1 Welcome to the foreword to freeze-dried
Lyophilized is widely used in the preparation of therapeutic protein preparations, protein lyophilized preparations can provide better shelf life, facilitate the storage and transportation of drugs, however, there are many stresses in the lyophilization process of proteins, including low temperature stress, freezing stress (formation of dendritic ice crystals, increase in ionic strength, change of pH, phase separation, etc.), drying stress (loss of water molecules on the surface of proteins), etc., these stresses often directly or indirectly lead to the loss of natural conformation of protein drugs and thus denaturation or inactivation. Therefore, even if the gentle drying method of freeze drying is adopted, it is necessary to add a suitable lyophilized protective agent to protect the stability of the protein.
The principle of protection of proteins by freeze-dried protectors has been studied and discussed for decades, forming some generally accepted consensus. For example, in the freezing phase, the main hypothesis is "prioritization"; There are two main hypotheses in the drying phase, namely "vitrification" and "water displacement".
There are many ways to classify freeze-drying protective agents, and a formulaic model is proposed in the literature to classify freeze-drying protective agents according to their functions, including 5 categories: (1) pH buffers, such as Tris, histidine, citric acid, etc.; (2) Ligands, which can optimize the thermodynamic stability of proteins; (3) Stabilizers, generally disaccharides, such as sucrose, trehalose, etc., can play a protective role by inhibiting the unfolding of proteins and providing glass matrix; (4) nonionic surfactants, which can reduce the aggregation of proteins; (5) Fillers, such as mannitol, glycine, hydroxyethyl starch, serum albumin, etc., can improve the physical formability of the product. Here we briefly introduce several of them.
2 buffer salts
First, protein stability is affected by environmental pH, and buffer selection is critical during the development of protein formulations and must be established during the formulation phase. When selecting buffer pH, it is recommended that the pH not be too close to the pI (isoelectric point) of the protein to avoid aggregation.
Phosphate is one of the most common buffers in protein formulations, especially aqueous protein drugs, with an effective pH range of 5.8-8.0 and biocompatibility. However, as discussed in "pH changes during freezing", phosphate buffers, especially disodium hydrogen phosphate, undergo significant pH changes during the freezing process and are therefore not recommended for pH-sensitive proteins. Conversely, potassium phosphate, histidine, trimethylolaminemethane (Tris), and citrate buffers show minimal pH changes during freezing. In addition, low buffer concentrations can help reduce pH changes. Histidine is an amino acid with an effective pH range of 5.5-7.4, which is compatible with biological pH and is often used for freeze-dried proteins. However, the selection of appropriate freeze-dried aqueous formulations, including buffers, depends on the requirements of the protein and its route of administration. Therefore, many studies and various biophysical methods are required to sift out a list of concentrations of several buffers.
3 fillers
In order to ensure that a good formula structure is formed after freeze-drying to prevent the product from collapsing, and at the same time improve the solubility of the formula, a filler is also needed to be added to the freeze-drying protector. First, the filler should be sufficiently soluble and compatible with protein drugs in the formulation when resolved. In addition, they have no toxicity problems and have a high eutectic/confluent temperature (Teu), which effectively improves freeze-drying efficiency.
Glycine and mannitol are often used as fillers in lyophilized protein preparations. Glycine has an advantage when used as a filler because it is non-toxic. It has a high eutectic/confluent temperature (Teu) for efficient freeze drying and high solubility.
Mannitol has the same advantages as glycine and has a stabilizing effect on specific protein drugs such as LDH and transforming growth factor-b1, the stabilizing effect of which depends on its concentration. Therefore, when choosing a filler, the stabilization effect as well as the appearance of the powder should be carefully considered. Most amino acids are easy to crystallize and can potentially be used as fillers to stabilize proteins, however, the formation of acids hinders their crystallization. NaCl is another crystallization agent, but its co-fusion temperature (Teu) is too low relative to other fillers and is therefore not recommended.
For fillers, annealing steps should be considered. Glycine and mannitol should be present in crystalline form during freeze-drying. However, during the freezing step, supercooling induces the two fillers to freeze in an amorphous form, which can easily lead to the collapse of the powder. In addition, in the absence of annealing, the filler can recrystallize during primary drying causing the vial to rupture, especially when the preparation contains a high concentration of mannitol. Fillers should be effectively crystallized between the glass transition temperature of the amorphous phase (Tg') and the co-fusion temperature (Teu) of the filler, while the annealing time depends on the filler concentration and its properties. For mannitol and glycine, at a filling height of 1 cm, it is recommended at -20 or -25 °C and 2 h.
4 saccharides
Sugars are the most commonly used excipients in lyophilized protein preparations because they have many hydroxyl groups in their structure and can therefore provide a stabilizing effect by replacing the hydrogen bond between the protein and water. In addition, most sugars do not crystallize under normal freeze-drying conditions, resulting in faster re-dissolution of lyophilized protein preparations. Reducing sugars are not usually used for freeze-drying because they react with proteins through maillard reactions, however, they can be used when their stability effect is superior to other excipients. Sucrose has been widely used in protein formulation formulations, and trehalose is considered an excellent lyophilized protector than sucrose because it has a higher vitrification transition temperature (Tg'). Trehalose also has other advantages, such as low hygroscopicity, the absence of internal hydrogen bonds, resulting in more flexible hydrogen bonds with proteins and low chemical reactivity. The high viscosity of sucrose and trehalose also reduces the rate of protein denaturation during drying at temperatures above the glass transition temperature (Tg'), thereby improving freeze-drying efficiency.
5 Other
Polyols are widely used in protein stabilizers because they can stabilize proteins through their hydroxyl groups. Polyols have been used as cryoprotectants and lyophilized protectors. For example, during freeze-thaw, LDH is protected to varying degrees by glycerol, xylitol, sorbitol, and mannitol.
Polymers have been used as protectors for protein solutions and lyophilized preparations. Serum albumin is one of the most widely used polymers for protein drug development as a lyophilized protector and cryoprotectant. Many marketed protein drugs contain varying amounts of albumin (Table 1). However, serum albumin has potential contaminants associated with bloodborne pathogens, limiting its use in protein drug formulations. Therefore, rHA is recommended to replace serum albumin. Ultimately, it is recommended to develop protein drugs in the absence of albumin.
During the freezing step, the formation of an ice-water interface may lead to denaturation of the protein surface. Surfactants reduce the surface tension of protein solutions, protecting them from surface denaturation. One of the most widely used surfactants is Tween 80.
6 summary
More than 50% of protein drug products have chosen freeze-drying because in solid conditions, the physical and chemical degradation of protein drugs is reduced, thus ensuring their long-term stability. Different structures and properties
Freeze-dried protective agents have different protection mechanisms for protecting proteins at different stages of freeze-drying, and have synergistic protective effects on certain proteins when used in combination, so understanding the types and properties of commonly used freeze-protectants is crucial for the development of more effective protein preparations.