A Technology Roadmap For Today's Gene Therapy Manufacturing Challenges
By Ruben Carbonell, Arpan Mukherjee, Jonathan Dordick, and Christopher J. Roberts
In Nov. 2018, The National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) published technology roadmaps addressing needs and gaps in three key product areas: gene therapy, antibody-dug conjugates, and vaccines. The roadmaps were developed with the collaborative input of industry, academic, and government experts. This piece highlights technology opportunities for gene therapy manufacturing. The NIIMBL roadmaps are available as a resource to the biopharmaceutical manufacturing community and can be downloaded at http://www.niimbl.org/roadmaps.
Gene therapy encompasses any treatment involving delivery of genetic material to a patient to modify a gene causing a disease or to redesign living cells so they are more effective in treating the disease. Several mechanisms can be employed to affect these changes: adding a gene that has been lost, replacing a disease-causing defective gene with a correct version, and adding a new gene (natural or unnatural) to treat a disease. This is one of the most important new therapeutic modalities for the future, as it can be curative and promises to aid patients suffering from a wide range of disabling and deadly diseases.
Several approaches to enable the genetic transformations required for gene therapy are being considered, including:
- introducing plasmid DNA or RNA into cells using a suitable carrier such as lipid nanoparticle, viral, or bacterial vectors1, 2
- introducing gene-editing technologies such as CRISPR-Cas9, zinc-finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs) into the patient’s cells to modify their genetic makeup3, 4
- removing cells from a patient, modifying their genes using a viral vector, and then re-injecting these modified genes into the patient. Autologous chimeric antigen receptor (CAR) T cell therapy is an example of the latter approach, where the patient’s T cells are modified to express CAR-targeting cancer cells.5
In this early stage of development, gene therapies are extremely expensive, but they offer the promise of being curative in a single step, thus reducing overall healthcare costs. Recent developments in identifying genes responsible for diseases and use of gene-editing technologies for genetic modification could treat many diseases. As a result, biopharmaceutical manufacturers are focused on rapid deployment of products into the marketplace. Their efforts include developing facilities and supply chains to handle increasing patient demands worldwide, reducing manufacturing costs, increasing the efficacy and potency of these novel modalities, and increasing safety to patients by reducing potential side effects of gene therapy products.
Major components that address these challenges involve the implementation of novel, efficient, and robust manufacturing and analytical technologies for the production of gene therapy products and developing a well-defined global regulatory framework for process validation and batch release. Most developing processes for viral vector production rely on transient transfection of the complex set of plasmids containing the desired gene of interest (GOI) in HEK-293 or HEK-293T adherent cells6 or Sf9 insect cells.7 The host cells express desired viral vectors incorporating the plasmids, which then need to be harvested and purified for further use either by direct injection into a patient or by modifying the patient’s cells. Formulation and fill/finish operations also present great challenges for viral vector products, and there are significant issues regarding the analysis and characterization of viral vectors for safety and potency. The sections that follow describe some of the major challenges and needs associated with viral vector production for gene therapy.
Production Of Plasmids And Host Cells
In the context of gene therapy production, plasmid DNA is essentially a raw starting material. Plasmid production is commonly done by recombinant E. coli fermentation, during which the appropriate genetic sequences are amplified and then harvested, purified, and tested for safety. There are significant issues with lot-to-lot consistency in the fermentation, and large lot-to-lot variability in yield and purity of the resulting product, which needs to be greater than 95 percent pure plasmid DNA, free of process-related impurities and variants. In some cases, viral vectors have small payloads, e.g., adeno-associated virus (AAV), which may necessitate other vectors, including lentiviruses, which pose human health concerns. Thus, removing such concerns through vector engineering is necessary.
Even though most production processes for viral vectors use adherent HEK-293T cells, the FDA suggests other cell lines be used for production of AAV vectors since the DNA of these cells comprise the DNA sequence of the SV40 large T antigen. This construct is an oncogene that may induce tumors in patients.8 Currently, there is significant interest in developing more efficient techniques for clone development and enhanced transfection capabilities, as well as suspension cultures for higher virus titers. Longer term, it is desirable to develop stable suspension cell cultures that can achieve higher cell volume density and increase productivity and process reliability. Integrating the gene package into the cellular DNA of the stable cell lines and increasing their auto-resistance to the vector product would also result in significant improvements in cell performance over current approaches.
