Thermal Characterization as Part of an Empirical Process for Developing Optimized Formulations and Lyophilization Cycles
Jeff Schwegman, CEOLabyrinth BioPharma, [email protected]
Lyophilization
What is Lyophilization?
Lyophilization is the process we use to remove water from a formulation at low temperatures (prevents thermal degradation) through a process of sublimation.
Lyophilization
Why do we Lyophilize (Freeze-Dry)?
Products are not stable (< 10% degradation) in the solution state at controlled room temperature for at least 2 years*
What is Freeze-Drying?
Vapor
Liquid
Solid
Temperature, °C
Triple point(0oC, 4.5 mm Hg)
Pre
ssu
re (
atm
)
0 100
1
Choice of Dryer Parameters
“The choice of values for the operating parameters of the cycle must be based on sound physical characterization of the formulation and understanding of how these parameters impact the final product”
- John Carpenter/Byeong Chang
John Carpenter/Byeong Chang. 1996. Biotechnology and Biopharmaceutical Manufacturing, Processing and Preservation. Buffalo Grove, IL: Interpharm Press.
Why Lyophilization Works
Sublimation: 1: (chemistry) a change directly from the solid to the gaseous state without becoming liquid
Ice sublimes in an attempt to achieve vapor equilibrium. When chamber pressure = ice vapor pressure, sublimation stops
The Driving Force = ∆P
Every 5 degree C increase inice temperature results inroughly a 75% increase inthe vapor pressure of ice!
Describes perfectly whylyophilization cycles need tobe optimized…wasting timeand resources if drying too farbelow critical temperatures
Lyophilization
Steps of Lyophilization
1. Product Freezing (I & II)2. Thermal Treatment (Annealing)3. System Evacuation (Vacuum)4. Primary Drying (Removal of Ice)5. Secondary Drying (Removal of Unfrozen
Water)6. Backfill with Inert Gas
Freezing
What Occurs in the Freezing Phase?•Ice nucleation•Ice Crystal Growth•Solute Concentration•Formation of Glasses•Solutes Crystallize (pure crystals)•Formation of Eutectics
Freezing of Aqueous Systems
Solution
Solute crystallizes?
Ice Nucleation
Ice crystal growth, freezeconcentration
Solute Crystallizes,eutectic formation
MetastableGlass
Stable Glass
LyotropicLiquid Crystal
Temperature
Definitions
A eutectic mixture is an intimate mixture of 2 (or more) crystalline species that are in such close contact that they melt like a single, pure substance.
A glass is an amorphous species (randomly oriented molecules) which forms a solid, non flowing mass below its Tg’
Thermal Transition Terms Important in Freeze-Drying
•Eutectic temperature (Te)• Refers to crystalline systems and measured by thermal
or thermoelectric analyses
•Glass transition temperature (Tg’ and Tg)• Refers to amorphous systems and also measured by
thermal or thermoelectric analyses
•Collapse temperature (Tc)•Essentially the same as Tg’, but measure by freeze-dry
microscopy.
Eutectics and Glasses
Why are These Important?The eutectic or glass transition temperatures determine maximum temperature that the product can withstand during primary drying without loss of structure
Unfrozen Water
The amorphous solute, when in equilibrium with ice contains large amounts (10-50%) of unfrozen water which is dissolved in the solute phase
The crystalline solute contains no unfrozen water except surface adsorbed water on crystal surface or occluded water in crystals (<1%)
Eutectics and Glasses
These two different species dry very differently both in primary and secondary drying.
Cycle development conditions must take this into consideration
Eutectics and Glasses
In primary drying, must stay below Tg’ or Te or complete structural loss or the product will occur (Tg’typically much lower than Te)
Water embedded in glass while adsorbed to the surface of crystals
Eutectics and GlassesInterstitial space must be rigid enough to support itself after water (acting as the scaffolding) sublimes away
Eutectics and Glasses
In secondary drying:•Water must diffuse to surface of glass
before vaporization (very slow)•Water easily vaporized off of the
crystal surface (very fast)
DSCIn DSC, the sample and a reference are subjected to a carefully controlled temperature program each with their own furnace.
When a transition occurs that results in a change in sample temperature, heat is added or taken away from the sample so that both cells remain at the same temperature.
This heat is equal to the energy of the transition.
DSC
•Measures thermal transitions in materials
•Useful for characterization of both frozen samples and freeze-dried solids
DSC
When using DSC for conducting thermal analysis studies for lyophilization cycle development, discard the cooling curve and only focus on the heating curve.
The sample has the potential for “supercooling” and gives misleading data.
Freeze-Dry Microcopy
Direct examination of freezing and
freeze-drying via a special microscope
and thermal stage
Compliments and supports the
information gained from the DSC
PLM Theory
In addition to determining critical
temperatures, it is possible to tell if
your sample is crystalline or partially
crystalline due to the birefringence of
anisotropic crystals within the frozen
matrix
PLM Theory
Crystals are classified as being either isotropic or anisotropic.
Amorphous species have no ordered structure.
Equipment
Necessary equipment includes:
• Polarized Light Microscope
• Liquid nitrogen cooled thermal stage
• Vacuum pump
• Complete systems available at McCrone Microscopes in Chicago, IL
PL Microscopy
Necessary equipment includes:
• Optical Microscope (Olympus BX51)
• Bertrand Lens
• Long working distance objectives
• Polarizer and analyzer
• Condenser extension lens
What have we learned?
Thermal analysis studies (DSC/Freeze-Dry microscopy have given us several key pieces of information:• Tells us if the system is amorphous, crystalline,
or partially crystalline
• Tells us our critical temperatures (Tg’, Te)
• Tells us if we need to anneal the system and approximately what those conditions are
What have we learned?
Thermal analysis studies allow us to take an empirical approach to lyophilization cycle development as opposed to using trial-and-error which has been the dominant means of development in the past. These techniques also allow us to better troubleshoot problem cycles and formulations