The document details the principles, processes, advantages, and disadvantages of lyophilization, a technique used to preserve and extend the shelf life of sensitive products by removing moisture through sublimation. It describes the various stages of the lyophilization process, including freezing, primary drying, and secondary drying, as well as the essential components of a lyophilizer. Additionally, it outlines qualification tests and critical attributes necessary for successful lyophilization operations.

1. LYOPHILIZATION
Basic Principles of Lyophilization
Date - Sep 2022
Presented by – Mr. Pravin V. Daundkar
Asst. Manager Production
08-01-2023 1

2. Contents…
2
1 Definition, Principle and Objective of Lyophilization
2 Advantages and Disadvantages of Lyophilization
3 Steps of Lyophilization Process & End point determination
4 Various Components of Lyophilizer
5 Operation of Lyophilizer
6 Qualification tests of Lyophilizer
7 Media fill Process Simulation
8 Development of Lyophilization Cycle
9 Critical Material Attributes (CMA), Critical Quality Attributes (CQA) & Critical Process Parameters (CPP)
10 Types of Defects in Lyophilized Vials
11 Guidance
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3. Definition, Principle and Objective of Lyophilization
3
Definition of Lyophilization:-
• It is the process of removal of ice or other frozen solvents from a material through
the process of sublimation and the removal of bound water molecules through
the process of desorption.
• Lyophilization and freeze-drying are terms that are used interchangeably
depending on the industry and location where the drying is taking place.
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History of Lyophilization Process:-
• Freeze-drying was invented by Jacques- Arsene d’Arsonval at the College de
France in Paris in 1906.
• Lyophilization was first actively developed during world war – II to preserve and
transport of blood serum.
• The main aim was to store the products without refrigeration and to remove
moisture from thermolabile compounds.
• Apart from pharma, it has major use in food industry to produce clean, whole
and nutritious food ingredients with a highly prolonged shelf life.

4. Definition, Principle and Objective of Lyophilization
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Principle:-
• The process of Lyophilization involves a phenomenon
called sublimation in which water is directly passes from
solid state (ice) to vapor state without converting to the
liquid state.
• Lyophilization is performed at temperature and pressure
below the triple point , to enable sublimation of ice.
• The product that has to be dried is first frozen and then it
is subjected to heat (conduction, convection or radiation)
under a very high vacuum, so that frozen liquid sublimes
and only dried solid components of original liquid remains.
• The driving force for the process of sublimation of ice
during Lyophilization process is the pressure difference
between the vapor pressure of ice and partial pressure of
water in the chamber.
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5. Definition, Principle and Objective of Lyophilization
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Objectives:-
 To preserve the biological activity of product. Eg. Vaccine.
 To reduce the product weight to lower the transportation cost.
 To extend the shelf life or stability of product.
 To dry thermolabile materials.
 To eliminate the need for refrigerated storage.
 To get accurate, sterile dosing into the final product container.
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6. Advantages and Disadvantages of Lyophilization
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Advantages:
• Removal of water at low temperature.
• Improved product characteristics.
• Compatible with aseptic process.
• Enhancing the stability of a dry powder as well as the product stability in a dry state.
• Minimal changes in the properties because microbe growth and enzyme effect cannot be exerted under low
temperature.
• Transportation and storage under normal temperature.
• Rapid reconstitution time.
• Oxidizable substances are well protected under vacuum conditions.
• Constituents of the dried material remain homogenously dispersed.
• Product is process in the liquid form.
• Sterility of product can be achieved and maintained.
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7. Advantages and Disadvantages of Lyophilization
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Disadvantages:
• Single most expensive unit operation.
• Lyophilization is a time and energy consuming process, which leads to higher product cost of freeze dried
product.
• Biological molecules are damaged by the stress associated with freezing and drying.
• Long Lyophilization cycle, which leads to longer batch processing time.
• Sterile diluent needed upon reconstitution
• Volatile compounds may be removed by high vacuum.
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8. Steps of Lyophilization Process & its End Point Determination
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A. Steps of Lyophilization Process
 Sample Preparation
 Freezing (Solidification)
 Primary drying (Sublimation)
 Secondary drying (Desorption)
 Final Product
Steps of Lyophilization cycle

