Lyophilization

Senthamil selvan T
Introduction
ↂ Lyophilization is a science of drying which involves drying of parenteral drug products which
is unstable (possibly degradation) in aqueous media and sensitive to temperature.
ↂ Main principle of Lyophilization is to freeze a substance and removal of any ice or solvent
through sublimation, which turns it into vapor without passing through a liquid stage.
ↂ Ideal method for the preservation of many different heat-sensitive materials including
microbes, proteins, pharmaceuticals, plasma, and tissues.
Scope in Pharmaceutical industry:
ↂ Increased shelf-life of injectables and vaccines.
ↂ Improved stability and storage of labile drugs.
ↂ Sterility can be maintained
ↂ Ease of reconstitution.
ↂ Improved product life cycle
Requirements of lyophilization
To develop the nominal drying conditions for lyophilization cycle parameters.

Glass Transition (Tg’)

Collapse
Temperature (Tc)
Thermal
Property
Re-crystallization
Temperature (Tcry)

Eutectic Temperature
(Te)
Excipient selection
ↂ Formulating lyophilized product Physical & chemical stability of active ingredient to be
studied with excipients.
ↂ Measure the solubility under a range with excipients.
ↂ Determine the excipient option and concentration.

Tonicity Modifiers Miscellaneous

(Dextrose)
• Addition of tonicity modifiers
(Dextrose) can reduce the Tc of the
formulation and increase primary
drying times.
materials
• Antioxidants, co-solvents,
complexing agents, surfactants,
dispersing agents, antimicrobial
agents, and CCT modifiers are other
excipients used in lyophilized
products.
Excipient selection
• Improving physical structure.
• Product contain low solid content and potent compounds (where drug content is
Bulking low).
agents • Characterization of crystalline structure must be performed to detect changes
during processing, including polymorphic transitions and the formation of API-
excipient complexes.

• To stabilize pH during processing, storage, and reconstitution.

Buffers • Buffers that crystallize during the freeze-drying process may significantly alter
(Tris, salts, acids, bases) the pH,which can be especially detrimental to pH-sensitive compounds like
proteins. Amorphous buffers are highly preferable.

• Act as stabilizers to prevent drug degradation during lyophilization process

(when subjected to both thermal and physical stresses).
• Mechanism:Lyoprotectants form hydrogen bonds with proteins as water is
Lyoprotectants and
removed during lyophilization, helping to maintain their structure and biological
Cryoprotectants
activity
• Prevention of reaggregation of complex formulations in nanosuspensions and
lipid-based systems during lyophilization and reconstitution.
Determination of Tc
 Collapse temperature is the maximum temperature of product can withstand during lyophilization
without melting or collapse.
 Crystalline products have Eutectic or Collapse Temperature .Amorphous products have the
“Glass Transition temperature”.
  Tc of amorphous products is typically a few degrees warmer than its glass transition temperature.

Start of freezing Starting point of collapse Complete Collapse

Annealing
 Annealing is a step keeping the product above its final freezing temperature for stipulated time period (to
crystalize the crystalline compounds)
 Annealing allow the formulation to rearrange crystals (high stable state).
 This step highly preferable for the formulation contains bulking agents (glycine, mannitol).
 Bulking agent's crystallization occurred during primary drying will resulted in vial breakage.
 Annealing time is depends on ration and property of bulking agents used.
  ratio of bulking agent (>80% of total solute)= Crystalize very faster

How vial breakage can be prevented ?

