Although lyophilization (freeze-drying) is a costly, time-consuming process, it offers a number of major advantages over simpler dosage forms.
Tablets and capsules can create problems for patients with nausea or difficulty swallowing; syringes can carry risk of infection; and even certain drugs administered from vials can take excessive time to be broken down and absorbed into the body.
By lyophililizing pharmaceutical products such as vaccines, peptides and liposomes, it’s possible to prevent a significant amount of chemical degradation. Lyophilization can also provide long-term stability in storage, maximize biological activity, and produce a rapidly dissolving formulation that reliably retains the characteristics of the original dosage form.
But in order to take full advantage of the benefits of lyophilization, pharmaceutical developers must optimize the design of the process around the unique attributes of the compounds with which they’re working. Rather than handling all this research and design work in-house, many developers opt to partner with contract manufacturing organizations (CMOs) who have experience overcoming the challenges associated with lyophilized dosage forms.
A fully optimized effective lyophilization procedure requires precise alignment of several factors. Here are five major areas of concern regarding good laboratory practice (GLP) for any lyophilization method.
Ideal formulation conditions
Throughout the freezing, primary drying and secondary drying stages of the lyophilization process, risks of damage and destabilization abound. This is one area which the expertise of a CMO can prove useful, both for small molecules and more complex biologic products.
As the formulation cools, pure crystalline ice forms within it. This creates a freeze concentration of the non-frozen liquid, which transforms into a more viscous state, and does not crystallize. The goal is for this concentrated, viscous solution to solidify into a phase that may be amorphous, crystalline, or amorphous-crystalline.
But a rapid nucleation and growth rate can significantly impact the size and number of ice crystals that form. If supercooling takes place too rapidly, an unexpectedly large number of smaller ice crystals may develop. When proteins come into contact with this large ice-water interface, they can become desaturated. This means the structure of the formulation may be denatured, disrupting the liposome bilayer and emulsion structure. Denaturation can result in flaws throughout subsequent stages of the process.
In the primary drying stage, ice forms during freezing is removed through the use of sublimation at subambient temperatures, within a vacuum. This typically takes place at chamber pressures of 40 to 400 Torr, and shelf temperatures from -30° to -10° Celsius. In order to dry the product while retaining the structure established in the freezing stage, the product is maintained in a solid state below its collapse temperature. Throughout this stage of the process, stabilizers are essential to prevent the accidental removal of the hydration shell, and the destabilization of the formulation’s protein structure.
Finally, in the secondary drying stage, the relatively minor amount of bound water that remains in the matrix is removed by desorption. Meanwhile, the shelf and product temperature are raised at a rate that facilitates adequate desorption rates, and produces a good level of residual moisture. During this stage, GLP guidelines emphasize that it’s critical to maintain adequate moisture. This is because, if the water content becomes too low, the lack of moisture can also result in destabilization.
Even so, well-calibrated cooling, ice removal and moisture adsorption are only three of the vital components in effective lyophilization. The right recipe is equally crucial.
Correct excipients in optimal quantities
Every lyophilized formulation is structured around its active pharmaceutical ingredient (API). Still, a formulation may also include a number of excipients, such as stabilizers, buffers, pH adjusters, tonicity modifiers and bulking agents. A poorly planned array of excipients can put the efficacy of the API itself at risk. On the other hand, a well-chosen excipient formula can prepare a lyophilized product for a long shelf-life and a stable level of biological activity.
In many pharmaceutical formulations, buffers are used to stabilize pH. Choosing a correct buffer is especially crucial in lyophilized formulations, since some buffers (such as phosphates, especially sodium phosphate) can undergo dramatic pH shifts during the freezing process. Thus, GLP guidelines recommend choose a buffer and undergoes minimal pH change (such as a citrate or histidine buffer). A complementary tactic, used in many lyophilization procedures, is simply to use low concentrations of the buffer, to prevent it from impacting the pH of the formulation as a whole.
When a formulation contains a low concentration of API, a bulking agent helps create a larger, firmer cake structure. Many formulations use crystalline bulking agents, which produce well-formed cakes with good mechanical properties. However, these agents can be dangerous for lyophilized products, because they tend to destabilize emulsions, proteins and liposomes. For this reason, GLP guidelines dictate the use of disaccharide bulking agents like sucrose for many formulations. Or, if a crystalline phase won’t disrupt the structure of the API, mannitol can be used as a bulking agent.
In fact, disaccharides are helpful for more than just bulking. They also form an amorphous sugar glass, which helps stabilize liposomes and proteins throughout the lyophilization process. Sucrose and trehalose have proven especially stabilizing in liposome and virus formulations. This property makes disaccharides highly desirable as stabilizers throughout the lyophilization process.