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Basic Lyophilization cycle:-

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1. Freezing (Solidification):-
Freezing, also known as solidification, is a phase
transition where a liquid turns into a solid when its
temperature is lowered below its freezing point. Typically,
the product freezing done below 4-5 degrees of Eutectic
point.
• Nucleation - formation of a ice crystal from a
solution/liquid on foreign surface and additional
particles are deposited as the crystal grows.
• Super cooling - is the process of lowering the
temperature of a liquid or a gas below its freezing point
without it becoming a solid.

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Freezing curve of water and solute:-

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• Triple point - The temperature and pressure at
which three different phases, such as gaseous,
liquid, and solid phases, of a particular substance
can coexist in equilibrium.
• Eutectic point - The eutectic point is the lowest
temperature at which the liquid phase is stable at
a given pressure. The "Eutectic Point" is the lowest
freezing point that can be reached for a eutectic
combination (which means lowest melting point).

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• Crystalline freezing:
If the solute is crystallize, the temperature at
which crystalline (eutectic) phase melts is
referred as the eutectic temp (te).
• Amorphous freezing:
If the solutes forms an amorphous glass the
temperature at which amorphous (GLASS)
phase soften is referred as the glass transition
temperature (tg).
• Annealing
Annealing is the process of warming a sample above its glass transition temperature (but below the eutectic
temperature and/or ice melting temperature ) and allowing the glass to relax and crystalize. E.g. Mannitol
and Glycine.

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Correlation of Freezing rate Vs Sublimation rate:-
The rate of ice crystallization define the freezing process and efficiency of primary drying.

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2. Primary Drying (Sublimation):-
• Primary drying is the process of converting ice directly into a
gas without melting. The product is held at a temperature
lower than the eutectic point of the ice and controlled
vacuum conditions are used to remove the vapor.
• Driving force of sublimation - is the pressure and
temperature difference between the product chamber and
the ice condenser.
• Larger temperature and pressure differences, which allow for
a faster process.
• Application of Heat - It is essential to heat the product as much as possible (without passing the eutectic
point) in order to increase the pressure differential between the product and the condenser.
• Typically, shelf temperatures during Primary Drying are ramped from –40 to +20°C during the process time.
• Application of Vacuum - The vacuum speeds up the process by removing air molecules to allow sample
vapor molecules to move easier from the sample, through the chamber and into the condenser.
• About 85-90% of water removed in this phase.

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• Heat transfer mechanism in Lyophilization cycle
In Lyophilization process Heat brought mainly by Conduction, Convection and Radiation.

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• Sublimation curve:-

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3. Secondary Drying (Desorption):-
• The process of removing the amount of bound (un-
freeze) water in a freeze dried product after primary
drying is complete with the goal to achieve the target
value of residual moisture in the final product.
• The bound water is removed by Desorption
phenomenon.
• “Desorption” is the release of an adsorbed substance
from a surface.
• The process is called “Isothermal Desorption” as the
bound water is desorbed from the product, desorption
drying facilitated by increasing shelf temperature and
reducing chamber pressure to minimum.

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• At the end of this secondary drying, final residual water content in final product is around 1 - 4 %.
• Typically the product temperature is maintaining between 20°C and 40°C for several hours.
• The duration of the secondary drying phase is dictated by the desired residual moisture level in the freeze-
dried product.

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• Cryo-microscope (Determines the shape of flash-frozen samples
by firing electrons at them and recording the resulting images.)
b. Primary Drying
• Product temperature with a thermocouple probe
• Comparative pressure measurement (i.e., Pirani vs. MKS Baratron)
• Dew point monitor (Electronic moisture sensor)
• Lyotrack (Gas plasma spectroscopy) – measures water vapour
concentration during drying.
B. End Point Determination
a. Freezing
• Product temperature with a thermocouple probe – Product temperature decreases gradually and equals to
shelf temperature.
• DSC (Differential Scanning Calorimetry) – DSC cooling & warming thermogram.
• Measurement of temperature and electric resistance helps to determine the Freezing point.
(The electric resistance of the dried product is always rises with the transfer from the liquid to the solid state
due to the reduced mobility of the ions and electrons.)

