Anhydrobiotic Process And Excipients For Anhydrobiotic Process And Excipients For Anhydrobiotic Process And Excipients For Anhydrobiotic Process And Excipients For Anhydrobiotic Process And Excipients For Anhydrobiotic Process And Excipients For Anhydrobiotic Process And Excipients For Anhydrobiotic 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. HajareProfessor and Head, Dept. of Pharm. Tech.

BHARATI VIDYAPEETH

COLLEGE OF PHARMACY, KOLHAPUR

ashok.hajare@bharatividyapeeth.edu

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

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

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

SOME BIOMOLECULES SOME 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

BIOMOLECULES DELIVERYBIOMOLECULES DELIVERY

Source of protein

Physicochemical and storage stability

Physiological barriers

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Route of delivery

Pharmacokinetic factors

Formulation type

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.

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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

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.

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.

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.

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.

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.

NONNON--COVALENT PROCESSESCOVALENT PROCESSES

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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.

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.

pH

Ionic strength

Oxidants

Free radicals

DENATURANTSDENATURANTS

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Free radicals

Heat

Mechanical stress: Shear, Shaking

Pressure

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.

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.

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.

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.

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.

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).

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

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.

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

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.

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

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.

FREEZE FREEZE -- 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.

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%

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.

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.

VACUUM FOAM DRYING (VFDVACUUM FOAM DRYING (VFD))

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‘Scalable long-term shelf preservation technique for sensitive biologicals’

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.

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.

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.

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.

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.

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.

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 sampleduring 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 cakeheight in container.

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height in container.

Allows high ambient temperature stabilization with minimum loss of activityduring 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 orfrozen storage & spoilage due to handling & power failures.

Distribution in areas where refrigeration and freezing facilities are not availableor inadequate (under developed countries)

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 byusing 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.

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.

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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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.

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.

120

140

160

180

200

Fo

am

he

igh

t (m

m)

0.5%- F108 1%-F108 3%-F108

0.5%-F68 1%-F68 3%-F 68

0.5%-F87 1%-F87 3%-F87

VFD OF LASOTA VFD 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)

Fo

am

he

igh

t (m

m)

VFD CYCLE OPTMIZATION VFD 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 - - - - - -

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.

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.

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