Pharmaceutical Technology

1. Anhydrobiotic Process And Excipients ForAnhydrobiotic Process And Excipients ForAnhydrobiotic Process And Excipients ForAnhydrobiotic Process And Excipients ForAnhydrobiotic Process And Excipients ForAnhydrobiotic Process And Excipients ForAnhydrobiotic Process And Excipients ForAnhydrobiotic Process And Excipients For
Preservation Of BiomoleculesPreservation Of BiomoleculesPreservation Of BiomoleculesPreservation Of BiomoleculesPreservation Of BiomoleculesPreservation Of BiomoleculesPreservation Of BiomoleculesPreservation Of Biomolecules
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A. A. Hajare
Professor and Head, Dept. of Pharm. Tech.
BHARATI VIDYAPEETH
COLLEGE OF PHARMACY, KOLHAPUR
ashok.hajare@bharatividyapeeth.edu

2. Recombinant DNA and Hybridoma technologies …..
- Production of commercially viable enzymes, proteins,
etc.
Result - Definite need for skill in formulation
Protein pharmaceuticals development is
challenging area
BIOMOLECULE THERAPEUTICSBIOMOLECULE THERAPEUTICS
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challenging area
- Production
- Purification and
- Physical and chemical stability of proteins
Loss of activity during …..
- Processing
- Packaging
- Shipping and
- Long-term storage

3. PROTEIN MARKETPROTEIN MARKET
Protein pharmaceuticals are (and will be) the most rapidly growing
sector in the pharmaceutical repertoire.
Most “cures” for difficult diseases (Alzheimer's, cancer, auto-immune
diseases, etc.) will probably be found through protein drugs.
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>210 FDA approved protein drugs
>35% are recombinant proteins
Protein pharmaceutical sale (2009) : $60 billion
Expected to reach (by 2015) : $200 billion/year

4. PROBLEMS IDENTIFIED BY WHOPROBLEMS IDENTIFIED BY WHO
All vaccines are unstable and need refrigeration
$200 million PA cost of "cold chain"
8 billion injections required p.a. by 2015
Lack of medically trained staff
Non-compliance: Patients refuse painful booster injections
12 vaccines per child by 2008
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12 vaccines per child by 2008
Cross infections - HIV, Hepatitis, septic anemia
Solution Required:
Completely stable vaccines
Single dose units
Ready-to-inject

5. SOME BIOMOLECULESSOME BIOMOLECULES
1. Insulin
2. Blood products
3. Herceptin, humulin, alferon
4. Human growth hormone
5. Erythropoietin
6. Antibodies
7. Cytokines
16. Immunomodulators
17. Actimmune
18. Activase
19. BeneFix
20. Betaseron
21. Humulin
22. Novolin
23. Pegademase
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7. Cytokines
8. Tissue plasminogen activator
9. Urokinase
10. Vaccines
11. Microorganisms
12. Streptokinase
13. Cyclosporine
14. Hormones
15. Immunomodulators
23. Pegademase
24. Epogen
25. Regranex
26. Novoseven
27. Intron-A
28. Neupogen
29. Pulmozyme
30. Infergen

6. BIOMOLECULES DELIVERYBIOMOLECULES DELIVERY
Source of protein
Physicochemical and storage stability
Physiological barriers
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Route of delivery
Pharmacokinetic factors
Formulation type

7. ANHYDROBIOSISANHYDROBIOSIS
All living organisms require water (75% of most organisms is water)
Number of creatures which can survive in a dry state after losing all of their
body water e.g. bacteria, fungi, animals and plants.
Anhydrobiosis was first recorded by Antoine van Leeuwenhoek, in 1720.
A familiar example is baker’s yeast (Saccharomyces cerevisiae) which exist as
a dry powder and recovered alive and active by simple rehydration.
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a dry powder and recovered alive and active by simple rehydration.
Organisms are animals : Soil nematodes
Plants : Selaginella lepidophylla and
Craterostigma plantagineum.
All these living things preserve their biological molecules without
refrigeration/freezing.

