PHARMACEUTICAL TECHNOLOGY AND BIOPHARMACY 3 (PTB3)
Lecture 1: Therapeutic Proteins – W. Hinrichs
Therapeutic proteins are highly effective and highly specific compared to conventional low molecular
weight drugs.
Structure of proteins
- Primary structure sequence amino acids
Proteins only contain α-amino acids have only 1 central
carbon atom.
Amino acids form peptides bonds through condensation
reactions where water splits off (COOH reacts with NH 2)
C-terminal = carboxyl side, N-terminal = amine side.
Order from N-terminal to C-terminal is important (Gly-Ala ≠
Ala-Gly)
- Secondary structure specific 3D structure elements (α-helix and β-sheets).
- Tertiary structure relative orientation of secondary structural elements.
- Quaternary structure relative orientation of various polypeptide chains.
Not all proteins have a quaternary structure.
Simple proteins composed of amino acid residues only
Conjugated proteins also contains e.g. metal ions, carbohydrates (glycoproteins) etc.
Peptide <40 amino acids, do not have a tertiary structure and (smaller than 4,5 kDa)
Proteins >40 amino acids (and larger than 4,5 kDa).
How many proteins can exist protein with 100 amino acids (small) and 20 different amino acids
exist = 20100 combinations.
Cold chain
- Problem The action of a protein is determined by its 3D structure. The 3D structure is
maintained by relatively weak interactions hydrogen bonds, van der waals forces,
hydrophobic interactions, ionic interactions (all non-covalent), disulfide bridges (weak
covalent). Proteins are produced as aqueous solutions, but in this environment proteins are
generally unstable (not always).
- Solution Cold chain = between all steps of transport and storing, the product should be
kept between 2-8 oC.
But this comes with problems expensive, health care workers lack sufficient
knowledge and in remote areas difficult to maintain (particularly last steps)
Freezing is not an option because proteins will aggregate 3D structure is lost.
Cold chain monitor card when the cold chain temperature is exceeded, the color changes. Does
not record freezing temperatures. More expensive devices can also record exposure to freezing
temperatures.
Degradation mechanisms
- Chemical instabilities chemical reactions that make or break covalent bonds, generating
new chemical entities. E.g. via deamidation, oxidation, hydrolysis etc.
- Physical instabilities the chemical composition remains unaltered, but the 3D structure is
changed. E.g. aggregation, denaturation, adsorption etc.
1
,Can be synergistic chemical instabilities can trigger physical instabilities and vice versa.
Assessment stability
Unacceptable when over 5% potency loss from the initial value, exceeds pH limits, specified
degradant exceeding its specification limit.
Drying techniques
Proteins can be stabilized by bringing the protein in the dry state to reduce the mobility.
- Freeze drying freeze the solution, reduce pressure and apply heat to remove ice and water
by sublimation, dried particles.
- Spray drying bring solution in small droplets, heat them, dried particles.
- Spray freeze drying bring solution into small droplets, freeze them, reduce pressure and
apply heat to remove ice and water, dried particles.
Damage due to stresses
- Freeze drying freezing and dehydration stresses.
- Spray drying shear, thermal and dehydration stresses.
- Spray freeze drying shear, freezing and dehydration stresses.
Stabilizing excipient is needed to prevent disruption of the protein against all these stresses
sugars. For sugars to stabilize the proteins, the sugars need to be in the glassy state.
States of matter
Thermodynamically stable (equilibrium states)
- Crystal molecules are arranged in a lattice (structured, specific orientation) and no
translational mobility of the molecules (only vibrations and rotations). When T > melting
temperature (Tm) liquid.
- Liquid random orientation of molecules (not structured) and high translational mobility.
Thermodynamically unstable (non-equilibrium states)
- Glass random orientation of molecules, no translational mobility and
kinetically stable (= crystallization cannot occur). When T > glass
transition temperature (Tg) rubber.
- Rubber random orientation of molecules, high translational mobility,
kinetically unstable (= crystallization can occur).
Also referred to as super-cooled liquid.
Always Tg << Tm
Crystallization requires molecular mobility because molecules have to have a specific
orientation towards each other to form a crystalline lattice.
Glassy state
- Viscos
12 14
ity is so high (10 – 10 Pa.s) that the materials does not flow (no translational mobility).