Two main hypotheses have been put forward to explain this stabilizing effect of disaccharides. The water replacement hypothesis posits that disaccharides interact with liposomes and proteins by hydrogen-bonding in a similar way to the water they replace. The vitrification hypothesis, on the other hand, suggests that disaccharides form sugar glasses of extremely high viscosity, which immobilize the drug and water molecules and create the extremely high activation energies required for reactions to take place.
Finally, isotonic formulations tend to require tonicity adjusters (such as mannitol, sucrose, glycine, glycerol and sodium chloride) to improve their solubility and rate of dissolution. The tonicity adjuster of choice can be dictated by the stability requirements of the bulk solution, or by the route of administration. In either case, excipients like mannitol, sucrose, glycine, glycerol and sodium chloride can all serve as useful tonicity adjusters. They can be included in the diluent rather than in the formulation itself. At the same time, they’re major contributors to the rapid solubility and absorption that have proven to be such major selling point for lyophilized products.
Along with the excipients, the lyophilization process can impact the formulation as a whole, in a variety of ways. The use of disaccharides as a stabilizer and/or bulking agent, for example, will result in a low collapse temperature, which lengthens the drying process by requiring primary drying to take place at lower temperatures. A large volume fill, or a high percentage of solids content in the formulation, will provide heightened resistance to mass transfer, also lengthening the process.
As a matter of fact, the process can even shape the properties of the formulation. The freezing stage can impact the crystallization of excipients like glycine and mannitol. Incomplete crystallization, on the other hand, will lower the collapse temperature. If a significant amount of crystallization takes place within the bulking agent, this can quicken the drying time. However, large amounts of crystalline bulking agent can reduce the stabilizing effects of an amorphous stabilizer, particularly in regard to proteins within the formulation.
In short, a careful balance of buffers, stabilizers, bulking agents and tonicity adjusters is necessary to ensure beneficial interactions with the API throughout each stage of the lyophilization process. But throughout the process, potency, safety and sterility remain crucial concerns for dosage preparations of lyophilized drugs, where the potential for contamination abounds.
Quality-assured handling
Several recent inspections by the US Food and Drug Administration (FDA) have revealed potency and sterility problems with the manufacture and distribution of lyophilized drugs. These can creep in at many stages of the production process. They can emerge during the formulation of solutions, during the filling of vials, in the validation of the filling operation, in the sterilization of the lyophilization equipment, and in the testing of the end product.
In light of these concerns, it’s essential to perform research and development on good laboratory practices (GLP) for each lyophilization process, and to optimize around good manufacturing practices (GMP) when scaling up to the pilot-scale manufacturing stage.
For example, GLP dictates that the form of the pharmaceutical dosage itself should be carefully chosen to maximize stability, biological activity, safety and marketability. The exact approach for addressing these potential problems varies according to the facility and formulation. Even so, it’s best to plan carefully for each of these factors, to avoid problems throughout the production and distribution phases.
As the FDA has acknowledged, one of the main reasons for lyophilizing a drug product is because of that product’s instability in the solution form. This is precisely the reason why lyophilization is such a popular approach in the manufacture of antibiotics like semi-synthetic penicillins, cephalosporins, and certain salts of erythromycin, doxycycline and chloramphenicol. For many of these antibiotics, one would naturally expect a low bioburden in the batching stage. However, some other lyophilized dosage forms, such as hydrocortisone sodium succinate and methylprednisolone sodium succinate, actually lose their antibacterial effect in solution form. This makes their bioburden minimal, and necessitates that the bioburden be predetermined before sterilization and filling.
For all these reasons, tight control is essential throughout the batching and compounding phases. Controls can help prevent the potential for microbiological levels to increase at any point before the solutions are sterilized. One major concern, for any microbiological level, is the potential that endotoxins may develop at an accelerated rate. Thus, GLP for the compounding of lyophilized products should include batching in a controlled environment, in sealed tanks. This is especially vital if the solution is stored for any period of time before being sterilized.
After sterilization, a lyophilized product is loaded into vials. This stage presents its own unique set of challenges. At the beginning of the filling stage, a stopper is placed on top of the vial, and seated in the lyophilizer. However, due to variations in stoppering equipment, some stoppers may be placed on vials by manual operators rather than by machinery. These human operations obviously invite much greater risk of contamination. For this reason, it’s crucial to validate each filling operation with media fills, as well as sampling of critical surfaces and air during the dynamic conditions of active filling.
