Chapter 6 Protective agent and additives for freeze-drying of pharmaceutical products

1. Chapter 6 Protective agent and additives for freeze-drying of pharmaceutical products

2. During freeze-drying process and in the storage period of foods, drugs and organisms, many factors, e.g., chemical composition, cooling rate , freeze and dehydrated stress, glass transition temperature, residual moisture content, temperature, humidity of the storage environment and so on, can affect the stability of active constituent or even cause the loss of activation. • The massive experimental study indicated that except minority materials, such as some foods, the human blood plasma, the milk, may directly carry on the freeze-drying process; the majority of drugs and the biological preparations need addition of some appropriate protecting agents and some chemical additive for freeze-drying and storage.

3. Classification of protecting agents by their molecular weight • Low-molecular weight compounds: may also be classified as the acidic, the neutral and the alkaline. Acidic materials include glutanic acid, aspartic acid, apple ammonia acid, lactic acid and so on. Neutral materials include glucose, phaseomannite, lactose, sucrose, raffinose, trohalose, sorbitol DL- threonine, phaseomannite, xylitol and so on. Alkaline materials include arginine and histidine and so on. • High-molecular weight compounds: such as albumin, gelatin, protein peptone, soluble starch, dextrin, meat broth, pectin, Arabic gum, methylol cellulose, algae and some natural mixtures like degreasing milk, blood serum and so on. • As widely acknowledged, functional mechanism of protecting agent is that the low molecular compounds function directly on freezing and drying process while the high molecular compounds promote the protective function of low molecular compound. Therefore, when preparing a protecting agent formula, low-molecular and high-molecular compounds are used together.

4. Classification of protecting agents by their function • Lyoprotectant namely freeze drying protecting agent, is a kind of substance which can prevent the active constituent from denaturation during the freezing and drying processes, such as glycerine, DMSO, trohalose, sucrose, polyvinylpyrrolidone (PVP) and so on. • Bulking agent (bulking compound) namely bulking compound, is a kind of substance which can prevent the effective components of the formula from escaping along with the water vapor, and can promote the fixation of the effective components in the material, such as mannitol, lactose, gelatin and so on.

5. Antioxidant namely oxidation inhibitor, is a kind of substance which can prevent the effective components of the material from oxidation and denaturation during freeze drying and storage, such as Vitamin D, Vitamin E, protein hydrolysate, sodium hyposulfite and so on. • Buffer agent ( PH modifier) also called PH modifier, is a kind of substance which can regulate the acid and alkali value of the material to the most stable region for active material, such as phosphoric acid, sorbitol, EDTA (ethylenediamine tetraacetic acid), amino acid and so on

6. Classification of protecting agents by their material kinds • Sugars/polysaccharides • Polymers • Surfactants • Amino acids • Salts

7. In freeze-drying formula, there are some chemical additives playing one specific role and other chemical additives play several roles simultaneously. For example PVP (Polyvinylpyrrolidone) may serve as the low temperature protecting agent and bulking agent at the same time. • The specific functions of a chemical additive in the formula are difficult to identify strictly. Sometimes an identical additive may display quite different functions in several different freeze dried products.

8. 6.1 Sugars/polyols-type protective agents 6.1.1 The definition of sugars and polyols • Sugar is made of carbon, hydrogen and oxygen. The proportion of hydrogen and oxygen inside sugar is always 2:1 which is the same as water. Sugar is classified as monosaccharides, oligosaccharide and polysaccharide. Monosaccharide, with the smallest molecule, is the compound which cannot be further hydrolyzed. The typical types of monosaccharide include glucose, fructose, galactose and ribose. Oligosaccharide can be hydrolyzed into 2 to 10 monosaccharides molecules. Sucrose, maltose, monohydrate, lactose, trehalose dihydrate and raffinose pentahydrate belong to oligosaccharide. Polysaccharide can be hydrolyzed into monosaccharides and oligosaccharides. Starches, cellulose and pectin are polysaccharide.

