Yingtai:Application of Vacuum Freeze-Drying Technology in Biological Products
Using vacuum freeze-drying technology to preserve microorganisms (e.g., bacterial strains, viruses) or biological systems (e.g., blood cells, corneas) is currently one of the most convenient methods. Freeze-dried pharmaceuticals are porous, can be stored for extended periods, have excellent rehydration properties, and can restore activity. Therefore, freeze-drying technology is widely used to prepare solid protein drugs, orally fast-dissolving drugs, and drug-encapsulated liposomes. According to the database of the National Medical Products Administration, several freeze-dried drugs have been approved for market use in China, including injectable recombinant human granulocyte-macrophage colony-stimulating factor, recombinant human interferon α2b, freeze-dried murine epidermal growth factor, topical freeze-dried recombinant human epidermal growth factor, injectable recombinant streptokinase, recombinant human interleukin-2, recombinant human growth hormone, group A streptococcus injections, freeze-dried human coagulation factor VIII, and freeze-dried human fibrinogen.
1. Effects of Freeze-Drying on Bioactive Substances
Currently, vacuum freeze-drying has been successfully applied mainly to preserve prokaryotic cells, such as bacteria and lactic acid bacteria. This limitation arises because freeze-drying subjects cells to two intense physical changes—low temperatures and dehydration—which can severely damage cell membranes and intracellular components, leading to cell death or impaired biological functionality.
Mechanisms of Freezing Damage: Solute Effect and Mechanical Effect
Freezing damage to cells is primarily attributed to solute and mechanical effects. When the temperature of a cell solution falls below its equilibrium freezing point, extracellular fluid crystallizes first. The formation of pure ice concentrates the remaining solution, significantly increasing electrolyte concentration and altering pH, ionic strength, and osmotic pressure. This results in protein denaturation and cellular dehydration, a phenomenon known as the solute effect.
Additionally, during the pre-freezing phase, water inside the cell crystallizes, leading to volume expansion. The growth of intracellular ice crystals mechanically disrupts cell membranes and organelle membranes, a process referred to as the mechanical effect. Faster freezing rates result in more numerous intracellular ice crystals, exacerbating mechanical damage.
The freezing process is also critical for the overall freeze-drying outcome. If optimal pre-freezing conditions are not established, subsequent drying steps can further accelerate cellular damage and death. Glassy preservation of biological materials is a key strategy to prevent ice crystal formation and is crucial for low-temperature preservation. For example, red blood cells have been successfully vitrified using trehalose as a cryoprotectant, meeting clinical transfusion standards.
Membrane Fusion and Lipid Phase Transition
Membrane fusion and lipid phase transitions are two primary causes of cell damage during vacuum freeze-drying. Under normal physiological conditions, the polar head groups of phospholipids in cell membranes are separated by water molecules. However, during dehydration, these polar groups become densely packed, leading to interactions between membrane proteins and lipids. Hydrogen bonds formed between these groups compensate for the loss of water, resulting in membrane fusion, rupture, and leakage of intracellular contents.
Such intense interactions also increase the phase transition temperature. For example, the phase transition temperature of lecithin rises from approximately -7°C when fully hydrated to around 70°C when completely dehydrated. As a result, dehydrated lipid membranes are in a gel phase at room temperature, making them prone to irreversible phase separation. Furthermore, during rehydration, gel-phase membranes transition to the liquid-crystalline phase, increasing membrane permeability and causing metabolic dysregulation.
Role of Cryoprotectants
To mitigate these effects, cryoprotectants should be selected that possess both high glass transition temperatures and the ability to replace water molecules during drying, stabilizing the cellular membrane framework and maintaining its integrity.
2. Protective Mechanisms of Cryoprotectants
For cryoprotection, the "preferential exclusion" hypothesis is one of the mechanisms explaining protein stability in liquid states. Proposed by Timasheff and others, this hypothesis suggests that sugars do not directly interact with biomolecular structures but preferentially bind to water molecules on their surfaces. This stabilizes biomolecules by increasing surface hydration and chemical potential. Protectants are preferentially excluded from protein surfaces, enhancing structural stability.
Preferential exclusion also applies to freeze-thaw cycles, as protectants stabilize proteins during freezing by being excluded from protein surfaces. However, during freeze-drying, as the hydration layer is removed, this mechanism no longer applies.
"Water Replacement" Hypothesis
Studies have shown that organisms capable of surviving in low-temperature dehydration states often accumulate large amounts of disaccharides, such as trehalose and sucrose, in their cells. The "water replacement" hypothesis posits that the hydroxyl groups in disaccharides form hydrogen bonds with biomolecules in place of lost water, creating a hydration-like layer. This preserves the three-dimensional structure and functionality of biomolecules, preventing denaturation even during freeze-drying. Disaccharides also reduce lipid phase transition temperatures. For instance, sucrose or trehalose lowers lecithin's phase transition temperature to -20°C, preventing phase separation and leakage during rehydration.
"Vitrification" Hypothesis
Vitrification refers to the transition of a liquid into a non-crystalline, glassy solid. In the glassy state, substances exhibit both solid and fluid characteristics, with extremely high viscosity and low molecular diffusion rates. This inhibits membrane protein movement and maintains the stability of three-dimensional protein structures.
The vitrification hypothesis suggests that maintaining a glassy state throughout the freeze-drying process is essential for preserving cell viability. The system’s temperature during freezing and drying must remain below the glass transition temperature (T'g). High T'g sugars and polymers, such as trehalose, sucrose, hydroxyethyl starch, polyvinylpyrrolidone, polyethylene glycol, albumin, and dextran, provide effective protection. For instance, trehalose offers superior protection and extended shelf life for freeze-dried products.
Combining cryoprotectants with different mechanisms enhances protection. For example, hydroxyethyl starch (high T'g) and glucose (direct interaction with membrane groups) are ineffective individually in preventing lipid membrane leakage but work synergistically when combined. Therefore, a mixture of disaccharides and polymers is recommended for preserving blood cells during freeze-drying.
