Universita’ degli Studi di Milano

Corso di Laurea in Chimica e Tecnologia

Farmaceutiche
Fabbricazione Industriale dei Medicinali - 8 CFU

Prof. Andrea Gazzaniga

slides Dott. Carlo Vecchio

PROCESSO DI LIOFILIZZAZIONE
06/01/2010 1
 FREEZE-DRYING PROCESS
Principle and Practice

Carlo VECCHIO

Pharmaceutical Technologies
& Development

06/01/2010 2
 INTRODUCTION

Lyophilization, or freeze drying, is a

process in which the solvent (usually
water) is:

- first frozen and then

- removed by sublimation

in a vacuum environmental.

06/01/2010 3
 INTRODUCTION

Freeze drying is a widely used method for

the stabilization of otherwise easily
degraded substances:

- microorganisms

- foods

- biological products and

- pharmaceutical products.

06/01/2010 4
 INTRODUCTION

While the common application of pharmaceutical freeze drying is

in the production of:
- injectable dosage forms.

The process is also used in the production:

- of diagnostics and
- for oral solid dosage forms,
where a very fast dissolution rate is desired.

06/01/2010 5
 INTRODUCTION

Characteristics of the freeze dry process are:

1. minimization of chemical decomposition
(drying takes place at low temperatures)

2. complete dissolution of dried product

(resulting product has a very high surface area)

3. More compatibility with sterile operations

(solution is sterile-filtered before filling vials)

4. precise filling weight

(fill weight control is more precise for liquid)

5. absence of powder
(particulate contamination is minimized).
06/01/2010 6
 PROCESS OVERVIEW

Vials are aseptically filled

with the solution (or
suspension) to be freeze
dried and partially stoppered
with a special rubber closure
that allows to escape the
water vapor.

Then, vial are transferred

under aseptic conditions to
the freeze drier.

06/01/2010 7
 Schematic diagram of freeze drier

06/01/2010 8
 PROCESS OVERVIEW

Trays of product are placed on shelves containing internal

channels allowing circulation of silicone oil or another heat
transfer fluid.

Shelves may be pre-chilled or not.

The tray may have a removable bottom for direct contact

on the shelf eliminating one resistance to heat transfer.

A temperature-measuring device may be placed in some

vials for process monitoring/sequencing.

06/01/2010 9
 PROCESS OVERVIEW

The product is first frozen to a low enough

temperature to allow complete solidification of
the content of each vial.

Then, the chamber is evacuated until the

pressure is less than the vapor pressure of ice
at the temperature of the product.

06/01/2010 10
06/01/2010 11
 PROCESS OVERVIEW
DIAGRAM OF VIAL DURING PRIMARY
DRYING After this pressure is reached,
heat is applied to the shelves
to provide the energy required
for sublimation of ice.

As drying proceeds, a
receding boundary can be
observed in the vial as the
frozen layer decreases in
thickness and the thickness of
partially dried solids increases.

This phase is called primary

drying
06/01/2010 12
 PROCESS OVERVIEW

When the ice is gone, additional drying time

is request to remove water

- adsorbed to, or

- trapped by,

the solid matrix.

This phase is called secondary drying.

06/01/2010 13
PLOT OF PROCESS VARIABLES DURING FREEZE DRY CYCLE

06/01/2010 14
 PROCESS OVERVIEW

When the product is dry, the vials are

stoppered in place within the drier by
hydraulic compression of the shelf stack,
pushing the stoppers to the fully inserted
position.

This occurs either under a full vacuum or by

back-filling the chamber with inert gas

06/01/2010 15
 PROCESS OVERVIEW

The most important objective in the developing a freeze

dried product is to assure the quality requirements as:
- the original chemical or biological potency after
reconstitution
- rapid and complete dissolution
- appropriate residual moisture level, and
- acceptable cake appearance.

This requirements have to be met not only initially but

throughout the shelf life of the product.

06/01/2010 16
 PROCESS OVERVIEW

In addition, however, process conditions should

be chosen to maximize process efficiency.

Of all drying operations, freeze drying is the most

expensive both in:
- the capital investment and
- operating expense.

06/01/2010 17
 PROCESS OVERVIEW

Success in this challenge requires an

understanding of:
- the physical chemistry of “frozen solution”
- heat and mass transfer under conditions
encountered in freeze drying
- temperature and pressure measurement
- process monitoring
- general freeze drying system design
considerations.

06/01/2010 18
 THE FREEZING PROCESS

Freezing is a critical step in the freeze drying process, since the

microstructure (of both ice and solute) formed during freezing
determines both:
- the quality of the final product, and
- its processing characteristics such as the rates of
primary and secondary drying.

It is essential to know the physical events associated with

freezing process-supercooling, ice crystallization (primary),
concentration of the solutes during ice crystal growth and
crystallization of solute (secondary crystallization).

06/01/2010 19
 PHASE DIAGRAM OF WATER

At the triple point

(0.0098°C and 4.58
mm Hg), ice , water
and water vapor
coexist in equilibrium.

Freeze drying takes

place below the triple
point, where water
passes from solid
phase directly to the
vapor phase.

06/01/2010 20
 PHASE DIAGRAM OF WATER
The 4.58 mm Hg refers
to the water vapor
pressure, not the total
system pressure.

Sublimation can occur at

atmospheric pressure as
long as the water vapor
pressure is below 4.58
mm Hg.

This “atmospheric freeze

drying” is the phenomenon that
causes “freezer burn” in home
freezer.
It has been used by the
Eskimos as an effective
method of meat preservation.

06/01/2010 21
 FREEZING OF AQUEOUS SOLUTIONS
Temperature Vs. Time for Freezing of NaCl/water

In the segment ab, the

product temperature
decreases to below the
equilibrium freezing
temperature (Tf) of the
product.