8. 8

9. Anhydrobiosis : Net result of production and accumulation of
simple non-reducing sugars (sucrose / trehalose)
Many organisms : Uses trehalose
Resurrection plant : e.g. Craterostigma uses sucrose
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R. T. : Many vaccines (e.g. measles), Mcb’s, glucagon,
human growth hormones.
At 70 C : Enzymes

10. SUGAR GLASS AND STABILITYSUGAR GLASS AND STABILITY
Sugar forms a glass on drying in which biomolecules are embedded in
solid solution of extremely high viscosity.
Therefore, the molecular diffusion required for chemical reaction and
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degradation is therefore negligible.
They are non-reducing and very stable sugars so that the glass matrix
cannot participate in chemical reactions with the product, including the
Maillard reaction.

11. ANHYDROBIOTIC MECHANISMANHYDROBIOTIC MECHANISM
Viscous sugar syrup : during drying forms glass on removal of water
Transition - From freely mobile solution in the liquid phase to an
immobile solid solution in the glass phase.
Stabilization does not occur, if the sugar crystallizes.
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Molecules are excluded from the sugar crystals.

12. PROTEIN STRUCTUREPROTEIN STRUCTURE
Refers to sequence of amino acids & location of
disulfide bonds
Derived from stearic relations of amino acid
residues that are close to one another
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Refers to overall three dimensional architecture of
polypeptide chain
The arrangement of two or more polypeptide
chains to form a functional protein molecule.

13. In the context of protein structure, the term stability can be defined as
‘The tendency to maintain a native (biologically active) conformation’.
Proteins are only marginally stable
X- ray structure analysis of water-soluble proteins –
Hydrophobic cores of nonpolar amino acids groups surrounded by
THE PROBLEMS WITH PROTEINSTHE PROBLEMS WITH PROTEINS
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Hydrophobic cores of nonpolar amino acids groups surrounded by
hydrophilic shell of polar amino acids
Structure is held together by weak non-covalent forces.
When these forces becomes weak, get broken apart leading to unfolding and
inactivation of protein.
Highly susceptible to both chemical and physical degradation.

14. Formation or destruction of covalent bonds, within a polypeptide or
protein molecule.
- Alter the primary structure and impact higher level of its
structure.
Chemical instability (Covalent):
Deamidation, Oxidation. Disulfide exchange and Proteolysis.
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Deamidation, Oxidation. Disulfide exchange and Proteolysis.
Physical instabilitiy (Non-covalent):
Aggregation and precipitation, Adsorption to surface, and Protein
unfolding.
Deamidation and disulphide bond cleavage, may also lead to physical
instabilities.
Every protein is unique, both physically and chemically, and therefore
exhibits unique stability behavior.
Physical and chemical instability- May observed in final pack.

15. NONNON--COVALENT PROCESSESCOVALENT PROCESSES
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16. PROBLEMS WITH BIOMOLECULESPROBLEMS WITH BIOMOLECULES
Chemical complexity and marginal stability of higher order structures
of therapeutic biomolecules present critical problems in the stability of
their formulations.
Scientists are working hard to develop a technology that can formulate
and deliver life-saving and cheaper biological drugs like vaccines,
proteins, enzymes and hormones without refrigeration.
About 2 million children die every year from diseases that could be
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About 2 million children die every year from diseases that could be
treated with biomolecule products.
About 50% of these life-saving biopharmaceuticals are damaged due to
improper storage as well as unavailability of facilities for storing them
properly, specifically for temperature effects.
To be effective, biomolecules require some mechanism that can
maintain their potency and effectiveness at ambient temperature for a
sufficiently long time.
Biomolecules in a liquid state are stable only for a short period due to
molecular movement that may result in degradation.

17. Pharmaceutical products …
- Adequate stability over storage periods of several years.
- Many biomolecules are unstable in aqueous state at ambient
temperature at long-term stability .
To attain extended stability at ambient temperature
- Molecular movement needs to be arrested by some method that stops
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- Molecular movement needs to be arrested by some method that stops
degradation by transforming liquid into a highly immobile, noncrystalline
(amorphous solid) state during storage, called verification.
The system below its glass transition temperature (Tg) is stable due to
immobilization of the reactive entity in a solid glass-like system.

