Plan Early: Optimizing Stability During Lyophilization

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Lyophilization remains an integral step in the manufacturing process for certain biotherapeutics, as was demonstrated with the COVID-19 vaccines.

Lyophilization is also important in traditional biologic manufacturing and for emerging therapies. However, there remains inherent risk in subjecting these biomolecules through successive freeze-thaw cycles, which call for best-practice procedures to ensure that product viability and stability are maintained during the lyophilization process. Furthermore, questions have arisen about the long-term sustainability of current lyophilization techniques as the development of new biologic and emerging therapy drug candidates continues to grow.

Challenges with emerging therapies

One big bioprocessing challenge posed by the lyophilization of emerging biotherapeutics, such as cell therapy, gene therapy, and nucleic-acid-based therapies (e.g., the mRNA-based vaccines), is a general impact on machine technology. The shift from small-molecule pharmaceuticals to biologics has also changed the manufacturing requirements because of the nature of the products, explains Jörg Rosenbaum, director product management at Hesse, Germany-based Optima Pharma.

“Biologics are fragile products that cannot tolerate heat and have a limited stability. Lyophilization is a very important process in biotherapeutics manufacturing to improve product stability,” Rosenbaum states.

Factors that have made the lyophilization of biologic drug products challenging include the equipment requirements. The equipment used must be advanced, user-friendly, efficient, and applicable from development to scale-up, notes Rosenbaum. The increasing demand for biologics requires smaller batch sizes and greater flexibility in manufacturing solutions, which necessitates frequent line and equipment changes, Rosenbaum observes. The requirement for smaller batch sizes and manufacturing flexibility is leading to a shift from large manufacturing areas to small and flexible footprints. As a result of the shift towards biologics, there is also a growing trend towards the use of single-use technology, he adds.

“The driving forces are not only cost reduction and improved production efficiency, but also the ability to process potent compounds and produce smaller batch sizes,” says Rosenbaum.

Rosenbaum goes on to discuss some of the manufacturing challenges in the lyophilization of biologic drug product. “Freeze-dried and liquid drug vials for biologics are filled in a similar manner, but the different formulations present different manufacturing challenges,” he says.

Liquid products, for instance, require less time for development and therefore have shorter lead times. The lead time for lyophilized products accounts for the lyophilization cycle and the characterization of that cycle. Characterization is important because the parameters of the lyophilization cycle affect the production of a cake with high integrity, and the production of a cake without collapse, re-melting, or shrinkage requires additional development, Rosenbaum explains. “In both cases, it is necessary to understand the stability of the formulation, but for freeze-dried products, we need to develop the cycle through calorimetric measurements, refine the process parameters, and optimize the cycle. The freeze-dried product needs to be characterized and the stability profile needs to be established,” Rosenbaum states.

Meanwhile, a major technical challenge lies in the development work that takes place before a clinical batch is filled, as freeze-dried products require more preparation in selecting the right excipients, fillers, and cryoprotectants, as well as in cycle development. “It is also important to note that some of the COVID-19 vaccines are multi-dose and require the use of an evaluated preservative,” Rosenbaum says.

By way of example, Rosenbaum points out that messenger RNA (mRNA) vaccines use nanoparticle technology to increase stability, as opposed to the aqueous-soluble molecules typical of standard biologics. Recombinant protein vaccines may also require the use of an adjuvant to stimulate the immune response, and there are unique differences between live and attenuated or inactivated vaccines. “These differences require considerations related to plant classification and engineering controls, as live viruses must be separated from other types of vaccines. At the end of the day, COVID-19 vaccines are no more difficult than other biologics from a filling/manufacturing perspective. However, the real problem lies in managing the time that vaccines are out of controlled storage,” Rosenbaum cautions.

He does note that transferring liquid formulations to freeze-dried products can improve thermal stability and reduce the need for cold chain handling. There are already results showing that freeze-drying cycles can be developed and implemented that can be used to freeze-dry a COVID-19 mRNA vaccine.

Meanwhile, Matthew Bourassa, process development manager at LSNE Contract Manufacturing, a PCI Pharma Services company, notes that, in the past 36 months, oligonucleotides and mRNAs have been among the fastest growing market segments (particularly for LSNE). “As the biologics market continues to mature and new products enter the clinic, LSNE’s techniques for minimizing line-loss continue to gain efficiency. [The company] has seen a significant increase in the number of companies developing RNAi [RNA interference] or siRNA [small interfering] products, and the cost of the oligonucleotide drug substance is high as well,” Bourassa says.

LSNE is currently working on lipid nanoparticle (LNP) formulations, which is a common formulation technology used in mRNA vaccines. Traditional critical quality attributes (CQAs) such as residual moisture, cycle time, and an elegant cake are important, but, additionally, because of the lipid component in the LNP, special consideration must be taken for potential reconstitution issues. LSNE is taking an iterative approach to developing a robust lyophilization cycle with optimized reconstitution characteristics, according to Bourassa.

