Over the past year we’ve witnessed an explosion of activity in both vaccine research and manufacturing due to SARS-CoV-2. We’ve also seen an increased appreciation of what had been some rather obscure, though highly significant, technologies. These include vaccine types and sub-types, ranging from exosome encapsulated AAV vectored vaccines in a prime-boost system, to the lipid nanoparticle delivered mRNA of a viral antigen. While traditional platforms remain in use, innovative delivery systems now include entirely synthetic antigenic structures, sub-viral particles, and chimeric bacterial ghosts. Antigen production platforms employ such diverse approaches as animal tissues, cell-free expression, whole plants, cell culture and direct chemical synthesis.
Even limiting the discussion to cell-based manufacturing yields a diversity of platforms, from E. coli to yeast to fungi to animal cells. Operational approaches range from single-use (SU) to fed-batch microcarrier to fixed-bed continuous systems. Furthermore, approved legacy production processes need to keep up because such components as ﬁlters and resins change over time. Also, we see many different types of cell products being addressed, such as antigenic proteins, attenuated or subsequently inactivated viruses, virus-like particles (VLP), vectors or delivery packages such as extracellular vesicles (EV), and bacterial or bacterial vectored vaccines.
mRNA vaccines are a new type of vaccine designed to protect against infectious and chronic diseases. Many classical vaccines insert isolated natural or recombinant antigen assemblies into our bodies to elicit an immune response. But mRNA vaccines program our cells to make a protein—or even just a piece of a protein—to trigger the immune response. For example, COVID-19 mRNA vaccines employ mRNA coding for multiple immunogenic epitopes of a harmless COVID virus component called the spike protein. They deliver the mRNA to our cells in a vehicle such as a polymer- or lipid-based nanoparticle. After the particle delivers the mRNA, our cell’s machinery produces the spike protein antigen and then breaks down the mRNA. The cell then displays the protein on its surface, our immune system recognizes it as foreign, and it builds an immune response against it. This blog describes steps of the production process and identifies some key considerations in the design of a facility for mRNA vaccine production.
mRNA Vaccine Production
mRNA Drug Substance Production
The nucleotide portion of an mRNA vaccine can be prepared via four different means. It can be produced 1) chemically, that is, synthesized in a chemical reactor, 2) through in vitro transcription, using a linearized DNA template obtained from a bacterial plasmid, 3) using a DNA template amplified by PCR, or 4) now, even via commercially available double-stranded DNA fragments as the template. For DNA template-based in vitro transcription (IVT) production, ribonucleotides and a T7, a T3 or an Sp6 phage DNA dependent RNA polymerase that supplies all the factors for transcription (initiation, elongation, or termination) are used. We will here focus on this plasmid template-based approach.
Because mRNA vaccines are a completely different type of product, facilities harbor a smaller number of, and smaller sized, production suites. Unlike facilities producing egg-based vaccines or even monoclonal antibodies, full commercial scale mRNA vaccine manufacturing sites often contain between 3 and 5 production suites including one suite supporting 5 – 50L production fermentors to produce the plasmid (DNA template).
Commonly, frozen cells from a central cell bank are thawed and expanded in a shake flask for production fermentor seeding. A fermentor is charged with culture medium and inoculated with the flask contents. The fermentation process then takes 1-2 days. This culture is then chilled at the end of fermentation and the harvested culture is centrifuged to separate the cell mass (including plasmid template for the mRNA) from the spent medium. Following cell lysis, the harvest can be clarified by e.g., depth filtration. The DNA plasmid can be purified from the clarified lysate by column chromatography, enzyme digestion, a series of ultrafiltration/diafiltration filtrations and a final sterile filtration step.
mRNA Production from Plasmid
Purified plasmid is then transferred to a reaction vessel, along with in vitro transcription (IVT) reagents and enzymes from the buffer prep area, and the reaction is complete in a matter of hours. The plasmid can then be removed by digesting with DNase incubation in a matter of minutes. The quenched IVT product is concentrated, purified, and conditioned by chromatography and filtrations. Finally, the mRNA is processed with a capping reaction which occurs within some few hours − followed, by purification, buffer conditioning, dilution and sterile filtration.
Drug Substance Formulation & Bulk Fill
A delivery system, such as a polymer- or lipid-based nanoparticle (LNP), is prepared, and the purified mRNA is then encapsulated within it, using methodologies specific to different manufacturers. The resulting mRNA/delivery nanoparticle assembly undergoes concentration, buffer exchange and sterile-filtration. It is then finally assessed and packaged into bags/bottles in this sequence as bulk drug substance. The bulk drug substance is often stored frozen ahead of a final formation (although not applicable for some processes) and final drug product fill, followed in some cases by lyophilization.
Modern Vaccine Facilities
Today, facilities are providing a greater digital or “4.0” production environment enabling high throughput and robust manufacturing for a diverse array of products. Enterprise control systems enable both ease of technology transfer and manufacturing flexibility in highly automated processing landscapes. In fact, digital technologies are now integrated throughout modern plants and include enterprise resource planning systems with electronic production records, process control systems, data historian, and laboratory information management systems. Such digital platforms streamline product manufacturing, testing, and release. They also enable robust data management to support process development, characterization, and transfer.
We are seeing more extensive digital systems integration in all aspects of a facility, including elements of procurement, process, quality, and distribution systems delivering a more integrated manufacturing and supply chain. This will result in an automated cloud-based system that manages the planning and execution of a vaccine pipeline from design and scale-up, to manufacturing and distribution, to the regulated post market surveillance of the product.
