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P RENTER L PRODUCTS

MANJUL PRATAP SINGH ANITA SINGH

KAILASH INSTITUTE OF PHARMACY MANAGEMENT, GIDA,

GORAKHPUR

09628373561

K.I.P.M. GIDA, GORAKHPUR, U.P.

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DEFINATION:

Parenteralrefers injectable route of administration.

It derived from Greek words Para (Outside) and enteron (Intestine). So it

is a route of administration other than the oral route. This route of

administration bypasses the alimentary canal

Pyrogens, fever-producing substances are primarily lipid polysaccharide

product of metabolism of microorganism; they may be soluble,insoluble, or colloid.


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Parenteral Dose Forms

Parenteral preparations must be sterile

free of microorganisms

To ensure sterility, parenterals are prepared using

aseptic techniques

special clothing (gowns, masks, hair net, gloves) laminar flow hoodsplaced in special rooms


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Advantages of the Parenteral Route

The IV route is the fastest method for delivering systemic drugs

preferred administration in an emergency situation

It can provide fluids, electrolytes, and nutrition

patients who cannot take food or have serious problems with the

GI tract

It provides higher concentration of drug to bloodstream or tissues advantageous in serious bacterial infection

IV infusion provides a continuous amount of needed medication

without fluctuation in blood levels of other routes

infusion rate can be adjusted

to provide more or less medication as the situation dictates

Drug action can be prolonged by modifying the formulation.


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Disadvantages of the Parenteral Route

Traumatic injury from the insertion of needle

Potential for introducing:

Toxic agents

Microbes

Pyrogens Impossible to retrieve if adverse reaction occurs

injected directly into the body

Correct syringe, needle, and technique must be used

Rotation of injection sites with long-term use

prevents scarring and other skin changes

can influence drug absorption


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Routes of Administration of parenteral products

Various types of route of administration of parenteral products are:

Intradermal injection Subcutaneous (Hypodermis) injection

Intramuscular injection

Intravenous injection

Intra-arterial injection

Intracardiac injection

Intrathecal injection

Intracisternal injection

Peridural injection

Intra- articular injection

Intracerebral injection


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Subcutaneous Injections

Given at a 45-degree angle

25- or 26-gauge needle, 3/8 to 5/8

inch length

No more then 1.5 ml should be injected

into the site

to avoid pressure on sensory nerves

causing pain and discomfort

Administer medications below the skin into the subcutaneous fat

outside of the upper arm

top of the thigh

lower portion of each side of the abdomen

not into grossly adipose, hardened, inflamed, or swollen tissue

Often have a longer onset of action and a longer duration of action

compared with IM or IV injection


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Intramuscular Injections

Care must be taken with deep IM injections to avoid hitting a vein,

artery, or nerve In adults, IM injections are given into upper, outer portion of the

gluteus maximus

large muscle on either side of the buttocks

For children and some adults, IM injections are given into the deltoidmuscles of the shoulders

Typical needle is 22- 25 gauge - to 1-inch needle

IM injections are administered at a 900angle

volume limited to less than 3 ml


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Intravenous Injections or Infusions

Fast-acting route because the drug goes directly into the

bloodstream

often used in the emergency department and in critical care

areas

Commonly used

for fluid and electrolyte replacement to provide necessary nutrition to the patient who is critically ill

Intravenous (IV) injections are

administered at a 15- to 20-degree

angle


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Intra-arterial injection

Intracardiac injection

Intrathecal injectionThese are given into the subachonoid space the

surround the spinal cord. This route is used for

spinal anesthesia.

These are given into the heart muscle or ventricle

at the time of emergency only.

The inaction are given directly in to the artery


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Intracisternal injection

These are given in b/w first & second cervical nerve.

Used for CSF for diagnostic purpose.

Peridural injectionThese are given in b/w the dura matter & inner aspect

of vertebra.

Used for given spinal anesthesia.