At the point b, nucleation of

ice crystals occurs.
As nucleation and crystal
growth of ice begins at b,
energy is released (the latent
heat of fusion) and the
temperature increases to Tf.
06/01/2010 22
 TEMPERATURE VS. TIME FOR FREEZING OF NACL / WATER

Cooling continues with ice

crystal growing and the
interstitial fluid becoming
more concentrated.

At the point c,
crystallization of
concentrated interstitial
fluid is initiated: an
eutectic mixture of
crystalline NaCl/ice.

A eutectic is an intimate physical

mixture of two or more
crystalline solids that melts as
single pure compound
06/01/2010 23
 TEMPERATURE VS. TIME FOR FREEZING OF NACL / WATER

When eutectic crystallization is

initiated, the temperature of the
product increases to the
eutectic temperature (Te).

After eutectic crystallization is

completed at the point Te, no
more liquid is present and no
changes in microstructure of
frozen system take place.

Then, the product temperature

decrease more rapidly toward
the shelf temperature.

06/01/2010 24
 Phase Diagram of NaCl/water

The line ab represents the

equilibrium freezing
temperature of water (Tf)
as a function of NaCl
concentration.

The line bc represents the

solubility of NaCl in water.

The intersections of lines

at point b is the eutectic
point.

06/01/2010 25
 EUTECTIC TEMPERATURES FOR AQUEOUS
SOLUTIONS OF VARIOUS COMPOUNDS

Citric acid - 12.2°C

Glycine - 3.5°C
Mannitol - 1°C
Sodium acetate - 18°C
Sodium carbonate - 18°C
Sodiun chloride - 21.5°C
Sodium phosphate, dibasic - 0.5°C

06/01/2010 26
 EUTECTIC TEMPERATURE

The eutectic temperature (eutectic is an intimate physical

mixture of two or more crystalline solids that melts as a single
pure compound) is important in freeze drying because
represents the maximum allowable product temperature during
primary drying.

If the product exceeds the Te, drying takes place from liquid
instead of the solid.

However, eutectic behavior is only observed when the solute

crystallizes.

06/01/2010 27
TEMPERATURE VS. TIME FOR FREEZING OF AMORPHOUS SOLUTE

In the most cases, the

solute does not readily
crystallize during freezing.

The first part of curve is the

same, then a secondary
(eutectic) crystallization
does not occur, but a slight
change in slope of the
temperature vs. time curve
is observed at Tg (glass
transition temperature).

06/01/2010 28
 GLASS TRANSITION
(OR COLLASPE) TEMPERATURE

For amorphous system, glass transition temperature

(Tg) corresponds to a change in the viscosity of solution
from a viscous liquid to a glass or an essentially solid
solution of solute in water.

Tg is important for amorphous solute as Te for

crystalline solute. It represents the maximum allowable
product temperature during the primary drying.

If product temperature exceeds the glass transition

temperature, the product will undergo collapse.

06/01/2010 29
DRAWING OF MICROSTRUCTURE FOR CRYSTALLINE AND
AMORPHOUS SOLUTES UPON FREEZING

For the crystalline (a), the

interstitial material consists of a
mixture of eutectic ice and
crystalline solute.
When the ice is removed by
sublimation, a crystalline solid with
very little water is left.

For the amorphous system (b), the

interstitial glassy material must be
rigid enough to support its own
weight after the ice is removed
in order to keep the microstructure
established during freezing.

06/01/2010 30
 PHASE DIAGRAM FOR AN AMORPHOUS SOLUTE

The line ab represents

the freezing temperature
of water as a function of
solute concentration.

Instead of the solute

crystallizing at point b,
the interstitial material
remains as liquid, or
freeze concentrate, and
continues along line b-
Tg.

06/01/2010 31
 PHASE DIAGRAM FOR AN AMORPHOUS SOLUTE

Ice crystals continue to grow, and

the freeze concentrate become
more concentrated and more
viscous.

The family of curves shown by the

dashed lines are iso-viscosity
curves, i.e., combinations of solute
concentration and temperature that
result in the same fluid viscosity.

The solid line is the glass transition

point of amorphous solid as a
function of water content and is itself
an iso-viscosity curve representing a
viscosity of about 1014poise.
06/01/2010 32
 PHASE DIAGRAM FOR AN AMORPHOUS SOLUTE

As freezing proceeds, the freeze

concentrate becomes more
viscous until the system reaches
point Tg and the growth of ice
crystal stops.

At the point Tg (the glass

transition temperature of the
freeze concentrate) the interstitial
fluid changes from a viscous
liquid or rubber to an elastic solid.

The concentration of unfrozen water

in glass is represented by Wg.

06/01/2010 33
 GLASS TRANSITION TEMPERATURE OF
VARIOUS COMPOUNDS

Dextran - 9°C
Ficoll - 19.5°C
Fructose - 48°C
Glucose -40 to 43°C
Sucrose - 32 to 34°C
Maltose -32°C
Trehalose - 29.5°C
Sorbitol -45 to 51°C
Lactose - 32°C
Ovalbumin - 10°C
Gelatin - 8 to 10°C
Polyvinylpyrrolidone -23 to 24°C
Methylcellulose -9°C

06/01/2010 34
 THE PROCESS

The process is conventionally divided into three stages:

Freezing
Cooling the material until completely frozen

Primary drying
Sublimation of ice from product reducing
pressure in the chamber and providing heat to
the product

Secondary drying
Desorption of residual moisture from the product

06/01/2010 35
 PROCESS

The process of freezing involves:

(1) dissolving the drug and excipients in a suitable solvent,

generally water

(2) sterilizing the bulk solution by passing it through bacteria-

retentive filter (0.2 microns)

(3) filling into individual sterile containers

(4) freezing the solution by placing the open container on cooled

shelves in a freeze drying chamber or pre-freezing into
another chamber

(5) applying a vacuum to the chamber and heating the shelves in

order to sublime the water from the frozen state.