9. The alcohol containing two or more hydroxyls is called polyol or sugar alcohol.Glycerol, sorbitol and mannitol are polyols widely used in the freeze-drying of pharmaceutical products. Sugar and polyol have similar protective mechanisms because they have the same functional groups (hydroxyls).

11. 6.1.2. The function of sugars/polyols in freeze-drying • Monosaccharides • Theoretically, monosaccharides can protect the biological products if the hydrogen bonding, which formed between the molecules of saccharides and the active components of biological products, replace the position of water molecules. • However, experiment results show that monosaccharides (such as glucose、galacrose) cannot protect protein during the freeze-drying process, because the weak stabilization provided by monosaccharide in the freezing step will result in the irreversible denaturation of protein before the drying begin. But trehaloses can provide effective protection during both freezing and drying process. • Generally the monosaccharides are usually mixed with other excipients as the protective agents for the freeze-drying of biological products.

12. 2. Oligosaccharides • Oligosaccharides, especially disaccharides, are usually used as protective agents because the disaccharide can be used as both the cryoprotective agents in freezing and the protective agents in drying process. • Disaccharide can be classified as reducing disaccharide (lactose, maltose) and non-reducing disaccharide (trehalose, sucrose). However, the disaccharide will cause the Maillard reaction (protein browning reaction) and result in the deterioration of freeze-dried product in storage. Consequently, sucrose and trehalose are the most widely used protective agents .

13. Trehalose

14. 3. Polyols • Mannitol is white, odorless and sweet crystallization powder. It is easy to dissolve in water, but hardly dissolve in ethanol and ether. The melting point and boiling point of mannitol are 166-170℃ and 290-295℃, respecitvely. It is very stable in the sterile solution and difficult to be oxidized. Mannitol is usually used as bulking agent because it can form supporting structure for the active components by crystallizing during the slow freezing process. Mannitol does not react with the active components. • Sorbitol is the isomer of mannitol, but its solubility is larger than mannitol. At room temperature, it is transparent viscous liquid that is optically active and hygroscopicity. It can dissolve many kinds of metal, but it is unstable at high temperature. Sorbital is normally used as bulking agent in freeze-drying.

15. Glycerol (or glycerin) is viscous liquid that is transparent, odorless and sweet. Its melting point and boiling point are 17.9℃ and 290℃, respectively. The water absorption ability of glycerol is strong. Glycerol can mix with water and ethanol in any proportion. It is slightly soluble in ether and hardly soluble in organic solvent such as benzene, chloroform, carbon tetrachloride and carbon disulfide. But it is not soluble in fats. Glycerol is usually used as cryoprotective agents.

16. Recovery of PFK（%） Concentration of trehalose（mM） 6.1.3 Effect of sugar/polyol concentration on the protection • The protective effect related with the concentration of sugar/polyol. the recovery of PFK does not increase when the initial concentration of sugar-type protective agents reach a fixed value which is called the most effective concentration. Too high concentration of sugar/polyol even cause the protein degeneration.

17. 6.2 Polymer-type protective agents6.2.1 The definition of Polymer • Polymer is a large molecule (macromolecule) composed of repeating structural units typically connected by covalent chemical bonds. One polymer molecular consists of thousands to millions of atoms, so its molecular weight is large. Polymers are mainly classified as chain polymers and net polymers. • In the freeze-drying process of biological products, polymer-type protective agents act as both cryoprotective agents and dehydration protective agents. The typical polymer-type protective agents are PVP, BSA, dextran, PEG and so on.