Intra- articular injection

These are given in into the articulating ends of bones in

a joint.

Used for lubricating the joints.Intracerebral injection

These are given into the cerebrum.


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Official types of injections

Injection: Liquid preparation there are drug substance ordrug solution thereof e.g. insulin injection USP.

For injection: Dry solid that upon addition of suitable vehicles yieldsolutions confirming in all respect to the requirements to the

injection. e.g. Cefuroxime injection USP.

Injectable emulsions: Liquid preparation of drug substancedispersed in a suitable emulsion medium. e.g. Propofol USP.

Injectable suspension: Liquid preparation of solid suspended in a

suitable medium. e.g. Methyl Prednisolone Acetate Suspension USP.

For injectable suspension: Dry solid that upon addition ofsuitable vehicle yields preparation confirming in all respect

to the requirements for Injectable suspension.

e.g. Imipenem and Cilastatin injectable suspension USP.K.I.P.M. GIDA, GORAKHPUR, U.P.

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General requirements of parenteral preparations

Stability

Sterility

Free from Pyrogens

Free from foreign particles

Isotonicity

Specific gravity

Chemical purity


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Formulation of parenteral products In the preparation of parenteral products, the following substances

are added to make a stable preparation:

The active drug

Vehicles

Aqueous vehicle (e.g. water for injection, water for injection free from CO2 )

Non-aqueous vehicle (e.g. Ethyl alcohol, propylene glycol, almond oil)

Adjuvants

Solubilizing agents (e.g. Tweens & polysorbates)

Stabilizers & antioxidants (e.g. thiourea, ascorbic acid, tocopherol)

Buffering agents (e.g. citric acid, sodium citrate)

Antibacterial agents (e.g. benzyl alcohol, metacresol, phenol)

Chelating agents (e.g. EDTA)

Suspending, emulsifying & wetting agents (e.g. MC, CMC)

Tonicity factor (e.g. sodium chloride, dextrose)


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PREFORMULATION FACTORS

It is study about physical & chemical properties of drug substance

prior formulation is called as preformulation.

They are

pH /pka

Solubility

Thermal/heat effect

Dissociation constant

Compatabilty studies- FTIR / DSC

Oxidation & reduction

particle sizeK.I.P.M. GIDA, GORAKHPUR, U.P.

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pH and pKa SOLUBILITY PROFILE

pKa Determination: The Henderson Hasseslebach equation

provides an estimate of the ionized and un ionized durg concentration

at a particular pH.

For acidic drugs,

pH = pKa + log (ionized drug) / un-ionized drug)Forbasic drugs,

pH = pKa + log (unionized drug / ionized drug )

Buffers, temperature, ionic strength and cosolvent can affect the pKa

value.

Potentiometric titration offers maximum sensitivity for

compounds with pKa values in the range of 3-10.K.I.P.M. GIDA, GORAKHPUR, U.P.

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SOLUBILIZATION

Solubilization is increased by co solvent addition.

E.g.- propylene glycol solubilize drug molecules by

disrupting the hydrophobic interactions of water.

More non polar the solute

greater is the solubilisation

achieved by co solvent addition


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THERMAL/HEAT EFFECTS

Drugs which are unstable to heat requires refrigerative storage or

lyophilisation (these products must be used within short periods)

If it is endothermic---> H is +ve

increase in temp ---> increase in drug solubility

If it is exothermic---> H is ve

increase in temp ---> decrease in drug solubility

For determining H

ln S= - H /RT + C

S=molar solubility at temperature, T=temperature in Kelvin,

R= gas constant K.I.P.M. GIDA, GORAKHPUR, U.P.

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Particle size

FINE PARTICLE CHARACTERISATION Very imp. Property and

here smallest particle should be tested to facilitate homogeneous

sample preparation.

