In the ever-evolving world of molecular biology and genetic research, oligonucleotides play a pivotal role in a vast array of applications, from DNA sequencing to gene synthesis and targeted therapy development.
The demand for these short, customized DNA or RNA sequences has been steadily rising, driven by advances in biotechnology and our expanding understanding of genetics. They have a wide range of applications in genetic testing, research, and forensics. Because they can be manufactured as single-stranded molecules with any user-specified sequence, they are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, molecular cloning and as molecular probes. The result is a class of pharmaceuticals revolutionizing the treatment of diseases that other therapies cannot touch.
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Oligonucleotide manufacturing is a highly specialized process requiring state-of-the-art facilities. Oligonucleotide facility design presents unique challenges, as the manufacturing process requires a blend of active pharmaceutical ingredient (API) facility design and biopharmaceutical facility design that requires High-hazard space (H space) limitations. In other words, oligonucleotide facilities are a mix of chemistry and biology and must meet requirements for both types of spaces. Designers must combine heavy solvent usage requirements with cleanroom environments, resulting in distinct challenges to compliance, containment, and scalability.
Oligonucleotides and “untreatable” diseases
Oligonucleotides are short strands of modified DNA and RNA sequences that are usually about 20 nucleotides in length. Thanks to DNA synthesis, oligonucleotides can be manufactured synthetically with modified nucleotides to improve their therapeutic effects. They are designed to bind to specific DNA or RNA sequences in the same manner as naturally occurring oligonucleotides, preventing genetic errors from translating into proteins. However, they do not affect the genome itself. Such therapeutics target the genetic code to reach the root cause of diseases, as opposed to biologic drugs that target proteins.
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In recent years, significant advances in oligonucleotide drug design resulted in therapeutics with greatly improved stability. We also saw progress in efficient delivery to target organs and tissues (other than the liver), better cellular uptake, and a reduction in undesired side effects. Modifications led to an increased ability to target patient-specific sequences causative of rare diseases, specific alleles, distinct transcript isoforms, pathogenic fusion, and viral sequences that evolve resistance to an oligonucleotide therapy.
Overcoming these and other challenges means new forms of oligonucleotide drugs will pave the way for cures of rare diseases previously thought untreatable using traditional therapeutics. In fact, as of December 2021, approximately 15 oligonucleotide drugs have received regulatory approval globally (USA, EU, JP), primarily focusing on:
Neurodegenerative diseases Neuromuscular diseases Ocular diseases Cardiovascular diseases Oncology Infectious diseases Renal disease
Until recently, most oligonucleotide development has been lab-oriented on a very small scale. However, the rapid growth of this niche means manufacturers need facilities to handle large-scale processes. To successfully do so, operations must scale up and change to meet compliance requirements.
Upstream process with oligonucleotides
The upstream process with oligonucleotides differs from traditional pharmaceutical processes due to the unique combination of chemistry and biology required. The upstream process begins with a phosphoramidite chemical reaction (i.e., the addition of bases through a repeated sequence of reactions) to grow the chain of modified nucleotides. The drug substance is then synthesized on a solid substrate in a synthesis column. These reactions involve several thousand liters of flammable solvent (ethanol, acetonitrile, and toluene) per kilogram of produced product. After synthesis, oligonucleotides are separated from the solid support by heating them in concentrated aqueous ammonium hydroxide, eliminating protecting groups from the bases and phosphates. Deprotection then removes the blocks used to ensure linear chains versus split chains. Finally, the drug undergoes a crude ultrafiltration process before moving to downstream processing.
Challenges in the oligonucleotide upstream process
An oligonucleotide facility must safely handle the large quantities of flammable solvents required. Once these solvents are used, they are removed as hazardous waste. In addition, systems using fire-safe valves, interlocks, and single-pass airflows need to be in place to minimize the risk of fire and contain potential solvent spills. Any electrical components used in areas with these solvents also require proper classification.
Downstream process with oligonucleotides
The purification process varies among manufacturers, but most use Reverse Phase Chromatography (RPC) and Anion Exchange (AEX) chromatography to separate components, ensuring impurities are removed prior to the concentration process. Depending on the type of process used, either a solvent- or water-based solution is used. Post-purification, the concentration process uses ultrafiltration/diafiltration (UF/DF), followed by lyophilization (freeze drying) of the final substance before the final formulation.
Challenges in the oligonucleotide downstream process
Bacterial or microbial contamination is a significant factor in facility design. Special care is necessary to minimize the danger to workers and the product. Facilities must incorporate aspects of biologic drug production plants to ensure a suitable environment for sterile formulation and fill-finish of the final parenteral oligonucleotide drug product.
Scaling challenges
Thinking long-term when designing an oligonucleotide facility is crucial to getting the most out of it. If you think you will make the same drug in five years, plan for potential expansion during the design phase. You will appreciate this extra work when you need to expand and spend less time and money than if you had no plan.