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• TDLAS (Water concentration from tunable diode laser absorption spectroscopy).
• Condenser pressure
• Pressure rise test (Manometric temperature measurement)
• Determination of residual moisture either gravimetrically or by Karl Fischer.
• Visual inspection of cake structure for melt-back, collapse, and retention of cake
structure at the apparent end point of primary drying.
c. Secondary Drying
• Product temperature with a thermocouple probe – Product temperature
increases gradually and equals to shelf temperature.
• Comparative pressure measurement (i.e., Pirani vs. MKS Baratron)
• Pressure rise test. (Manometric temperature measurement)
• Dew point monitor (Electronic moisture sensor)
• Lyotrack (Gas plasma spectroscopy) – measures water vapour concentration
during drying.
• Determination of residual moisture either gravimetrically or by Karl Fischer.

22. Various Components of Lyophilizer
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Lyophilizer comprises of following main components:
1. Chamber
2. Shelf Stack
3. Condenser
4. Circulation System
5. Vacuum System
6. Hydraulic System
7. Refrigeration System
8. MKS and Pirani gauge

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1. Chamber
• The chamber is a welded fabrication manufactured from
stainless steel optimized to provide adequate access to
components within the chamber.
• View glass - is mounted at the side of the chamber to view of
the product during Lyophilization cycle.
• Validation port – is provided to allow the introduction of
validation probes into the chamber.
• Pressure sensor & Vacuum sensors - to measure the
chamber pressure and vacuum.
• Pirani gauge - to measure the thermal conductivity of gas
sample.
• Chamber insulation - with glass fiber and polymer foam.
• Nozzles & Spray ball - for CIP and SIP of Lyophilizer.
• MOC of internal chamber - SS 316 L
• Safety Valve.

24. Various Components of Lyophilizer
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• Chamber Door – Pizza door/Load door/Full size door/Door-in-Door

25. Various Components of Lyophilizer
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2. Shelf Stack
• The shelf stack comprises the product shelves,
compensation shelf, pressure plate, counter-
pressure plate etc.
• Shelves - made from SS 316L with the hollow
construction.
• Inside the shelf are baffles to channel the
diathermia silicone oil through the shelves, in
order to provide uniform distribution of heating
and cooling to all areas of the shelves.
• The silicone oil flows from each shelf through
flexible braided SS hoses.(SS 316L)
• Dimensions of Lyophilizer – Shelf area
5/20/30/45 m2.
• Total shelf – 12+1 (Radiating plate) with 110
mm inter shelf distance.

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• Silicone oil flow from product shelves.

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3. Condenser
• Condenser has condenser coils to trap the vapors generated from
product and these coils are cooled by direct expansion of the
refrigerant.
• Mushroom valve/Main valve – to isolate and connect the chamber
and condenser.
• Condenser coils – to trap the vapors generated during sublimation and
these coils can be cooled to an ultimate low temp. of -75°C.
• Temperature probes - to monitor the condenser coil temperatures.
• Pressure sensor & MKS sensors - to measure the chamber pressure.
• Spray system - to spray the steam or the hot water into the condenser.
• View glass – to view the ice formation on the condenser coils.
• Validation port - is proved for insertion of validation probes.
• Low pressure & high pressure relief valves are equipped on the
condenser.
• The external surface of the condenser is fully insulated and cladded
with SS.

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4. Circulation System
• The main purpose of this system to provide sufficient
heating/cooling rate to maintain the required shelf
temperature.
• This system comprises of following main components.
• Expansion tank- to accommodate the volume changes between
the various stages of processes.
• Circulation pump - rate of flow through the system will be
created to meet the heating/cooling capacity requirements.
• Heater – to heat the oil at the specified rate. SSR PID control.
• Cooling exchangers - to disperse heat from a fluid that has
passed over a cold plate attached to the heat-producing
component.
• Moisture filter - to prevent water being absorbed by the
circulation medium.
• Heat transfer fluid – Generally Low viscosity 1 - 5 cSt
(centistokes) silicone oil is used.