Coulter Counter Technique To check particle size and particle volume

BET (BRUNAUER, EMMET, TELLER) NITROGEN

ADSORPTION APPARATUS Measurement of surface area

SEM( SCANNING ELECTRON MICROSCOPY) to check surface

morphology


O id i & d i

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Oxidation & reduction

Adding oxygen to form an oxide (oxidation) or 2H2+ O2-> 2H2O

Removing oxygen (reduction).

Due to oxidation ,

- shelf life reduces

- changes in color

- Potency of drug becomes less

So antioxidants are used to prevent oxidation e.g sodium bisulphate,

sodium metabisulphate


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Processing of parenteral preparationFollowing steps are involved in the processing of parenteral

preparation:

1) Cleaning of containers, closures & equipments

2) Collection of materials

3) Preparation of parenteral products

4) Filtration

5) Filling the preparation in final container

6) Sealing the container

7) Sterilization

8) Evaluation of the parenteral preparation

9) Labeling & packagingK.I.P.M. GIDA, GORAKHPUR, U.P.

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1. Cleaning of containers, closures & equipments: Thoroughly cleanedwith detergents with tap water distilled water finallyrinsed with water for injection.

Rubber closures are washed with 0.5% sod. pyrophosphate in water.

2. Collection of materials: All raw material of preparation should becollect from warehouse after accurate weighed.

Water for injection should be Pyrogens free.

3. Preparation of parenteral products: The parenteral preparationmust be prepared in aseptic conditions.

The ingredients are accurately weighed separately and dissolved in

vehicle as per method of preparation to be followed.

4. Filtration: The parenteral preparation must be filtered by

bacteria proof filter such as, filter candle, membrane filter.


5 Filli th ti i fi l t i Th filli ti i

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5. Filling the preparation in final container: The filling operation is

carried out under strict aseptic precautions.

6. Sealing the container: Sealing should be done immediate after filling

in aseptic environment.

7. Sterilization: For thermostable substances the parenteral products

are sterilized by autoclaving method at different temp. & pressure.

10 lb pressure (115.50C, or 2400F) for 30 minutes

15 lb pressure (121.50C, or 2500F ) for 20 minutes

20 lb pressure (126.50C, or 2600F) for 15 minutes

Heat sensitive or moisture sensitive material are sterilized by

exposure to ethylene oxide or propylene oxide gas .

8. Evaluation of the parenteral preparation: The following tests areperformed in order to maintain quality control:

1. Sterility test 2. Clarity test 3. Leakage test

4. Pyrogen test 5. Assay

9. Labeling & packaging K.I.P.M. GIDA, GORAKHPUR, U.P.

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Evaluation of Parenteral products

Sterility testing

Particulate matter monitoring Faculty seal packaging or leakage test

Pyrogen testing

LAL test

Assay or drug content uniformity


S ili i

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Sterility testing DEFINITION: Sterility Testing: It is a procedure carried out to detect and

conform absence of any viable form of microbes in or on pharmacopeia

preparation or product.

PRINCIPLE : Sterility testing only shows that organisms capable of growing

in selected conditions are absent from the fraction of batch that has been

tested. If the microorganism are present in the product can be indicated by

a turbidity in the clear medium.

OBJECTIVE OF STERILITY TESTING:

For validation of sterilization process.

To check presence of microorganisms in preparation which are sterile.

To prevent issue of contaminated product in market.K.I.P.M. GIDA, GORAKHPUR, U.P.

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STEPS INVOLVED IN STERILITY TE TESTING

1) Sampling

2) Selection of the quantity of the product to be used

3) Method of sterility testing

i ) METHOD 1 Membrane filtration method

ii) METHOD 2 Direct inoculation method

4) Observation and interpretation Must be carried out under

aseptic condition.


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Sampling The sample must be representative of the whole of the bulk material

& a lot of final containers.

MAINLY FOLLOWED BY TWO RULES:

A fixed percentage of the final container are selected.

A fixed number of container are taken independent of the lotor batch size.