06/01/2010 36
 CHARACTERISTICS

The desired characteristics of a freeze-dried

pharmaceutical dosage form include:

(1) an intact cake occupying the same shape and size as

the original frozen mass

(2) sufficient strength to prevent cracking, powdering, or

collapse

(3) uniform color and consistency

(4) sufficient dryness to maintain stability (<2%)

(5) sufficient porosity and surface area to permit a rapid

reconstitution.

06/01/2010 37
 CHARACTERISTICS

And, of course, freedom from contamination

such as:

- micro-organisms (sterile),

- pyrogens (5 Endotoxins Units/Kg), and

- particulates (less than 50 particles of 10 μm per

container and less than 5 particles of 25 μm per
container result)

is an essential attribute.

The desired characteristics can be achieved by

proper formulation of the product and by employing
optimum freeze-drying cycles.
06/01/2010 38
 DEVELOPMENT

The development of a suitable formulation and a freeze-dry

cycle requires knowledge of some basic properties, such as:

- eutectic temperature

- temperature effect on solubility

- thermal properties of the frozen solution

- degree of super-cooling

- heat transfer properties of the freeze-dryer shelves, the

metal trays, the containers and the frozen product

- equipment design and equipment capability.

06/01/2010 39
 DEVELOPMENT A SUITABLE FORMULATION
FORMULATION

In developing a formulation of freeze drying, the

optimal formula will permit the overall cycle to be
carried out in the least amount of time, while
providing a stable and efficacious product which:

- contains a low moisture content,

- undergoes rapid reconstitution, and

- possesses the desired appearance.

06/01/2010 40
 SOLVENTS

The solvent is generally water.

Organic solvents can be added up to 20%:

- to promote wetting and/or solubilization of

the drug

- to reduce the degradation rate of the drug in

water during processing

- to induce crystallization of the drug in the

frozen state.

06/01/2010 41
 ORGANIC SOLVENTS

Organic solvents may be present as a residual

impurity (less than 2% in pharmaceutical
powder).

Most commonly organic solvents include:

- ethanol,
- n-propanol,
- n- and tert-butanol,
- iso-propanol,
- ethyl acetate and
- dimethyl carbonate.

06/01/2010 42
 ORGANIC SOLVENTS

The presence of organic solvents affects the efficiency of

the freeze-drying process:

- with frozen liquid residue (e.g., tert-butanol) the

products dry mainly by direct sublimation and the
drying process is usually rapid

- with unfrozen liquid residue (e.g., ethanol, iso-

propanol) the products dry more slowly and there is a
risk of meltback or collapse.

Solvent melting points:

- tert-butanol, 25.5°C
- ethanol, - 110.5°C
- iso-propanol, - 85.8°C.
06/01/2010 43
 ORGANIC SOLVENTS

Moreover, when lyophilizing organic-aqueous systems, the

freeze-dryer needs to be modified to handle such solvents:

- the condenser have to be linked to a refrigerated

solvent trap, otherwise an extra condenser is needed,
more effective as refrigerating capacity

- the unit system must be equipped with selected

solvent-resistant gaskets.

Although non-aqueous solvents have certain useful properties, their use

is limited because they are not as easy to handle as water and give
serious toxicity problems for the products administered parenterally.

06/01/2010 44
 ADDED SUBSTANCES

Bulking agents
For very low dose products, the minimum practical fill volume
results in a solute concentration so low that the dried product
layer formed does not have sufficient mechanical strength to
withstand the force of flowing water vapor during primary
drying: the dried product is “blown” all over the drier.

Too low an initial solids concentration may also result in dry

cake of fluffy consistency, that fails to cohere and which is
extremely hygroscopic.

The need for a suitable of bulking agent is often indicated to

provide the necessary bulk and desired characteristics.

Generally, bulking agent should be above 2% and not exceed

30%, with 5 to 15% content being optimum.

06/01/2010 45
 ADDED SUBSTANCES

Bulking agents
Most commonly used bulking agents include:
- mannitol,
- lactose,
- dextran,
- sorbitol,
- sucrose,
- dulcitol,
- gelatin,
- bovine serum albumin,
- glycine,
- polyvinylpyrrolidone,
- sodium chloride,
- ficoll 70 (a branched polymer synthesized from sucrose and epichlorohydrin).

06/01/2010 46
 ADDED SUBSTANCES

Rigidizers or collapse temperature modifiers

If a substance is vulnerable to collapse, a rigidizer such as
glycine or mannitol may need to added.
It is important to point out that dilution with a bulking agent is
also a way to avoid meltback or collapse.
One may also try to raise the collapse temperature of a
formulation.

Potential collapse temperature modifiers with the respective

collapse temperature are:
- dextran (-10°C),
- ficoll (-20°C),
- human serum albumin (-9.5°C),
- gelatin (-8°C),
Sugar may contribute towards collapse because they do not
crystallize.
06/01/2010 47
 ADDED SUBSTANCES

Cryoprotectant agents
If the damage during freezing is a problem, a cryo-protective
agent such as sugars (sucrose, trehalose) or bovine serum
albumin may be added.

Buffer agents
If degradation is a risk during freezing due pH change, buffers
(acetate, citrate, phosphate, glutamate) may be added to
maintain the pH in a region desirable for the stability of a drug.

Tonicity adjusters
Tonicity adjusters to avoid hemolysis or crenation of red blood
cells in isotonic solution, e.g., NaCl 0.9% and dextrose 5%, the
cell maintain their “tone”
(hypotonic solution < 280 mOsm/kg; Hypertonic solution >360 mOsm/kg).
06/01/2010 48
 ADDED SUBSTANCES

Antioxidants
They prevent oxidation of the drug (sodium bisufite and
metabisulfite, tocopherols, butylhydroxyanisole, ascorbic
acid,)

Preservatives
They avoid risk of contamination in containers intended for
multiple injections (benzalkonium and benzethonium
chloride, benzyl alcohol, methyl and propyl parabens,
chlorobutanol)

Surfactants
They help solubilization (poloxyethylene sorbitan
monooleate, sorbitan monooleate).

