Lyophilizer history, Lyophilization and it's validation aspects are discussed in this slide. Triple point, temperature maping etc.

2. Introduction & History
Freeze-drying is a kind of preservation technology, by which the material is cooled below its
eutectic temperature or glass transition temperature firstly to be solidified completely, then
dried in vacuum space at low temperature by sublimation drying and desorption drying till
95-99% of moisture is removed.
• 1250 BC to 850 BC :The basic process of freeze drying food was known to the
ancient Peruvian Incas of the Andes.
• 1905: Benedict and Manning reported the drying of animal tissue at pressures less
than 1 atm. by means of a chemical pump.
• 1910’s: Shackell took the basic design of Benedict and Manning and used an
electrically driven vacuum pump, instead of the displacement of the air with ethyl
ether, to produce the necessary vacuum.
• 1920’s: Lyophilization was established as a stabilizing process for heat-labile
materials.
• Freeze-dried coffee was first produced in 1938, and lead to the development of
powdered food products.
• 1940’s: During World War II, the freeze-dried process was developed commercially
when it was used to preserve blood plasma and penicillin.

3. General Design Consideration
• Basically design of the
Lyophilizer based on the
temperature and pressure
with respect to time.
• Major Components
 Chamber
 Shelves
 Vacuum pump
 Vacuum break/vent filter
 Compressor
 Condenser
 Hydraulics
 Isolation valve
 Temperature & pressure
control system
SCHEMATIC DIAGRAM

4. Working Principle
• The fundamental principle in freeze-drying
is sublimation, in this solid phase directly shift
into a gas phase under lower pressure and
temperature.
• Lyophilization is performed at temperature and
pressure conditions below the triple point, to
enable sublimation of ice.
• For a substance to take any particular phase,
the temperature and pressure must be within a
certain range. Without these conditions, that
phase of the substance can't exist.
• if water at atmosphere pressure, then freezing
point of water is 32°F or 0°C and boiling point is
212° F or 100° C.
• if increase the temperature of water above 32°
F while keeping the atmospheric pressure
below 0.06 atmospheres (ATM), but there is no
enough pressure for a solid to form in liquid. It
becomes a gas.

5. Working Principle
• The concentration gradient of water
vapour between the drying front and
condenser is the driving force for removal
of water during lyophilization.
• At atmospheric pressure (approx. 1,000
mbar) water can have three physical
states.
1. Solid;
2. Liquid;
3. Gaseous.
• Below the triple-point (for pure water: 6.1
mbar at 0°C), only the solid and the
gaseous states exist.
• Lyophilization process follows heat
transfer and Mass transfer methods for
drying of the product.
Vapor Pressure (mbar) Temperature (C)
6.104 0
2.599 ‐10
1.034 ‐20
0.381 ‐30
0.129 ‐40
0.036 ‐50
0.011 ‐60
0.0025 ‐70
0.0005 ‐80
Pressure ‐ Temperature for sublimation

6. Heat Transfer
• The transfer of the heat through the product is
generally done by means of circulating a fluid
through the shelf on which vials /product trays are
placed.
• Heat transfer to the product can be divided into
three components:
 Direct conduction,
 Gas conduction
 Radiation.
• Heat transfer during freeze drying occurs in three
ways:
 Heat transfer through the frozen layer.
 Heat transfer through the dried layer.
 Heating by microwave.

7. Mass Transfer
• Mass transfer occurs, when heat
reaches the sublimation front, it raises
the temperature and the water vapor
pressure of the ice. Then vapor move
through the dried product to a region of
low vapor pressure in the drying
chamber.
• The mass transfer of water vapour from
the product to the condenser is
determined by several resistances to
vapour flow that limit the flow rate. The
most important factor is the resistance
of the already dried layer to mass
transfer, it is called product resistance
(Rp).
• 1 g of ice forms 2 m3 of vapor at 67 Pa.
Heat & Mass Transfer in Freeze Drying

8. Coupling between heat and mass transfer
• During the steady state of primary drying, the amount of heat introduced in
product is equilibrium with amount of heat removed in sublimation of ice. During
freeze-drying Heat and mass transfer are coupled which can be described by
. ∆ .
Where,
dQ/dt - Flow to heat in to product, dm/dt - Removal mass by sublimation, ∆HS -
Temperature-dependent heat of sublimation of ice (cal/g), ms - Sample mass
(g), cv - Specific heat of the sample (cal/K*g) and dT/dt - Change of product
temperature (K/s).
• Unbound water removed in primary drying and bound water removed during
secondary drying process.