29. Various Components of Lyophilizer
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5. Vacuum System
• The vacuum system comprises of primary vacuum pumps, isolation valves and a Mechanical booster pump
and they are attached to the main vacuum line that connects to condenser via butterfly valve.
• Two types of primary vacuum pumps i.e. Oil and Dry vacuum pumps are used.
• This system is used to evacuate the chamber and condenser during the process of Lyophilization and bellows
integrity test.
• This reduces the pressure of non condensable gases within the chamber and condenser.
Oil sealed rotary vane vacuum pump Schematic diagram of oil sealed vacuum pump

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• Mechanical booster pump compresses air prior to the vacuum pump, enabling it to operate more efficiently.
• It helps to increase the evacuation speed.
• It increases the pumping speed and ultimate pressure of vacuum pumps.
Mechanical booster pump

31. Various Components of Lyophilizer
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6. Hydraulic System
• This system comprises the following
components,
a. Hydraulic power station unit
b. Shelf ram - is used for shelf and vial full
stoppering.
c. Main valve ram - is used for main valve
movement.
• Stoppering pressure - 90 -120 bar pressure used
for vial full stoppering and its pressure shall be
set manually from switch.
• Bellow - The shelf and main valve ram equipped
with optional bellow made of 316L stainless
steel are installed to prevent any possible
contamination of the product by hydraulic oil
that may leak through the hydraulic piston seals.
Basic hydraulic system

32. Various Components of Lyophilizer
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7. Refrigeration System
• All refrigeration circuits on the Lyophilizer
can be used to cool the shelves or the
condenser. This system comprises the
following components.
a. Compressor
b. Condenser
c. Expansion valve
d. Evaporator
e. Refrigerant gas
Refrigeration system

33. Various Components of Lyophilizer
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a. Compressor
• Compression is the first step in the refrigeration cycle, and a
compressor is the piece of equipment that increases the
pressure of the working gas. Refrigerant enters the compressor
as low-pressure, low-temperature gas, and leaves the
compressor as a high-pressure, high-temperature gas.
• Single Stage compressor – the refrigerant gas is compressed
one time.
• Two Stage compressor - It is two compressors built into one. Its
function is to be able to attain low temperatures by being able
to operate at low pressures. A low stage connected internally
and a high stage connected externally with piping, called
Interstage.
Three Types of compressors
1. Screw compressor
2. Reciprocating compressor
3. Rotary Compressor
Compressor

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1. Screw compressor 2. Reciprocating compressor 3. Rotary compressor

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b. Condenser
• The condenser, or condenser coil, is one of two types
of heat exchangers used in a basic refrigeration loop.
• This component is supplied with high-temperature
high-pressure, vaporized refrigerant coming off the
compressor.
• The condenser removes heat from the hot refrigerant
vapor gas vapor until it condenses into a saturated
liquid state.
• After condensing, the refrigerant is a high-pressure,
low-temperature liquid, at which point it’s routed to
the loop’s expansion device.
Water Condenser

36. Various Components of Lyophilizer
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c. Expansion valve
• The expansion valve reduces the pressure of the refrigerant fluid upstream of the evaporator. This drop in
pressure cools the fluid, it is then sprayed into the evaporator.
• Mainly two types of expansion valves in Lyophilizer.
i. Thermostatic expansion valves (TXV) or thermal expansion valves – mechanically controlled.
ii.Electronic expansion valves (EEV) – PLC controlled.
Thermostatic Expansion valve(TXV) Electronic Expansion valve(EEV)