06/01/2010 49
 ADDED SUBSTANCES

Especially for potent drugs, adhesion of the active

substance to the glass surface is experienced.

In such cases, the vials may be internally coated

with silicone.

The depth of fill in a container is critical.

While this depends on the volume of the container, a

rule of thumb has been 1 to 2 cm in depth, but never
exceed one-half the capacity of the container.

06/01/2010 50
 FREEZING OF AN AQUEOUS SOLUTION

Freezing is the reduction of the temperature of the product

to induce crystallization of the bulk of the contained water
before primary drying.

The freezing process can have a very important effect on

the appearance and the properties of the final product.

The crystal size formed during freezing can significantly

affect the dissolution rate of the dried material.

06/01/2010 51
 FREEZING OF AN AQUEOUS SOLUTION

Generally the slower rate of freezing, the larger the ice

crystal that form.

Slow freezing can subject the drug to concentrated

solutions for longer periods of time, permitting maximum
chance for crystal growth.

Usually, the small crystals formed during rapid freezing

result in a product which has a fast solution rate.

A fast ice growth also help to prevent the denaturation of proteins (if
present) which may result from prolonged exposure to strong
concentrations of salts because of slow ice growth.

06/01/2010 52
 FREEZING OF AN AQUEOUS SOLUTION

On the other hand, the main

pores in the solid residue after
freeze-drying are those left by
the sublimation of pure ice and
they form the principal
channels for the escape of
vapour.

During sublimation, very small

ice crystals form smaller pores
and pathways, which are more
restrictive to vapour flow than
those formed after slower
freezing.
06/01/2010 53
 FREEZING OF AN AQUEOUS SOLUTION

Freezing of the solution to be freeze-drying is most

conveniently accomplished in the chamber to be
employed for drying (internal freezing), by placing the
containers of solution (tray, vials ampoules) on a shelf
that is cooled by a circulating refrigerant, such as Freon,
Cellosolve or thrichloroethynene)

It is sometimes economical to carry out the freezing in a

separate installation the frozen material is then
transferred to the shelves of the freeze-dryer.

06/01/2010 54
 FREEZING OF AN AQUEOUS SOLUTION

Freezing in a separate installation is usually done for one of

the following reasons:
- to store unstable material until a complete freeze-dryer
load is ready
- to achieve maximum utilization of the freeze-dryer for
drying
- successive freezing of layers of different fluids is more
easily carried out
- filling time into the containers can be prolonged if filling
machine capacity is limited.

06/01/2010 55
 FREEZING OF AN AQUEOUS SOLUTION
However, freezing products outside the chamber has certain
disadvantages:
- increased chance of contaminating the product because of the
need for extra handling
- possibility that the frozen product may partially melt during
transfer to the freeze-dryer
- moisture from the atmosphere may condense and form frost on
the containers of the frozen product or on the necessarily pre-
cooled shelves of the freeze-dryer while it is being loaded.
This frost:
-makes loading difficult,
-increases the quantity of ice to be sublimed in the early stages of
primary drying,
-may cause the containers to make poor thermal contact with the
shelves in the freeze-dryer

- increased risk of breakage of the containers and loss of product

- increased labour is required, increased floor area is needed and
more electrical power is consumed.
06/01/2010 56
 FREEZING OF AN AQUEOUS SOLUTION

Thus, in general, these are good reasons for preferring an

installation in which freezing is carried out in the freeze-
dryer.

In any case, where practicable, the depth or thickness of

the material should be minimized for short freezing and
freeze-drying cycles.

A typical thickness is 10-15 mm.

06/01/2010 57
 STAGES OF FREEZING

The initial freezing process is of critical importance since

it will influence the pattern of the sublimation phase.

The latter phase must occur from the solid state

throughout the cycle.

Thus, appropriate cooling cycles must be determined in

order to obtain an appropriate structure of the frozen
mass, which is a function of the rate of freezing and the
final freezing temperature.

06/01/2010 58
 STAGES OF FREEZING

In general, the freezing of an aqueous binary solution

consisting of a solute in water may be considered to occur
as follow:

- assuming the solubility of the solute is high enough

so that it is not deposited on cooling, ice crystals first
form at a temperature usually below 0°C as a
consequence of supercooling effect

- as the ice crystals form and grow, the remaining

solution ("interstitial fluid") becomes more and more
concentrated in the solute

06/01/2010 59
 FREEZING OF AN AQUEOUS SOLUTION

- if the solute forms a true eutectic with water,

an eutectic phase - consisting of finely divided
crystals of the solute and ice - crystallizes out.

The highest temperature at which the whole

system becomes solidified is termed the
maximum temperature of complete
solidification, Tcs.

This is the temperature at which no liquid

states exist in the product and it is a state
that must be achieved if a solution is to be
considered a freeze-dried.

06/01/2010 60
 FREEZING OF AN AQUEOUS SOLUTION

- If the solute does not crystallize (i.e., does

not form a true eutectic), it is transformed
into a rigid glass when the system is brought
below the glass transition temperature of the
amorphous phase (Tg).

This parameter describes the temperature at which there

is a fundamental change in the physical properties of the
product, which does not reflect a change in state, but
rather a change in the macromolecular mobility: below
the Tg product mobility is severely restricted.

The amorphous phase consists of

uncrystallized solute and uncrystallized water.

06/01/2010 61
 THERMAL TREATMENT

Compounds that do not form true eutectic are

difficult to dry successfully because during the
drying process, as the product temperature rises,
the glassy structures soften and the matrix
collapses making it necessary to reject the batch.

It is often possible to crystallize a solute system that

tends to remain amorphous or to promote the
growth of the ice crystals by following a freezing
procedure referred to as thermal treatment (or
tempering or annealing).

