By Herman F. Bozenhardt and Erich H. Bozenhardt
Discovered in 1961, liposomes have been around for several decades as a drug delivery platform that has achieved varying levels of applications and popularity. With their biocompatibility and well-understood chemistry of encapsulation of a wide variety of APIs (active pharmaceutical ingredients), liposomes make it through the screening process for many potential products. Most recently, API payloads have expanded beyond small molecules to include mRNA and other gene editing tools. The carrying capacity of liposomes allows for larger/more complex edits than viral or cell membrane disruption techniques. However, liposomes’ production complexity, “shell” deterioration, and particle size variability and distribution make them a production nightmare that is often more of an art than a science. This has traditionally manifested itself with extended development times, start-up challenges, and mandatory BE (bioequivalency) studies when making any changes to the process, even one as simple as a facility relocation. The advantage of the liposomal-based product is the ability of the liposome to deliver the API in a very targeted manner. The targeting precision of the “homing” exterior of the phospholipid/liposome can be readily designed for a wide level of applications.
Regardless of the challenges, liposomes have seen a resurgence in use for biosimilars as well as new products, especially in the area of oncology. This article is focused on the key production processes, their engineering, and the design/construction of a successful, repeatable, and safe manufacturing plant for injectable therapies.
This article is the first of a two-part series that discusses liposomes from the process equipment and implementation viewpoint. The key focus here is to look at older liposome process requirements that are being adapted to a more modern approach using single-use systems (SUSs), as well as micro particle collision mixing systems, and highlight new processes that are being considered for the next generation of therapies.
Process Concepts And Basic Equipment
The process to develop and encapsulate a liposomal-based pharmaceutical has several major steps.
Material Dispensing, Dissolution, And Solution Sequencing
This aspect of the process is often overlooked, primarily because it seems mundane and often tedious. This, however, is the single most operator-intensive effort and requires the greatest level of detail. Most liposome operations require the dispensing and dissolution of anywhere between 5 and 10 rounds of phosphate salts (for buffers), amino acids, sucrose, solvents, PEG, saline, and additional chemicals to build the “homing” layer, as well as between one and five raw lipids. All these materials are used in three stages of the process: 1) emulsion preparation and solvent dissolution of the lipids, 2) infusion of the API into the lipid and creating the homing layer, and 3) stabilizing the final solution into a NaCl-based solution and removing all the impurities in the final formulation. The dissolution of many materials also requires dispensing many quantities of water for injection (WFI). Although not a glamorous part of the process, every improvement in this area will reduce time and the staff size required. This type of operation is a natural for single-use systems (SUSs) utilizing disposable bags, pallet tanks (possibly jacketed), and an automated dispensing system that follows an automated recipe system synchronized with the bill of materials (BOM). As an optimizing step, the quantities of WFI needed for dissolution could be purchased pre-dispensed, speeding up the process, or, for that matter, all the phosphate buffers, salt solutions, and commodity solutions could be premade/procured in a disposable unit. This filled disposable unit could then be connected to a manifold and pumped into the process when required. Furthermore, these liquids could be filtered through a .22-micron filter as they enter the process, in an effort to reduce bioburden and reduce or eliminate downstream bioburden filtrations, which occur over the many days of processing.
The liposome is created by dissolving the raw lipids in ethanol (generally) and impacting them physically to create a liposome particle between 50 nm and 100 nm. The size range is dependent on the product’s target organ in the body and the physical biology required. The method always begins with a stable emulsion of water, solvents, and dissolved lipids. There are several methods of lipid transformation:
- Sonication is the ultrasonic irradiation of the liquid emulsion of lipids. This energy shatters the lipids and creates the spherical vesicles characterized by a bilayer of lipids with internal aqueous cavities. This has been generally successful for small volumes of liposomes and for use in labs and pilot plants. Sonication is often effective at the 1- to 10-liter scale, but it is often replaced upon scale-up operations. Sonication systems are typically self-contained modules that can be transferred to a single-use bag.
- High-shear mixing/high-shear dispersion/homogenization is a method first introduced by Szoka in 1983, in which energy is imparted to the emulsion and a spectrum of particles is formed. The careful control of energy levels, temperatures, pressures, and residence time in the mixing system is critical to the average particle size, median particle size, and the distribution. The high-shear mixing systems will require stainless-steel vessels to contain the temperature, pressure, and the byproduct recycling of the high-shear liquid/foam.
