Research Progress on Freeze-Drying Technology of Protein Drugs
1. Introduction
Freeze-drying, also known as lyophilization, is a widely used technique in the pharmaceutical industry for preserving protein drugs. This method allows for the production of stable, long-lasting solid formulations that can be easily rehydrated to restore their original activity. Due to its ability to maintain the structural and functional integrity of sensitive biological molecules, freeze-drying has become essential in the development of various drug products, including injectable protein therapeutics, oral instant formulations, and liposomal drug carriers.
According to data from the State Drug Administration, several protein-based drugs have been approved for commercial use, such as recombinant human granulocyte macrophage colony-stimulating factor, recombinant human interferon α2b, lyophilized mouse epidermal growth factor, and lyophilized recombinant human epidermal growth factor. Other examples include recombinant streptokinase, recombinant human interleukin-2, and lyophilized human factor VIII. In the United States, as of February 2000, the FDA had approved a total of 76 biotech drugs using this technology.
The origins of freeze-drying trace back to 1813 when it was first developed by the English scientist Wollaston. It gained significant attention in 1909 when Schickel applied it to preserve biological materials such as toxins, bacterial cultures, and rabies virus with promising results. During World War II, the demand for blood products accelerated the industrial application of freeze-drying. Since then, advancements in refrigeration and vacuum technologies have further propelled the development of this technique.
In the late 20th century, the rapid advancement of science and growing public awareness of health issues provided strong momentum for the development of freeze-drying technology. While significant progress has been made in understanding the mechanisms of drug damage and protection during lyophilization, as well as in the design of freeze-drying equipment, the field remains interdisciplinary, requiring knowledge from biology, pharmacy, refrigeration, and control systems. Therefore, there are still many challenges to address.
2. Principle and Characteristics of Drug Freeze Drying
Lyophilization involves freezing the drug solution at low temperatures, followed by sublimation under vacuum to remove ice crystals, and finally desorption drying to eliminate residual bound water. The process typically consists of five steps: formulation preparation, pre-freezing, primary drying (sublimation), secondary drying (desorption), and sealing. Once lyophilized, the drug can be stored at room temperature for extended periods and reconstituted with water or saline when needed.
Compared to traditional drying methods, freeze-drying offers several advantages:
a) Accurate dosing due to liquid dispensing before freezing;
b) Preservation of heat-sensitive compounds through low-temperature drying;
c) Reduced oxidation and microbial contamination due to low-pressure conditions;
d) Maintenance of the original shape and color due to the porous structure formed during freezing;
e) Excellent rehydration properties;
f) Thorough dehydration suitable for long-term storage and transportation.
Despite these benefits, freeze-drying is energy-intensive, time-consuming, and requires expensive equipment, which limits its widespread use.
3. Mechanism of Drug Damage and Protection During Freeze Drying
Freeze-drying is a multi-step process that exposes the drug to various stresses, including low-temperature stress, freezing stress, and drying stress. These stresses can lead to denaturation, aggregation, or loss of activity in the protein drug. To mitigate these effects, protective agents are often added to the formulation. Common protective agents include sugars, polymers, anhydrous solvents, surfactants, amino acids, and salts.
Protective agents function through different mechanisms, such as preferential hydration or glass formation. The "water substitution hypothesis" suggests that protective agents replace water molecules on the protein surface, forming a hydrated layer that stabilizes the protein structure. Alternatively, the "glass state hypothesis" proposes that the protective agent forms a glassy matrix around the protein, preventing molecular movement and maintaining stability.
Understanding these mechanisms is crucial for developing effective formulations that ensure the quality and efficacy of lyophilized drugs.
4. Freeze-Drying Process and Optimization
Optimizing the freeze-drying process is critical to minimizing damage and improving the quality of the final product. Key factors include freezing rate, freezing mode, annealing, and drying parameters.
Freezing mode significantly affects ice crystal morphology and subsequent drying efficiency. Rapid freezing leads to small ice crystals and higher mass transfer resistance, while slow freezing produces larger crystals and faster drying rates. Directed crystallization techniques, such as vertical freezing, can enhance uniformity and reduce drying time.
Annealing, the process of heating the frozen drug below the eutectic point, helps promote crystallization, increase glass transition temperature, and improve drying efficiency. However, the exact mechanism of annealing is still not fully understood, and parameter selection remains challenging.
Drying involves two stages: primary drying (removal of free water) and secondary drying (removal of bound water). Controlling temperature, vacuum level, and shelf conditions is essential to optimize the drying rate and prevent product degradation.
5. Freeze-Dryer Equipment
Freeze dryers come in various sizes and configurations, including experimental, pilot, and industrial models. They typically consist of a drying chamber, vacuum system, refrigeration system, cold trap, heating system, capping system, and automatic control unit. Advanced systems may also include steam-in-place (SIP) and clean-in-place (CIP) capabilities for sterilization and cleaning.
Modern freeze dryers must comply with Good Manufacturing Practices (GMP) to ensure sterility, reliability, and ease of maintenance. Future developments will focus on improving automation, reducing costs, and enhancing the efficiency of heat and mass transfer processes under low-temperature and low-pressure conditions.
With the continued emergence of peptide and protein-based drugs, the importance of freeze-drying technology will only grow. Despite its many advantages, there are still challenges to overcome, such as improving stability, reducing energy consumption, and optimizing the freeze-drying process. Ongoing research into the mechanisms of drug damage and protection, along with the development of advanced freeze-drying equipment, will be key to advancing this vital pharmaceutical technology.
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