PHARMACEUTICAL TECHNOLOGY AND BIOPHARMACY 3
Lecture 1 – therapeutic proteins
Compared to conventional low molecular weight drugs, therapeutic proteins are
highly effective and highly specific. Generally, there are little to no side effects.
There is an increase in the production of therapeutic proteins over the years, with
monoclonal antibodies being the most prominent type.
Proteins are composed of amino acids linked together in a specific sequence. This
amino acid sequence, also known as polypeptide, forms the primary structure of the
protein. As the chain of amino acids folds, it forms three-dimensional structural
elements such as alpha-helices and beta-sheets, which are the secondary
structure. These structures are stabilized by hydrogen bonds. The relative
orientation of these structural elements within a single polypeptide chain gives rise to
the tertiary structure, which defines the overall three-dimensional conformation of
the protein.
In some cases, a protein is made up of multiple polypeptide chains. The relative
orientation of these polypeptide chains is referred to as the quaternary structure. Not
all proteins have a quaternary structure.
Each amino acid consists of a central carbon atom, known as the alpha
(α) carbon, bonded to four groups: an amino group (–NH₂), a carboxyl
group (–COOH), a hydrogen atom, and a variable side chain, known as
the R group. Because both the amino and carboxyl groups are attached to
the same central carbon, these compounds are formally referred to as α-
amino acids.
While there are other types of amino acids, such as ε-
amino acids, which have five carbon atoms separating
the amino and carboxyl groups, these are not
incorporated into proteins. Only α-amino acids are
found in proteins.
When two amino acids combine, they form a peptide bond through a
condensation reaction, where a molecule of water is released. This bond
links the carboxyl group of one amino acid to the amino group of another.
The resulting peptide has two ends: the N-terminal, which has a free
amino group, and the C-terminal, which has a free carboxyl group.
When combining two amino acids, there are 2 possible
formations.
Amino acids are categorized based on the properties of their R groups. They can be
nonpolar (hydrophobic), polar (hydrophilic), or electrically charged, which includes
acidic (negatively charged) and basic (positively charged) side chains. All amino
acids contain C, H, O, and N. However, methionine and cysteine also contain S.
1
,Simple proteins are only composed of amino acid residues. Conjugated proteins
consist of a protein part combined with non-protein components, such as metal ions,
carbohydrates (as in glycoproteins), or lipids (lipoproteins).
• Peptides do not have a tertiary structure while proteins do
• Peptides < 40 amino acids, proteins > 40 amino acids
How many different proteins can theoretically exist? There are 20 different amino
acids, let’s assume a protein composed of 100 amino acids → 20100 = 10130
possibilities.
The major difference between conventional drugs and therapeutic proteins lies in
their size or molecular weight. Conventional drugs are typically small molecules,
where therapeutic proteins are large, complex molecules.
A protein's function is determined by its three-dimensional structure,
which is maintained by relatively weak interactions. These include
hydrogen bonds, van der Waals forces, hydrophobic interactions,
and ionic interactions, all of which are non-covalent. In addition,
disulfide bridges also maintain the structure; they are covalent bonds,
though relatively weak.
Proteins are typically produced as aqueous solutions. However, in
aqueous environments, proteins are often unstable. To preserve their structure and
activity, protein solutions should be kept refrigerated at 2–8 °C, a practice known as
the cold chain. Despite refrigeration, the shelf life of therapeutic proteins can still be
limited.
The cold chain method is a problem in tropical developing countries due to
expensiveness and health care workers lacking sufficient knowledge of cold chain
management. The last steps in the cold chain, such as transport to clinics, are
particularly difficult to maintain.
Cold chain failure can result from inadequate temperature control, particularly when
cold packs at –20 °C are used in cool boxes. This can lead to the formation of ice
and the creation of an ice/protein interface. At this interface, proteins may adsorb to
the surface, where they are more prone to unfolding and aggregation. This disrupts
their three-dimensional structure and leads to a loss of activity.
Following freeze-thawing, the protein solution becomes
turbid, a visible indication of protein aggregation and structural
degradation.