18. pH
Ionic strength
Oxidants
Free radicals
DENATURANTSDENATURANTS
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Free radicals
Heat
Mechanical stress: Shear, Shaking
Pressure

19. PROTECTANTSPROTECTANTS
Formulating biomolecule:
Fundamental understanding of the mechanisms to stabilize proteins.
Cryo and Lyo protection:
Nature protects life from freezing by accumulating selected
compounds to high concentration within organisms.
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Cryoprotectants are preferentially excluded from surface of proteins
and act as structure stabilizers.
Both freezing and dehydration can induce protein denaturation.
To protect a protein from freezing (cryoprotection) and from
dehydration (lyoprotection) denaturation, a protein stabilizer/s is
incorporated in the formulation.

20. BUFFERSBUFFERS
In the development of lyophilized formulations, the choice of buffer
can be critical.
Phosphate buffers particularly phosphate; undergo drastic pH changes
during freezing.
A good approach is to use low concentration of a buffer that undergoes
minimal pH changes during freezing such as Tris, citrate and histidine
buffers.
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For example:
8.4 µg/ml of ox liver catalase in 10 mM phosphate buffer (pH 7.0)
freezing at - 15ºC to -75ºC retained 80% of activity.
About 0.5 mg/ml of LDH in 0.1 M NaCl and 10mM phosphate buffer
(pH 7.5) retained 76% of the activity13.
For stabilizing recombinant factor IX, histidine is found to be the best.

21. BULKING AGENTSBULKING AGENTS
Bulking agents are added to provide bulk to the formulation.
Important at very low concentrations of biomolecules.
Crystalline bulking agents produce an elegant cake structure with good
mechanical properties.
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Mannitol, sucrose or any other disaccharides are suitable.
For example,
Sucrose (34.5% w/v) : Rabbit muscle lactate dehydrogenase.

22. SUGARSSUGARS
Disaccharides form an amorphous sugar glass.
Most effective in lyophilization.
Sugars like glycerol, xylitol, sorbitol, lactose, mannitol, sucrose, trehalose and
inulin – used as cryoprotectant and lyoprotectant.
In comparison with monosaccharide, disaccharides are found to be most
effective.
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For example:
Sucrose (30 mM) : Chymotrypsin and growth factors
Glucose and sucrose (1:10) : Glucose-6-phosphatedehydrogenase
Trehalose : β-galactosidase, S- adenosyl - L-
methionine, E. coli and B.Thuringienesis.

23. TONICITY ADJUSTERSTONICITY ADJUSTERS
Needed either for stability or for route of administration.
Mannitol, sucrose, glycine, glycerol, sodium chloride, polymers, etc.
Increased concentrations showed increased activity.
For example:
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For example:
BSA (1%) : Rabbit muscle LDH during freezing.
Polyvinyl pyrrolidone : LDH with increased concentrations.
Dextran in sucrose : Actin during lyophilization.

24. METAL IONSMETAL IONS
Metal ions can protect some proteins during lyophilization.
Salts and amines have been used as cryoprotectants.
For example:
Zn+ : Insulin protection.
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Divalent metal ions (In presence of sugars)
: Preserves PFK activity.
Potassium phosphate : Higher recovery of LDH
(sodium cholate and sucrose monolaurate - synergistic effects).

25. SURFACTANTSSURFACTANTS
Use of surfactants to reduce adsorption and aggregation.
Help in foam formation.
Act as solubilisers
Tween 80, Pluronic F-68, and Brij 35
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For example:
Pluronics : Lysozyme, Lasota virus
: Reduce adsorption of calcitonin

26. BIOMOLECULE PROTECTIONBIOMOLECULE PROTECTION
Stresses in solutions - heating, hydrolysis, agitation,
freezing, pH changes and exposure
to denaturants.
The net result - inactivation or aggregation
- less clinical efficacy
- high risk of adverse side effects
The practical solution - remove the water.
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To develop formulation - specific conditions and proper
stabilizing additives
Uniqueness of protein - responsible for specific routes of
chemical and physical degradation
during lyophilization and storage.
Difficult to predict degradation pathway by simply designing formulation.

