INNOVATIVE THERAPEUTICS
Lecture 1: Protein structure and stability
Proteins are referred to as biopharmaceuticals, biologicals, biologics, therapeutic
proteins, or recombinant proteins. Proteins are macromolecules with molecular
weights ranging from 1 to 150 kDa.
The production of proteins requires biological systems, typically living cells such as
bacteria, yeast, or mammalian cell cultures. These systems are used because
proteins must be folded correctly and may require post-translational
modifications, such as glycosylation, to be biologically active.
Biologicals are derived from living systems and depend on them for their production.
Proteins are biologicals, but not all biologicals are proteins. Vaccines, for instance,
are also classified as biologicals. They can be based on weakened living organisms,
inactivated organisms, subunits of pathogens, or mRNA. Biologicals also include
advanced therapy medicinal products, or ATMPs. This comprises gene therapy
agents, somatic cell therapy products and tissue-engineered products.
In contrast to proteins, peptides are shorter chains of amino acids. Their size and
structure make them simpler to produce, and unlike proteins, they can be
synthesized without the need for biological systems. Because of this, peptides are
not necessarily biologicals.
Proteins are always biologicals, but peptides do not have to be!
Recombinant proteins are proteins that are produced through genetic engineering.
The gene that codes for a specific protein is inserted into the DNA of a host cell via a
vector, such as bacteria (like E. coli). Once the host cell takes up this DNA, it begins
to produce the protein encoded by the inserted gene. Recombinant proteins can be
made in large quantities.
An example is insulin, which used to be extracted from pig or cow pancreases, but is
almost entirely produced as recombinant insulin in E. coli cells, ensuring treatment for
diabetes.
Most therapeutic targets are proteins. Diseases caused by a protein’s gain of
function are treated by blocking or inactivating that protein, as seen with inhibitors
used in cancer or autoimmune diseases. In contrast, diseases caused by protein
loss of function are treated by replacing the impaired protein, such as giving insulin
in diabetes.
Protein therapeutics –
Antibodies
• Produced by B lymphocytes
• Binding to specific molecules
• Vehicles for targeted delivery
• Monoclonal antibodies are the most common type
1
,Enzymes
• Catalyse a wide variety of biochemical reactions
• Catalyse the degradation, cleavage or modification of therapeutic targets
• Replacement therapy
Coagulation factors
• Blood clotting process
• Plasma-derived or produced recombinantly
Protein hormones
• Secreted by endocrine glands
• Binding to receptors and triggering signalling cascade
• Insulin is the classic example
Cytokines
• Mediate cell-to-cell communication during immune response
• Immunomodulatory agents
The market position of protein therapeutics is increasing. Most registered products
are engineered (recombinant) proteins, but there are also originators under patent
protection and biosimilars.
Monoclonal antibodies represent the largest group of protein therapeutics. A
monoclonal antibody is designed to recognize a single specific antigen, and these
are predominantly used for oncology and autoimmunity/immunology.
Protein therapeutics are composed of amino acids and often undergo post-
translational modifications such as glycosylation or the formation of disulfide bridges.
They can also be chemically modified, for instance through pegylation. These
therapeutics originate from microorganisms, animal, or human sources. They can be
produced using recombinant DNA technology.
The characterization of protein therapeutics is complex. It requires physicochemical
tests alongside biological testing of both the active ingredient and the final product. In
addition, there is control of the production process and continuous quality monitoring.
Preserving the structure of therapeutic proteins is essential as their activity depends
on their three-dimensional conformation. The correct folding of primary, secondary,
tertiary, and quaternary structures must be maintained throughout production,
purification, storage, and administration to ensure therapeutic effectiveness and
patient safety.
The primary structure is the linear sequence of amino acids linked by peptide bonds
(covalent). It is a determining factor for folding into alpha-helices and beta-sheets in
secondary structure.
There are 20 naturally occurring amino acids, each with a unique side chain that
influences the properties of the resulting polypeptide. These side chains affect
molecular weight, charge (pH dependent), polarity, and pKa.
2
,(pKa is a measure of an acid's strength. A lower pKa indicates a stronger acid,
meaning it donates protons (H+) in a solution more readily.)
