of cell types with high efficiency. And Unlike other retroviral systems that require a mitotic event for viral gene integration, LV systems have an active integrase system that allows integration of it’s genomic payload into both dividing , and slowly dividing and non-dividing cells. Therefore, long term (i.e. permanent) integration and persistence of the gene transfer is one of LV’s main advantage as a gene therapy vector. However such can also be recognized as a potential liability as the genomic integration is mainly random throughout the genome, and therefore functional gene silencing and/or activation of nearby genes that may lead to oncogenesis may occur. This was problematic with early retroviral based systems….but present day transfer vectors that include SIN sequences or self inactivtating sequences which reduce the spurious transcription and activation of nearby genes post integration …….are now found in most current vector designs. AAV viral particles are non-enveloped virions that are much smaller in size and have been found to be less immunogenic then other viral vectors. While the genome for AAV vectors is typically non-integrating into host cell chromosome, transgenes delivered by AAV have been seen to persist for multiple generations. Cell specificity is dictated by the use of different capsid proteins which can target different cell types. Several “ hybrid” and “archaic” capsids designs……… that can infect a wider variety of cell types are also being developed in order to make the use of such more versatile and ubiquitous. Lastly, while LV systems can package and deliver some 8-10kb of genomic material, the small 4-5Kb packaging size of the AAV ssDNA can be problematic when delivering gene or genes of large size.
- This slide reviews the typical present day production process for LV systems. While relatively simple and straightforward for small scale needs, large scale manufacturing using such techniques becomes cumbersome and expensive resulting in a scale out mode of production rather then a scale up.
- So the industry is looking for solutions to these scale up issues. Obviously one of our key challenges is to then find processes that take us from “complicated” and costly scale out methods…..to a processes which are more straightforward….. and easily scalable to multi-liter reactors Like the early days of Mab production in CHO cells in the 1980’s when that industry grappled with low yields, difficult production processes and high costs…..is the current state of viral vector production Therefore the viral vector manufacturing industry is actively looking for technology improvements, reagents, cell lines and processes that will increase efficiencies and drive down overall production costs.
- A starting point for improvements is the optimization of the current transient Lentiviral vector production process. This can be done by breaking down…….and looking at the different parts of the viral transient production process and optimizing each of the components of such a system. The first can be performed by screening and selection of better producer cell lines with improved growth and viral production characteristics. Moving away from adherent cultures into a suspension based system is a major driver……which now allows for more efficient scalability and use of well known and in place bioreactor engineering practices learned from performing years of such processes by the Mab production world. Similarly, optimization of the downstream purification systems……..in which current losses during such processing are estimated to be as high as 80%........the development of new assemblies (that is … filtration and chromatography modules and systems) that can lead to improved efficiencies and recoveries……. will further reduce costs and improve overall yields.