06/01/2010 62
 THERMAL TREATMENT

Thermal treatment consists of:

- first freezing the product at low enough temperature

- warming it gradually to a predetermined temperature well

above the glass transition temperature

- holding there for a sufficient period of time to allow any

metastable state to crystalize out,

- and then cooling it again to suitable temperature before

initiating primary drying .

06/01/2010 63
 TRANSITIONS OF PHASE

The phase transitions in the frozen state occur and

influence the properties of the dried product.

A better understanding of the transitions which

occur during the warming of frozen systems would
permit better control and optimization of freeze-
drying cycles in order to provide a finished product
of higher quality.

06/01/2010 64
 TRANSITIONS OF PHASE
Differential Scanning Calorimetry
(DSC) can be usefully employed to
determine the phase transitions in the
frozen systems.

In DSC, the temperatures of the

sample and a suitable reference
(which does not undergo any
transition in the temperature range of
interest) are compared as both are
heated or cooled simultaneously and
at the same rate.

The aim is to keep the temperatures

of the sample and reference equal.

06/01/2010 65
 TRANSITIONS OF PHASE
When the sample undergoes a transition, its temperature
differs from that of the reference and heat either flows to or
away from the sample.

If heat flow occurs toward the sample, then it must have

undergone an endothermic change and viceversa. The heat
flow is displayed as a peak in the thermogram.

Second order transitions (e.g., glass transition), involving only

a change in the heat capacity of the sample, are displayed as
a change in the slope of the baseline.

The output signal is proportional to the difference in energy

needed to keep the sample and reference temperatures equal.
Thus, the DSC monitors thermal events quantitatively as well
as qualitatively.

06/01/2010 66
 TRANSITIONS OF PHASE

Transitions such as melting involve an

equilibrium and therefore are reversible.
A reversible thermal event is one which following warming
and recooling, the thermal event will reoccur when
warmed again.

Transitions such as crystallization and

polymorphic changes may not be in
equilibrium and thus are irreversible.
An irreversible thermal event is one which following
warming and recooling, the thermal event will not reoccur
when warmed again.

06/01/2010 67
 THERMAL TREATMENT

Gatlin and De Luca

observed some
features of the low
temperature DSC
thermograms of some
antibiotic solutions,
which provide a base
for thermal treatment
them to obtain
crystals of the drug.

06/01/2010 68
 THERMAL TREATMENT

The thermogram shows:

- a first endothermic shift
occurring at -20°C (Point A)
- an irreversible exotherm
beginning at -11°C (Point B)
(which can be interpreted as
representing the crystallization of
the solute during warming)
- melting of ice
(endothermic shift)
beginning at -4°C (Point E)

06/01/2010 69
 THERMAL TREATMENT

Considering the portion of the

curve beginning just below the
initial endotherm and to just above
the irreversible exotherm, if the
frozen solution is warmed to just
beyond the exotherm but below
-4°C (say -6°C) and then the
system is recooled to -25°C, upon
rewarming, the thermogram in
dashed line is obtained.

06/01/2010 70
 THERMAL TREATMENT

Material submitted to thermal treatment exhibits:

- birefringence under optical microscopy
- defined shape by SEM
- X-ray diffraction pattern consisting of peaks of
various intensity.

All of these are indications of a crystalline structure.

06/01/2010 71
 X-RAY DIFFRACTION SPECTRA OF FREEZE DRIED
CEFAZOLIN

The upper tracing

is of material dried
without thermal
treatment.

The lower is of
material which was
thermally treated
before drying.

06/01/2010 72
 INDUCTION TO THE CRYSTALLIZATION

Induction to the crystallization of

pharmaceutical compound can be also
accomplished by:
- humidity treatment
- excipient addition
- solvent addition.

06/01/2010 73
 CRYSTALLIZATION OF ACTIVE COMPOUND

Crystallization of active compound is desirable for the following

reasons:
- firstly, the stability of a drug is usually greater if it is present
in the crystalline form than the amorphous form
- secondly, the crystalline drug can be dried at a higher
temperature than the amorphous
- thirdly, a crystalline solid can be dried faster than the
corresponding amorphous form because of the higher
melting temperature

Increase of the sublimation rate of the ice, thereby decrease the

drying time.
On the contrary, a advantage is a possible decrease in solubility
or an increase in reconstitution time.
06/01/2010 74
 CRITICAL TEMPERATURES

Before designing an optimum freeze drying cycle for a

solution, two critical temperatures need to be determined:

- Tcs or temperature of complete solidification

It is the highest temperature at which any liquid state
ceases to be present during cooling

- Tim or temperature of incipient melting

It is lowest temperature at which liquid state begin to appear
during warming.

06/01/2010 75
 CRITICAL TEMPERATURES

These two values are used in the freeze drying

cycle as follow:

- Tcs is the minimum temperature at which the

solution must be cooled to have a cake
completely frozen

- Tim is the maximum temperature at which

the product must be kept during sublimation to
avoid melting or other damages

06/01/2010 76
 METHODS FOR STUDYING FREEZING
CHATACTERISTICS

The methods currently used to study the freezing drying

characteristics of solutions are :

-Thermal Analysis
(DSC cooling and warming)

- Electrical Resistance
(mobility of the ions)

- Freezing/Freezing Drying microscope.

06/01/2010 77
 METHODS FOR STUDYING FREEZING
CHATACTERISTICS

Thermal Analysis
It may be usefully employed:
- DSC cooling thermograms may be used to
determine the temperature of complete
solidification (Tcs)
- DSC warming thermograms may give a direct
measurement of Tg and the eutectic temperature

06/01/2010 78
 METHODS FOR STUDYING FREEZING
CHATACTERISTICS

Electrical Resistance
This method involves the simultaneous monitoring of
resistance and temperature of a frozen sample.

Resistance measurements offer an advantage over thermal

analysis in the estimation of Tcs and Tim because
conductance (and, therefore, resistance) in solution
depends on the mobility of the conducting species.