According to Indian Pharmacopoeia following guidelines for

determining the minimum number of items are:


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Selection of the quantity of the product to be used

Selection of the quantity of the product to be used for sterility testing

depends mainly on the volume or weight in the container.


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Method of sterility testing

Membrane filtration method (METHOD 1):

Membrane filtration Appropriate for : (advantage)

Filterable aqueous preparations

Alcoholic preparations

Oily preparations

Preparations miscible with or soluble in aqueous or oily (solvents

with no antimicrobial effect)

All steps of this procedure are performed aseptically in a Class 100

Laminar Flow Hood


M b filt 0 45 it

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Membrane filter 0.45porosity

Filter the test solution

After filtration remove the filter

Cut the filter in to two halves

First halves (For Bacteria) Second halves (For Fungi)

Transfer in 100 ml culture media

(Fluid Thioglycollate medium)

Incubate at 30-350 C for not less then 7 days


(Soyabeans-Casein Digest medium)


Observe the growth in the media Observe the growth in the mediaK.I.P.M. GIDA, GORAKHPUR, U.P.

Di i l i h d (METHOD 2)

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Suitable for samples with small

volumes

volume of the product is not morethan 10% of the volume of the

medium

suitable method for aqueous

solutions, oily liquids, ointments ancreams

Direct inoculation of the culture

medium suitable quantity of the

preparation to be examined istransferred directly into the

appropriate culture medium &

incubate for not less than 14 days.

Direct inoculation method (METHOD 2):


Ob ti d lt

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Observation and results

Culture media is examined during and after at the end of incubation.

The following observations are possible:

1) No evidence of growth Pass the test for sterility.

2) There is evidence of growth Re-testing is performedsame no.

of sample, volume & media as in original test No evidence of

growth Pass the test for sterility.

3) There is evidence of growth isolate & identify the organism.

Re-testing is performed with twice no. of sample if:

No evidence of growth Pass the test for sterility.

There is evidence of growth Fail the test for sterilityK.I.P.M. GIDA, GORAKHPUR, U.P.

Particulate matter monitoring

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Particulate matter monitoring Particulate matter is defined as unwanted mobile insoluble matter

other than gas bubble present in the product.

If the particle size of foreign matter is larger than the size of R.B.C.. Itcan block the blood vessel.

The permit limits of particulate matter as per I.P.are follows:

Source of particulate matter:

1. Intrinsic contamination: The material which are originally

present in the parenteral solution e.g. Barium ions leach inparenteral & react with sulphur ions in the product to formbarium sulphate crystals.

2. Extrinsic contamination: The material which comes from theenvironment e.g. Shedding of material from cloth, body, &

cotton, paper, rubber, tissue etc.K.I.P.M. GIDA, GORAKHPUR, U.P.

M th d f it i ti l t tt t i ti

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Methods for monitoring particulate matter contamination:

1) Visual method

2) Coulter counter method

3) Filtration method4) Light blockage method

Identification of particulate matter:

1) Microscopy

2) X-ray powder diffraction

3) Mass spectroscopy

4) Polarized light spectroscopy

5) Scanning electron microscopy (SEM)


F lt l k i / l ki t ti

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Faculty seal packaging / leaking testing

The sealed ampoules are subjected to small cracks which occur due

to rapid temperature changes or due to mechanical shocks.

Vials & bottles are not suitable for this test because the sealing

material used is not rigid.

Filled & sealed ampoules

Dipped in 1% Methylene blue solution

Under negative pressure in vacuum chamber

Vacuum released colored solution enter into the ampoule

Defective sealing


Pyrogen Testing

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Pyrogen Testing

Pyrogen = Pyro(Greek = Fire) + gen(Greek = beginning).

Fever producing, metabolic by-products of microbial growth and

death.

Bacterial pyrogens are called Endotoxins. Gram negative bacteria

produce more potent endotoxins than gram + bacteria and fungi.

