Prioritizing Formulation Strategies for Temperature-Sensitive Biotherapeutics

Biopharmaceutical manufacturing largely involves biomolecules whose stability depends on temperature. Antibodies, insulin, and, more recently, messenger RNA (mRNA)-based vaccines are examples of biotherapeutics that require cold storage. This article explores the difficulties with developing stability-enhancing formulations for temperature-sensitive biomolecules and technology innovations that are enabling their formulation.

Factors that have been most challenging in developing formulations that enhance stability in temperature-sensitive biomolecules include buffer, pH, and sugar. These are usually the typical factors that need to be evaluated to thermodynamically stabilize monoclonal antibodies (mAbs), notes Joseph Cao, PhD, senior scientist, Analytical and Formulations Development, Bionova Scientific. He explains that there are different types of buffers, sugars, and a range of pH to choose from when developing a proper formulation for mAbs that will prevent aggregation, degradation (such as deamidation, oxidation), and particle formation. Besides those factors, amino acids, salts for ionic strength, and excipients are also important in developing a formulation recipe, he adds.

In addition, the limited amount of therapeutic protein to work with poses another challenge to formulation development. “With design of experiment (DoE), it has allowed us to cut the amount of protein and help us efficiently screen for factors,” Cao says. “Freeze-thaw cycles, container combability, and manufacturing impurities should also be considered during formulation development.”

Heonchang Lim, director, Formulation Development, Samsung Biologics, agrees that finding the ideal range of pH and buffer conditions while selecting the optimal formulation condition to get a good thermal stability property are primary factors that challenge formulation development in these molecules. However, he notes, the thermal stability problem can mostly be solved by using optimal pH buffer and excipient combination.

“Nowadays, we bump into another challenge to develop an optimal formulation condition. That is regarding solubility and hydrophobicity of the molecule. Though clients would prefer higher concentrations, when taking unique characteristics of molecules into consideration, formulation scientists cannot always get high enough concentrations. In addition, pH shift may occur due to the Donnan effect,” Lim says. “Consequently, when a client requests a highly concentrated formulation, particularly above 100 mg/mL, [the] formulation team at Samsung Biologics implements rigorous gate checks to prevent such challenges from arising during formulation stages.”

Caroline Ailhas, Pharmaceutical Development director, CARBOGEN AMCIS says that, for her, the main factors challenging formulation development for temperature-sensitive molecules revolve around maintaining the structure and functionality of the molecule and defining the required duration of stabilization. The latter can vary from a few days—to allow for manufacturing and visual inspection before storage at 2–8 °C—to several months, or even years. “The definition of this duration significantly impacts the formulation strategy,” Ailhas says.

“One of the key performance indicators for the development of biotherapeutic formulations is colloidal stability to prevent protein aggregation and potentially precipitation,” Greg Chrimes, site head and vice-president, Analytical and Formulation Science, KBI Biopharma, adds. He points out that proteins may denature and aggregate at interfaces such as air/liquid, liquid/liquid, and liquid/solid. Chemical modifications and degradation may also impact effector function, efficacy, conformational stability, and other important parameters, he emphasizes.

“Excipients are evaluated during formulation development to limit product denaturation that may lead to particle formulation or chemical modification. Those may include buffering components, polyols, surfactants, amino acids, and antioxidants, to name a few. Finding the optimal combination components requires a methodical approach with matched analytics such as differential scanning calorimetry (DSC), dynamic light scattering (DLS), differential scanning fluorometry (DSF), and other product-specific techniques to down-select to a final formulation,” Chrimes states.

Innovations targeting stability

Lipid nanoparticles (LNPs) are emerging as a formidable technology that can facilitate molecule stability in biologic-based medicines. “LNPs facilitate the stabilization of the molecule due to their amphiphilic nature. This property allows large molecules to maintain their structure by interacting with either the polar head of the lipid or with the hydrophobic part of the lipid,” says Ailhas. Ailhas also points out that the variety of available lipids offers numerous options for LNP composition and stabilization strategies. Because of this variety, LNPs composed of different lipids are highly versatile, enabling them to stabilize various molecules, such as peptides or nucleic acids, with different charges.