When a solution is frozen, irrespective of whether it is in a

glassy or crystalline form, its resistance increases because
of the reduced mobility of the ions.

06/01/2010 79
 Electrical Resistance

06/01/2010 80
 METHODS FOR STUDYING FREEZING
CHATACTERISTICS
Freezing/Freezing Drying microscope
The freezing microscope, unlike all the above
methods, allows direct observation of the sample
being frozen or warmed.

Typically, the solution is trapped between two glass

slides or cover slips, and cooled (or warmed) while
being observed.
A vacuum pump and a temperature control unit may be
connected to the slide and the whole device becomes a freeze-
drying microscope which permits observation of the entire
lyophilization process.

This device has found very useful application in lyophilization,

especially in the study of collapse in freeze-dried preparations.

06/01/2010 81
 SUPERCOOLING AND DEGREE OF
CRYSTALLIZATION

When an aqueous solution is cooled, the water in the

solution almost undergoes some degree of supercooling
before crystallizing out as ice.

This means that no ice forms at the thermodynamic or

equilibrium freezing point.

The ice usually nucleates and crystallizes after

supercooling below the equilibrium freezing point.

The degree of supercooling depends on:

- the nature of the solutes
- the freezing procedure
- the container, and
- the presence of particulate matter.
06/01/2010 82
 SUPERCOOLING AND DEGREE OF
CRYSTALLIZATION

The degree of supercooling is important in determining the

size of ice crystals formed: a higher degree of supercooling
produces smaller ice crystals.

The size of the ice crystals determines the size of the pores
(or channels) created during ice sublimation and
determines the surface area of the porous solid produced
by the sublimation process.

Thus, the degree of supercooling affects:

- the rate of sublimation (large ice crystals create large
pores, leading to rapid sublimation), and
- the rate of secondary drying (large ice crystals create a
small surface area, leading to slow secondary drying).

06/01/2010 83
 DEGREE OF SUPERCOOLING

According to Jennings:
- a high degree of supercooling leads
to homogeneous network of fine pores
- a low degree of supercooling yields
a heterogeneous plug structure with a
thick skin on the surface and a very
fine pores on the bottom.

The presence of a glaze on the

surface of the product (which may
result from slow ice growth from the
bottom of the vial) may retard the
sublimation of ice from product.

The presence of a fine

structure/coarse structure boundary
may be responsible for collapse at the
boundary.
06/01/2010 84
 DEGREE OF SUPERCOOLING

Thus, in general, a moderate/high degree of supercooling

(10-15°C) is desirable.

Most important, the degree of supercooling should be

uniform, both within a given vial and within the entire batch
of vials.

In practice, this is not so easy to obtain, because of the

variations in the cooling process and in the product.
The following is a tentative methodology to obtain a
moderate degree of uniform supercooling:
- Minimize solution depth
- Moderate shelf/solution temperature difference (~20C).

06/01/2010 85
 DEGREE OF SUPERCOOLING

If necessary, a tempering process may be followed to assure

uniformity in degree of supercooling.
This procedure involves first cooling all product to a temperature lower
than 0°C but higher than the temperature that will cause nucleation
and crystallization (typically, -5° to -10°C).
The shelf temperature is then decreased (typically -20° to -30°C) to
induce crystallization of ice in all containers.
When sufficient time has elapsed to result in ice crystallization in all
containers, the shelf temperature is lowered below the temperature of
complete solidification.

The final product temperature during freezing is typically about

-40°C.

Once the system is completely solidified, primary drying may

begin.
06/01/2010 86
 PRIMARY DRYING

Primary drying is a problem in coupled heat and mass

transfer, and heat and mass transfer issues must be
recognized to achieve process optimization.

Mass transfer may be discussed in terms of resistance to the

flow water vapor through the various mass transfer barriers
(which are partially dried product, stopper openings, chamber-
to-condenser pathway).

The heat is supplied from shelf (by means of circulating fluid)

and is transferred mainly by conduction through the frozen
matrix to the sublimating front.

06/01/2010 87
 RESISTANCE TO THE FLOW

The resistance to the flow may be defined as the ratio

- of driving force for sublimation expressed by the pressure
difference across the barrier
- to the flow through the barrier represented by the sublimation
rate.

Pressure difference across the barrier

Resistance to flow = -------------------------------------------------------------
Sublimation rate

06/01/2010 88
 Typical pressures in primary drying and
various resistances

Pi and Pcd are determined by

the vapor pressure of ice at
the front of sublimation and
the condenser surface,
respectively.

The resistance of dried

product typically accounts
for over 90 % of the total
resistance to mass transfer

06/01/2010 89
 RESISTANCE OF THE DRIED PRODUCT

The resistance of the dried product accounts for over 90% of

the total resistance and depends:
- the nature of the product
- the cross-sectional area of the product
- the thickness of dried product.

Dried product resistance decreases as:

- vials diameter increases
- product thickness decreases
- solute concentration decreases.

It also generally decreases:

- as the temperature of frozen product approaches the eutectic temperature or
the glass transition temperature
- if larger ice crystals are produced by a tempering process during freezing.

06/01/2010 90
 SUBLIMATION RATE

The sublimation rate per vial (dm/dt) may be expressed in

terms of driving force for transport of water vapor from ice-
vapor interface to the chamber (po - pc):

dm/dt = (po-pc) / (rp-rs)

where:
po = equilibrium vapor pressure of ice at the
temperature of the frozen product
pc = pressure in the drying chamber
rp = dried-product resistance
rs = stopper resistance.

06/01/2010 91
 SUBLIMATION RATE

Because po - which is equilibrium vapor pressure of ice at the

temperature of the frozen product - increases exponentially with the
temperature, the sublimation rate increases dramatically as the
product temperature increases

About 13% for each 1°C increase in temperature.