ↂ Slow freezing of bulking agents during freezing phase.
ↂ Freeze the bulking agents to -25C until complete crystallization occur (Don’t lower the temperature till
completion of crystallization).
ↂ Annealing temperature must lies b/w Tg of amorphous and Teu of bulking agents (Higher crystallization rate
and completion of crystallization).
ↂ Annealing above Tg results growth of ice crystals and decrease product resistance flow of water vapor (
drying time - cost effective !)
ↂ Low annealing temperature = high crystallinity due to super saturation (crystallization rate
is low and higher viscosity)
Effect of heating and cooling rate
• Cooling rate
 Slower crystal growth (Time) 
Larger crystals (Size of crystals)
 Slower Cooling  Uniform
temperature (Throughout the Vial)
 Slow cooling is preferable.
 Rapid cooling  Heterogenous
distribution of temperature (Colder
at Bottom & Warm at top portion)
Larger ice crystals results large
pores  Less resistance and dry
faster.
• Heating rate
 Heating rate (Time)  dry of
product.
 Rapid heating may result in product
collapse or cause melt back.
 Determine the Tc/Tg or Teu of bulk
and heat the product accordingly.
 Heating rate 0.1 to 0.15°C/min
(amorphous products)
 Heating rate 0.3-0.4°C/min
(Crystalline products )
Vapor pressure
ↂ Vapor pressure is the point at which the vapor of a substance is in equilibrium with its liquid or solid
form.
ↂ if the atmosphere surrounding substance is below its vapor pressure, boiling (liquid) or sublimation
(solid) takes place. 
ↂ Atmosphere surrounding a substance is above its vapor pressure results Condensation. 
Primary drying factors
ↂ Fill Volume : 35% of Vial capacity recommended (maximum).
ↂ Chamber pressure : 0.060 to 0.260 mBar is generally following range.
ↂ Product temperature : Below the collapse temperature (5-7 °C below) is ideal.
ↂ Cake dimension : Fill height and % solid content allows the dimension of cake/powder/plug.
ↂ Ramping rate : 1°C/min
ↂ Drying temperature : 5 °C to 40 °C (Should be Lower than Shelf temperature)
ↂ Sufficient vial spacing or vial arrangement during loading
ↂ Half stoppering height of vial
Nucleation
ↂ Development of a critical sized nucleus through the random aggregation of molecules (ice).
ↂ Occurs at temperature of <-15C. Water cooling rapidly (1 million /sec)
ↂ Phase change stage (Change from liquid to solid) starts once formulation reaches its ice
nucleation temp (Tn).
ↂ First ice nucleus appears  primary nucleation. (extremely fast process).
ↂ Secondary nucleation immediately follows primary nucleation (addition of ice nucleation sites).

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Cooling Primary Secondary Solidification
below Tf nucleation nucleation stage

Temp: -5°C Temp: -15°C Temp: -35°C Temp: -40°C

999.999 mbar 999.999 mbar 999.999 mbar 999.999 mbar

Nucleation (Phase change)
ↂ Formation of ice crystals is an exothermic process .In this process to continue, the heat given
off must be removed.
ↂ This heat is removed by the supercooled solution.
ↂ The amount of heat absorbed during secondary nucleation determines the total number of
ice crystals formed.
ↂ Higher degree of supercooling allows bigger amount of heat to be absorbed during the
exothermic crystallization process and this grants the formation of many ice crystals.
ↂ Higher degree of supercooling (Lower Tn) results faster ice crystal growth due to colder
shelves of lyophilizer remove the heat generated during ice formation.
Properties of Ice

Polymeric
Lyophilization is performed at form
Crystal Density
Pressure Temperature
pressure < 1 atm & temperature of structure (g/cm3)
types
> -80°C, the crystal structure of ice Ic Cubic 0.92 < 5,000 atm > -80°C
will be limited to Ih . Ih Hexagonal 0.92 < 5,000 atm > -80°C
II Rhombohedral 1.17
Rate of ice growth increase when
III Tetragonal 1.14
increasing in degree of
IV - 1.28
supercooling.
V Monoclinic 1.23 20,000 atm 20°C -
 Supercooling of 20°C, the ice 81.6°C
growth rate about 27.8 cm/sec. VI Tetragonal 1.31
VII Cubic 1.50
 WFI in glass vials will have a VIII Cubic 1.50
degree of supercooling that is within IX Tetragonal 1.14
the range of 10±3°C (5.2cm/sec)
Rate of freezing /water property