18. 6.2.2 The polymer-type protective agents for freeze-drying

19. Generally, the polymer-type protective agents have the following characteristics: • Crystallize firstly during freezing process • Have higher surface activity • Produce steric hindrance effect among protein molecules • Increase viscosity of the solution • Increase the glass transition temperature significantly • Restrain the crystallization of excipients with small molecules (eg. sucrose). • Keep the PH value of solutions

20. 6.3 Surfactants and amino acid-type protective agents6.3.1 Surfactant-type protective agents • Definition of surfactant • Surfactants are wetting agents composed of hydrophilic and oleophilic groups that can reduce the surface tension of a liquid and reduce the interfacial tension between two liquids. It is soluble in oil because of the C-H chains and soluble in water because of polar groups (-COOH，-OH）. When these molecules are located at air-water or oil-water interface, the hydrophilic groups face the water phase, while the oleophilic groups point at gas and oil phase.

21. Surfactants can be divided into two main types: ionized and non-ionized. The ionized surfactants can be ionized into ions when dissolving in water. • In the freeze-drying of biological products, surfactants can reduce the denaturation during freezing and dehydration which is caused by the stress in ice-water boundary. The surfactants also can act as humid agents during rehydration process.

22. 2. The typical surfactants in freeze-drying

23. 6.3.2. Amino acid-type protective agents • The definition of amino acids • An amino acid is a molecule containing both amine and carboxyl functional groups. Amino acids are the building blocks of proteins. The α-Amino acid is the most important type. α-Amino acid consists of a amino, a carboxyl, a R group and a hydrogen atom that attach to the same α-carbon atom • Because amino acid ion is characterized by basic and acidic groups, it can inhibit the change of PH value of the solution during the low temperature storage and freeze-drying of biological products to protect the active components.

24. 2. Amino acid-type protective agents

25. 6.3.3. Other additives • Antioxidants • There are many types of antioxidants. The characteristics and mechanisms of antioxidation are different. Some types of antioxidants consume the O2 inside the samples by self-oxidations to protect the sample from oxidizing. Some types of antioxidants inhibit the oxidation chain reaction of freeze-drying samples by providing electric and hydrogen atom. And some types of antioxidants restrain the activity of oxidase to prevent the oxidizing determination. • the mixture of different antioxidants have better anti-oxidation effect than single antioxidant.

27. 2. Buffer agents • Proteins can react with both acids and bases. The specific pH value, at which the number of positive charges is equal to the negative charges in the solution, is named the isoelectric point of the protein. The protein is more stable at its isoelectric point. At neutral pH values, most of the proteins carry negative charges while a few proteins carry positive charges. But at extreme pH values, the high electrostatical charge can cause strong intramolecular repulsion and split the proteins structures. • The concentration of the protein solution increases gradually with the freezing of solution. High concentration of protein solution may change the pH values and cause the denaturation of the protein. So right amounts of buffer agents are usually added into the lyoprotectants.

29. 3. Agents for acceleration of freeze drying • Freeze drying is a time and energy consuming process. It is necessary to optimize the protocol of freeze-drying to shorten the freeze drying time. In recent years, it was found that needle-like crystal formed when tertiary butyl alcohol (TBC) was added into the lyoprotectants. The tube-shape channel formed with the sublimation of needle-like ice, resulting in the reduction of flowing resistance of vapor and increment of sublimating rate. Such kind of additives as TBC is named as acceleration agents. • Tertiary butyl alcohol is a type of alcohol with small molecular. Its molecular formula is (CH3)3COH. It dissolves in water, has low toxicity and high vapor pressure. It can be used as freeze-drying solvent and compound solvent by combining with water. The compound solvent of tertiary butyl alcohol-water is being widely investigated nowadays.

30. Addition of tertiary butyl alcohol into the pharmaceutical solution has several purposes: • reduce resistance of drying layer, accelerate freeze-drying process and shorten freeze drying time. • dissolve pharmaceutical products that are difficult to dissolve in water. • increase the surface area of the products and make it easy to be rehydrated. • increase the stability of pharmaceutical solution and freeze-dried products. • inhibit the growing of bacteria.