27. MECHANISMS OF PROTECTIONMECHANISMS OF PROTECTION
Lyophilization / Rehydration:
a) Thermodynamic Mechanism
b) Protein Cryoprotectant Complex Mechanism
c) Diffusion Restriction Mechanism
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Drying:
a) Water Replacement Mechanism
b) Single Amorphous State Immobilization Mechanism
c) Viscosity Mechanism
d) Hydration Protection Mechanism

28. Techniques:
Spray drying, freeze drying or lyophilisation, freeze thawing,
precipitations with organic solvents, air drying and rotors evaporation
Major limitations:
Freezing and moderate low temperatures cause damage to
TECHNIQUES AND LIMITATIONSTECHNIQUES AND LIMITATIONS
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Freezing and moderate low temperatures cause damage to
thermolabile biomolecules, reducing their clinical efficacy and
increasing the risk of adverse effects.
Process is lengthy and time-consuming.
If formulated successfully, storage facility such as cold chain storage
transport is a must to maintain stability.
Not suitable for bulk aseptic production.

29. Protein solution atomised and particles dried in seconds in an air stream.
Major advantage
- Spherical particles produced
- Good flow properties with control over particle size
- Very useful for design of non-parenteral dosage forms
SPRAYSPRAY--DRYINGDRYING
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Use - Materials that can withstand high temperatures during drying
Unsuitable:
Damage to sensitive biologicals and pharmaceuticals
High temperature requirement

30. During cryopreservation by freezing
- Damage with formation of ice crystals
During preservation by cryovitrification, the specimen are
subjected to toxic effects of concentrated vitrification
CRYOPRESERVATIONCRYOPRESERVATION
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subjected to toxic effects of concentrated vitrification
solutions
Damage caused during freezing and cryopreservation limits
survival or activity yielded after preservation.

31. FREEZEFREEZE -- DRYINGDRYING
Cost-effective, and produces chemically stable and active
protein.
Best for long term storage.
Removes a considerable amount of water.
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Freezing of specimens before lyophilization and equilibrium of
specimens in partially frozen state can be very damaging.
Cryoprotectants are used to prevent damage.
After lyophilization needs refrigerated storage conditions.

32. FREEZEFREEZE--DRYING PROCESSDRYING PROCESS
Biological items are first frozen in container.
Place under strong vacuum.
Solvent sublimates leaving only solid at
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Solvent sublimates leaving only solid at
intermediately low temperatures (above 50ºC)
Reduces moisture content to <0.1%

33. STORAGESTORAGE
Refrigeration:
Freezing is best for long-term storage.
Low temperature:
Reduces microbial growth and metabolism.
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Reduces microbial growth and metabolism.
Reduces thermal or spontaneous denaturation.
Reduces adsorption on to the container wall.

34. Smooth glass walls best to reduce adsorption or precipitation.
Avoid polystyrene or containers with silanyl or plasticizer
coatings.
PACKAGINGPACKAGING
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Dark, opaque walls reduce chances of oxidation.
Air-tight containers or argon atmosphere reduces air oxidation.

35. VACUUM FOAM DRYING (VFDVACUUM FOAM DRYING (VFD))
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‘Scalable long-term shelf preservation technique for sensitive biologicals’

36. EVAPORATIVE Vs FREEZE DRYINGEVAPORATIVE Vs FREEZE DRYING
Very few scientists working…
Annear, Bronshtein, Roser, Pisal, etc.
Preservation of biological fluids and components, proteins, enzymes
and micro-organisms
Evaporative drying for long periods at ambient temperature without
significant loss of activity.
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significant loss of activity.
Observations:
1. Stability is better than that of freeze-dried samples.
2. Dehydrated solutions with protectants are viscous.
3. The process is under industrial scale up stage.

37. FOAM FORMATIONFOAM FORMATION
For the last 50 years…
Freeze drying has been the best method for stabilization due to belief
that low temperatures cause minimum damage.
Preservation by foam formation (PFF) is a new technology…
- Proposed by Bronshtein in 1996.
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According to Bronshtein, this belief in low-temperature drying with
minimum damage is not correct.
Before Bronshtein’s invention of foam formation, no scalable technology
had been proposed to preserve thermolabile biomolecules at ambient
temperature.

38. Preserved bacteria in a dried state.
Claim: Viscous solutions and biological liquids can be dried by
forming foam by applying a vacuum.
ANNEAR et. al.
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He used this FFP for a only small volume of sample.
FFP was not used until recently, because it was considered to be a
process that damages biologicals.