The key to successful drying is to remove the water vapor from the
frozen cake without allowing liquid water to form.

In other words, operate conditions at or just below the eutectic or

collapse point.

06/01/2010 92
 EUTECTIC MELTING AND COLLAPSE

Eutectic melting involves the melting of the eutectic

phase and therefore occur throughout the frozen
matrix. It results in drying by evaporation of water from
liquid phase.

Collapse is essentially the amorphous system analog of

a eutectic melt.
If the product temperature rises above the collapse
temperature, the amorphous solute-water system gains
sufficient fluidity to undergo viscous flow once the ice in
that region has sublimed.
Thus, the dried region adjacent to the ice will “flow” and
lose the structure.

06/01/2010 93
 EUTECTIC MELTING AND COLLAPSE

The collapse temperature and the glass temperature are

closed related (for practical purposes are identical)

Methods of preventing collapse include:

- addition of a solute which crystallizes, or one with a high
collapse temperature
- thermal treatment to cause the metastable water to
crystallize out
- careful control of freeze-drying conditions.

06/01/2010 94
 HEAT TRANSFER

The transfer of heat to the product is generally done by means

of circulating a fluid through the shelf on which vials
(ampoules or trays) are placed.

Thus the heat is supplied from below and transferred mainly

by conduction through the frozen matrix to sublimating front.
It has to pass through four barriers:
- the shelf
- the tray (if present)
- the glass vial
- the frozen solution.

The temperature of the frozen interface determines the vapor

pressure of the ice and the driving force for sublimation.
This temperature is different from that of the supporting shelf, or the
product container, as a temperature gradient is needed to ensure the
flow of heat to the product.
06/01/2010 95
 Temperature profile in primary drying

• The temperature difference

between the shelf surface and
interior, 8°C, is a thermal barrier
that represents imperfect heat
transfer within the shelf itself.
• The temperature difference
between the shelf surface and
the top surface of the tray
bottom is 20 °C,
• that between the product in the
bottom of the vial and the pan
surface is 30°C
and
• that between the ice at the vial
bottom and the ice at the
sublimation interface is about 2
06/01/2010
°C. 96
 HEAT FLOW

For vials resting directly on the freeze-drier shelf, the vials

heat coefficient, kv, is defined by:

dq/dt = av kv(ts - tb)

where:
dq/dt = heat flow (cal/s) from shelves to the product in a given vial,
av = cross-sectional area of the vials calculated from the vial outer
diameter,
ts = temperature of the shelf surface, and
tb = temperature of the product at the bottom center vial.

Therefore, the heat-transfer coefficient (Kv) is defined as the

ratio of the area-normalized heat flow to the temperature
difference between heat source (the shelf) and heat sink (the
frozen product).

06/01/2010 97
 HEAT-TRANSFER COEFFICIENT

The vial heat-transfer coefficient is the sum of three

contributions representing three parallel heat-transfer
mechanisms:
kv = kc + kr + kg
where:
kc= contribution from direct conduction between shelf and glass at the
points of actual contact,
kr= contribution from irradiative heat transfer, and
kg= contribution from conduction through the gas between the shelf
and the vial bottom.

kg increases with increasing pressure, due to the increased number of

gas molecules to conduct heat through collisions between gas
molecules and the two surfaces (vial bottom and shelf surfaces)

06/01/2010 98
 EFFECT OF CHAMBER PRESSURE ON PRIMARY DRYING

As the chamber pressure increases, the vial heat-transfer

coefficient increases, thereby transporting more heat to the
product at a fixed shelf temperature (more gas molecules
conducting heat) and increasing the product temperature
(increasing po, therefore the driving force).

For a given formulation, fill volume, container, and freezing

process, the chamber pressure and shelf temperature
sequence with time determine the product temperature
(essentially constant at a safe level below the eutectic or
collapse temperature; safe margin is 2-5°C).

06/01/2010 99
 EFFECT OF CHAMBER PRESSURE ON
PRIMARY DRYING

Primary drying rate is

more dependent upon
pressure at low
pressure than at higher
pressure.

The primary drying rate

at 0.5 mm Hg is
approximately twice as
fast as at 0.05 mm Hg
at a constant shelf
temperature.

06/01/2010 100
 EFFECT OF CHAMBER PRESSURE ON
PRIMARY DRYING

The general rule is that chamber pressure should

be significant lower than the vapor pressure of ice
at the target product temperature (in the range of
10-30% of the vapor pressure of ice).

If the target temperature is -33°C (that is, collapse

-30°C) the vapor pressure of ice is 0.21 mm Hg,
and the chamber pressure should be about 0.06
mm Hg.

06/01/2010 101
 END POINT OF THE PRIMARY DRYING

Primary drying ends when all ice in all product containers has
been removed.

Indications of the end of primary drying are:

- product temperature:
The product begins to warm up and its temperature rises to
roughly reach that of the shelf
- pressure rise test:
The valve separating the driving chamber and the condenser
chamber is periodically closed, and the rate pressure increase
is monitored. If the rate significantly exceed the leak rate, ice
is must still be present, and the valve is opened to continue
with primary drying.

06/01/2010 102
 SECONDARY DRYING

Secondary drying involves the removal of absorbed water

(water which did not separate out as ice during freezing,
even 20%) from the product, to reduce the residual moisture
to an optimum value for stability (usually 0.5-2.0).

Typical additional times are 0.35 to 0.5 times the primary

drying times.

The product temperature is usually raised (by increasing

shelf temperature) and the chamber pressure further
reduced.

06/01/2010 103
 END POINT DETERMINATION OF SECONDARY DRYING

During the secondary drying the product temperature

generally rises gradually and equals the shelf
temperature.
Pressure rise test is an effective method to indicate the
end point of secondary drying.

The chamber isolated from the condenser and pumping

system and the rise in the chamber measured.

The rise in chamber pressure is directly proportional to

the residual moisture of the product.