37. Various Components of Lyophilizer
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d. Evaporator
• This is the part of the refrigeration system is doing the actual cooling and its function to collect the water
vapor released by the sublimation of ice within product, and it must have sufficient surface and cooling
capacity to freeze all the vapor generated during the sublimation process.
• The evaporator consists of finned tubes, which absorbs heat from their surrounding environment.
• Fins and tubes are made of metals with high thermal conductivity to maximize heat transfer.
e. Refrigerant gas
• CFCs: Chlorofluorocarbons, commonly called R-11, R-12, R-502, R-500, have chlorine content which presents
a high risk to the ozone layer. CFCs are being phased out completely.
• HCFCs: Hydro chlorofluorocarbons R-22, R-401A, R-402A, have a low chlorine content and present a low risk
to the ozone layer. These are service refrigerants used in existing equipment.
• HFCs: Hydrofluorocarbons, such as HFC-134a, HFC-143a, HFC-125, R-404A, R-407C, R-410A, R507 are
chlorine free and ozone safe. These are commonly used in Lyophilizer.

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General Refrigeration cycle:-

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8. MKS and Pirani Gauge
Description MKS Gauge Pirani Gauge
Type Capacitance manometer gauge Thermal conductivity gauge
Principle
Measures the change in capacitance of the capacitor. Sense
the deflection of diaphragm by change in capacitance
between electrode and diaphragm. Pressure is determined
by measuring the change in capacitance between the metal
diaphragm and an adjacent, fixed dual electrode.
Measures the electrical resistance of a filament wire whose resistance
varies with filament temperature. The Pirani gauge measures the
vacuum pressure dependent thermal conductivity from the heated
wire to the surrounding gas.
Operating range 0 to about 2 torr 0 to 1000 mbar
MOC
It consists of a stretched, flexible circular metal diaphragm
that is welded in place symmetrically between two
fixed electrode plates effectively isolating two regions of
space.
Pirani gauge consists of a metal sensor wire/filament suspended in a
tube which is connected to the system whose vacuum is to be
measured. The heated Pirani sensor wire/filament is typically made of
a thin (<25 µm) Tungsten, Nickel or Platinum wire.
Applications
During the drying step, the chamber pressure is controlled
using a capacitance manometer, which measures the
absolute pressure in the drying chamber.
Pirani vacuum gauge can be used to detect the end of primary drying.