9. FREEZE DRYING PROCESS
• Lyophilization process has be
involved in 4 stages.
 Pretreatment (Sample
Preparation)
 Freezing (Solidification)
 Primary Drying (Sublimation)
 Secondary Drying (Desorption)

10. FREEZE DRYING PROCESS
Transfer
to Filling
Unloading
for sealing
& Packing
Loading
Pretreatment Filling
LyophilizationFinal Product

11. Pretreatment (Sample Preparation)
• Method of treating of the product prior to the
freezing. This may include concentrating the
product, formulation revision (i.e., addition of
components to increase stability and/or improve
processing), decreasing a high vapor pressure
solvent or increasing the surface area.
 Definite Freeze concentration,
 Define the Solution phase concentration,
 Formulation to Preserve Product
Appearance,
 Formulation to Stabilize Reactive Products,
 Formulation to Increase the Surface Area,
 Decreasing High Vapor Pressure Solvents.

12. Freezing (Solidification)
• The product must be frozen at a enough low
temperature to completely solidify the product.
• During freezing low temperature and low
atmospheric pressure should be maintained for
adequately pre-frozen product.
• Pre-freezing and the final temperature of the
frozen product can affect the ability to
successfully freeze dry the product.
• The rate of ice crystallization define the
freezing process and efficiency of primary
drying.
• Unfrozen product may expand outside of the
container when placed under drying with high
vacuum.
• Slower cooling results in large ice crystals and
less restrictive channel in the matrix during the
drying process.

13. Primary drying (Sublimation)
• After completion of product freezing phase, secondary drying phase started,
in which ice can be removed from the frozen product via sublimation,
resulting in a dry, structurally intact product.
• In this phase chamber pressure lowered , and enough heat is supplied to
the product for the sublimation of ice.
• H2O molecules drift from the high- pressure area to a lower pressure area.
Where vapor pressure of product is related to temperature, it is essential
that the product temperature is warmer than the cold trap (ice collector)
temperature.
• Primary drying is complete when all frozen bulk (Unbound/Loose) water is
removed via sublimation. At this point, the product still contains some bound
unfrozen water that has to be removed by desorption at higher
temperatures during secondary drying.
• The time at which there is no more frozen layer is taken to represent the
end of the primary drying stage.

14. Primary drying (Sublimation)
• Heat enters the products by one of numerous mechanisms: -
 By direct contact between the container base and the shelf, so here the shape of
the container is important.
 By conduction across the container base and then through the frozen mass to
the drying front (also called the sublimation interface)
 By gaseous convection between the product and residual gas molecules in the
chamber.
 By radiation, this is low due to low temperature encountered in freeze-drying.

15. Secondary Drying (Desorption)
• After primary drying phase completion, Secondary drying phase started.
• In this phase, bound water (moisture) which was not removed during primary
drying, shall be removed by desorption.
• Secondary drying is normally continued at a (low) more than primary drying
temperature to product temperature (Stable).
• This process is called ‘Isothermal Desorption’ as the bound water is desorbed
from the product, desorption drying is facilitated by raising shelf temperature
and reducing chamber pressure to a minimum.
• After completion of cycle Vacuum should be break through filtered nitrogen
gas.
• Secondary drying is usually carried out for approximately 1/3 or 1/2 the time
required for primary drying.

16. Process Variables of Lyophilization

17. Validation Approach
• DQ, FAT, IQ, OQ and PQ is
the part of Lyophilizer
qualification.
• Instruments and equipment s
used for validation of
Lyophilizer is mentioned
below.
 Validator
 Temperature
sensor/Thermocouple
 Pressure sensor
 Biological Indicator
 Riboflavin/NaCl

18. Validation Approach
1. Chamber Vacuum Leak Test
2. Chamber Evacuation Rate and Ultimate Vacuum Verification
3. Filter Integrity test (Vacuum Break/Vent Filter)
4. CIP
5. SIP (with vacuum)
6. Shelf temperature Uniformity Test
7. De-Icing Test (Condenser Defrosting)
8. Media Fill
• Performance Qualification:

19. Validation Approach
• Chamber Leak Test:
Calculate the volume of chamber and condenser for calculation of rise
in pressure.
Chamber leak rate: Q = (P2 – P1) X V/t Pa.m3/s
Where: P1: Initial pressure, P2: Final pressure, V: Chamber Volume
 Close the pump isolation valve, stop vacuum pump, and record P1
and the time; record P2 and the time after 30 minutes, and then
calculate the leak test.
 Acceptance Criteria:
 Entire leak rate of the chamber ≤ 0.03 mbar L/s
• Note: Bellow Leak test is an important part of Lyophilization Qualification. It
minimize risk rate of product contamination due to the bellow leak.