- The more common industrial method is extrusion of the emulsion through a uniform pore-size polycarbonate membrane (N x 10,000 Dalton), such as the Millipore Millipak, EMD Millipore Ultra Filter units, Amicon Ultra units, and the Microncon Ultra filter units. These filters extrude the dissolved lipids, which are recirculated through the filter a number of times (often called “passes”) under specific time, temperature, pressure, and flow rates. This method appears to have the most appeal since it requires lower energy and fewer mechanical systems (which could cause contamination), and the consistency of the filter generates a tighter particle size distribution. The older extrusion systems used high-pressure nitrogen (95 to 120 psig) to push the lipid solution through the membrane units in batches. However, today there are higher pressure pumps that provide the pressure without the shear force, such as the Quattroflow pump systems. These pumps need to be explored for the liposome formation process. If they meet the needs of the formation filtration, the entire extrusion process could be a disposable system.
- Another high-energy application of liposome manufacturing was developed with ultra-high pressure/ultra-high-shear micro fluidization. This technology grew out of the homogenizer equipment arena and uses a mixing chamber that operates at 30,000 to 40,000 psi. These units are popular at the lab scale because they produce liposomes quickly. The high pressure of the equipment, compounded with the handling of solvents, has not made it a popular choice for scale up due to the energy and risk levels, along with the support systems required to operate the equipment.
- The ability of our industry to use computing technology to model flow characteristics at the finite element level has given rise to a new series of flow technologies that demonstrate an alternative method to generate the particle interaction necessary to create the “collisions” and forces needed to generate the liposomes from the basic materials. This mechanism provides a more controlled, focused, and precision mechanism (rather than the random nature of the high-energy systems) to create these particles in a very tight distribution pattern. The first of these are the micro-chip/micro channel flow systems. Introduced by companies like Precision Nanosystems and Dolomite Microfluidics, this technology is based upon a micro-sized “zig zag” flow path compacted into modularized “cassettes” that fit into a skid with standard operational connections. The turbulent collision flow path directed by these microchip systems creates a high-shear environment similar to that of the edges of an impeller blade or the interstitial area of a filter. This technology creates the liposomal particles without the high energy and the high volume of solvents and buffer systems required by the technologies previously discussed. This provides a unique situation in which the liquid handling is reduced while achieving a higher liposome concentration. Depending on the applications, these units can be scaled by “stacking” many in parallel to provide the product flow needed. These systems are moving from stand-alone lab-based systems toward commercial-scale capability. The only drawback of these units is their inability to be cleaned for GMP application. However, they will fall into the same category as “single use systems,” and will come pre-sterilized and ready to use in manufacturing.
- The next series of newer, innovative systems for liposome generation is classified as “jet injection” systems, which provide a definitive and continuous flow path. The jet injection technology creates a highly precise and calculated jet stream to create a localized high-shear/collision environment to produce the liposomes in a continuous stream. The two innovators in the jet injection arena are Polymun (Austria) and the University of Connecticut (UConn). Polymun uses its technology in its proprietary CMO application, which involves radial jet injection into a vessel to form the liposomes. For more information, see this article: https://pubmed.ncbi.nlm.nih.gov/26428671/.
UConn developed its jet injection technology based upon funding from the FDA focused on continuous flow manufacturing of critical care products (liposome encapsulated therapies). UConn’s technology is based upon co-axial turbulent jet in co-flow injection with built-in particle size analysis for parametric release. This technology, while a breakthrough in methods that are unique, uses standard piping and standard instrumentation/controls, making it ideal for scale up and incorporation into a GMP facility. The jet injection technologies also provide an ideal scenario where the amount of solvent is reduced dramatically and the entire liquid required is also reduced, providing a higher liposome concentration. We believe this technology presents an optimized approach to future liposome manufacturing.
In summary, the computing-based technologies have enabled manufacturing organizations to use less energy and solvents, reduce liquid handling, and achieve higher concentrations while improving precision, particle size and distribution, and yield. Clearly, the manufacturing market will be shifting and so will the engineering of these facilities.
Liposomal Solution Purification
After the liposome generation, the solution contains a spectrum of liposomes along with ethanol, salts, and any other chemical additive that has aided the shearing of the lipid and preservation of the spherical module. This purification generally takes the form of a selective filtration method that eliminates lower particle sizes and dissolved material. This has been done by large traditional ultrafiltration (UF) skids. Today, this can be handled by smaller and more efficient tangential flow filtration (TFF) systems, where the smaller liposomes, lipid fragments, solvents, and other dissolved materials are purged or passed out of the system by passing through the selective membrane (the permeate). The TFF system requires the lipid-bearing solution to be diluted several-fold and pumped around the TFF membrane to assure dissolution and dilution of the ethanol (must be < 5% in the final formulation). Ultimately, the permeate stream of the TFF will carry away the impurities and the solution with be concentrated to less than the original raw liposome volume. The popular TFF systems used today are the Millipore “stacked” TFF module system, which allows the capacity or scale-up change by simply adding more Pellicon units, and the new Pall TFF systems such as the Allegro and the Cadence modules, which inject the continuous phase solution during the “pump around” to effectively dilute, replace, filter, and concentrate in a time-saving fashion. GE and SciLog also offer standard TFF skids. This entire operation should be done in a SUS bag and tubing system. In the past, the traditional peristaltic pump served the TFF and disposables systems; however, the newer Quattroflow pumps could also be used to reduce the processing/filtration time.