Viral Vector Production
A typical viral vector production process includes several upstream steps comprising cell thaw and seed train, cell expansion, cell transfection, and viral production. Downstream purification of viral vectors includes nucleic acid removal achieved commonly with Benzonase; a harvest/clarification step normally achieved by microfiltration; a tangential flow filtration (TFF) concentration step; two purification steps involving affinity, ion exchange, and size exclusion chromatography, followed by concentration and diafiltration using TFF; and finally sterile filtration.9, 10
There are several manufacturing challenges in upstream operations. The first is the need to optimize the transient transfection step to reduce the number of plasmids required. As mentioned earlier, there is a critical need for suspension cell lines that can increase the productivity of viral particles and minimize the amount of costly plasmid DNA and other reagents used in the transfection process. There is a need to maximize the viral titer that contains GOI as opposed to empty capsids. Finally, there is an absolute need to minimize adventitious agent (AA) contaminations (viruses, microbial) in the cell culture step. Because the viral vectors are similar in size and characteristics to adventitious viruses, it is not possible to have separate viral inactivation in the viral removal step without affecting product yields and efficacies. Chemically defined media, as well as more complex biomolecular separation strategies downstream, would significantly reduce the risk of AA contamination.
Downstream operations in current viral vector production processes result in very low yields in terms of virus recovery, in both primary recovery as well as purification processes. Alternative methods for clarification and particle capture that do not affect viral potency are needed. Typical chromatographic methods involving affinity and ion exchange chromatography use elution conditions, such as low pH and high conductivity, which can damage the viral vectors.10 Size exclusion chromatography as used currently has inherently low productivity. Flow-through processes that do not involve viral product capture and elution could go a long way toward increasing both productivity and product yield. Novel resins that can selectively capture host cell proteins and other impurities would also contribute greatly to achieving the high purities necessary for approval and could be used in a flow-through model. Particular effort needs to be made in the development of platform purification processes for the most common viral vectors such as AAV and lentiviruses (commonly used in the development of CAR T cell therapy products).
Formulation, Stability, And Fill/Finish
Methods for formulation and stabilization of viral vector products are still being developed. More information is available for formulating the more robust non-enveloped adenoviruses, but much less information is available on lentiviruses (enveloped viruses that are more sensitive to temperature and pH).
Storage and shipping of viral vectors at temperatures of -70 degrees Celsius or below create supply chain issues. Routes to develop room temperature (or higher temperature) viral vector stabilization (e.g., through the use of lyophilization in the presence of tailored excipients) are necessary to stabilize these products to increase shelf life. A compounding issue for assessing improved product stability is that there remains a lack of standardized potency tests for viral vectors. Shear effects on viral structure and potency are important considerations during filling, and completely closed systems are desired to reduce viral and microbial contamination. Low-shear filling operations that do not rely on needles and can be done in closed systems would be a major breakthrough. These closed filling devices need to ensure sterility, given that repetitive sterile filtrations are impossible to execute for viral vectors due to product losses. Real-time sterility testing and closed end-to-end processes would offer a potential solution to these issues long-term.
Facilities Of The Future
The production of viral vector intermediates such as plasmid DNA requires different facilities than those producing gene therapy drug substances and drug products. There is an urgent need for increased viral vector manufacturing capacity in contract manufacturing facilities to meet current demand. Production facilities need cleanroom BSL-2 spaces that are segregated and have modular capacity that can be increased as needed.
Analytics, Regulatory Science, And Standards
Analytical methods are available for characterizing the RNA and DNA of a viral vector product since these rely mainly on identifying the oligonucleotide sequence and any chemical variants. However, the viral vector as a product also needs to be characterized for its efficacy, toxicity, and potency, and the approaches to doing this are, for the most part, still in development.11
Some of the many properties of the viral vector product that need to be addressed include viral vector titer, percent-filled virus, genome copy number, vector dimensions and composition, vector identity, purity, and potency. Methods for measuring particle size such as ultracentrifugation and transmission electron microscopy are difficult and need to be performed offline. Viral vector titrations also rely on methods such as qPCR (quantitative polymerase chain reaction) and ELISA (enzyme-linked immunosorbent assay) that have not been standardized and are subject to significant variations.12, 13 Potency assays also pose their own separate challenges given the complex mechanism of action involving transfection/infection, transcription/translation, and activity of the translated protein in the targeted cells. There are also challenges measuring and characterizing the potential for cytotoxicity and/or immunogenicity of the products. Critical quality attributes of viral vectors need to be identified, as well as the most important process- and product-related impurities that can affect safety, efficacy, and potency.