Other examples of cold chain failure:
• Storage in the door of a refrigerator – when frequently opened, the
temperature in the door can be (much) higher than on the shelves
• Failure of electricity supply
• Delayed repackaging – arrival of package in the airport just before the
weekend, repackaging on Monday
2
,The temperature can be controlled with a cold chain
monitor card; the etiquette changes colour when the
cold chain temperature is exceeded. The cold chain
monitor card does not record exposure to freezing
temperatures. More complicated temperature monitoring
devices are required to determine exposure to freezing
temperatures.
A solution for tropical developing countries is to use solar panels to supply electricity
to a fridge. But this only works if the sun shines.
Degradation mechanisms –
• Chemical degradation : chemical reactions that make or break covalent bonds,
generating new chemical entities (e.g. oxidation, hydrolysis)
• Physical degradation : the chemical composition remains unchanged, but the
three-dimensional structure is changed (e.g. aggregation, denaturation)
Degradation pathways can be synergistic, where one type of degradation promotes
the other. Chemical degradation can trigger physical degradation, and physical
degradation can trigger chemical degradation, e.g. denaturation can expose reactive
sites within the protein, making it more susceptible to chemical degradation.
Relative humidity (RH) is a measure of the amount of water vapor present in the air
compared to the maximum amount the air can hold at a given temperature,
expressed as a percentage. For example, 60% RH means the air is holding 60% of
the maximum water vapor it can contain at that temperature. At 100% RH, the air is
fully saturated with water vapor.
To evaluate a drug’s stability over time, both long-term and
accelerated stability testing are conducted under
controlled temperature and humidity conditions, following
guidelines set by the ICH for different climate zones.
A commonly recommended protocol includes:
• Long-term testing at 25 °C and 60% RH for 12 months
• Accelerated testing at 40 °C and 75% RH for 6 months
A formulation is considered unacceptable when:
• A potency loss of more than 5% compared to the initial batch value
• Any specified degradant exceeds its predefined specification limit
• The pH of the product falls outside its range
Most degradation reactions in proteins require molecular mobility. Therefore, an
effective strategy to improve protein stability is to reduce this mobility by removing
water and bringing the protein into a dry state.
In addition to enhanced stability, drying proteins opens the door to the development
of non-parenteral dosage forms, such as oral tablets or inhalable powders.
3
, Several drying techniques are used to achieve this, including freeze drying, spray
drying, and spray freeze drying.
Freeze drying involves freezing the aqueous protein solution to convert it into ice.
Under reduced pressure, there is sublimation, where the ice is directly converted
into vapor without passing through the liquid phase.
• Freezing and dehydration stresses
Spray drying is a method where the protein solution is atomized into a hot air
chamber. As the droplets fall, the water rapidly evaporates, leaving behind dry
particles.
• Shear, thermal and dehydration stresses
Spray-freeze drying combines aspects of both techniques. The protein solution is
atomized into a very cold environment, like liquid nitrogen (N2), instantly freezing the
droplets. The frozen particles are then transferred to a freeze dryer, where
sublimation takes place under vacuum.
• Shear, freezing and dehydration stresses
Drying proteins without protection causes damage. To prevent this, a stabilizing
excipient is required, most commonly sugars.
Animals like the wood frog can survive extreme environments (freezing
temperatures) due to the accumulation of high amounts of sugars in their cells.
These sugars act as protectants for biomacromolecules like proteins and DNA.
Many plant cells, microorganisms, and other organisms can survive freezing and
desiccation (uitdroging) due to the accumulation of high sugar concentrations.
Mechanisms of stabilization by sugars:
• Water replacement
• Particle isolation
• Vitrification / shielding
For all three mechanisms, the sugar should be in the glassy state.
Thermodynamic stability refers to a system in its lowest possible energy state. When
a system is thermodynamically stable, it is in equilibrium, meaning there is no natural
tendency for it to change. A thermodynamically unstable system is not in its lowest
energy state and is therefore not in equilibrium. Such a system has an inherent drive
to change into a more stable form.
Thermodynamically stable states (equilibrium) –
Crystal
• Molecules are arranged in a highly structured lattice with a specific orientation
• Molecules do not have translational mobility, only vibrational and rotational
movements
• Exists at temperatures below the melting temperature (T < Tm)
4
Lecture 1 – therapeutic proteins
Compared to conventional low molecular weight drugs, therapeutic proteins are
highly effective and highly specific. Generally, there are little to no side effects.