The secondary structure of a protein refers to the local folding
of its polypeptide chain, stabilized by hydrogen bonds
between the amide (NH) groups, which act as hydrogen
donors, and carbonyl (C=O) groups, which act as hydrogen
acceptors. The two most common secondary structures are
alpha-helices and beta-sheets.
In an alpha-helix, hydrogen bonds form between the NH and
C=O groups of every fourth amino acid, creating a helical turn
with 3.6 amino acids per turn. Side chains extend outward,
allowing long-range interactions, with hydrophobic regions facing inward and
hydrophilic regions facing outward.
Beta-sheets arise from hydrogen bonds between multiple polypeptide chains
arranged in parallel or antiparallel orientations, resulting in a flat structure.
Tertiary structure is the three-dimensional shape of a single polypeptide chain,
formed by the folding of secondary structures and stabilized by interactions between
the amino acid side chains. These interactions include hydrophobic interactions,
hydrogen bonds, ionic bonds, and disulfide bonds, all of which contribute to the
protein's three-dimensional conformation, which is essential for the
protein’s function. Larger proteins are often folded into distinct
structural domains, as seen in immunoglobulins and Factor VIII.
Domains are independently folded regions within a single polypeptide
chain that often have specific functional roles. Immunoglobulins
(antibodies) consist of multiple domains: the variable domains of the
heavy and light chains bind to antigens, while the constant domains
mediate effector functions like interacting with immune cells.
The quaternary structure of a protein refers to the assembly of multiple tertiary-
structured polypeptide chains (subunits) into a functional protein complex. These
subunits are held together mainly by non-covalent interactions and sometimes
stabilized by disulfide bridges. An example is hemoglobin, which consists of four
subunits: two alpha chains and two beta chains.
Peptides are short chains of amino acids (2-50). They do not form a tertiary
structure.
Protein structure is maintained by a combination of covalent and non-covalent
interactions. Covalent bonds include peptide bonds and disulfide bridges. Non-
covalent interactions occur between chemical groups within the protein and are
responsible for secondary, tertiary, and quaternary folding.
Hydrophobic interactions arise between nonpolar groups. Hydrogen bonds form
between electropositive hydrogen atoms and electronegative oxygen or nitrogen
atoms, either within the molecule or with water.
3
, Electrostatic interactions occur between oppositely charged groups, with strength
influenced by distance and the surrounding environment. Water has a high dielectric
constant, which weakens electrostatic interactions because the water molecules
shield the charges from each other. However, the interior of a protein is nonpolar, with
a very low dielectric constant, so there is little shielding. As a result, electrostatic
interactions are much stronger inside the hydrophobic core of a protein because the
low-dielectric environment allows charges to interact more directly.
Van der Waals interactions involve attractions between permanent or induced
dipoles.
On the exterior, water is loosely bound from the surrounding environment, while
inside the protein, some water molecules are trapped more tightly, where the
dielectric constant is lower. These bound water molecules are essential because they
help maintain the protein’s structure and therefore function. A small amount of water
is needed for enzymes to work, just enough to cover the protein’s polar groups.
In solution, proteins are further stabilized by interactions with small molecules such
as sugars and amino acids. These stabilizers, when added, protect proteins by
interacting with their surfaces and maintaining their functional conformation.
In the endoplasmic reticulum and Golgi apparatus, proteins undergo post-
translational modifications. These include the enzymatic addition of sugar groups
through glycosylation, the attachment of phosphate groups through
phosphorylation, and the addition of sulfate groups through sulfation. Another
modification is the formation of disulfide bridges.
Correct glycosylation is essential for therapeutic proteins because it affects their
folding, stability of 3D conformation, solubility, resistance to hydrolysis and
denaturation, immunogenicity, binding affinity for the target, interactions with other
proteins, half-life in circulation, and even localization within the body.
Glycosylation can occur at asparagine residues, known as N-linked glycosylation,
or at serine and threonine residues, known as O-linked glycosylation.
There are differences in glycosylation depending on the living system, which can
have major consequences for the final therapeutic protein. In bacteria, there are
almost no post-translational modifications like glycosylation. This makes bacterial
systems mainly suitable for simple proteins such as insulin.
Protein folding is essential for stability and function, enabling interactions with
substrates, receptors, and other proteins. In the body, water, ions, and solutes help
maintain this structure, but during production the protective environment is often lost.
To counter this, stabilizers such as sugars are added, which interact with the protein
surface and help preserve its functional conformation.