- Material is able to carry its own weight.
- No change of structure.
- Viscosity rapidly decreases over and above the Tg (T > Tg rubber: high mobility)
2
,Mechanisms of stabilization by sugars
- Water replacement H-bonds between the protein and H2O are gradually replaced by H-
bond between the protein and sugar-OH’s.
Sugars should be in the amorphous state to enable tight coating, because then they
are faced towards the irregular surface of the protein and not towards each other.
Sugars should be in the glassy state because in the rubbery state crystallisation can
occur where H-bonds are broken and mechanical stress is applied damage to
protein
- Particle isolation sugar matrix acts as a physical barrier between the particles, so they do
not aggregate.
Sugars should have a low translational mobility glassy state.
- Vitrification / shielding formation of protective sugar coating around the protein shields it
from the environment (oxygen and light). Sugar matrix should form a tight coating in which
diffusion on a realistic time scale is inhibited.
The sugar should be in the amorphous state for tight coating.
The sugar should be in the glassy state to prevent crystallization which ruins the right
coating and also to prevent diffusion.
Critical point
Phase diagram of water
With (spray) freeze drying the solid-gas transition curve is passed
sublimation
- Decrease the pressure at low temperature.
With spray drying the liquid-gas transition curve is passed evaporation Triple point
- Increase the temperature at atmospheric pressure.
State diagram water/sugar
In this system, only solid and liquids are considered, vapor phase is ignored. Pressure is fixed at 1 atm.
Thermodynamically stable phases:
- Left curve is the crystallization curve water the higher the amount of sugar,
the lower the temperature is needed to crystalize the water. Pure water
freezes/crystallizes at 0oC = 273 K starting point.
- Right curve is the crystallization curve sugar the purer the sugar, the higher
temp is needed to crystalize the sugar.
- The two curves meet each other at the eutectic point (E). Te is the temperature
at E and Ce the composition E.
3
, Thermodynamically unstable phases (slide 81)
- From B to C ice formation concentration of the solution is increasing.
- During C to D? fast cooling to prevent crystallization of the sugar (avoid rubbery state)
- When D is reached remaining solution becomes glassy (solution with ice
crystals and water in the glass composition (Cg’)
The glass/ice mass ratio increases A < A’ < A’’.
Freeze drying
Freeze drying in 3 steps:
1. Freezing the solution is cooled to blow Tg’.
Should not be too slow or too fast
2. Removal of water in the form of ice crystals (primary drying).
Sublimation of ice. Pressure is reduced. Should not be too low slow sublimation.
The temperature should be as high as possible (faster process) but not above Tg’.
3. Removal of water in glass (secondary drying).
Desorption of water from glass. Pressure should be
decreased. Temperature can be gradually increased but not
above Tg.
Slow vs fast freezing
- Slow freezing low nucleation rate, large crystals, small interface.
- Fast freezing high nucleation rate, small crystals, large interface
Proteins unfold at interfaces. Therefor slow freezing is favourable. However, during freezing the sugar
crystallizes = phase separation between stabilizer and protein = increased degradation rate fast
freezing is favourable.
- Rule of thumb: about 1 oC/min not too slow and not too fast.
Primary drying first and then secondary drying because sublimation of ice if much faster than
evaporation of water from glass.
Primary drying
- Sublimation of ice crystals.
- Temperature as high as possible but below Tg’ (avoid the yellow area!).
If T > Tg’ the cake will collapse.
- Pressure as high as possible but below sublimation curve.
- Composition of freeze concentrated fraction remains the same Cg’.
- Glass transition temperature remains the same (Tg’)
- Ratio ice/glass decreases.
Secondary drying
- After all ice has been sublimation, water from glass now evaporates.
- Composition glass gradually changes to the right.
- Temperature can be increased but always below Tg (avoid yellow area), so temperature
should be below the glass transition curve (blue curve).
Sublimation/evaporation is an endothermal event.
Freeze dried product mirror image of u=ice crystals formed during freezing. It is a
highly porous product. Never shake a protein solution vigorously to avoid high air-
protein interfacial surface area where protein denaturation can easily occur.
Spray drying
Solution is aerolized using a nozzle aerosol is exposed to hot air (evaporation is
4