Endotoxins are heat stable lipopolysaccharides (LPS) present in

bacterial cell walls, not present in cell-free bacterial filtrates

Stable to at least 175oC; steam sterilization ineffective

Water soluble; monomer unit of LPS can be 10,000 Daltons (1.8 nm)

so endotoxins can easily pass through 0.22mfilters

Sources: Water (main), raw materials, equipment, process

environment, people, and protein expression systems if using gram

negative bacteria.


Biological properties of endotoxin

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Pyrogen elevate the circulatory levels of inflammatory cytokines

which may be followed by fever, blood coagulation, hypotension

Low doses of Pyrogen: asymptomatic inflammation reaction

Moderate doses: fever & changes in plasma composition

High doses: cardiovascular dysfunction, vasodilation,

vasoconstriction, endothelium dysfunction, multiple organ failure

& finally death.

Sources of pyrogen1) Equipment

2) Containers (Glass , plastic , metal)

3) Solvent

4) Solute K.I.P.M. GIDA, GORAKHPUR, U.P.

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Elimination of pyrogens

Dry heat sterilization : For glass wares, metal equipments,

powders, waxes, oils, heat stable drugs

650 oC temp - 1 min

250 oC temp - 30 min


Ultra filtration

Reverse osmosis : RO membrane is composed of cellulose acetate

phthalate/ polyamide

Distillation

Adsorption methodK.I.P.M. GIDA, GORAKHPUR, U.P.

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Principal:

Rabbits are used to perform this test because their body temp

increases when pyrogen are introduced into their bodies by

parenteral route 3 healthy adult rabbits of either sex, each weighing NLT 1.5 kg are

selected

Do not use any rabbit

having a temp higher than 39.8 oC

Showing temp variation >0.2 oC between two successive reading

in the determination of initial temp

Sham test is performed within 7 days of actual test

Animal showing temp increase over 0.6 oC should be removed from

pyrogen testing


Method :

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Method :

Dissolve the subs being examined in, or dilute it with a pyrogen freesaline solution

Warm the liquid being examined to approx. 38.5o C temp beforeinjection

The volume of injection is NLT 0.5ml/kg & NMT 10ml/kg of bodyweight

Withhold water during test

Clinical thermometer is inserted into the rectum of rabbit to recordbody temp

2 normal reading of rectal temp are should be taken prior to the testinjection at an interval of half an hr & its mean is calculated- initialtemp

The solution under test is injected through an ear vein

Record the temp of each rabbit in an interval of 30 min for 3 hrs

The difference between initial temp & maximum temp is recorded-taken as response


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Interpretation of results


Bacterial endotoxin (LAL) test )

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To detect or quantify endotoxins of gram-ve bacterial origin

Reagent: amoebocyte lysate from horseshoe crab (Limuluspolyphemus or Tachypleus tridentatus).

The name of the test is also Limulus amebocyte lysate (LAL) test

Mechanism of LAL Test:

The test is based on the primitive blood-clotting mechanism of

the horseshoe crab

enzymes located with the crab's amebocyte blood cells endotoxin

Initiation of an enzymatic coagulation cascade

proteinaceous gelK.I.P.M. GIDA, GORAKHPUR, U.P.

Test performance (short)

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Avoid endotoxin contamination

Before the test:

interfering factors should not be present

equipment should be depyrogenated the sensitivity of the lysate

should be known

Test:

equal Volume of LAL reagent and test solution (usually 0.1 ml of

each) are mixed in a depyrogenated test-tube

Incubation at 37C, 1 hour

remove the tube - invert at (180) observe the result

pass-fail test K.I.P.M. GIDA, GORAKHPUR, U.P.

LAL test

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LAL test

Three different techniques:

1. The gel-clot technique - gel formation

2. The turbidimetric technique - the development of Turbidity aftercleavage of an endogenous substrate

3. The chromogenic technique - the development of color after

cleavage of a synthetic peptide- chromogen complex

Advantages of LAL testFast - 60 minutes vs. 180 minutes

Greater Sensitivity ,Less Variability

Much Less False, Positives ,Much Less Expensive

Alternative to Animal Model, cheaper,particularly useful for:

Radiopharmaceuticals and cytotoxic agents

Blood products

Water for injectionK.I.P.M. GIDA, GORAKHPUR, U.P.

