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. However, liposomes’ production complexity, “shell” deterioration, and particle size variability and particle size distribution make them a production nightmare that is often more of an art than a science. This has 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 the older liposome process requirements and discuss how the process can be adapted to a more modern approach using single-use systems (SUSs), migrating away from the stainless-steel world liposomes have been accustomed to.
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 not simple. 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 salts, several rounds of phosphate salts (for buffers), amino acids, sucrose, solvents, PEG, 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 many dispensing 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 predispensed, 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 125 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 three methods of the lipid transformation:
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, that injects 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.
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 this 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. 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 is serviced by a TCU (temperature control unit) that uses chilled process water and a quick reacting thermal heater.
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) 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.
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.
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.