06/01/2010 104
 OPTIMIZATION OF THE LYOPHILIZATION PROCESS

Development of an optimized lyophilization cycle,

designed on a scientific basis through the
knowledge of the most important physico-chemical
parameters which may affect product quality and
stability.

The objective of freeze-drying process

development is to minimize the process time while
maintaining high product quality.

06/01/2010 105
 CYCLE TIME AND WORKING DAYS

In a typical industrial situation the working will

allow for 220 to 240 days.

An ideal cycle is 22 hours, which gives 4 cycles

per week.

34 hours or 46 hours cycles give respectively 3

or 2 cycles week.

06/01/2010 106
 USE OF MICROPROCESSOR

The use of microprocessor now makes it possible to run

freeze-drying cycles automatically from beginning to end
while controlling the heating rate and the chamber
pressure.

While this is an important advancement, it does not remove

the need for a thorough understanding of freezing
characteristics of each drug solution, and the factors which
influence the rate drying.

Understanding the freeze-drying process thoroughly can

significantly reduce processing time and therefore reduce
costs, ensuring the quality of the final product.

06/01/2010 107
 CONCLUSIONS

Freeze-drying has often been carried out in an

empirical "trial and error" manner.

The preceding discussion attempts to show however

that every stage of lyophilization is governed by
certain principles which need to be understood if the
process is to be optimized.

The study of thermal events and related phase

transitions as well as the use of microprocessors are
the foundations to approach rationally the freeze-
drying process.

06/01/2010 108
 CONCLUSIONS

A strict cooperation a pharmaceutical

technologist, thermal analysis scientist and
the designer of freeze-dryer is essential to
achieve the goal:
a freeze-dried product with the best
attributes and stability.

06/01/2010 109
 FREEZE DRY EQUIPMENT

A freeze dry system for production of pharmaceutical

dosage forms consists of:
- a chamber containing shelves through which a heat
transfer fluid can be circulated
- a system for pumping, heating, and cooling the fluid
- a vacuum pumping system
- a condenser for trapping water vapor, and
- a refrigeration system for cooling the condenser.

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 FREEZE DRY EQUIPMENT

In addition these essential components,

pharmaceutical freeze dryers may incorporate
systems for:
- sterilization of the chamber/condenser
- stoppering vials within the chamber
- automatic cleaning in place (CIP), and
- automatic loading and unloading of vials
- computerized monitoring and control.

06/01/2010 111
 VACUUM SYSTEM

The most common type of vacuum

pump in freeze drying is the rotary oil
pump.

It consists of a steel cylinder rotating

eccentrically within a round casing.

The gas being pumped is admitted into

the casing via an inlet valve,
compressed, and forced out a
discharge valve.

Oil serves both as a lubricant and a

sealant to prevent back diffusion of gas
past the rotating cylinder.

Rotary oil pump are able to achieve

vacuum as low as about 1 μm Hg.

06/01/2010 112
 SPEED OF A VACUUM PUMP

The speed of a vacuum pump is given by

pumping speed (S) in the equation:

dP/dt = S / C (P-Ps)

where:
dP/dt= rate of change of pressure with time
S=pumping speed (volume/time)
C=volume of system to be evacuated
Ps=lowest attainable pressure.

06/01/2010 113
 VACUUM PUMP

Roots pumps are frequently used and

comprise two figure eight-shaped rotors
that counter-rotate without touching each
other or the chamber walls.

There are no inlet or discharge valves, and

no oil or other fluid for lubrication.

The function of roots pumps is to increase

the speed of the pumping system by about
a factor 10, and to also increase the lowest
attainable vacuum.

They are always used in combination with

another pump such as a rotary oil pump.

06/01/2010 114
 REFRIGERATION

It is required both for cooling the shelves during

freezing of product and for cooling the
condenser during drying.

The condenser is generally cooled by direct

expansion of the refrigerant, usually a fluoro-
hydrocarbon, in the condenser coils.

Generally, refrigeration can be switched from a

condenser to the heat transfer fluid during
freezing of product, and back to the condenser
during drying.

06/01/2010 115
 REFRIGERATION

The refrigerant evaporates in the condenser coils,

withdrawing the latent heat of sublimation from the
condenser.

Vapor is drawn from the freeze drying condenser by a

compressor and pumped to a condenser at a higher
pressure.

In the condenser, cooling water causes the compressed

vapor to liquefy, and the condensed refrigerant is
collected in a receiver.

The liquid refrigerant is returned to the cooling coils via

an expansion valve and the cycle is repeated.

06/01/2010 116
 HEAT TRANSFER FLUID

The most common types of heat transfer fluid

are silicone oil, trichloroethylene (TCE) and
Lexol, an oil similar to kerosene.

Silicon oil is by far the most common; TCE has

been phased out due to safety concerns.

06/01/2010 117
 STERILIZATION OF FREEZE DRYERS

The most common method of sterilization of freeze

dryers is steam under a pressure of about 15 psi,
which corresponds to a temperature of about
121°C.

Some units are sterilized by ethylene oxide (under

regulatory scrutiny).

06/01/2010 118
 VACUUM INTEGRITY TEST

The vacuum integrity of the freeze dryer

chamber/condenser should be monitored, since a
leak of non-sterile air into the system will compromise
asepsis.

This is easily done by evacuating the system to a

known pressure (100 μm), closing the valve between
the vacuum pump and the freeze dryer, and
monitoring the increase in pressure for at least 15
min.

06/01/2010 119
 SUMMARY

Freeze drying provides a valuable tool to the

pharmaceutical scientist by permitted dehydration
of heat-sensitive drug or biologicals at low
temperature.
The final product is quickly and easily
reconstituted, and the process is compatible with
aseptic operations.

The trend in parenteral manufacturing is toward

developing technology that automatically monitors
the critical variables (temperature and pressure)
and controls throughout the process removing the
operators from direct interaction with products .

06/01/2010 120