With the introduction of the micro-chip and jet injection systems, which use less solvent and deliver higher concentration of liposomes, the ability to use single-pass TFF disposable cartridges becomes a reality and the process becomes continuous. The opportunity to execute an entirely continuous process presents itself, from liposome formation through TFF and potentially to stabilization.
After the initial UF or TFF, the liposome solution now is generally NaCl surrounding the liposome, with a particle size distribution at and above the target range. At this point, the solution is diluted again with WFI, and a bioburden filtration is done at .22 microns. This effectively eliminates all the particles above 200 nm, thus narrowing the distribution. A variety of disposable filters are used here, with the filtration time being a factor only from an efficiency standpoint. After this filtration, the liposome solution is generally refrigerated in order to prevent product degradation. In fact, the products now need to be kept at 0 to 5 degrees C when not being directly processed.
This is actually the most chemically complex and significant part of the process. In this step the empty liposome spheres are infused with the key pharmaceutical entity that is the basis for the therapy.
In the traditional process the liposome solution is heated up to formation or higher temperatures while undergoing a pH change to essentially open the aqueous interior. The API is then introduced (generally a stochiometric amount) into the solution, wherein it chemically penetrates the bilayer of the liposome. The target within the liposome could be the aqueous center or the organic annular area between the layers. The target is based upon the API’s stability and whether it is hydrophilic or hydrophobic. This happens under a very controlled time period to allow the process to complete. Once the process (usually derived from key development research) is completed, the liposome is cooled, the pH is brought to neutral, and the homing molecules and homing layers are added quickly in series in an effort to bind to the liposome sphere. Typically, these chemicals (amino acids, sugars, PEG, and other chemicals used to control biophysical assimilation and stability) are added to the exterior of the liposome in order for it to be attracted to the target area in the body, attach, and assimilate. This process requires the liposome shell solution to be rapidly heated from a cold state to a range of 60 to 80 degrees C, pH changes, titrations, and a rapid cooldown. This type and sequence of processing is done in a stainless-steel jacketed “reactor type” vessel with multiple ports for material addition. It is also required that the jacket be serviced by a TCU (temperature control unit) that uses chilled process water and a quick reacting thermal heater.
Within the context of the newer micro-chip or jet injection technologies, the micro level of the interactive mixing provides another use for the technologies, that is, an energy-free loading of the API. The jet injection technology applicable to loading (also from UConn) can be found in here: https://pubmed.ncbi.nlm.nih.gov/26428671/. The low-energy nature of these methods makes them preferable for large-molecule APIs for gene editing. Assuming the liposome is correctly sized and the target within the liposome can be properly characterized chemically, the lipid could be loaded in a continuous flow path.
Final Product Stabilization
Depending on the liposome stability, the stoichiometry, and how the liposome loads (actively or passively), the solution may need to undergo an additional TFF or ultrafiltration to purge any non-liposomal material, such as “free” or non-reacted API. In addition, if during the previous processes chemicals or ingredients that are bioburden-prone (e.g., bulk sugars) are used, a bioburden filtration step will be required prior to refrigeration. This entire step can be conducted using a disposable SUS, including tubing, pump contact parts, and filter units.
The final step in the manufacturing process is the sterilizing filtration and filling. Although the density and viscosity of the typical liposomal-based drug is nearly the same as water, the nature of the liposomal solution makes the filtration process very slow. In this case, manufacturers have experimented with elevated temperature (e.g., 30 degrees C or greater), increased filtration area, and increased delta pressure (via nitrogen pressure or Quattroflow pumps) across the filter to speed up the effort and maintain pace with a filling machine. However, the lower filtration area, higher pressure, and increased temperature can deform the liposome, cause re-agglomeration, skew the particle size distribution, and undo the previous processing. Several filtration runs are needed to verify re-agglomeration has not occurred.
One of the final points of sterile filtration is the need to perform a bacterial challenge test and a bacterial retention test. Dependent on the liposomal concentration a single pass through a .2 micron filter may not achieve the 6 log reduction, this is common. Therefore, bacterial challenge testing is necessary before finalizing the sterile filtration process. The result you may find is that a single sterile filtration may require two .2 micron filters in series.
As you can plainly see, the use of liposomes as a drug delivery vehicle has many moving parts and a lot of options that will be explored in the process development. With this as a basis, the next article in our series will cover the engineering aspects of building a manufacturing facility to accommodate the liposome process.
In Part 2, we cover factors in installing a liposome manufacturing process in a facility and the engineering points to consider in doing so.
About The Authors:
Herman Bozenhardt has 42 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 12 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.