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Pirani Gauge MKS Gauge

41. Operation of Lyophilizer
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Operation of Lyophilizer includes following cycles:
1. CIP (Clean In Place)
2. FIT (Filter integrity test)
3. SIP (Sterilization In Place)
4. VLT (Vacuum Leakage Test)
5. Lyophilization cycle
6. Defrosting
1. CIP (Clean In Place)
• The CIP cycle is used to wash particular matter, dried water soluble stains and product residues from the
chamber and condenser.
• The CIP water is pumped into the freeze dryer through a series of pipes, valves and nozzles.
• The CIP system shall clean the chamber, condenser, main isolation valve, shelf stack, view glass ports etc.,
with 99% coverage within the chamber.
• During CIP wash shelf up and down movement is done to cover the top and bottom of the product loading
shelves. Purified water or WFI used for CIP of Lyophilizer.
• Frequency – Pre and post Lyophilization cycle.
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42. Operation of Lyophilizer
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2. FIT (Filter integrity test)
• The filter integrity is performed to confirm the structural integrity of a sterilizing filter.
• 0.2 µ hydrophobic filter is used for vacuum break.
• Integrity testing of the filter is done through software program itself or by using filter integrity test
apparatus.
• Filter is sterilized during SIP (Sterilization in place) cycle of the Lyophilizer.
• Filter integrity test is mainly performed by water intrusion test.
• Frequency – Pre and post Lyophilization cycle.
3. SIP (Sterilization In Place)
• The purpose of the sterilization process is to sterilize the product and ice condenser chambers, including all
chamber fixtures and piping systems of the freeze dryer that come in contact with the product.
• Pure steam shall be used to achieve minimum exposure of 121°C for 15 minutes or the equivalent
temperature / time combination for effective sterilization.
• The SIP sterilization temp. is measured at the drain line of chamber, condenser, and sterile filter if applicable.
• Frequency – Before every Lyophilization cycle.
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4. VLT (Vacuum Leakage Test)
• The purpose of leakage test is to confirm the integrity Lyophilizer chamber and condenser including all ports,
drain line, vacuum line, steam line etc. of the freeze dryer.
• Frequency – Before every Lyophilization cycle.
• Formula for Leak rate calculation:
Allowable Pressure Rise In System = (Leak Rate X Time )/Total Chamber volume
E.g. Acceptable Leak rate calculation of Lyophilizer
= (0.02 X 3600)/16300
= 0.004 mbar = 4 µbar
• Types of Leak
a. Virtual leaks are sourced within the system. They may be due to out gassing of moisture or solvents
absorbed from materials in the system such as elastomer seals, or tapped volumes of air gradually being
drawn back into the system. Contamination from cleaning fluids or water may show up as a virtual leak.
b. Real leaks are genuine leaks from outside to inside of the system and usually shown up on test as a constant
leak rate i.e., linear pressure rise.
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44. Operation of Lyophilizer
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Graphical representation of Virtual Leak vs Real leak:-
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5. Lyophilization cycle: Lyophilization cycle can be run loaded semi-automatically or fully automatically.
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Steps Description
Loading Half stoppered liquid filled vials are loaded semi-automatically or fully automatically into the Lyophilizer.
Freezing Product loaded on the shelves is frozen to a specified temperature, usually below 4-5 degrees of eutectic point.
Evacuation The chamber and condenser is evacuated to achieve the vacuum set point immediately without ramping.
Drying
In primary drying frozen ice is sublimed with application of heat under high vacuum while in secondary drying unfrozen liquid is
adsorbed with high temperature and vacuum than primary drying.
Pressure rise test
This test is performed in the chamber to determine the amount of moisture that remains in the product by closing main valve
for one minute.
Pre-aeration Chamber vacuum is break with OFN grade 0.2 µ filtered nitrogen gas, usually vacuum break up to 800mbar.
Full Stoppering
Half stoppered vials are fully stoppered by means of hydraulic press under partial vacuum inside the vial. 90-120 bar stoppering
pressure applied for vial full stoppering.
Final Aeration Aeration done up to atmospheric pressure with 0.2 µ filtered compressed air/nitrogen gas.
Storage Freeze dried product kept at its final storage temperature till its unloading.

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• Rubbers stoppers used for Lyophilized products
6. Defrosting
• This cycle is mainly performed after completion of Lyophilization cycle to melt and remove the trapped ice
from condenser coils.
• Usually defrosting is performed at 50-100°C for specified time.
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Two leg stoppers Three leg stoppers Igloo stoppers

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Qualification Test Rationale
CIP coverage verification
To ensure that cleaning fluid is sufficiently covering all the surfaces of chamber, wall, shelves & bottom surfaces.
Riboflavin is used to ensure cleaning efficiency and 99 % of the surface area of the Lyophilizer shall be free from the
Riboflavin.
SIP effectiveness verification
The test includes validation of sterilization process of chamber, condenser & vacuum break filter in order to ensure
that components of freeze dryer are rendered sterile after exposure of defined sterilization cycle. Sterilization hold
time set here by assuming biological indicator D value of 2.5 min at 121 °C and 12 log reduction cycle hence
sterilization hold period will be 30 min. Geobacillus stearothermophilus biological indicator is used to challenge the
sterilization effectiveness.
Shelf temperature performance
verification
To verify the shelf heating & cooling rate, ultimate high & low temperatures are as per the system specification.
Heating and cooling rate shall be >= 1°C / min
Shelf temperature set point
control
To evaluates the performance of temperature control (shelf inlet temperature) at various set points. Shelf temperature
shall be maintained within ±2 °C.
Shelf temperature uniformity
verification
To analyse and evaluate the uniformity of temperature distribution across all Lyophilizer product loading shelves. Shelf
uniformity between all shelves ≤ 2 °C.
Vacuum Performance
verification
To verify the vacuum system is capable of producing vacuum in accordance with system specification. The time
required to reach chamber vacuum to 100 µbar shall be less than or equal to 30 minutes.