Sustainability in lyophilization

Given the fact that there is a large number of new biologics and emerging biotherapeutics currently in clinical development, the sustainability of the lyophilization process, for such products, is coming under greater scrutiny. Some industry participants have been looking towards solutions to keeping lyophilization sustainable.

There are potential pros and cons pertaining to the sustainability implications of lyophilization when it comes to certain aspects, such as packaging, according to Matt Hall, principal technical affairs manager, Corning.

“For example, removal of water from the drug product reduces the overall weight, which can reduce the energy required for shipping. In addition, freeze drying should significantly improve the stability of the drug product, allowing for room temperature storage and avoiding the energy needed to maintain a cold supply chain. Another potential sustainability benefit is reduced usage of expanded polystyrene insulation materials that are bulky and difficult to recycle,” Hall states.

The process of lyophilization does come with some potential negative implications in terms of sustainability, Hall observes. While the energy cost of the cold chain storage is avoided, the freeze-drying process itself can be energy intensive, he explains. “In addition, sterile diluent (and its associated energy costs for production and distribution) is still required to reconstitute the lyophilized drug. Finally, the use of fluorinated refrigerant gases could have negative impacts on the environment and increase adverse reactions related to global warming,” says Hall.

Hall enumerates the innovations below that can be used to improve sustainability of lyophilization. These innovations include, but are not limited to:

• New refrigerant technologies that reduce or eliminate the use of gases with a high global warming potential
• Development of pretreatments and/or simultaneous processes (e.g., infrared, microwave, etc.) that reduce the time to complete conventional freeze-drying cycles
• Development of process analytical technologies that accurately determine the endpoint of the drying cycle and potentially reduce additional time that is routinely used to ensure sufficient water removal
• Use of parenteral packaging that reduces the likelihood of vial breakage, which could result in a high loss of drug product, during the lyophilization process. Such parenteral packaging includes certain glass types used in vials (e.g., Valor Glass, Corning) that are treated in such a way as to enhance the protective properties of the glass (e.g., in the case of Valor Glass, the glass is ion-exchanged and externally coated, which provides up to 10x the strength of conventional glass vials).

Product quality is influenced by a number of factors, including the primary packaging components. Externally coated glass vials can improve quality by reducing the generation of glass particulate that can contaminate the product and the introduction of glass damage that can create cosmetic defects, which make vials more susceptible to breaking during the freeze-drying process. “Consistent vial geometry can also improve product quality by reducing the variability of the vial-heat-transfer-coefficient and its subsequent impact on the drying rate of product within a given vial,” says Hall.

Another approach that companies are taking to address the challenge of product stability and cold-chain management for biotherapeutics is by developing a lyophilized dosage form for that product. “The benefit of a lyophilized dosage form is to increase product stability at preferred storage conditions; for example, developing a lyophilized dosage form with refrigerated or ambient [temperature] for a liquid formulation, where previously -20 °C or -80 °C storage was required,” says Bourassa.

“A lyophilized product can dramatically reduce the product’s carbon footprint and the overall cost of cold chain management for parenteral products due to the lower shipping weight and ambient shipping temperatures,” Bourassa adds.

Meanwhile, newer refrigeration systems are designed to be particularly energy efficient. These new systems (such as those designed by Optima) achieve the defined temperatures in the ice condenser and set-up areas with extreme precision, emphasizes Rosenbaum. “With the F-Gas regulation [European Union Fluorinated Greenhouse Gases Regulation (1)] in Europe, for example, new framework conditions apply to the design of freeze-drying plants in regard to the refrigeration technology. Different solutions are possible to reach the required temperatures of approximately -100 °F/-70 °C at the ice-condenser and -60 °F/-50 °C on the shelves,” Rosenbaum explains.

The solutions being referred to earlier include LN2 refrigerant, which is a technically proven solution that has been known for years and is future-proof in terms of the F-gas Regulation, according to Rosenbaum. He explains that the investment costs in relation to the refrigeration system are lower here compared to the following solutions, but the consumption costs increase the more often the system is used. “This means that freeze-drying plants with LN2 refrigerants must be adapted to meet the operator’s operating scenario. This solution is suitable for new projects. However, with minimal effort, existing plants for climate-damaging refrigerants could also be converted to an LN2 system,” he states.

Meanwhile, for the use of refrigerants that are expected to be available in the medium term, such as R410A, and refrigerants available in the longer term, such as R448A, both refrigerants are non-flammable, but neither is completely environmentally friendly. Thus, refrigerants with a low global warming potential (compared to R404A/R507) can still be used in leak-tight, two-stage refrigeration systems. “There are two variations that can be considered: direct and indirect cooling of the ice condenser. From a technical point of view, the indirect cooling of the ice condenser makes the system future-proof. Here the refrigeration system is designed to provide indirect cooling of the ice condenser,” says Rosenbaum.

Flammable refrigerants can also be used at a later date by replacing the refrigeration system, Rosenbaum continues. It is not yet possible to say, however, whether the classic design—where the ice condenser is cooled directly—will require future design changes to the ice condenser to comply in the long term with the F-gas Regulation. “First and foremost, with this solution the investment costs are lower than with the solution referred to above. However, retrofitting could be technically complex or even technically impossible. Should a retrofit of the ice condenser be deemed necessary and technically possible, the total investment will be greater than for indirect cooling of the ice condenser, which will be implemented immediately. The operating costs for both variants are higher (approx. +10% to +30% compared to conventional freeze-drying systems),” says Rosenbaum.