4
Lecture 1: Protein structure and stability
Proteins are referred to as biopharmaceuticals, biologicals, biologics, therapeutic
proteins, or recombinant proteins. Proteins are macromolecules with molecular
weights ranging from 1 to 150 kDa.
The production of proteins requires biological systems, typically living cells such as
bacteria, yeast, or mammalian cell cultures. These systems are used because
proteins must be folded correctly and may require post-translational
modifications, such as glycosylation, to be biologically active.
Biologicals are derived from living systems and depend on them for their production.
Proteins are biologicals, but not all biologicals are proteins. Vaccines, for instance,
are also classified as biologicals. They can be based on weakened living organisms,
inactivated organisms, subunits of pathogens, or mRNA. Biologicals also include
advanced therapy medicinal products, or ATMPs. This comprises gene therapy
agents, somatic cell therapy products and tissue-engineered products.
In contrast to proteins, peptides are shorter chains of amino acids. Their size and
structure make them simpler to produce, and unlike proteins, they can be
synthesized without the need for biological systems. Because of this, peptides are
not necessarily biologicals.
Proteins are always biologicals, but peptides do not have to be!
Recombinant proteins are proteins that are produced through genetic engineering.
The gene that codes for a specific protein is inserted into the DNA of a host cell via a
vector, such as bacteria (like E. coli). Once the host cell takes up this DNA, it begins
to produce the protein encoded by the inserted gene. Recombinant proteins can be
made in large quantities.
An example is insulin, which used to be extracted from pig or cow pancreases, but is
almost entirely produced as recombinant insulin in E. coli cells, ensuring treatment for
diabetes.
Most therapeutic targets are proteins. Diseases caused by a protein’s gain of
function are treated by blocking or inactivating that protein, as seen with inhibitors
used in cancer or autoimmune diseases. In contrast, diseases caused by protein
loss of function are treated by replacing the impaired protein, such as giving insulin
in diabetes.
Protein therapeutics –
Antibodies
• Produced by B lymphocytes
• Binding to specific molecules
• Vehicles for targeted delivery
• Monoclonal antibodies are the most common type
1
,Enzymes
• Catalyse a wide variety of biochemical reactions
• Catalyse the degradation, cleavage or modification of therapeutic targets
• Replacement therapy
Coagulation factors
• Blood clotting process
• Plasma-derived or produced recombinantly
Protein hormones
• Secreted by endocrine glands
• Binding to receptors and triggering signalling cascade
• Insulin is the classic example
Cytokines
• Mediate cell-to-cell communication during immune response
• Immunomodulatory agents
The market position of protein therapeutics is increasing. Most registered products
are engineered (recombinant) proteins, but there are also originators under patent
protection and biosimilars.
Monoclonal antibodies represent the largest group of protein therapeutics. A
monoclonal antibody is designed to recognize a single specific antigen, and these
are predominantly used for oncology and autoimmunity/immunology.
Protein therapeutics are composed of amino acids and often undergo post-
translational modifications such as glycosylation or the formation of disulfide bridges.
They can also be chemically modified, for instance through pegylation. These
therapeutics originate from microorganisms, animal, or human sources. They can be
produced using recombinant DNA technology.
The characterization of protein therapeutics is complex. It requires physicochemical
tests alongside biological testing of both the active ingredient and the final product. In
addition, there is control of the production process and continuous quality monitoring.
Preserving the structure of therapeutic proteins is essential as their activity depends
on their three-dimensional conformation. The correct folding of primary, secondary,
tertiary, and quaternary structures must be maintained throughout production,
purification, storage, and administration to ensure therapeutic effectiveness and
patient safety.
The primary structure is the linear sequence of amino acids linked by peptide bonds
(covalent). It is a determining factor for folding into alpha-helices and beta-sheets in
secondary structure.
There are 20 naturally occurring amino acids, each with a unique side chain that
influences the properties of the resulting polypeptide. These side chains affect
molecular weight, charge (pH dependent), polarity, and pKa.
2
,(pKa is a measure of an acid's strength. A lower pKa indicates a stronger acid,
meaning it donates protons (H+) in a solution more readily.)
The secondary structure of a protein refers to the local folding
of its polypeptide chain, stabilized by hydrogen bonds
between the amide (NH) groups, which act as hydrogen
donors, and carbonyl (C=O) groups, which act as hydrogen
acceptors. The two most common secondary structures are
alpha-helices and beta-sheets.
