By Herman F. Bozenhardt and Erich H. Bozenhardt
In our first article on liposome manufacturing we discussed the various processes and equipment used in the many liposome manufacturing processes. Our key point is to recognize the wide spectrum of the process requirements: traditional processes trying to adapt single-use systems (SUSs) and newer technologies better suited for mRNA and gene editing therapies. Because liposomes have been around since the 1960s and came into process “vogue” in the 1990s, we need to move in the direction of the modern biotech systems to increase efficiencies and decrease the complexity of the processes.
In this installment we will cover factors in installing a liposome manufacturing process in a facility and the engineering points to consider in doing so.
Manufacturing liposomes has traditionally been a multi-day, multi-step process that involves many long days of processing combined with clumsy logistics. The sheer volume of steel tanks, plumbing systems, flexible hoses, specialty vessels, components, and materials creates a nightmare for the operators. This, combined with batch records that have hundreds of pages, makes for an error-prone process.
Risk Factors In Liposome Manufacturing
We also must consider the various risk factors that rarely are even expressed in design:
- The probability of production failure exists as each day of processing increases, and after the investment of N days, the N+1 day’s failure causes a loss of materials, time, labor, and utilities. In addition, the traditional process will force the cleaning of N+1 days’ worth of vessels and parts, and re-dispensing of the N+1 days’ worth of materials. This makes the restart of the process another nearly N days. Given a five-day or a 10-day process, the impact (essentially an outage of 10 or 20 days) on the supply chain could be catastrophic.
- Operator fatigue is another consideration. Most of these processes range from four to 20 days in the cycle, with several days running 12 to 16 hours consecutively. With the pressure for production, we have a scenario where the operators will be processing and cleaning for extended hours, day in and day out. This creates an environment that invites mistakes, errors, and lost batches. As designers, it is very easy to just say let the operators work longer, harder, and under mandatory overtime. That would be a mistake!
- Operator involvement, training, and water batching are the keys to piloting the process and understanding the time, motion, logistics, operational critical events, and risks we take and make as engineers. We must make every effort to focus our training on critical activities, ease logistics (e.g., cleaning equipment), and making the process operations no longer than 8 hours per day. The solution to this is often automation, additional self-contained process skids, SUS (single use systems), and operator involvement in design.
There is an important additional dimension to the engineering requirements: safety. The liposome manufacturing process will require dispensing of and interacting with various amounts of solvents, nitrogen, high pressures, high-speed rotating equipment, and, potentially, hazardous APIs. Unlike the typical biotech plant, in which water for injection (WFI) is the main component, the liposome plant faces real engineering challenges and material logistics.
Key safety points of focus include the following:
- Protecting the operators from nitrogen leaks
- Protecting operators from high-pressure fluid processing (e.g., microfluidizers, extrusion skids)
- Removing ethanol as a source of fuel in the air, and simultaneously eliminating the potential of an electrical discharge as a source of ignition of an explosion
- Protecting operators from high-speed rotating equipment
- Assuring the vessels and lines are rated for the pressures they will encounter
- Protecting all personnel from hazardous APIs such as steroids.
As a secondary task, the process flow and logistics should be designed to add the operators in:
- Verifying and labeling the SUS to reduce confusion and reduce batch time
- Standardizing the connections from step to step
- Providing an open EU Grade C operating environment that can pre-position the equipment, skids, and disposables in a logical manner and easily move from one process to the next without the previous one cluttering up the path.
Beyond the above discussion, what we have learned about the advancements in liposome formation and loading technology via the “micro-chip” and “jet-injection” systems is that the operation will become simpler, use less energy, and potentially be faster to execute. This potentially will shorten cycles and reduce production time to complete multiple formulations on a shift. In addition, these new technologies are continuous in nature and our definition of a “batch” will change. These “soft benefits” are very significant as this will lower our risk in production, allow campaigning to progress quickly, and ultimately increase capacity in our fixed equipment assets and facility.
Design And Construction
To meet the requirements of the process in a safe and efficient manner, the following features must be included in the design and construction:
At this point in time, most liposomal products are manufactured (upstream of fill finish) in an EU Grade C environment because of the number of SUS assemblies being built in situ, ingredients being added in open vessels, and cleaning operations. If this is the case, the operating suite for the pre-API addition needs to be built to the EU specifications, including air changes (i.e., 30 to 45 per hour for Grade D or 60 to 90 per hour for Grade C), ceiling coverage, downward velocity, as well as particle count. If all SUS systems are implemented and all solutions are added via aseptic connections, and the process is completely closed, then the suite could be built to EU Grade D standards. In the case where the API is a potent compound, the design and build-out must consider using an isolator for API handling and building a separate suite to complete the process downstream of the API addition. While the API may be a small amount, the potential of a spill, leaking tubes with free API, and other potential handling problems make a separate suite a prudent move. A potent compound evaluation, based upon the AIChE and OSHA guidelines, must be made to judge the level of containment (see “Handling & Processing Of Potent Compounds: A Holistic Approach”).
Avoiding Nitrogen Leaks
All nitrogen lines must be evaluated for their pressure rating, and hand-operated wingnut tri-clamp connections may not be adequate. Higher-rated clamps with “nut and bolt” fastenings will be necessary. In addition, the room must be fitted with oxygen sensors at the core of the process and away from the core where operators may congregate. Specialized high-pressure nitrogen cylinders, if used, need to be placed in an unoccupied case and only the supply line in the operating suite.
