Transcript University of Shanghai for Science and Technology School of

University of Shanghai for Science and Technology
Chapter 6
Protective agent and additives for freeze-drying
of pharmaceutical products
School of Medical Instrument and Food Engineering
University of Shanghai for Science and Technology
•
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 freezedrying and storage.
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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.
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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.
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• 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
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Classification of protecting agents by their material kinds
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•
Sugars/polysaccharides
Polymers
Surfactants
Amino acids
Salts
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• 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.
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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.
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• 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).
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6.1.2. The function of sugars/polyols in freeze-drying
1. 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.
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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 .
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• Trehalose
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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 290295℃, 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.
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• 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.
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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.
Recovery of PFK %
（
）
Concentration of trehalose（mM）
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6.2 Polymer-type protective agents
6.2.1 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.
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6.2.2 The polymer-type protective agents for freeze-drying
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Generally, the polymer-type protective agents have the following
characteristics:
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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
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6.3 Surfactants and amino acid-type protective agents
6.3.1 Surfactant-type protective agents
1. 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 CH 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.
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• Surfactants can be divided into two main types: ionized and nonionized. 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.
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2. The typical surfactants in freeze-drying
Name
Tween 80
Triton X-100
sucrose fatty acid monoester
3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS)
Hydroxypropyl-β-cyclodextrin (HP-β-CD)
sodium dodecanesulphonate(SDS)
Brij35， Brij30
Lubrol-px
Pluronic F127
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6.3.2. Amino acid-type protective agents
1. The definition of amino acids
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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.
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2. Amino acid-type protective agents
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6.3.3. Other additives
1. 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.
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Name
Chemical formula
Relative molecular weight
antisterility factor (Vitamin E)
C29H50O2
430.72
ascorbic acid (Vitamin C)
C6H8O6
176.13
Lecithin
C40H82NO9P
752.08
D(-)-Isoascorbic acid
C6H8O6
176.13
L- ascorbic sodium
C6H7 NaO6
198.11
sodium thiosulfate anhydrous
Na2S2O3
158.11
3-tert-Butyl-4
-hydroxyanisole
C11H16O2
180.25
butyl atea hydroxy toluene
CH3C6H2(OH)
[C(CH3)3]2C15H24O
220.36
propyl gallate
(HO)3C6H2COOCH2CH3
212.20
Ethylenediamine tetraacetic acid
disodium salt dehydrate,
Na2EDTA
C10H14N2Na2O8.2H2O
372.24
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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.
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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.
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•
•
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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.
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The change of mass of water with the freeze drying time
(a: with TBC；b：without TBC)
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a
b
The SEM photos of freeze dried sucrose solution
(a: with TBC；b：without TBC)
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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.
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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 freezedrying 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.
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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).
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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.
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• 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 freezedrying process of biological products. However, salts may decrease
the glass transition temperature (Tg’) of biological products.
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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.
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