There are new designs providing additional value while maintaining GMP methods, facility, and control requirements. They are a result of many advances, including those occurring in materials of construction, workflow, equipment, utility, and HVAC. Single use, closed, continuous and modular process designs are changing paradigms in space allocation and classification.
The rapid delivery of flexible and adaptable facilities is currently being supplied by qualified vendors of advanced aseptic processing solutions, facility designs and build support. Such vendors are providing design of compliant materials, space allocation, workflow, and equipment for research, PD, and manufacturing. Understanding of high throughput and straight-through manufacturing approaches with both product and process flexibility is now a requirement, and the ability to establish flexible manufacturing processes and spaces for the rapid changeover between either existing product campaigns or new development candidates is expected.
Modular and podular prefabricated facility designs have become a regular consideration for many projects. They can provide reduced footprints, time-to-market, service requirements, and operating expenses. Multi- product, -process designs and process trains, which can be less expensive to build and operate, can also offer increased flexibility and heightened environmental sustainability. There is also a move toward a nesting of classified space. Prefabricated podular suites can be installed in unclassified (gray space) environments, and with the addition of closed systems within, can themselves be operated at a reduced classification.
Even traditional warehouse design and operations are being transformed. Automated storage and retrieval systems can greatly increase storage capacity and picking efficiency. New warehouse design, software and automated retrieval equipment can increase productivity, recover floorspace and improve picking accuracy with absolute ergonomic performance.
mRNA Vaccine Drug Substance Facilities
Many aspects of the development and production of an mRNA vaccine coincide with that of many other regulated biologicals− for example, much of the service, supplies and QC techniques. But some elements can be quite distinct. In the case of a mRNA vaccine, entity production occurring in relatively small-scale fermentors / reactors differentiates them from more voluminous biomanufacturing.
From a regulatory perspective, because of the novel composition of the vaccine, it’s difficult to know even with what division or department of an agency to begin a filing. For example, the FDA has two basic divisions to register a biologic vs a drug compound, CBER and CDER respectively. Distinctions require specific study, as they aren’t necessarily intuitive from the terminology. It turns out that current mRNA vaccines are not being considered an Advanced Therapy Medicinal Product (ATMP), and in the US are being filed in a BLA under the auspice of CBER.
With respect to the facility, the FDA’s Development and Licensure of Vaccines to Prevent COVID-19 Guidance for Industry (FDA-2020-D-1137) provides rather standard guidelines for the facility per se, such as that operations should be adequately designed to prevent contamination; that such processes as facility and equipment cleaning and maintenance, sterile filtration and aseptic activities must be validated; that HVAC systems must provide adequate control; and that manufacturing equipment should be qualified.
It does clearly recommend communication with CBER’s Office of Compliance and Biologics Quality to discuss inspections, and that they are generally conducted following the acceptance of a BLA filing (21 CFR 601.20). But, it states that during the COVID-19 public health emergency, the FDA is utilizing using additional tools to determine even the need for an on-site inspection.
An mRNA vaccine drug substance manufacturing facility will support three basic activities, following the outline of production activities above. They are: 1) fermentation of a transfected bacterium, such as E. coli, to produce the DNA plasmid, followed by purification of the DNA plasmid, 2) production and purification of the mRNA bulk drug substance from that plasmid, its purification (and freezing) and, 3) production of the polymeric- or lipid-based nanoparticle and mRNA drug product/vehicle conjugation. The manufacturing facility will directly support all of this, as well as, of course, require such standard areas as buffer/media preparation, weigh/dispense, service, etc.
Typically, an individual manufacturing suite (or appropriate accommodation) is dedicated to the purpose of 1) buffer and media preparation, 2) mRNA production, 3) nanoparticle formulation, 4) mRNA / nanoparticle assembly, and 5) final bulk drug product formulation / sterilization / packaging. Some sponsors will operate steps 3 and 4 within the same area. Depending upon scale, buffer can be distributed from the buffer prep room by one of two ways: it can be piped (fixed or flexible single use tubing) into the production suite directly from the buffer prep room, or totes and bags can be brought to the equipment and transferred via flexible connections. It is common to have utility panels available, which include instrument and process air, oxygen, and nitrogen, as well as solvent aqueous waste sump stations. One must be cognizant of solvent use requirements and any process steps requiring attention to ATEX or XP standards with NEC Class 1 or 2 rated rooms or zones.
These are exciting times for vaccine entity sponsors, material suppliers and facility designers alike. The array of possibilities becoming available for even mRNA vaccine process and suite design is illustrated by CureVac’s collaboration with Elon Musk to make “mobile molecule printers.” While the details of the dimensions and requirements of these “mRNA microfactories” are yet to be disclosed, we do know they are described as being both highly automated and designed to be shipped to remote locations. This might indicate unique service support and reduced footprint / personnel requirements as compared to even current mRNA vaccine facilities.
Header Image: Moderna’s Norwood facility took 2019 ISPE FOYA Facility of the Future Category Award. Adapted from image courtesy Andy Ryan Photography Inc.
About the Author
William Whitford, Life Science Strategic Solutions Leader, DPS Group
Bill Whitford has recently joined DPS Group as the Life Science Strategic Solutions Leader. Here he will assist in developing creative strategies supporting the manufacturing of both classical and innovative biotherapeutics.
Bill began his carrier as an R&D Leader, commercializing over 40 distinct products supporting biomedicine and biomanufacturing. Applications ranged from assisted reproduction to the culture of animal cells in protein biological and vaccine production.
Most recently Bill has been a thought leader identifying burgeoning biomedical products and processes. An invited lecturer at international conferences, he has published over 300 articles, book chapters, and patents in bioproduction; is a regular presenter at international conventions; and is an instructor in biomanufacturing.