In an alpha-helix, hydrogen bonds form between the NH and
C=O groups of every fourth amino acid, creating a helical turn
with 3.6 amino acids per turn. Side chains extend outward,
allowing long-range interactions, with hydrophobic regions facing inward and
hydrophilic regions facing outward.
Beta-sheets arise from hydrogen bonds between multiple polypeptide chains
arranged in parallel or antiparallel orientations, resulting in a flat structure.
Tertiary structure is the three-dimensional shape of a single polypeptide chain,
formed by the folding of secondary structures and stabilized by interactions between
the amino acid side chains. These interactions include hydrophobic interactions,
hydrogen bonds, ionic bonds, and disulfide bonds, all of which contribute to the
protein's three-dimensional conformation, which is essential for the
protein’s function. Larger proteins are often folded into distinct
structural domains, as seen in immunoglobulins and Factor VIII.
Domains are independently folded regions within a single polypeptide
chain that often have specific functional roles. Immunoglobulins
(antibodies) consist of multiple domains: the variable domains of the
heavy and light chains bind to antigens, while the constant domains
mediate effector functions like interacting with immune cells.
The quaternary structure of a protein refers to the assembly of multiple tertiary-
structured polypeptide chains (subunits) into a functional protein complex. These
subunits are held together mainly by non-covalent interactions and sometimes
stabilized by disulfide bridges. An example is hemoglobin, which consists of four
subunits: two alpha chains and two beta chains.
Peptides are short chains of amino acids (2-50). They do not form a tertiary
structure.
Protein structure is maintained by a combination of covalent and non-covalent
interactions. Covalent bonds include peptide bonds and disulfide bridges. Non-
covalent interactions occur between chemical groups within the protein and are
responsible for secondary, tertiary, and quaternary folding.
Hydrophobic interactions arise between nonpolar groups. Hydrogen bonds form
between electropositive hydrogen atoms and electronegative oxygen or nitrogen
atoms, either within the molecule or with water.
3
, Electrostatic interactions occur between oppositely charged groups, with strength
influenced by distance and the surrounding environment. Water has a high dielectric
constant, which weakens electrostatic interactions because the water molecules
shield the charges from each other. However, the interior of a protein is nonpolar, with
a very low dielectric constant, so there is little shielding. As a result, electrostatic
interactions are much stronger inside the hydrophobic core of a protein because the
low-dielectric environment allows charges to interact more directly.
Van der Waals interactions involve attractions between permanent or induced
dipoles.
On the exterior, water is loosely bound from the surrounding environment, while
inside the protein, some water molecules are trapped more tightly, where the
dielectric constant is lower. These bound water molecules are essential because they
help maintain the protein’s structure and therefore function. A small amount of water
is needed for enzymes to work, just enough to cover the protein’s polar groups.
In solution, proteins are further stabilized by interactions with small molecules such
as sugars and amino acids. These stabilizers, when added, protect proteins by
interacting with their surfaces and maintaining their functional conformation.
In the endoplasmic reticulum and Golgi apparatus, proteins undergo post-
translational modifications. These include the enzymatic addition of sugar groups
through glycosylation, the attachment of phosphate groups through
phosphorylation, and the addition of sulfate groups through sulfation. Another
modification is the formation of disulfide bridges.
Correct glycosylation is essential for therapeutic proteins because it affects their
folding, stability of 3D conformation, solubility, resistance to hydrolysis and
denaturation, immunogenicity, binding affinity for the target, interactions with other
proteins, half-life in circulation, and even localization within the body.
Glycosylation can occur at asparagine residues, known as N-linked glycosylation,
or at serine and threonine residues, known as O-linked glycosylation.
There are differences in glycosylation depending on the living system, which can
have major consequences for the final therapeutic protein. In bacteria, there are
almost no post-translational modifications like glycosylation. This makes bacterial
systems mainly suitable for simple proteins such as insulin.
Protein folding is essential for stability and function, enabling interactions with
substrates, receptors, and other proteins. In the body, water, ions, and solutes help
maintain this structure, but during production the protective environment is often lost.
To counter this, stabilizers such as sugars are added, which interact with the protein
surface and help preserve its functional conformation.
4