- We’ve started to look at improvement of the HEK293T cell line via a variety of research approaches The cell line HEK293 is the workhorse line of choice for viral vector production ……..with or without the inclusion of the large T antigen In comparisons to the parental 293 cell line……. the 293T line Shows improve transfection and r-protein production capabilities most likely due to the plethora of effects in parted by the expression of the T antigen……and improved episomal maintenance of plasmids that carry the SV40 replication origin. Therefore we started our cell enginneeing studies to look at improving the HEK293T parental cell line by performing single cell cloning and screening for improved growth and production characteristics Once we have identified such a cell line, we wanted to Suspension adapt the cells in a series of unique Chemically Defined mediums formulations that were compatible with a manufacturing acceptable transfection system and reagents. On the next few slides I’ll show you some data that we obtained post screening for such features. In addition……we have undertaken Gene editing technologies .which now offers us the ability to perform targeted cell engineering against potential cellular or viral production liability genes As an example…..genes involved in the apoptotic pathways- such as Bax, Bak, caspases, etc……………we speculate that high viral production may induce some of these genes ……..and therefore limiting these may enhance overall production levels. Other genes that are attractive targets are the Viral “defense” genes; that is INF response genes, and nucleic acid sensor genes that again can activate such apoptotic pathways leading to early cellular death. We have also implemented a strategy to look for gene targets that Reduce the cellular toxicity caused by the expression of toxic viral proteins. (VSV-G receptor) While the above approaches target specific genes of potential interest such screens are relatively slower as compared to performing……..Large scale KD and/or KO screens again using libraries generated against endogenous genes and pathways….. These screens may help to identity novel gene targets and pathways which could then be engineered for improved viral production performance
- Here’s an example of LV production on either the parental HEK293T population and several clonal variants. While the initial screen we isolated and looked at several hundred clones, we have been able to identify several clones that show improved r-viral production as compared back to the parental population. We have found this phenotype to be stable across multiple generations and we are currently trying to identify the genetic and metabolic cause or causes that led to such an improved LV production response. To The left side of the middle red bar are clones assayed for the production of p24 (one of the viral capsid proteins that is produced and hence easily assayed by ELISA)…. The arrow points to several clones that showed improved p24 production as compared to other clones and the parental cell line (those in the middle of the graph) To the right of the red bar….are the same clones analyzed by functional titer (or tranducing unit). Dark bars represent viral production at 48 hour harvest post transfection whereas light bars represent a harvest time at 72 hours post transfection. TU titers for our improved cell line was found to be in the mid 10e7 range……with the parental cell line showing nearly 10X less production.
- As mentioned previously, moving away from an adherent process to one where the cells can be grown and transfected in suspension culture would allow for more scalable solution for viral manufacturing by allowing for larger and more economical batch production sizes. In addition to screening clones for improved LV production, we also looked at the ability of cells to grow well in suspension growth conditions. (i.e. maintain similar or even faster cell doubling times) show minimal cell clumping (as seen in the picture below) especially at higher cell densities, and maintain the LV production phenotype stbly over multiple generations …….and be able to scale up what we found in shaker flasks to larger scale suspension bioreactors and produce in bioreactor conditions as shown in the graph
- Another key feature of growing cells in suspension and the high titer production of virus will be the selection of the improved medium formulations that can support not only the good growth profiles, but allow for high transfection efficiency and viral production. Therefore the design features we sought to create in our media formulations are: The ability to Support fast growth rates, show minimal clumping at high cell densities, have high transfection compatibility using linear PEI……. and support high viral productivity (when using either 2nd or 3rd generation packaging plasmids. However it does no manufacturer any good if one cannot support cGMP supply and delivery at such large scales. Therefore, Need vendors that can supply at scales required to support long term clinical and end product manufacturing. Can your supplier support your clinical and production manufacturing needs ? Also important will be Vendors that source from qualified raw materials with supporting regulatory documentation Other media development efforts that are ongoing throughout the industry include: The development of Media supplements, feeds, and viral “boost” reagents/enhancers Will these add costs and be regulatory acceptable ? Are there reagents be added that improve viral stability, accumulation and show minimal interference downstream processes? Recall that the Measured functional stability of LV particle is less then 12 hours at 37C. So unlike Mabs’ that are stable for days and even weeks……LV particles in particular are most labile.
- While not much effort or overall designs have changed in the last several years surrounding the development of better viral packaging plasmids i.e. 2nd generation, 3rd generation 4th generation SIN vectors, etc.)……there are several groups that are currently exploring and developing some new plasmid features and designs and cost effective manufacturing processes for large scale manufacturing These include plasmids that have Improved viral plasmids (i.e. packaging and transfer vectors) (i.e. stronger promoters, inducible promoters, synthetic promoters that are stronger and smaller in size) Elimination or reduction of “extraneous” vector sequences to improve transfection efficiency/viral production The old cliché of garbage in equal garbage out…..is certainly most relevant when talking about plasmid quality for viral production needs. High quality manufacturing and characterization of said plasmids is critical Quality features such as endotoxin levels and % supercoiling…….are a couple of features that ensure higher viral production Certainly the cGMP production capability and scalability of such plasmids will lead to more stable and uniform production process. And finally processes that Establish optimized packaging plasmids (viral gene) ratios and quantities per cell with the appropriate transfection reagent…..at various bioreactor scales. The latter will require some empirical testing and optimization based on the multiple variables such as cell line of choice, medium , transfection reagent, etc…….. that are present in final manufacturing process.