There is an increase in the production of therapeutic proteins over the years, with
monoclonal antibodies being the most prominent type.
Proteins are composed of amino acids linked together in a specific sequence. This
amino acid sequence, also known as polypeptide, forms the primary structure of the
protein. As the chain of amino acids folds, it forms three-dimensional structural
elements such as alpha-helices and beta-sheets, which are the secondary
structure. These structures are stabilized by hydrogen bonds. The relative
orientation of these structural elements within a single polypeptide chain gives rise to
the tertiary structure, which defines the overall three-dimensional conformation of
the protein.
In some cases, a protein is made up of multiple polypeptide chains. The relative
orientation of these polypeptide chains is referred to as the quaternary structure. Not
all proteins have a quaternary structure.
Each amino acid consists of a central carbon atom, known as the alpha
(α) carbon, bonded to four groups: an amino group (–NH₂), a carboxyl
group (–COOH), a hydrogen atom, and a variable side chain, known as
the R group. Because both the amino and carboxyl groups are attached to
the same central carbon, these compounds are formally referred to as α-
amino acids.
While there are other types of amino acids, such as ε-
amino acids, which have five carbon atoms separating
the amino and carboxyl groups, these are not
incorporated into proteins. Only α-amino acids are
found in proteins.
When two amino acids combine, they form a peptide bond through a
condensation reaction, where a molecule of water is released. This bond
links the carboxyl group of one amino acid to the amino group of another.
The resulting peptide has two ends: the N-terminal, which has a free
amino group, and the C-terminal, which has a free carboxyl group.
When combining two amino acids, there are 2 possible
formations.
Amino acids are categorized based on the properties of their R groups. They can be
nonpolar (hydrophobic), polar (hydrophilic), or electrically charged, which includes
acidic (negatively charged) and basic (positively charged) side chains. All amino
acids contain C, H, O, and N. However, methionine and cysteine also contain S.
1
,Simple proteins are only composed of amino acid residues. Conjugated proteins
consist of a protein part combined with non-protein components, such as metal ions,
carbohydrates (as in glycoproteins), or lipids (lipoproteins).
• Peptides do not have a tertiary structure while proteins do
• Peptides < 40 amino acids, proteins > 40 amino acids
How many different proteins can theoretically exist? There are 20 different amino
acids, let’s assume a protein composed of 100 amino acids → 20100 = 10130
possibilities.
The major difference between conventional drugs and therapeutic proteins lies in
their size or molecular weight. Conventional drugs are typically small molecules,
where therapeutic proteins are large, complex molecules.
A protein's function is determined by its three-dimensional structure,
which is maintained by relatively weak interactions. These include
hydrogen bonds, van der Waals forces, hydrophobic interactions,
and ionic interactions, all of which are non-covalent. In addition,
disulfide bridges also maintain the structure; they are covalent bonds,
though relatively weak.
Proteins are typically produced as aqueous solutions. However, in
aqueous environments, proteins are often unstable. To preserve their structure and
activity, protein solutions should be kept refrigerated at 2–8 °C, a practice known as
the cold chain. Despite refrigeration, the shelf life of therapeutic proteins can still be
limited.
The cold chain method is a problem in tropical developing countries due to
expensiveness and health care workers lacking sufficient knowledge of cold chain
management. The last steps in the cold chain, such as transport to clinics, are
particularly difficult to maintain.
Cold chain failure can result from inadequate temperature control, particularly when
cold packs at –20 °C are used in cool boxes. This can lead to the formation of ice
and the creation of an ice/protein interface. At this interface, proteins may adsorb to
the surface, where they are more prone to unfolding and aggregation. This disrupts
their three-dimensional structure and leads to a loss of activity.
Following freeze-thawing, the protein solution becomes
turbid, a visible indication of protein aggregation and structural
degradation.
