Important Parameters and

Basics in Lyophilisation
Process of Lyophilisation
 Phases of Matter
Solids
Molecules with both orientation and
positional order, and are held to each
other strongly.
At high pressures and low
temperatures, the substance is in the
solid phase Phase diagram showing the triple point of water at 0.01°C, 0.00603
atm. Lyophilization is take place below the triple point
Liquid
Molecules with no orientation and
positional orders, but are held together
by weak intermolecular forces
Gas:
No ordering, no intermolecular
attraction
 Solids and Types
Two types, based on
• Atomic arrangement,
• Binding energy,
• physical & chemical properties
1.Crystalline
Particles of crystalline solids are arranged in a regular
repeating pattern. The repeating particles form a
geometric shape called a crystal.
2. Amorphous
Amorphous means “shapeless.” Particles of amorphous
solids are arranged more-or-less at random and do not
form crystals
A change in the coordination number and bonding is
likely to result in important differences in the physical
properties of amorphous and crystalline phases of the
same material.
 Crystalline solids

Atoms in crystalline solids arrange in 3D repeating

pattern known as lattice.
Lattice system:
Triclinic, Monoclinic, Orthorhombic, Tetragonal,
Hexagonal and cubic
Lattice Energy: The energy that is lost when
separated ions (gaseous), positive or negative ions
come together to form a solid ionic compound
 Amorphous Solids
• Non-crystalline solid is a solid that lacks the long-range order that is characteristic
of a crystal.
• constituent particles of matter are arranged in a random manner
• Amorphous materials have an internal structure made of interconnected structural
blocks. Whether a material is liquid or solid depends primarily on the connectivity
between its elementary building blocks so that solids are characterized by a high
degree of connectivity whereas structural blocks in fluids have lower connectivity
• According to the temperature, the amorphous regions can be either in the glassy or
rubbery state.
• The hard or brittle state is called Glassy state.
• The soft flexible state is called rubbery state.
Differences between Crystalline and Amorphous
Solids
Property Crystalline
Characteristic Particles (atoms, ions, or molécules) are
Geometry definitely and orderly arranged
Melting Point Sharp melting point, i.e., it changes into
liquid state at a definite temperature.
Isotropy and Crystalline solids show these physical
Anisotropy: properties different in different directions.
Therefore crystalline solids are called
anisotropic. The anisotropy itself is a strong
evidence for the existence of orderly
molecular arrangement in crystals.
Amorphous
Do not have characteristic geometry.
No sharp melting point. glass is heated, it softens and then
starts flowing without undergoing any abrupt or sharp change
from solid to liquid state. Therefore, amorphous solids are
regarded as “liquids at all temperatures”.
The properties of amorphous solids, such as, electrical
conductivity, thermal conductivity, mechanical strength,
refractive index, coefficient of thermal expansion etc. are
same in all directions. Such solids are known as isotropic.
Gases and liquids are also isotropic.
 Differences between Crystalline and
Amorphous Solids
Property Crystalline Amorphous
Cooling curve: while crystalline solids show two breaks Amorphous solids
in cooling curve. In the case of crystalline show smooth
solids two break points ‘a’ and ‘b’ cooling curve
appear. These points indicate the
beginning and the end of the process of
crystallization. In this time interval
temperature remains constant. This is due
to the fact that during crystallization
process energy is liberated which
compensates for the loss of heat, thus the
temperature remains constant.
Cutting clean cleavage Irregular cut, due to
conchoidal fracture
on cutting with a
sharp edged tool.
 What Occurs in the Freezing Phase?
• Ice nucleation : Nucleation with Exotherm at super cooling temperature

• Ice Crystal Growth : Separation of ice and crystal growth during freezing

• Solute Concentration : Solute get concentrated due to separation of ice crystals

• Formation of Glasses : Frozen solute or water in non-crystallized state

• Solutes Crystallize : Pure crystals

• Lyotrophs: Mixture of amphiphilic solutes and ice crystal forms intermediate phase of liquid and
Solid.

• Formation of Eutectics : Mixture of ice crystals and solute crystals forms eutectic

• Formation of Glass : Mixture of ice crystals and amorphous solute forms glass
 Nucleation
Nucleation is typically defined to be the process that determines how
long we have to wait before the new phase or self-organised structure
appears.
Explanation:
Due to Density fluctuations from Brownian motions in the supercooled
liquid water and water molecules they form clusters almost same
molecular arrangement like ice crystals but this process is energetically
unfavourable for these clusters and they will breakup rapidly.
These nuclei grow both in number and size at lower temperature once
critical mass of nuclei is reached ice crystallisation occurs rapidly in
entire system.
Nucleation is stochastic, describes something that was randomly
determined.
Primary Nucleation and Secondary nucleation:

The time until the appearance of the first crystal is

called primary nucleation time.