39. BRONSHTEIN et. al.BRONSHTEIN et. al.BRONSHTEIN et. al.BRONSHTEIN et. al.
First to report that biologicals could be effectively stabilized by foam
drying.
Claim:
PFF has been used successfully to dry various volumes of biological
liquids from 1-100,000ml.
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liquids from 1-100,000ml.
The only limitation of this technology is that the volume of liquid to
be dried must not be more than 20-25% of the container volume,
because the sample expands during foam formation.
The time required for this process is much shorter than other
processes due to intensive boiling.

40. FOAM FORMATION PROCESSFOAM FORMATION PROCESS
In this process, the biological solutions or suspensions are first
transformed into mechanically stable dry foams by boiling them under
vacuum at ambient temperature above freezing point but significantly
below 100ºC (primary drying).
Samples are then subjected to stability drying at an elevated
temperature to increase glass transition temperature (Tg).
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g
Activity yield after the rehydration of the foam-dried sample is
achieved by proper selection of protectants (sugars like sucrose and
trehalose), which are dissolved in the suspension before processing.
Proper selection and use of vacuum, as well as temperature protocols
during drying, help to produce elegant and therapeutically active
products that remain stable at an ambient storage temperature.

41. Suspension containing a biologically active agent is dehydrated or
concentrated by evaporation to high vacuum of pressure higher than 7.6
Torr.
Then pressure adjusted in between 0- 7.6 Torr.
This is sufficient to cause boiling and this lead to mechanically stable dried
foam during boiling.
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Secondary drying is carried out by applying vacuum or dry air to form
stable at elevated temperature
Surfactant is added to enhance foam stability during secondary drying.
Protectant is selected from a group consisting of sugar, carbohydrate,
polysaccharide, polymer, peptide, protein or their mixture.

42. ADVANTAGES OF PFFADVANTAGES OF PFF
Scalable and turbulent process with efficient preservation capability.
Stability of sensitive biologicals at room temperatures.
Lends itself as an aseptic process due to higher vapor pressure above the sample
during PFF, leading to less surface area exposure and less exposure time.
Does not require freezing of sample before drying, therefore more efficient,
gentle and less damaging.
Less time consuming and more energy efficient.
More scalable process compared to freeze drying, which has limitation of cake
height in container.
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height in container.
Allows high ambient temperature stabilization with minimum loss of activity
during drying and storage.
Offers the potential to deliver biomolecules outside the cold chain storage.
High production yields and Long shelf life.
Materials are shipped at ambient temperatures, eliminating refrigerated or
frozen storage & spoilage due to handling & power failures.
Distribution in areas where refrigeration and freezing facilities are not available
or inadequate (under developed countries)

43. APPLICATIONS OF VFDAPPLICATIONS OF VFD
PFF has been used successfully for stabilisation of thermolabile
enzymes and pharmaceuticals:
Amphotericin, urokinase, luciferase, ß-galactosidase, lactate
dehydrogenase, isocitric dehydrogenase, Isocitrate
dehydrogenase, erythopoeitin, lysozyme and icenucleating
proteins at ambient or higher temperature.
Live viruses:
Lasota, herpesvividae, paramyxovividae, flaviviridae,
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Lasota, herpesvividae, paramyxovividae, flaviviridae,
parvoviridae and retroviruses can also be stabilised by
using this vacuum foam drying technique.
Gram-negative bacteria : E. coli and B. bronchiseptica
Gram-positive bacteria : Lactobacillus acidophilus and
Lactococcus lactis subspecies.
Thermolabile antibiotic such as doxorubicin.

44. India - Formulation and preservation research is limited.
Development of stable pharmaceuticals - Much slower pace.
Limitations of current technologies-
1. Retain less biological activity
2. Require long processing time
WHY VFD?WHY VFD?
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2. Require long processing time
3. Produce short shelf life products
4. Cold chain storage and transport systems
5. Induces stresses that denatures proteins.