Because LNPs can be used to encapsulate biotherapeutics, they protect the fragility of these therapies from accelerated degradation in vivo, Chrimes says. He explains that LNPs are biocompatible and naturally break down; however, the biodegradation needs to be timed to allow mRNA (as one example) to enter the target cells. “This type of delivery platform has accelerated the use of mRNA therapies. The control of lipid impurities generated during production of LNPs has also been reported to improve shelf life,” he adds.

LNP encapsulation shields molecules, such as mRNA, from temperature change, oxidation, and enzymatic degradation, Cao states. However, formulation development for mRNA-based therapies will still face the same challenges as mAb formulation development, Cao notes. “LNPs require different ratios of lipid components, such as ionizable lipids, PEG [polyethylene glycol]-lipids, cholesterol, and phospholipids, for their stability,” he emphasizes.

In addition to LNP technology, protein engineering and lyophilization are other techniques employed to facilitate the development of formulations that can offer better stability for temperature-sensitive molecules. “Development of stable products at ambient temperature is typically the preferred endpoint for biopharmaceutical formulations. However, it is not often obtained due to the physical forces experienced during transport, storage, and delivery,” says Chrimes. He explains that product liabilities can be removed in the primary sequence of a biotherapeutic, in some cases to eliminate the instability. He points to lyophilization as an often-successful technique to avoid colloidal instabilities that occur during shipment or long-term liquid storage.

Protein engineering and the use of machine learning, meanwhile, has been increasingly used to develop mAbs that are more thermostable, says Cao. Furthermore, the use of AI to analyze the local solvent-accessible surface area of the variable region of previously approved mAbs have been used to make predictions on aggregation, he notes.

“If proteins can get re-engineered, then the YTE mutations [M252Y/S254T/T256E] could be employed to achieve thermal stability,” adds Lim, who explains that once a molecule passes the discovery phase, a formulation scientist would then need to analyze basic characteristics of the molecule to identify its distinguished traits. “These analyses help us determine the onset temperature (Tonset) and melting point (Tm) of the molecules and provide us insight into how much the molecule can resist thermal stressors,” he says.

Furthermore, identifying pH/buffer levels is required to earn both thermal and chemical stability, Lim adds. “We can also add various saccharide excipients to formulations, such as sucrose and trehalose, to further enhance thermal stability by about 2–3 degrees Celsius. For sensitive/complex molecules [such as] antibody-drug conjugates, lyophilization may be the preferred technique to achieve maximum stability,” he states.

Meanwhile, other types of nanoparticles, such as polymeric ones, are being explored for thermal stability, says Ailhas. “Additionally, lyophilization combined with the appropriate selection of excipients should be investigated to enhance the stability of temperature-sensitive products,” she states. Unique excipients such as polyols, surfactants, amino acids, and antioxidants have been demonstrated to improve protein chemical stabilization and colloidal stability, Chrimes notes.

“Beyond LNPs, other type nanoparticle carriers such as polymeric nanoparticles, silica nanoparticles, and gold nanoparticles are being investigated for the capability to stabilize the molecules through encapsulation,” says Cao.

From formulation to delivery mechanism

At a certain point, once a formulation strategy is in place, focus shifts to the drug delivery method. Drug delivery systems are usually considered in later clinical stages of drug development, notes Lim, who emphasizes that in early stages of drug development, getting a fast investigational new drug (IND) application approval is a high priority for drug developers. Speed in securing an IND approval grants a competitive edge to drug developers, especially in a drug discovery market that is dynamic. “The emphasis during the early stages is less on selecting a delivery system and more on obtaining reliable thermal and physical stability results for the targeted molecule,” he says.