Secondary nucleation: Growth of crystal nuclei

produced from a pre-existing crystal
 Homogeneous nucleation
Homogeneous nucleation, which is not influenced in any way by solids.
These solids include the walls of the Vial and particles of any foreign
substance.

Homogeneous nucleation rarely occurs in practice due to the high

energy necessary to begin nucleation without a solid surface to catalyse
the nucleation
 Heterogeneous nucleation
This occurs when solid particles of foreign
substances cause an increase in the rate of
nucleation that would otherwise not be seen
without the existence of these foreign particles.
 Crystallization
Crystallization is the process by which a solid forms,
where the atoms or molecules are highly organized into
a structure known as a crystal. Some of the ways by
which crystals form are precipitating from a solution,
freezing, or more rarely deposition directly from a gas.
Crystallization occurs in two major steps
nucleation,
crystal growth,
 Supercooling
Supercooling, is the process of lowering the temperature of a liquid
below its freezing point without it becoming a solid.
Explanation:
A liquid crossing its standard freezing point will crystalize in the
presence of a seed crystal or nucleus around which a crystal structure
can form creating a solid. Lacking any such nuclei, the liquid phase can
be maintained all the way down to the temperature at which crystal
homogeneous nucleation occurs. Homogeneous nucleation can occur
above the glass transition temperature, but if homogeneous nucleation
has not occurred above that temperature, an amorphous (non-
crystalline) solid will form.
 Degree of Supercooling

Equilibrium freezing temperature = Tf Tf

Nucleation temperature = Tn.

The exothermal nature of Degree
of
crystallisation leads to an increase Super
cooling
of product temperature up to Tf, and
thus stops secondary nucleation.
The temperature difference
between Tf and Tn is known as Tn
degree of supercooling and was
found to be as high as 30°C or more
in a GMP manufacturing facility with Typical product temperature profiles during uncontrolled
low particulate environment nucleation indicated by thermocouples placed centre
bottom within the vial.
 Types of Super cooling
• Global Supercooling: Entire liquid volume exhibits a similar level of
super cooling.
• Local Super cooling: Only small volume of liquid super cooled.
• Freezing time: The time from the complete ice nucleation to the
removal of latent heat.
 Parameters impacted by Supercooling
• Ice morphology (Size and shape of ice crystals )
• pores and cavities formation.
• Drying time
• Product resistance
How?
 How controlled
Modifying cooling rate by 8. Vacuum- induced surface
1. Shelf –ramped freezing freezing
2. Precooled shelf method 9. Depressurization technique
3. Annealing 10. Addition of ice nucleating
agents
4. Quench Freezing
11. Non-aqueous cosolvents
5. Directional Freezing
12. Pre-treatment of Vials
6. Electro freezing
7. Ultrasound controlled
nucleation
 Ice Structures in Aqueous Solutions

Regular Dendritic Ice Irregular Dendritic Ice Spherulitic Ice

Increasing rate of cooling

Freezing Induces Heterogeneity
 Liquid crystals
• Phase that exists between solid and liquid, Liquid crystals are
of
• Thermotropic LCs exhibit a phase transition into the
liquid-crystal phase as temperature is changed.
• Lyotropic LCs exhibit phase transitions as a function of both
temperature and concentration of the liquid-crystal
molecules in a solvent (typically water).
• Metallotropic LCs are composed of both organic and
inorganic molecules; their liquid-crystal transition depends not
only on temperature and concentration, but also on the
inorganic-organic composition ratio.
• Liquid crystal, substance that blends the structures and
properties of the normally disparate liquid and crystalline solid
states
 Lyotrops and Thermotrops transition
Lyotrops form as a function of concentration
and temperature, During freezing solution
temperature lowers , solvent crystalizes and
solute concentration rises according to
temperature which favours the formation of
intermediate Lyotropic liquid crystal phase.
It is common behaviour of materials composed of
amphiphilic molecules upon the addition of a
solvent. Such molecules comprise a water-loving
hydrophilic head-group (which may be ionic or
non-ionic) attached to a water-hating,
hydrophobic group.
The micro-phase segregation of two incompatible
components on a nanometer scale results in
different type of solvent-induced extended
anisotropic arrangement, depending on the
volume balances between the hydrophilic part
and hydrophobic part. In turn, they generate the
long-range order of the phases, with the solvent
molecules filling the space around the
compounds to provide fluidity to the system.
 Transitions of Nematic and Smectic liquid
crystal
• Not all molecules have liquid crystal phases. Water molecules, for example, melt directly from solid
crystalline ice into liquid water.
• As temperature decreases, matter tends to evolve from highly disordered states with continuous
symmetries toward ordered states with discrete symmetries. This can occur through a sequence of
symmetry-breaking phase transitions
• As a substance in the liquid state is reduced in temperature, rotational symmetry breaking creates the
nematic liquid crystal state in which molecules are aligned along a common axis. Their directors are all
nearly parallel.
• At lower temperatures continuous translational symmetries break into discrete symmetries. There are
three independent directions for translational symmetry. translational symmetry is broken along only one
direction, the Smectic liquid crystal is obtained when continuous .
• At temperatures sufficiently low to break continuous translational symmetry in all directions, the ordinary
crystal is formed.
 Annealing
•Annealing is the process of warming a
sample above it’s glass transition
temperature (but below the eutectic
and/or ice melting temperature)and
allowing the glass to relax and crystallize
(mannitol and glycine are good
examples).
 Advantages of Annealing
• Complete crystallization of drug and excipients.
• Uniform pore size distribution
• Improvement of sublimation rate to reduce lyophilization time.
• Reduction of products reconstitution time
• Enchantment of products stability
 Critical temperatures