Unfortunately, methods for analyses of gene therapy products have no suitable reference standards and are expensive, time-consuming, and not harmonized across the industry. Innovative technologies that can help meet these challenges are needed, especially if they can be implemented in-line and in real time in a cGMP environment. Specific reference standards are needed for AAV strains, adenovirus, lentivirus, and gamma-retrovirus. Reference standards for AAV serotype 2 (rAAV2) and serotype 8 are available to the scientific community.14, 15 Such standards might facilitate regulatory science based decision making at agencies such as the USFDA, the European Medicines Agency, and other regulators by helping to benchmark viral vector manufacturing processes and product testing.
The advent of gene therapies has resulted in significant new challenges and opportunities in workforce development to ensure the industry has adequate support, at all levels, for what promises to be a major sector of biopharmaceutical manufacturing in the future. Process scientists and engineers must become familiar with the basic research associated with viral vector product development, and bench scientists and engineers need a better appreciation of the issues involved in commercialization, cGMP, and scale-up or scale-out processes. Academic institutions, particularly at the university-community college interface, need to provide additional hands-on training, instruction, and contract services to help industry partners educate employees entering the field. Such education is needed in integrating novel process and analytical technologies to develop what will ultimately become process platforms for these exciting new biologics that offer such great promise to patients throughout the world.
The technology roadmaps were developed with support from the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) and financial assistance award 70NANB17H002 from the U.S. Department of Commerce, National Institute of Standards and Technology.
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- J. A. Kulkarni, P. R. Cullis and R. van Der Meel, "Lipid Nanoparticles Enabling Gene Therapies: From Concepts to Clinical Utility.," Nucleic Acids Therapeutics, vol. 28, no. 3, pp. 146-157, 2018.
- P. D. Hsu, E. S. Lander and F. Zhang, "Development and Applications of CRISPR-Cas9 for Genome Engineering," Cell, vol. 167, no. 5, pp. 1262-1278, 2015.
- R. M. Gupta and K. Musunuru, "Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9," The Journal of Clinical Investigation, vol. 124, no. 10, pp. 4154-4161, 2014.
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- E. Ayuso, M. Federico and F. Bosch, "Production, Purification and Characterization of Adeno-Associated Vectors," Current Gene Therapy, vol. 10, no. 6, pp. 423-436, 2010.
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- M. R. Veldwijk et al, "Development and optimization of a real-time quantitative PCR-based method for the titration of AAV-2 vector stocks," Molecular Therapy, vol. 6, no. 2, pp. 272-278, 2002.
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NIIMBL is a public-private partnership with the goal of advancing innovation in biopharmaceutical manufacturing. NIIMBL is part of Manufacturing USA®, a network of 14 manufacturing institutes across the country that brings together industry, academia, and the public sector to propel promising research and technology developments, accelerate new products to market, and train tomorrow’s workforce to secure America’s future. NIIMBL is funded through a cooperative agreement with the National Institute of Standards and Technology (NIST) in the U.S. Department of Commerce and leverages additional support from industry, academic institutions, non-profit organizations, and several state governments. The NIIMBL mission is to accelerate biopharmaceutical innovation, support the development of standards that enable more efficient and rapid manufacturing capabilities, and educate and train a world-leading biopharmaceutical manufacturing workforce, fundamentally advancing U.S. competitiveness in this industry.
About The Authors:
Ruben Carbonell is chief technology officer at NIIMBL and the Frank Hawkins Kenan Distinguished Professor of Chemical Engineering at North Carolina State University.
Arpan Mukherjee is a scientific project manager at NIIMBL.
Jonathan Dordick is the Howard P. Isermann Professor of Chemical and Biological Engineering at Rensselaer Polytechnic Institute.
Christopher J. Roberts is the associate institute director at NIIMBL and a professor of Chemical and Biomolecular Engineering at the University of Delaware.