Formulation development is usually initiated at the early stages of drug development as well, says Cao, who explains that early formulation development ensures the stability of the product and reduces the need for cold storage with, for example, techniques such as lyophilization. Formulations that result in stability can then inform the design of the delivery mechanism, he notes.

“A study for delivery can also be conducted first to test which delivery method (intravenous [IV] bag, subcutaneous injection, etc.) is effective for the therapeutical drug, followed by formulation development,” Cao also says. “Different modes of delivering the drug will require different drug concentrations. IV-bags do not require high concentrations of drug, but sub-cutaneous does. Challenges and limitations observed in the delivery mechanism can drive further optimization of the formulation,” he adds.

“In my opinion,” says Ailhas, “it is more beneficial to prioritize an optimized formulation that can function in a non-cold environment. This approach simplifies production and storage, eliminating the need to find specific drug product manufacturers capable of handling cold manufacturing processes, which can be challenging to locate.” Ailhas also points out that, although a formulation-first strategy may incur slightly higher costs at the beginning of a project, the long-term cost savings during the transition to good manufacturing practice manufacturing, either for clinical or commercial supply, will be significant.

Chrimes agrees that is it important to prioritize a formulation-first strategy: “Optimizing the formulation to limit the impact of temperature excursions would be the preferred development path for biotherapeutics,” he says, adding that every effort should be explored to limit potential liabilities as well as screen applicable excipients and explore presentations to “lock” the product in a stable state through techniques such as lyophilization, for example. Lyophilization removes water from sensitive products without damaging them. “Developing liquid formulations that are compatible with lyophilization can be an effective strategy for cost savings during clinical development where lyophilization cycles are deferred to commercial development,” he states.

Technical considerations

Finally, what technical considerations remain to be solved that can conceivably facilitate a breakthrough to realistically realize temperature-stable biomolecules that no longer require stringent cold-chain handling?

Cao answers the question by pointing out that developing advanced stabilizers and excipients, as well as improving lyophilization and spray drying techniques, can aid in maintaining biological molecule stability without compromising efficacy or safety. Such advancements would allow biological molecules to be less dependent on stringent storage and transportation conditions.

“Don’t be afraid of lyophilization,” stresses Ailhas. “It can significantly help solve this problem, and it is not so difficult to manage, at least for mAbs. For mRNA vaccines formed with LNPs, the breakthrough will involve finding a way to remove water from the inner part of LNPs during lyophilization without damaging the LNP structure and ensuring they can return to their original form during reconstitution.”

Meanwhile, Chrimes says that the solutions are going to look different based on the modality, mechanism of action, mode of delivery, and more. Across the board, however, progress always starts with understanding what is causing the instabilities in a given biotherapeutic. “From there, we can work … to optimize not just for temperature stability, but shelf-life and other critical parameters that can impact commercial success. For the most part, it’s an ongoing process of optimization and refinement.” Chrimes adds that innovations in predictive modeling and machine learning could accelerate the development of unique excipients and formulation development.

“The key focus during the process development or clinical trial stages is to minimize the risk of failure,” adds Lim, who notes that, to minimize risk, a pre-molecular developability assessment is crucial. “Through this evaluation process, scientists can find any potential issues, such as lack of thermal stability, which can bring out exclusion or implementation of risk mitigation strategies for further development,” he says.

“This assessment can help researchers optimize strategies for thermal stability, including lyophilization as part of the formulation development process. Not only can this reduce the risk of failure but also save time during the development timeline,” Lim concludes.

About the author

Feliza Mirasol is the science editor for BioPharm International.

Article details

BioPharm International®
Vol. 37, No. 9
October 2024
Pages: 12–14

Citation

When referring to this article, please cite it as Mirasol, F. Prioritizing Formulation Strategies for Temperature-Sensitive Biotherapeutics. BioPharm International 2024, 37 (9), 12–14.