20. Validation Approach
• Chamber Evacuation Rate and Ultimate Vacuum
Verification:
 Start vacuum pump after condenser reaches -45°C,
 Record the time of chamber evacuation from 1 atm. (1013.2mbar)
 Pressure down to 1 X 10-1 mbar.
 After the chamber pressure is below 1 X 10-1heat the selves till
chamber pressure reaches the ultimate vacuum (≤1X 10-2 mbar).
 Acceptance Criteria:
 The evacuation rate: Chamber pressure should pump-down
to 1 X 10-1mbar within 30 min.
 Ultimate vacuum: ≤1X 10-2 mbar

21. Validation Approach
• Filter Integrity Test: Hydrophobic filters are installed with the
Lyophilizer for the vacuum break/vent for the cycle.
 Filter integrity will be performed with two methods:
 Bubble Point Test
 Water Intrusion Test
• CIP (Clean in Place):
Two type of solution used for verifying the CIP test of any vessel
or container, it is also called “Spray ball coverage test”.
 Riboflavin Test (0.2% solution used)
 NaCl Test (0.9% solution used)

22. Validation Approach
• SIP (Steam in Place):
 There should be uniform distribution of heat inside the chamber and
condenser during the sterilization hold period and the temperature at each
temperature mapping probe should be within the range of 121.6 °C to 124.0
°C during the sterilization hold period.
 Temperature uniformity at a given time of temperature recording between
all probes during hold period should not be more than ±1°C.
 Temperature spread during hold period at a certain monitoring location
NMT 2°C.
 The lag time (the time difference between the first sensor reaches
sterilization temperature and sterilization hold start inside the chamber)
should not be more than 30 second.
 The chemical indicators should change the colour from pink to green for at
least three compartments.
 All BI should show no growth after incubation as per vendor
recommendation.

23. Validation Approach
• Shelf temperature Uniformity Test:
 For validation of the chamber, the usual number of temperature
measuring points is five for each shelf – one in each corner plus one
in centre.
 One sensor required for the condenser temperature verification.
 Usually, the cross shelf stability (homogeneity) required is ±0.5°C to
1°C and shelf-to-shelf variation is 1°C to 2°C.
 As freeze dryers are often equipped with 10-15 shelves or more, it is
essential that the logging hardware and analyzing software can
handle data of that magnitude.
 The loggers have a storage capacity of up to 60,000 data points per
logger while the ValSuite™ Pro software can handle more than 100
channels in each session.

24. Validation Approach
• DE-ICING TEST (CONDENSER DEFROSTING):
 For initial validation after lyophilisation dummy trial, the condenser
defrosting cycle is to be carried out.
 For Revalidation after any production batch the defrosting cycle is
to be carried out
 After the cycle is over inspect visually for any ice in condenser
and dryness of condenser.
 Acceptance Criteria:
 No ice should observe in the condenser
 The condenser should be dried.
• Media Fill:
 For lyophilization operations, FDA recommends that unsealed
containers be exposed to partial evacuation of the chamber in a
manner that simulates the process. Vials should not be frozen,
and precautions should be taken that ensure that the medium
remains in an aerobic state to avoid potentially inhibiting the
growth of microorganisms.

25. Application
• The product can be stored at
room temperature or 4°C
for long time after freeze
drying.
 Pharmaceuticals Industry
 Biopharmaceutical
Industry
 Food Processing Industry
 Ceramics Industry
 Dairy Industry
 Chemical Industry
 Documents Recovery
 Flower Preservation
Freeze Dried CarrotFreeze Dried Meat
Freeze Dried Drugs

26. Routin Monitoring & Safety
• Periodic tests: Must be subject to a schedule of periodic tests at various levels and
intervals.
 Daily,
 Weekly,
 Quarterly
 Yearly
 The yearly test schedule is essentially a revalidation schedule. It provides for
performance requalification (PRQ) tests to confirm that data collected during
performance qualification remain valid.
• Safety Measures:
 Eye Protection
 Lab Coat, asseptic area garment
 Closed-toed Shoes
 Heat-resistant Gloves

27. Advantage & Disadvantage
• Advantage
 Chemical decomposition is
minimized.
 Removal of water without excessive
heating.
 Enhanced product stability in a dry
state.
 Low particulate contamination.
 Solid more stable than solution
 Low temperature process => less in-
process degradation
 Compatible with aseptic processing
 Can be easily reconstituted
• Disadvantage
 Increased handling and processing
time.
 Volatile compounds may be removed
by vacuum.
 Need for sterile diluents upon
reconstitution
 Cost => capital expenditures, process
long and expensive
 Difficult to produce crystalline material