45. VFD COMPARED TO LYOPHILIZATIONVFD COMPARED TO LYOPHILIZATION
CONSIDERATION VFD TECHNOLOGY LYOPHILIZATION
EFFICIENCY - Boiling materials under vacuum at temperature
above 0°C.
- Very efficient.
- Reduce spoilage due to handling and power failures
- Inefficient
- Time consuming
- comparatively less efficient
CYCLE TIME 24 Hours 2 - 10 days
SCALABILITY - Formation of stable foam to form thin films.
- Allow efficient removal of water at broad range of
volumes.
- Drying rate is limited by
cake-height in each container.
- Scalability is achieved by
- At room as well as at higher temperatures.
- Scalability is achieved by
using more containers.
YIELD - Water evaporates at temperatures above samples
freezing point
- Eliminates damage due to freezing.
- High production yields
- The need to freeze before
sublimation of water can
damage the material.
- Lead to lower yields.
TEMPERATURE
STABILITY
- Combination of protective fillers and dehydration
process allows high temperature stability.
- Preserves broad range of materials at up to 50°C
and can be shipped at room temperature.
- Long shelf life
- Most of freeze dried samples
are stable under refrigeration.
- In some cases at room
temperature
- Short shelf life
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46. FOAM FORMATION EQUIPMENTFOAM FORMATION EQUIPMENT
At the present time no special industrial equipment has been
designed and is available for the bulk production of powders or
market-ready vials by the vacuum foam drying technique.
Researchers have claimed that with a few modifications to the
controls and process cycle programming software, commercially
available freeze dryers could be modified for PFF in glass vials.
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available freeze dryers could be modified for PFF in glass vials.
The main requirement is simultaneous control of vacuum and
temperature during foam drying.
Thus, the pharmaceutical and other industries are suffering from an
absence of effective drying equipment that produces bulk products
stable at ambient temperature.

47. Optimization of temperature and pressure cycles:
1. Vacuum concentration
2. Stability drying and
PROCESS DEVELOPMENTPROCESS DEVELOPMENT
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3. Rapid cooling for glassy matrix.

48. 120
140
160
180
200
Foamheight(mm)
0.5%- F108 1%-F108 3%-F108
0.5%-F68 1%-F68 3%-F 68
0.5%-F87 1%-F87 3%-F87
VFD OF LASOTAVFD OF LASOTA : SCREENING OF FOAMING AGENTSCREENING OF FOAMING AGENT
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0
20
40
60
80
100
0 20 40 60 80 100 120 140
Tim e(m in)
Foamheight(mm)

49. VFD CYCLE OPTMIZATIONVFD CYCLE OPTMIZATION
STEP
CYCLE 1 CYCLE 2 CYCLE 3
Temp
(°C)
Vacuum
(mT)
Time
(Min)
Temp
(°C)
Vacuum
(mT)
Time
(Min)
Temp
(°C)
Vacuum
(mT)
Time
(Min)
1 8 4000 120 18 4000 120 -10 - 15
2 10 4000 120 20 4000 120 10 1200 60
3 10 1500 120 20 1500 120 15 1000 60
4 12 1500 120 22 1500 120 20 800 120
5 14 1500 120 24 1500 120 22 600 120
6 16 1200 60 26 1200 60 24 200 120
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6 16 1200 60 26 1200 60 24 200 120
7 18 1200 60 28 1200 60 26 100 120
8 20 400 120 30 400 120 28 25 120
9 22 200 60 32 200 60 28 25 120
10 24 200 60 34 200 60 28 25 120
11 26 100 120 36 100 120 28 25 120
12 28 25 120 38 25 120 30 25 240
13 30 25 120 40 25 120 40 25 120
14 40 25 120 26 25 120 26 25 120
15 26 25 120 - - - - - -

50. CURRENT STATUS OF VFDCURRENT STATUS OF VFD
It is a new processing technique.
Yet not much exploited but has better potential.
The concept is under investigation for larger scale.
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The products are stable at room temperatures.

51. LIMITATIONSLIMITATIONS
Although foam formation is an old invention, preservation by foam
formation under vacuum and controlled temperature is a new
technology in the embryonic stage, being used for only a few
pharmaceutical applications and needs some improvement.
Elimination of uncontrolled eruptions and spitting out of material from
vials or containers during boiling are the improvements required in this
technology.
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technology.
More parameters must be studied for its comparison with freeze drying
and other known and newly developed drying processes.

52. Preservation by foam formation may be a substitute to
freeze drying or lyophilisation and will stimulate
development of new processes and equipment for
preservation of thermolabile biologicals in a dry state.
COMMENTSCOMMENTS