Meanwhile, solutions for flammable, environmentally friendly refrigerants include developing another future-proof, environmentally friendly variant. In this case, the refrigeration system should be designed as a cascade system, for example. In addition, the indirect cooling of the ice condenser must also be performed via a second circuit. “Investment costs are higher than those for classic freeze-drying plants (up to +40% higher). Operating costs are also higher by [approximately] 30%,” Rosenbaum notes.

Where biomolecules are most at risk

From a pharmaceutical point of view, the thawing of the raw material (as in the case of proteins, for example) prior to formulation and filling plays a decisive role in determining where the biomolecule is most at risk. Concentration differences during the thawing process can negatively affect the stability of the drug substance, specifies Rosenbaum. During filling of the finished formulation into vials, factors such as pressure and shear stress due to pumping processes, among other things, can lead to aggregation of sensitive biotherapeutics. The choice of the right pumps as well as suitable tubing materials is therefore essential.

“During the freeze-drying process, special attention is paid to the freezing step. The cryoconcentration that takes place causes stress to the protein drug and can degrade its physical and conformational stability. Another essential step for maintaining the quality of the product is to find a right balance during primary drying between too conservative drying processes that increase production costs and too aggressive drying above the characteristic parameters Tg (glass transition temperature) or Tc (collapse temperature) leading to product loss,” Rosenbaum explains.

From a technological point of view, being able to determine a biomolecule’s vulnerability involves the design of the drying processes within the design space, Rosenbaum adds. With what he calls the ‘design space approach’, a biomanufacturer can develop an understanding of the boundaries within which freeze-drying for a certain product can be safely done. “The critical process and material attributes for the quality are unique to every product,” Rosenbaum says. “By specifying a design space, the conditions for a sustainable quality of the product are defined in advance.”

A threshold is the critical product temperature that is required before a collapse occurs, Rosenbaum explains. The second limit is the minimum controllable pressure of the plant. The course of the chamber pressure and the sublimation rate is dependent on the heat transfer coefficient of the vial and the product resistance, which determines the correlation between the controlled parameters and the product temperature. Changes in the design of the freeze-dryer as well as product deviations may alter the drying process and could lead to product loss, and irregularities in the shelf temperature or drying pressure can result in uncontrolled freeze-drying, he cautions.

Best practice approaches

Having a best-practice approach to the lyophilization of biologic drug product is a good way to optimize product stability during the lyophilization cycle. Approaching the problem early on in the drug product’s development cycle is one such best practice.

To develop a robust lyophilization cycle, special attention should be given in the early stages of development to comprehensively understand a product’s physical characteristics and thermal properties, says Bourassa. Bourassa explains that having a deep knowledge of the formulated product’s thermal profile is essential prior to developing a lyophilization cycle.

“The use of modulated differential scanning calorimetry (mDSC) and freeze-dry microscopy (FDM) identifies the physical characteristics of the formulated drug product, such as a product’s freezing point, glass transition, eutectic temperature, and collapse temperature, to tailor a lyophilization cycle that will produce a stable pharmaceutical product,” Bourassa states. LSNE’s Process Development team, for instance, will use a “sample thief” to collect numerous samples during development lyophilization runs. This allows the team to assess moisture content or residual solvents in real-time and ascertain an optimal secondary drying cycle and lyophilization process parameters, Bourassa explains.

Bourassa stresses the importance of taking proactive steps to ensure phase-appropriate development, which helps to speed up progress and minimize API or biologic drug substance loss. “If the clinical program is successful, clients may choose to spend additional time and money on expanded development. If time or budget is constrained, a lyophilization development program with fewer development runs for early clinical needs may be the best approach,” he says.

Bourassa adds that using a conservative lyophilization cycle and later optimizing once dose range studies are complete can save time and money in early clinical trials. “Progressing with an optimized cycle is vital as a company approaches Phase III/registration and process validation batchesby greatly reducing risk during manufacturing and saving costs over the life of the product,” he states.

A number of techniques exist that are new to the study of freeze-drying. For instance, modeling and simulation are advanced techniques that can provide a useful understanding of the process itself. “Integrating the known product characteristics into the simulation will shorten the necessary trial-and-error development runs,” says Rosenbaum. “If the freeze-drying parameters are well defined, the whole process will achieve the best efficiency and robustness.”

Reference

1. EC, “EU Legislation to Control F-gases,” ec.europa.eu, accessed Jan. 14, 2022.

About the author

Feliza Mirasol is the science editor for BioPharm International.

Article Details

BioPharm International
Vol. 35, No. 2
February 2022
Pages: 26–29

Citation

When referring to this article, please cite it as F. Mirasol, “Plan Early: Optimizing Stability During Lyophilization,” BioPharm International 35 (2) 26–29 (2022).