Ethanol And Other Flammables
Handling ethanol in an open fashion in any closed room forces the designer to classify the room as Class I Div. 1 of the NEC (National Electric Code), which imposes electrical design restrictions on the entire room, as it is considered potentially flammable. On the other hand, if the ethanol can be carried in a closed container and the transfer during the process is completely closed, as is the remainder of the process, the electrical classification can be reduced to Class I Div. 2. That requires the room to have its power outlets 3 feet off the floor, and the restrictive area for the room is reduced to an 8-foot “bubble” from the contained source of the ethanol. Within this bubble, we must eliminate or minimize other potentially flammable materials such as plastics. In all cases, the electrical equipment in the processing suite must be explosion-proof or encapsulated in a pressurized purged stainless-steel enclosure. In some cases, to comply with this rule, the agitators were pneumatically driven. Electrical classification of the area handling the ethanol is only one code consideration; depending on the quantity of flammables, fire sprinkler coverage may increase, and the building code may require architectural segregation.
High-Speed Rotating Equipment
The one area where high-speed rotating equipment is employed is when a high-shear mixer/dispersion/microfluidizer is used. In that case, sufficient shielding, anchoring, and cooling needs to be supplied to the system before, during, and after its use.
Although pharmaceutical businesses tend to not deal with high pressures, they are encountered in various extrusion, TFF, and filtration processes. The process design must take into account the ratings and state codes for traditional tanks and fixed vessels and assure they meet the ASME pressure rating for the working volume. In addition, when employing a system of mixed vessels and SUS, often interconnected, be sure you do not expose the disposable bag to pressures outside of its relatively low-pressure limits.
API (Small Molecule)
API material, especially if it is a CAT 3 (<100 µg/m3) or higher powder, needs serious built-in safety precautions to prevent operator exposure. At a minimum, a flexible/disposable polymer isolator for weighing and dispensing hazardous material, with enough interior room to load a disposable transfer SUS, is required. This SUS is made with ports for loading materials and adding WFI and provides a transfer port to the loading process. In cases where the API cannot be manipulated in a common operable SUS, a fixed isolator must be installed in the room, with a pneumatic or gravity feed device and isolating double butterfly valves. This will be needed to feed the API loading process vessel.
API materials can include steroids, which can have similar characteristics as potent compounds in terms of operator protection. Some steroids have a significant vapor pressure. In that case, the steroid must be treated as a potent compound, using an isolator and a segregated suite for processing. It is also recommended to use “once through” HEPA-filtered air for any suite where the steroid may be handled.
Finally, a new approach uses antibiotics as the API for targeted therapy. Depending on the operator sensitivity and class of the antibiotic, isolation may not be needed for these materials.
API (mRNA And Gene Editing)
Liposomes can be formulated to be absorbed into the cell membrane, similar to exosomes or viral envelopes. This makes them a natural candidate to deliver nucleic acids (mRNA, siRNA, DNA) into cells. Most candidates today are non-viral and can be handled in a manner similar to material from traditional biopharmaceuticals. There is consideration for using liposomes to create a viral envelope for in-vivo gene therapies where the immune system may react to the viral vector (e.g., adenovirus). In this case, the appropriate biological safety approaches need to be considered for the virus being handled (see Bozenhardt, Erich, and Herman Bozenhardt, “Biocontainment: An Introduction to Control Levels & Practical Design Concepts”).
This technology can be delivered to a whole population (mRNA vaccine) or as a personalized medicine (gene therapy targeting a patient’s specific cancer). Understanding the indications and population is key to having an efficient facility design.
Layout And Logistics
The problem of process layout is significant and perplexing with liposomes, and room logistics are rarely solved through a pen and paper exercise. The easiest way to solve this problem for each individual plant is to mock up the basic operating stations of the processes mentioned previously. The engineer needs to identify the amount and volume of solutions needed to service each process and run through the batch record execution with the operators. The insight into the logistics of pallet placement and material access, utility connections, and placement and assembly of the SUS will be enlightening. In addition, the concept of buy vs. make is critical here.
If you can buy a SUS assembly or system premade and sterilized and “ready to process,” buy it; it will save hours of operator time, warehouse logistics, procurement time, floor space, and tracking effort. For the same reasons, pre-made sterilized solutions and buffers should be bought and not prepared on-site. This will save time and reduce operator fatigue. It will also increase consistency and accuracy.
In summary, liposome manufacturing is not anything like today’s typical biotech projects. It has many process events and actions that are more akin to older-style chemical production processes. Liposome processes can be adapted directly to the SUS with a few exceptions and transformed into efficient manufacturing operations. Within this context, liposomes have their own characteristics, especially with formation of the empty liposomal shell, solvent handling, purification, and filtration, which the engineer/designer needs to recognize prior to completing the conceptual design.
The final aspect of the liposome project is the qualification, which will require significant time to fine-tune, qualify, and validate the process of the liposome shell formation. Lastly, the handling of any potent API must be reviewed, a risk assessment performed, and the logistics mocked up and run to really understand the risks and what could transpire.
Ultimately, the liposomal drug can be one of the most effective drug delivery mechanisms in our industry, and with care, simulation, and good judgement, we can design, build, and deploy these processes effectively.
About The Authors:
Herman Bozenhardt has 44 years of experience in pharmaceutical, biotechnology, and medical device manufacturing, engineering, and compliance. He is a recognized expert in the area of aseptic filling facilities and systems and has extensive experience in the manufacture of therapeutic biologicals and vaccines. His current consulting work focuses on the areas of aseptic systems, biological manufacturing, and automation/computer systems. He has a B.S. in chemical engineering and an M.S. in system engineering, both from the Polytechnic Institute of Brooklyn.
Erich Bozenhardt, PE, is the process manager for IPS-Integrated Project Services’ process group in Raleigh, NC. He has 14 years of experience in the biotechnology and aseptic processing business and has led several biological manufacturing projects, including cell therapies, mammalian cell culture, and novel delivery systems. He has a B.S. in chemical engineering and an MBA, both from the University of Delaware.