- Where I showed you that single cell cloning , expansion and selection via LV production assays can lead to cell lines with improved LV phenotypes, we now have as part of our research “toolbox” …….. a wide variety of Gene editing technologies which again offers us the ability to perform both targeted and large scale KD and KO screens against potential cellular or viral production liability genes. These include : use of chemical libraries that may identify viral boost reagents or cellular enhancers for improved viral production, stability or processing. Large scale KD screens using si, sh or microRNA libraries targeting various genes and pathways throughout the human genome….which may identity novel targets which could then be engineered for improved viral production performance Similarly, Gene editing tools such as CRISPR and ZFN (zinc finger nuclease) screens using libraries ……can lead to permanent gene modifications that again may lead to novel targets that can be stably engineered into a LV producer cell line. Laslty, While the above screens typically look for genes of that invoke some sort of liability of the sought after function, the use of an overexpression libraries …..and screens that can uncover pathway limited or “bottleneck” genes and protein functions (i.e. viral protein processing, trafficing, assembly, secretion) that again may lead to discovery of new of gene pathways to improve viral production.
- To this point, I’ve been mainly speaking about improvements for the transient based production of viral vectors……….but because of the variability that will occur batch to batch when using such…… researchers are seeking a more controlled process. This leads to the development of producer cells lines in which all of the viral packaging genes have been stably integrated into the host cell genome……and therefore produce such proteins under either a constiuitive (though unlikely) or more realistically an inducible expression system. Ideally, manufactures would then be able to “simply” grow up a cell line …from a well characterized working cell bank…………. that contains all of the viral packaging functions…… and if such production is constituitive and stable…….this process will be similar to current Mab production systems Elimination of the need for costly transfection reagents and purified plasmid preps will make the stable viral vector producer cell lines……a most attractive goal for future production needs. Unknowns at present will be if the inclusion of selective agents such as antibiotics….or inclusion of such genes in the stable producer cells will be acceptable by the regulatory agencies.
- However the construction and design of stable viral vector production systems to date have met their own specific challenges which are highlighted in this slide. Some of the reported systems are still in attached culture (though have been grown on microcarriers and/or fixed bed reactors) ………using serum containing mediums and in undefined medium. Most systems reported to date also use an inducible promoter system due to the toxicity issues due to overexpression of some of the viral proteins, most notably the toxicity of VSV-G production on the stable producer. Such inducible based systems will subsequently still force manufacturers to be limited to smaller batches post induction. Different env proteins have been reported to reduce toxicity, but some have limited tropism or less transduction efficiency on target cells (i.e. T cells) then VSV-g. While some improvements in processes have led to increased titers from such systems, overall titers are still typically a log or more lower then comparable transient production processes This slides highlights some of the features from two such reported systems. Whereas integration of viral genes using selectable markers and or viral integration relying on random integration and clone selection…… was built in a step wise manner….(St. Jude Medical)…..the system recently being described by GSK places all of the packaging and transfer GOI of interest onto a large bacmid based system and integration into target locations. This latter system has the advanatge it seems of faster construction and screening.
- I hope I’ve given you a small taste of the overall state of LV manufacturing…..and how we as an industry are trying to address such. Future LV and AAV manufacturing needs will require more efficient production processes. We’ve highlighted the many challenges that exists with the current processes In summary…….Some “low hanging fruit” solutions will be to move away from: Adherent into suspension based production processes……. That is growing cells under Bioreactor conditions from 200L to 2000L To develop better chemically defined mediums and supplements which can be supplied for Large scale manufacturing applications. Create reagents that create Higher cellular productivity : Such as Improved plasmid vectors And Engineered cell lines Stable producer cell lines will eventually replace transient production processes And lastly……optimized downstream processing via use of new chromatography and filtration systems will all lead to improve production efficiencies and lower costs Thanks for your attention Elie ?