Partially stoppered vials are next moved to the lyophilizer, where the lyophilization process itself will take place. GLP dictates that all transfer and handling of vials should take place under primary barriers. For example, laminar flow hoods should be in place to cover the vials to be filled.
It’s also critical to validate the handling through the use of vials filled with similar media. Assurance of fill volumes is a significant concern in the filling operation, for the obvious reason that a low fill will result in a vial whose potency is below standard. This poses a potentially serious risk for companies and patients alike. And unlike a powder or liquid fill, a low vial fill may not make itself apparent until after the long, expensive lyophilization process is completed. This is why GLP dictates that the volume of the fill should be monitored every 15 minutes. In addition, particular sections of the filling operation should be isolated and addressed as necessary, when unexpectedly high or low fills become apparent.
Once the filled, stoppered vials have been placed into the lyophilizer, sterilization of the lyophilization equipment itself presents a cause for concern.
Sterilized lyophilization equipment
Improperly sterilized lyophilizers are one of the most frequently encountered problems in the entire manufacturing process. Many older lyophilizers have poor tolerance for steam under pressure, creating environments in which sterilization is scarcely within reasonable margins.
One reason for this is that most lyophilizers simply have their interior surfaces wiped with a chemical agent, in an attempt to sterilize them. However, this technique produces mixed results, because these cleaning agents never come into contact with backfill piping for the administration of inert gas and sterile air. This makes it extremely difficult for a manufacturer to determine or demonstrate that these chemical cleanings actually sterilize their equipment at all.
However, other methods exist for more effective sterilization of lyophilization equipment. For example, some manufacturers introduce gaseous ethylene oxide into the lyophilizer. Even in this case, however, humidification is required (as with any ethylene oxide treatment) and it’s difficult to introduce this sterile moisture with any uniformity throughout the equipment.
Another sterilization option currently growing in popularity is to pump in moist steam under high pressure. In other words, this approach aims to imitate the effect of an autoclave, on the interior surfaces of the lyophilizer. This type of system needs to include two independent temperature-sensing systems: one to record and control temperatures throughout the cycle, and another to monitor temperature specifically in the cold spot within the chamber, to make sure it remains within acceptable margins.
In addition, such a system needs provisions for sterilizing the inert gas or air supply lines. As described above, these lines often fail to be properly sterilized by most methods. Measures also need to be implemented for sterilizing and assuring the integrity of vent filters or filters used to sterilize the inert gas and air, and for sterilizing the shelf support rods; ideally after every lyophilization cycle.
Correct dosage, sterility and stability
Once a finished batch of lyophilized product has been produced, GLP dictates that every dose should be tested for uniformity, sterility, and stability. The latter is particularly critical in cases of aged batches that have been reconstituted.
If the lyophilized product does not include any excipients or additives, and consists only of an API, weight variation is often the most effective method for ensuring each dose is uniform. And even if excipients or additive are present, weight variation testing can still help determine correlations between sample weight and potency.
Despite the fact that some products may be tagged for reconstitution with Bacteriostatic Water for Injection (BWFI), many hospitals only use Sterile Water for Injection (WFI), due to the toxicity risks associated with BWFI. However, standard WFI may actually kill some of the vegetative cells that are present as contaminants. For this reason, it’s crucial to test for this outcome before dispatching any product to a recipient who may reconstitute it differently.
Finally, it’s critical to review stability data, and create a product expiration date based on the batches that have been found to contain the highest moisture content. In truth, quite a few lyophilization products tend to be labeled with short expiration dates, for exactly this reason. Stability data should also incorporate provisions for assays of aged samples, and for the subsequent reconstitution of those samples, all the way up to the expiration date. It’s also important to test for stability of the least and most concentrated reconstituted solutions, and adjust expiration dates accordingly.
The advantages provided by a lyophilized drug product are certainly matched by the challenges and expense involved in creating it; as well as by the opportunities for shortfalls throughout the process. Mistakes in freezing, drying, excipient choice, stoppering, equipment sterilization, and dosage and stability analysis can create significant problems. That’s why it’s vital to have GLP in place for recognizing and addressing all these issues, and to partner with experts who have significant expertise in optimizing lyophilization processes for both clinical and commercial manufacturing.
About the author:
Raymond E Peck is the founder and CEO of Indianapolis-based VxP Pharma, Inc. and VxP Biologics. Inc. VxP offers contract development and manufacturing of lyophilized product, and currently has clients on almost every continent. VxP works with most of the top-20 global pharma companies, as well as countless small and virtual companies. Ray can be reached at Ray.Peck@VxPPharma.com
Bibliography
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