31. The change of mass of water with the freeze drying time (a: with TBC；b：without TBC)

32. b a The SEM photos of freeze dried sucrose solution (a: with TBC；b：without TBC)

33. 6.4 The formula issues of the freeze-drying products 6.4.1. Determination of the optimal PH value • The active components of the biological products (such as protein) are stable only in very small range of pH value, and the pH values affect the solubility of some types of protein. For example, the proteins in IL-2 expanded and denatured irreversibly when IL-2 was freeze dried at pH7, the proteins kept their native structure when freeze dried at pH5. • In addition, the PH value of the freeze-drying products will affect their long term storage and the physical/chemical stability of the solid-state protein. For example, the irreversible aggregation of the lyophilized RNase during the storage process was related to pH value of the protein solution before the freeze-drying process.

34. 6.4.2. The selection of buffer agents • Many buffer agents can be used in the formula of biological products for freeze drying. But each buffer agent can only be used to specific formula. For example, protein solution sensitive to pH should avoid using the buffer of sodium phosphate because Na2HPO4 crystallized first during the freezing process, inducing the decrease of pH and the denaturation of protein. • In addition, the correct selection of buffer concentration is also very important. For example, because of the crystallization of mannitol, it cannot protect the solution (2μg/ml) of β-galactosidase in the freeze-drying process. The addition of 10mM phosphate buffer (pH7.4) can partly inhibit the crystallization of mannitol, increasing the activity of enzyme to 95%. The crystallization of mannitol was completely inhibited when 200mM phosphate buffer (pH7.4) was added.

35. 6.4.3. The selection of bulking agents • The addition of bulking agents (easy to crystallize) into the formula of freeze drying products will have additional functions as: (1) providing adequate mechanical support for the final freeze-dried products. (2) improving the appearance of the freeze-dried products. (3) increasing the solubility of solutes. (4) preventing the freeze-died products from collapse or overflow. • The bulking agents should have the following characteristics: (1) good solubility. (2)compatetive with the active components of biological products. (3) nontoxic. (4) higher eutectic temperature. Mannitol and glycine are two typical bulking agents. • Some excipients may inhibit the crystallization of bulking agents, thus affecting the stability of biological products. In the mixture of mannitol with sucrose, trehalose, lactose or maltose, respectively, crystallization cannot be observed until the concentration of mannitol reached is greater than 30% (w/w).

36. 6.4.4 The selection of cryoprotectants and lyoprotectants • Sugars are the widely used protective agent in the freeze-drying process of biological products because sugars won’t crystallize at normal conditions of freeze drying. Reducing sugars are not chosen as protective agent since it may have react Maillard reaction with protein. • Some polymers is used as protective agents because they can increase glass transition temperature. However, the ability to form hydrogen bond between molecule of the polymers and protein is greatly lower than that between polymers and sugars. So, polymers and sugars are usually used together to not only increase the glass transition temperature of the formula but also form very strong hydrogen bond with the protein.

37. The active components of biological products (such as proteins )are also belonged to polymers, having high glass transition temperature. Therefore, improving the quality ratio between proteins and excipients can increase the glass transition temperature of freeze drying products and inhibit the crystallization of excipients as well. • Some salts can also be used as protective agent during the freeze-drying process of biological products. However, salts may decrease the glass transition temperature (Tg’) of biological products.

38. 6.5 Examples of formula • Generally, the contents of solid composition is between 2%-10% in the formula of protein. When the solid composition is less than 2%, hard freeze-dried products cannot be obtained. When the content of solid composition is more than 10%, it is difficult to completely dry the products, and even the freeze drying process is completed, the rehydration of freeze dried products is very difficult. • The physical/chemical function between the additives is mutual. It is important to maintain the suitable amount of additives in the formula. If a type of additive can play several functions simultaneously, it will be the additives fo first selection. For example, some sugars can be used as cryoprotectants and lyoprotectants. • In addition, large amount of buffer solutions or salts cannot be used because they may change the pH value during the freezing process or decrease the glass transition temperature of the formula.