 Freezing Point of Water: 32°F (0°C) [273.15 Kelvin]. Slow freezing Rapid freezing
 Freezing below 0 °C and 4.58 Torr. A Torr is (1/760th) of a
standard atmosphere. In millitorrs, or (1/760,000th) of a
standard atmosphere
 Water can be solidifying at 0°C (Nor Pure water), For
WFI (Purest) mayn’t Solidify even < -18 °C.
 Addition of Solutes & Excipients  Depression of
Freezing point .
 The latent heat of sublimation (change b/w state from
one to another) is 619 cal/g (1114 BTU/lb.*) for water.
* BTU – British thermal unit Dry very faster More resistance to dry
No Rp Small pores
Homogenous distribution Heterogenous distribution
Large ice crystals Smaller ice crystals
 Mass and Heat transfer
Heat radiation

Dried layer Sublimation

interface Drying of cake

Heat Convection
Sublimation Top layer
Radiation

Frozen front
matrix

Convection

Conduction
Heat Conduction

 Heat from shelf to vial and product (Direct conduction).

 Heat transfer from side wall (radiation).
 Heat transfer by convection
Mass and Heat transfer
Calculation

   

     
   

 
Freeze drying cycle

Lyophilization cycle
Freezing Primary Drying Secondary
50 1200
Phase Drying
40
1000
30 Atmospheric

Chamber pressure (mBar)

pressure Sublimation
20
Temperature (C)

Annealing 800
10
Nucleation
0 600
-10
400
-20
Desorption
-30
200
-40 Higher
Higher vaccum
vaccum
-50 0
Shelf Set temperature (°C) Probe -01 Probe-02 Chamber pressure
(°C) (°C) (mBar)
Freezing phase

 Freezing of drug solution under

<-40C to solidify the product.
 Water and ice have large specific
heats and low thermal conductivities,
both of which mitigate against rapid
heat transfer.
 In practice, on a freeze drier shelf, the
vial contents will cool at a rate of not
more than 0.3 to 0.5 ºC/minute, even if
the shelf is cooled more rapidly.
 Formation of ice crystals happen in the
vials and materials form the ice
structure with pores to release the
water molecules in later (drying)
stages.
Primary drying phase
 Sublimation process.
 Removal of 90-95 % of unbound water molecules
 Below the Teu , Tc & Tg
 Once frozen of product occur reduce the surrounding
the product pressure (below vapor pressure).
 Preferably cycle designed to 100-200 mTorr
(0.130 to 0.260 mBar).
 Condensor is much colder than the product, the
vapor pressure at the condenser is much lower than
the product.
 Water molecules that leave the product vial easily
(pressure gradient).
 ↑ Shelf temperature and ↓ Pressure = ↑ Sublimation rate ( Probably cause melt back due to overheat).
 Higher sublimation rate and product below its Tg’ = Decrease the drying time (Cost effective)
Secondary drying phase

 Desorption process.
 Removal of 2-5 % of bound water/adsorbed
molecules
 Temperature ranges from 20-30°C of primary
drying time.
 1/3 rd of primary drying time is suggested for
secondary drying process.
Full stoppering
 Full stoppering of vials shall be carried out inside of the lyophilization chamber and
sterility of product will be maintained.
 Full stoppering can be done after vacuum break by using nitrogen either under full
vacuum, partial vacuum, atmospheric pressure based on product nature.
Lyophilization transformation
 PAT tools
in freeze drying
technology
End point determination
 Capacitance manometer measures true pressure reading (always) pirani gauge will give
false high reading in presence of water vapor.
 When the Pirani reading reaches the CM reading , it indicates no water vapor present
(completion of primary dry).
 The Pirani gauge reads about 60% higher than the capacitance manometer.