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Oligonucleotide therapeutic manufacturing is a relatively new approach to pharmaceutical production, so working with them is not as predictable as traditional biopharmaceutical processes:
Production may change tremendously. Technology and intellectual property will evolve in ways we cannot foresee. A company may not produce the same drug in a few years.
Premier designers of modern oligonucleotide facilities understand relevant large-scale equipment and service design criteria, trends in flexible suite design, sustainability goals, and digital facility modeling.
The novelty of oligonucleotide manufacturing introduces additional challenges:
Lack of established best practices. Legacy technologies are less scalable for oligo-process development than for other types of processing, often requiring an adapted or unique process, equipment, and suite design. Service, regulatory, quality, safety, and ergonomic requirements must be fully understood. The production, purification, and concentration of oligonucleotide products are evolving, requiring creative solutions in designing manufacturing processes and facilities. Flexible suite design methods in this evolving field ensure a facility will rapidly and economically accommodate necessary equipment and process flows. When scaling up, synthesis and chromatography equipment (including skids and columns) increase in size and require valuable production floor space. Process skids, supporting feed vessels, distribution equipment, and utility systems must be viable across all production phases. Feed tanks are typically sized for the largest batch a facility produces. However, it is crucial to design the space to support multiple production sizes. The market expansion in the next 5 to 10 years might render a facility obsolete if it is not designed to meet the required capacity.
One of the best ways to plan for future expansion is to include so-called “shell” spaces, unfinished rooms built within the facility’s footprint. These spaces can be used for an intended purpose later, as needed. It is a cost-saving way to provide flexibility to a facility that may be small-scale now but is expected to reach market forecasts throughout its lifetime.
Supply chain challenges
The major raw material in oligo-based drug manufacturing is very expensive nucleic acids. Solvents used in this process are extremely hazardous. Manufacturers must provide safe on-site storage and guarantee safe transport to the facility. Designers must ask themselves:
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Where is the closest supplier? If the facility is in a populated area, chances are that a supplier is nearby. If the space is more rural, there may be geographic barriers between the lab and the supplier, increasing the need for on-site storage. How much must be stored onsite heavily impacts the facility design.
- If a facility is remote from its closest supplier, additional refrigerated warehousing may be necessary.
How will the product be shipped?
Solvents must be evaporated without ruining the product while freeze-drying the drug into a powder. This is often the most
expensive part of the process due to the equipment required.- If freeze-drying in-house, ship the powder in bags or bottles.
- If not, send it in liquid form to a sterilization and packaging site.
While there have been few issues with receiving raw materials for oligonucleotide processes, equipment procurement continues to be a challenge in 2023. Companies have been waiting a year or more for shipment of equipment, so planning is paramount.
Contamination and other safety concerns
Worker safety is paramount. While there is no risk of operators contaminating the product during synthesis, workers may be at great risk using the explosive chemicals necessary for the process. Oligonucleotide manufacturers must meet all code requirements to maximize safety.
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Most equipment in oligonucleotide facilities is on wheels and pushed from one room to another. Therefore, proper pathways must be considered early in the project:
Design corridors for ISO 8 and ISO 7 cleanliness levels. Do not send product through a clean, non-classified (CNC) space to control humidity and temperature. Ensure rooms coded for product have sufficient HVAC systems. Minimize transportation as much as possible by performing pressure transfers between rooms. Place product in cold storage at night if there is no overnight shift.
Other facility design challenges
Facilities using a substantial amount of flammable solvents must comply with the high-hazard occupancy requirements of the International Building Code (IBC). The typical solvents, such as acetonitrile, toluene, and pyridine, are all Class 1B solvents per the National Fire Protection Association (NFPA) code. Facilities may only store and process a limited amount of solvent before characterizing the space as a high-hazard area. Assuming a facility is designed according to the NFPA code, the maximum allowable quantity (MAQ) for Class 1B solvents is 480 gallons in storage and 240 gallons to be used in closed processes. Less is allowed for open processes. A large-scale facility may have 100 times that much in storage and use thousands of gallons of solvent for a single production run, requiring the space to be designated an H-rated facility. Designing an H-space is unlike designing other bioprocessing facilities, as code compliance is much more rigorous. For example, H-spaces require:
- Automatic fire detection and sprinkler systems meeting IBC and NFPA standards.
- Construct firewalls that separate controlled spaces (like labs) from non-controlled spaces (like office spaces).
Final thoughts
Designing oligonucleotide manufacturing facilities is a challenging prospect and designing them in a way that allows for flexibility and future scalability is even more daunting. However, the work is imperative to developing and distributing innovative therapies that can cure diseases previously thought of as uncurable. New oligonucleotide treatments continue to qualify for regulatory approval, giving hope to patients and those in the healthcare industry.