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Qualification Test Rationale
Vacuum set point control
To verify that vacuum system is capable of controlling the chamber vacuum at higher, middle & lower set point within
given acceptable range. vacuum shall be controlled within  5.0 µbar of the set point.
System leak rate test
The Lyophilization process take place in an evacuated chamber and it has challenge to maintain sterility of the product
in vacuum, hence this test is performed to verify integrity of the Lyophilizer chamber.
Condenser cooling verification
The cooling rate & ultimate temperature of condenser coils substantially determines the efficiency of coils to trap the
sublimed vapors. The temperature of condenser coils shall be achieved less than or equal to -75.0 °C (≤ -75.0 °C).
Shelf RAM/Main valve/Pusher
Bellows integrity test
The bellow is to be challenged for its integrity to avoid product contamination from bellow.
Redundancy check of MKS
vacuum sensor.
This test carried out to verify the redundancy of MKS vacuum sensor, if controlling MKS sensor gets fail, second MKS
vacuum sensor take over the controlling of the chamber vacuum.
Power failure verification
The impact of power failure on step control time in any auto cycle to be verified. The active step control time should
be paused after power failure & should be continued after resumption of power.
Ice condensing capacity
verification of Condenser
To verify and challenge the maximum ice condensing capacity during product processing.

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• Definition - A “media fill” (sometimes known as a “process
simulation”) is the performance of an aseptic manufacturing
procedure using a sterile microbiological growth medium in
place of the drug solution.
• Duration of Lyophilization cycle for media –
• Exact simulation of media fill as that of product
Lyophilization is not required, as we are simulating the
Lyophilization process not validating the Lyophilization cycle.
• The longest Lyophilization cycle will have possible reduction of microbiological levels after aseptic
manipulation which will not solve the purpose of aseptic process simulation.
• Lyophilizer chamber is tested for its integrity after SIP cycle, therefore no additional benefit is drawn by
holding the filled vials for longer duration since the Lyophilizer’s integrity is maintained throughout cycle.
• Shelves temperature - Lyophilizer shelves to be maintained at room temperature (20–25°C) to provide
favorable conditions for the growth of micro-organisms.
• Freezing of media - will form the ice crystals which is unfavorable to microorganisms growth and will reduce
microbial levels of some contaminants, hence freezing should be avoided.

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• Drying - drying of media will reduce microbial levels of some contaminants, hence drying should be avoided.
• Media hold in Lyophilizer - Media filled vials are hold in Lyophilizer chamber for NLT 12 hours.
• Vacuum Pulse –
• During Lyophilization process the chamber vacuum drawn range from 500 to 900 mbar to simulate the
Lyophilization.
• If complete vacuum drawn during media fill simulation, it may cause the media solution to get out from the
containers and contaminate the Lyophilizer as well fluid loss from the containers and hence a complete
vacuum as specified for the Lyophilization process should not be drawn during the media fill.
• Gas for vacuum break - The release of the vacuum must be done with a gas that shall not inhibit aerobic
micro-organisms growth. For Example sterile compressed air shall be substituted for sterile nitrogen gas in
case of lyophilized products.
• Maximum Sterilization hold of the equipment challenged for during aseptic process simulations.
a. Replicating the maximum interval between sterilization of the Lyophilizer and its use.
b. Replicating the maximum interval between sterilization of the Lyophilizer and till completion of Loading.
• Simulation of lyophilized product to be done considering its worst case process flow (i.e. from vial collection
unit to maximum path for the Lyophilizer loading and unloading area) and length of both loading and
unloading operations.