ↂ While sublimation product temperature is colder than shelf temperature (heat need from
shelf for phase change).
ↂ When shelf temperature equals to product temperature (end point of primary drying)
ↂ An electronic vacuum gauge can be used to measure condensable gases in the system.
When the pressure by the electronic gauge reaches the minimum pressure attainable by
the system, as no more water vapor is leaving the product
Primary drying end point detection
End point determination Pirani vs Capacitance manometer
180
45
Presence of water vapor (PG)
30 150

Chamber pressure
Temperature (°C)

15 120
End point Cycle end
0 True pressure (CM)
90
-15
60
-30
30
-45

-60 0
Time
Shelf set (°C) Pressure - PG (mTorr) Pressure - CM (mTorr)
Kv measurement
 Arrange the filled vials in the lyo trays as Relationship between chamber pressure and vapor
pressure of ice (I.e., ice temperature),
per (L* W).
Pc = chamber pressure/(above dried solute)
 Fill the vials with “X” Fill volume Rp = product resistance
 Stoppers pierced with fixed-length m =Ap (Po-Pc) = Ap (Po-Pc)
Rs =stopper resistance
Rp + Rs Rp^
precision bore stainless-steel tubes m = rate of sublimation
(d:“X”mm, L:“X”mm, equal resistance Po = vapor pressure of ice.
to mass flow); full stoppering.
 Weigh of every single vial before and Relationship between shelf temperature and ice
temperature m= Q/∆Hs = Av*Kv*(Ts-∆T-Ti)
after the sublimation test to determine
Av = surface of vial ∆Hs
the amount of sublimated water (X%
mass loss) Kv = vial heat transfer coefficient
Ts = shelf temperature
ΔHs = enthalpy of sublimation
ΔT = temperature difference across ice slab
Rp
Po Ti Ti = temperature at the ice interface
Ice
Kv Tb
Sin Ts Sout
Lyophilization modeling
 Area above the initial free surface of the drying
substance, consisting of vacuum and evaporated solvent
(water vapor).
 Porous region above the sublimation interface, consisting
of solid matrix with adsorbed solvent. Heat transfer
(from upper plate)
Heat transfer
(from upper plate)
 Porous region below the sublimation interface, consisting Water vapor
Dried material

of frozen solvent and solid matrix with adsorbed solvent. Desorption process Region-I
in dried region Porous region +
(free surface)

 Vial heating by heating plates in the freeze (one from Water vapor + gas Sublimation
surface
above another is below).Heat transfer to frozen Region-II

substance from the top, bottom and side of vials. Frozen material

 Heat transfer at top of vial occurrence by radiation. Ice +Porous

material
 Heat transfer from bottom by radiation and conduction
(Direct contact b/w vial and plate)
 Heat transfer to the wall of vials by radiation (depends on
no & position of surrounding vials)
Fill volume design considerations

• Drying time  Fill volume ( fill height)

• Fill volume of lyophilized product considered such as
Nominal volume, Extractable (withdrawable volume)

Control limit
Max. fill
and manufacturing tolerance. Target fill
Nominal fill

• Measure the cake height of RLD vial presentation.

Cake height
• By comparing the RLD, add the bulk solution to
equivalent height another vial and subject to freeze
RLD IH dry !
• Determine the fill volume and compare the fill height
Scaleup considerations