Other examples of cold chain failure:
• Storage in the door of a refrigerator – when frequently opened, the
temperature in the door can be (much) higher than on the shelves
• Failure of electricity supply
• Delayed repackaging – arrival of package in the airport just before the
weekend, repackaging on Monday
2
,The temperature can be controlled with a cold chain
monitor card; the etiquette changes colour when the
cold chain temperature is exceeded. The cold chain
monitor card does not record exposure to freezing
temperatures. More complicated temperature monitoring
devices are required to determine exposure to freezing
temperatures.
A solution for tropical developing countries is to use solar panels to supply electricity
to a fridge. But this only works if the sun shines.
Degradation mechanisms –
• Chemical degradation : chemical reactions that make or break covalent bonds,
generating new chemical entities (e.g. oxidation, hydrolysis)
• Physical degradation : the chemical composition remains unchanged, but the
three-dimensional structure is changed (e.g. aggregation, denaturation)
Degradation pathways can be synergistic, where one type of degradation promotes
the other. Chemical degradation can trigger physical degradation, and physical
degradation can trigger chemical degradation, e.g. denaturation can expose reactive
sites within the protein, making it more susceptible to chemical degradation.
Relative humidity (RH) is a measure of the amount of water vapor present in the air
compared to the maximum amount the air can hold at a given temperature,
expressed as a percentage. For example, 60% RH means the air is holding 60% of
the maximum water vapor it can contain at that temperature. At 100% RH, the air is
fully saturated with water vapor.
To evaluate a drug’s stability over time, both long-term and
accelerated stability testing are conducted under
controlled temperature and humidity conditions, following
guidelines set by the ICH for different climate zones.
A commonly recommended protocol includes:
• Long-term testing at 25 °C and 60% RH for 12 months
• Accelerated testing at 40 °C and 75% RH for 6 months
A formulation is considered unacceptable when:
• A potency loss of more than 5% compared to the initial batch value
• Any specified degradant exceeds its predefined specification limit
• The pH of the product falls outside its range
Most degradation reactions in proteins require molecular mobility. Therefore, an
effective strategy to improve protein stability is to reduce this mobility by removing
water and bringing the protein into a dry state.
In addition to enhanced stability, drying proteins opens the door to the development
of non-parenteral dosage forms, such as oral tablets or inhalable powders.
3
, Several drying techniques are used to achieve this, including freeze drying, spray
drying, and spray freeze drying.
Freeze drying involves freezing the aqueous protein solution to convert it into ice.
Under reduced pressure, there is sublimation, where the ice is directly converted
into vapor without passing through the liquid phase.
• Freezing and dehydration stresses
Spray drying is a method where the protein solution is atomized into a hot air
chamber. As the droplets fall, the water rapidly evaporates, leaving behind dry
particles.
• Shear, thermal and dehydration stresses
Spray-freeze drying combines aspects of both techniques. The protein solution is
atomized into a very cold environment, like liquid nitrogen (N2), instantly freezing the
droplets. The frozen particles are then transferred to a freeze dryer, where
sublimation takes place under vacuum.
• Shear, freezing and dehydration stresses
Drying proteins without protection causes damage. To prevent this, a stabilizing
excipient is required, most commonly sugars.
Animals like the wood frog can survive extreme environments (freezing
temperatures) due to the accumulation of high amounts of sugars in their cells.
These sugars act as protectants for biomacromolecules like proteins and DNA.
Many plant cells, microorganisms, and other organisms can survive freezing and
desiccation (uitdroging) due to the accumulation of high sugar concentrations.
Mechanisms of stabilization by sugars:
• Water replacement
• Particle isolation
• Vitrification / shielding
For all three mechanisms, the sugar should be in the glassy state.
Thermodynamic stability refers to a system in its lowest possible energy state. When
a system is thermodynamically stable, it is in equilibrium, meaning there is no natural
tendency for it to change. A thermodynamically unstable system is not in its lowest
energy state and is therefore not in equilibrium. Such a system has an inherent drive
to change into a more stable form.
Thermodynamically stable states (equilibrium) –
Crystal
• Molecules are arranged in a highly structured lattice with a specific orientation
• Molecules do not have translational mobility, only vibrational and rotational
movements
• Exists at temperatures below the melting temperature (T < Tm)
4