- characterization and safety testing of GTP follows the basic tenets of all biologics –Identity, purity, potency and freedom from residuals of the production process are the foundations of assuring product quality and safety Identity – Demonstration that the viral vector and construct is it what it is supposed to be? Titer – either by biological activity (tissue culture infectious dose) or particle enumeration by PCR Potency – how well does the genet therapy product work? This may be based on the mechanism of action or expression of the transgene, for example Purity - Verification that the product is free from impurities and adventitious agents; the gene therapy vector is identified and possible contaminants such as related vectors may be confirmed to be absent. Residuals - absence of process related contaminants such as host DNA and proteins
- As you can appreciate from Elie’s presentation, the manufacturing process is complex. There is not 1 size fits all approach to vector production. Regardless of the approach, however, the safety and identity profile must be addressed for all component of the process including the cells used to produce the virus, the viral seed and or helper virus and plasmids used for transfection of the cells. Raw materials that go into the manufacturing process must also be tested to insure safety of the patients and reduce risk to the manufacturing process. Throughout manufacturing, samples are taken for testing. One challenge faced in testing is that sample volumes are varied and in some points in the process may be quite low. Testing labs must take this in to consideration when designing methods. A well designed sampling plan with downstream testing in mind should be established during process development.
- Breaking down the testing into component parts, I want to first address the cell banks used for production. The cells form the foundation of any manufacturing program. It is worth noting that cell bank testing takes most of the time and should be planned for up front. This is a general outline of testing for any species used in production. The cells should be evaluated for sterility and to confirm the absence of mycoplasma. Freedom from adventitious viruses should also be demonstrated including non-vector retroviruses. Both broad spectrum and species specific methods may be employed. New technologies are coming regularly to the field to enable faster testing and release of cells for manufacturing.
- nikolay.korokhov10/9/2017 Progen ELISA kits available for AAV1, 2, 5, 6, 8, 9
- Gene Therapy is a rapidly growing field with numerous clinical candidates in development. Manufacturing of gene therapy products is complex and safety testing strategies for various vector systems can be challenging to design - especially when trying to address regulatory expectations. Characterization and safety testing of GTP follows the basic tenets of all biologics –Identity, purity, potency and freedom from residuals of the production process are the foundations of assuring product quality and safety. The testing process is continuous and includes all aspects of the process from cell bank and viral seed stock through manufacturing to the final gene therapy product. Significant Gene Therapy advances should be supported by implementation of robust tests which will guarantee safety and efficacy of gene therapy products.
- Commercial manufacturing Facility in Glasgow. GMP production of virus with regular regulatory inspection. Global company with GMP/GLP testing facilities on two continents; broad service capabilities in both Europe (2 sites in Scotland) and the US (Rockville). BioReliance is a Global Company with offices on three continents. Our Headquarters are located in Rockville, Maryland in the USA. This is a full service facility offering a comprehensive range of testing services. Our European Headquarters is based in Glasgow, Scotland in the UK. We also have facilities based in Stirling and Edinburgh, Scotland. These 4 campuses provide a full service complement in BioSafety testing. In addition to the US & UK facilities, BioReliance has recently established a commercial office in Tokyo, Japan. This is a significant development in BioReliance's history as it is the first physical footprint in the Asia Pacific market. This Global presence offers significant advantages to our organization and to our client base. Harmonization of quality systems, testing procedures, and processes allows for increased flexibility & capacity management. It also provides back-up capacity should it be required. Most significantly, it allows BioReliance to focus resource & personnel in several Centers of Excellence. For example, our US facility houses our R&D function whilst the Glasgow facility has advanced capabilities in Molecular Biology, Virology & Cell Biology.