Every formulation has a critical softens to the point of not being able
temperature, below which it should be to support its own structure.
cooled for complete solidification and • Teu - Eutectic temperature, this is the
maintained below during primary temperature at which the solute
drying in order to prevent processing material melts, preventing any
defects. structure forming after the solvent has
To design a freeze drying cycle on a been removed.
rational basis such information should • Tg’ - Glass transition, the temperature
be identified . of the frozen material changes from a
• Tc - Collapse temperature, this is the brittle to flexible structure.
temperature at which the material
 Critical temperatures
• For crystalline systems, the maximally tolerable temperature is the eutectic
temperature (Teut) which is commonly determined by Differential Scanning
Calorimetry (DSC). Eutectic temperatures are high relative to glass
transitions and processing of such formulations is easy.
• For amorphous systems, the critical temperature is represented by the glass
transition temperature of the maximally freeze concentrated solute (Tg’) or
the collapse temperature (Tc).
• Tc values are many times reported higher (2-5°C) than their corresponding
Tg’ values. Here, Tc represents the higher and more relevant temperature for
the optimization of freeze-drying cycles and formulations. Tc determined by
Freeze dried microscopy.
 Methods to determine Collapse - Freeze drying
microscopy:

Freeze drying microscopy sample prep Use of plane polarised light

 Novel Techniques – Impedance
Pioneered by the late ProfLouis Rey
• Comparatively simple technology
• Comparison of changes in temperature
between sample and reference material
(water or other solvent e.g methanol)during DTA
cooling and warming processes assembly

• Typically requires 2‐4ml of sample but

Sample heating
depends on equipment design block

Zsin
impedance
probe
 Glass transition temperature
• The temperature at which the transition in the amorphous regions between the glassy and
rubbery state occurs is called the glass transition temperature (or)
• The Temperature below which material is hard and brittle and above which it is soft called
glass transition temperature (Tg).
 3 Methods To Determine Glass Transition
Temperature

• Differential Scanning Calorimetry (DSC)

• Thermo Mechanical Analysis (TMA):
TMA estimates the glass transition temperature of a material by
tracking its coefficient of thermal expansion (CTE).
• Dynamic Mechanical Analysis (DMA)
 Tg’
DSC and mDSC
Small volume encapsulated
in metal pans
• Freeze rate
• Scan rates
• Empty pan reference
• Three thermal events
• Tm, Teu, Tg(‘)

Teu T melt
 Dynamic Mechanical Analysis DMA
DMA involves applying a periodic stress
(frequency/frequencies) to a sample and then
measuring the resultant strain.
A dynamic stiffness can then be calculated
and the damping (tan delta). When a sample
passes through its Tg the tan delta increases
dramatically.
 Importance of critical temperature
Freeze drying above the product critical Freeze drying too far below the product
temperature critical temperature
Loss of Physical structure Poor efficiency
Incomplete drying(High moisture content) High running cost

Decreased Solubility Longer cycles than necessary

Reduced activity and/or stability
 Melting during lyophilization
• Improper Freezing of the product.
• Proceeding to step secondary drying without completion of primary
drying.
• Completion of secondary drying step without proper drying.
Mass transfer resistance
 Mass transfer resistance
Mass transfer is the net movement of mass from one location, to another.
Mass transfer resistance is a Parameter which governs the relationship between independent variables
1. Shelf temperature,
2. Chamber pressure.
Dependent variables
1. Ice sublimation rate
2. Product temperature
MTR = (Po – Pv)/ m/Ap
m= Sublimation rate
Ap = cross section area of the product in vial
Po = Equilibrium vapour pressure of ice at temperature of sublimating ice
Pv= pressure in chamber
• Lower MTR higher sublimation rate.
Any Questions?
Thank you