 Perform thorough lyophilizer characterization in addition to the

common IQ/OQ on the ‘dry and empty dryer’ .
 Establish the relationship or ‘link’ between laboratory, pilot and
commercial lyophilizer, so that any cycle could be easily
transferred from location to location giving flexibility from the
commercial perspective.
  Keep the product temperature profile in the commercial
lyophilizer same development freeze dryer.
 Establish reasonable process tolerances (minimum
controllable pressure as function of sublimation rate, condenser
capacity, vial heat transfer coefficients, statistical information on
pressure and shelf temperature deviations)
Lyophilization cake acceptance level
Cake
Route cause Impact on CQA
appearance
Drying above critical product temperature Reconstitution time,
Total collapse
during primary or secondary drying residual moisture, stability, potency.
Primary drying above eutectic or ice melting Reconstitution time,
Melt back
point, or incomplete primary drying. residual moisture, stability, potency.
Major Reconstitution time & Container Closure
Filling , Manual loading.
splashing Integrity.
Reconstitution time if the
Cake shrinkage Low amounts of dissolved
Lifted cakes cake stays lifted, may impact
Solids.
product temperature history.
Higher residual moisture and abnormal
Vial not resting on the shelf
Slanted cake product temperature
completely due to loading issue.
History.
Lyophilization cake acceptance level
Cake appearance Route cause Impact on CQA
Cake shrinkage Formulation process, ramp to secondary drying too fast No
Chipping Shipping stress resulting in formation of loose powder/cake No
Fogging Glass surface property & Glass formation process No
Reconstitution time
Dripping from nozzle during fill & Agitation of vials after filling
Lyo ring depends on the
and before Loading
thickness of the ring
Bubble or foam
Foaming during filling No
formation
Volcano Process (freezing protocol) No
Drying above critical product temperature during primary
Partial collapse No
or secondary drying
Minor splashing Filling (Manual loading) No
Lyophilization cake acceptance level
Cake appearance Route cause Impact on CQA
Stability of proteins
High level of dissolved gases
Puffing sensitive to
Eutectic melting Freezing protocol.
Interface.
Non-uniform/ change in
Process (freezing protocol) No
cake texture
Non-uniform or change
Formulation Process No
in cake color
Splashing of very fine droplets
Glassy droplet No
during filling
Shipping stress resulting in formation of loose
Broken cake No
powder/cake
Macroscopic structural changes caused by the tensile
Cracked cake (“drying”) tension built up within a wet solid when water No
is removed
Lyophilization cake acceptance level
Cake appearance Acceptance level Cake appearance Acceptance level
Total collapse No Volcano Yes
Melt back No Partial collapse Yes
Major splashing No Minor splashing Yes
Lifted cakes No Non-uniform/change in
Yes
Slanted cake No cake texture
Puffing No Non-uniform or change
May be
in cake color
Cake shrinkage Yes
Yes (only if
Chipping Yes Glassy droplet confirmed
Fogging Yes to be a product)
Bubble or foam Broken cake Yes
Yes
formation Cracked cake Yes
Lyo ring Yes
Lyophilization defects
Collapse of product
֍ Cause: It is a phenomenon which occurs in amorphous solids when the product reaches a higher
temperature than the collapse temperature, Tc (closer to the glass transition temperature Tg’). The
amorphous phase of the solid experiences internal movement without reaching the melting point, with
the crumbling or collapse of the dried cake as a result.

֍ Solution: Maintain the product temperature below the collapse temperature during all the primary drying
time (while there is still presence of ice in the product). Avoid the formation of a “dried skin” at the top of
the surface.

Melt back
֍ Cause: It happens when the primary drying phase has ended but some of the vials still have some ice
at the bottom. During the secondary drying, the remaining ice is not removed. When the process
finished, and such ice has not been removed properly it melts and wets the dried product forming some
cavities.

֍ Solution: Set a longer primary drying. Increase the security time between the primary drying and the
secondary drying.
Lyophilization defects
Drops on vial
֍ Cause: It happens in eutectic and viscous products, when the primary drying is started, some parts of not frozen
product from the top layer boil, explode and get stuck to the internal lateral walls of the vial, which are at a lower
temperature than the product.
֍ Solution: Add an annealing step with thermal treatment. To use substances which prevent the migration of product to
the top layer.

Detached cake
֍ Cause: When the concentration of solids in the product’s solution is low, a reduction of the lyophilized mass may
occur, with the consequence of the cake detaching itself from the walls remaining free inside the vial with the
possibility of suffering some breaking.
֍ Solution: Increase the concentration of substance, concentration of solids, in order to obtain a more consistent cake.

Vial breakage
֍ Cause: A re-crystallization occurs during the freezing step, mainly in amorphous products. The cake suffers an
increase in volume. In concentrated solutions, the pressure of the vapor at the bottom of the vial may rise in case of
applying too much energy to the product at the beginning of the primary drying.
֍ Solution: Cool down the product and slowly freeze it to enhance the formation of larger and dendritic ice crystals or
add an annealing step with thermal treatment with a slow cooling at the end. Start the primary drying with a smooth
application of heat, to facilitate the exit of the water vapor through the lateral walls of the vial. Use thicker vials or vials
of more quality.
Thank You