51. Development of Lyophilization Cycle
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Development of Lyophilization cycle by QbD approach: A systematic approach to development that begins
with predefined objectives and highlight product and process understanding and process control, based on
sound science and quality risk management.
(Process Analytical Technology)
(Design of experiments)

52. Development of Lyophilization Cycle
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1. Quality Target Product Profile (QTPP)
The first step in the development of Lyophilization cycle using QbD principles is to identify QTPP
• Type of dosage form
• Fill volume
• Mode of administration
• Reconstitution media and its volume
• Reconstitution time
• Type of vial
• Shelf life
• Post reconstitution stability
• Biocompatibility
2. Identification & determination of following critical quality attributes & process parameters.
a) Critical Quality Attributes (CQA)
b) Critical Material Attributes (CMA)
c) Critical Process Parameters (CPP)

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3. Perform a Risk Assessment -
Link raw Critical material attributes (CMA) and Critical process parameters (CPP) to CQA’s and perform risk
assessment.

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4. Determine Design space:–
• The multidimensional combination and
interaction of input variables (e.g., material
attributes) and process parameters that
have been demonstrated to provide
assurance of quality.
• Simply it is the relation between process
inputs (CMA and CPP) and the CQA can be
described as design space.
5. Identification of control Strategy:–
• A control strategy is designed to ensure
that a product of required quality will be
produced consistently.

55. Critical Material Attributes (CMA), Critical Quality Attributes (CQA) &
Critical Process Parameters (CPP)
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CRITICAL MATERIAL ATTRIBUTES (CMA) CRITICAL QUALITY ATTRIBUTES (CQA) CRITICAL PROCESS PARAMETERS (CPP)
• Morphology - Crystalline/Amorphous • Cake Appearance • Freezing rate & annealing temp.
• Cake Collapse Temp. • Reconstitution Time • Freezing temp. and time
• Eutectic Temperature • Residual Moisture Content
• Primary drying – shelf temp./chamber
pressure /time/ end point
• Glass transition temp. • Stability/Potency • Sublimation Rate
• Product resistance • Colour uniformity
• Secondary drying – shelf temp. /chamber
pressure /time/end point
• Sterility • Heating rate
• Clarity after reconstitution • Product Temperature
• Post reconstitution stability
• Related substances

56. Types of Defects in Lyophilized Vials
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1. MELTBACK:
Cause:
It happens when 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 not
removed properly it gets melt.
Solution:
Set a longer primary drying. Increase the
security time between the primary drying
and the secondary drying.

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2. COLLAPSE:
Cause:
Collapse temperature is higher than the glass
transition temperature. As primary drying
proceed and ice sublime the glassy phase not
able to support its own weight resulting in a
loss of structural rigidity and collapse of
product.
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.

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3. DROPS ON THE VIAL WALL:
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.

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4. CAKE SHRINKAGE:
Cause:
If a Lyophilization cycle is finished but the product
still has a high percentage of residual moisture or if
the vials are stoppered with humid air inside, the
cake may suffer some shrinkage as it absorbs the
ambient from the vial’s atmosphere.
Solution:
Make sure the drying phases of the cycle have
properly finished. Break the existing vacuum in the
chamber with dry nitrogen gas.

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5. BREAKAGE OF THE BOTTOM OF THE VIAL:
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 vapors 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 vapor through the lateral walls of the vial.
Add a little fraction of alcohol.
Use thicker vials or vials of more quality.

61. Types of Defects in Lyophilized Vials
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6. 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.

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6. PUFFING (TOTAL BOILING):
Cause:
Appliance of excessive heat triggers the spontaneous
melting of the product. The product may end up
outside the vial. It may also happen because of a vial
being stoppered at the beginning of the cycle by
error.
Solution:
Reduce the heat input during the first phases of the
primary drying, increasing the time of the step. Also,
controlling the vacuum level.

63. Guidance
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• Guide to Inspection of Lyophilization of Parenterals (7/93).
• Inspection Technical Guide: Lyophilization of Parenterals (4/8/86).
• Guidance for industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing
Practice.
• Health Technical Memorandum 2010.
• EU guidelines - PIC/S Validation of Aseptic Processing (PI 007-6) 2011.
• ICH harmonised tripartite guideline - Pharmaceutical Development Q8(R2).
• ICH guideline Q9 on quality risk management.

64. Thank you…
pravin13687@gmail.com