Production facilities of parenterals

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The production area where the parenteral preparation are

manufactured can be divided into five sections:

Clean-up area

Preparation area

Aseptic area

Quarantine area

Finishing & packaging area


Clean up area:

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Clean-up area:

It is not aseptic area.

All the parenteral products must be free from foreign particles &

microorganism.

Clean-up area should be withstand moisture, dust & detergent.

This area should be kept clean so that contaminants may not be carried out

into aseptic area.

Preparation area:

In this area the ingredients of the parenteral preparation are mixed &

preparation is made for filling operation.

It is not essentially aseptic area but strict precautions are required to prevent

any contamination from outside.


Aseptic area:

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Aseptic area:

The parenteral preparations are filtered, filled into final container

& sealed should be in aseptic area.

The entry of personnel into aseptic area should be limited &

through an air lock.

Ceiling, wall & floor of that area should be sealed & painted.

The air in the aseptic area should be free from fibers, dust and

microorganism.

The High efficiency particulate air filters (HEPA) is used for air.

UV lamps are fitted in order to maintain sterility.


Quarantine area:

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Quarantine area:

After filling, sealing & sterilization the parenteral product are held

up in quarantine area.

Randomly samples were kept foe evaluation. The batch or product pass the evaluation tests are transfer in to

finishing or packaging area.

Finishing & packaging area:

Parenteral products are properly labelled and packed.

Properly packing is essential to provide protection against

physical damage.

The labelled container should be packed in cardboard or plastic

container.

Ampoules should be packed in partitioned boxes


Lyophilization or freeze drying

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Lyophilization or freeze drying is a process in which water is removed

from a product after it is frozen and placed under a vacuum, allowing

the ice to change directly from solid to vapor without passing through

a liquid phase.

The process consists of three separate, unique, and interdependent

processes;

Freezing,

Primary drying (sublimation), and

Secondary drying (desorption).


Th d t f L hili ti i l d

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The advantages of Lyophilization include:

Ease of processing a liquid, which simplifies aseptic handling

Enhanced stability of a dry powder

Removal of water without excessive heating of the product

Enhanced product stability in a dry state

Rapid and easy dissolution of reconstituted product

Disadvantages of Lyophilization include:

Increased handling and processing time

Need for sterile diluent upon reconstitution

Cost and complexity of equipment


The Lyophilization process generally includes the following steps

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The Lyophilization process generally includes the following steps:

Dissolving the drug and excipients in a suitable solvent, generally

water for injection (WFI).

Sterilizing the bulk solution by passing it through a 0.22 micronbacteria-retentive filter.

Filling into individual sterile containers and partially stoppering

the containers under aseptic conditions.

Transporting the partially stoppered containers to the lyophilizerand loading into the chamber under aseptic conditions.

Freezing the solution by placing the partially stoppered containers

on cooled shelves in a freeze-drying chamber or pre-freezing in

another chamber. Applying a vacuum to the chamber and heating the shelves in

order to evaporate the water from the frozen state.

Complete stoppering of the vials usually by hydraulic or screw rod

stoppering mechanisms installed in the lyophilizers.K.I.P.M. GIDA, GORAKHPUR, U.P.

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There are many new parenteral products, including anti-infectives,

biotechnology derived products, and in-vitro diagnostics which are

manufactured as lyophilized products.

Additionally, inspections have disclosed potency, sterility and stability

problems associated with the manufacture and control of lyophilized

products.


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hank you for listening me


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HIGH PERFORMANCE THIN

LAYER CHROMATOGRAPHY

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CHROMATOGRAPHY

Chromatography is a physical process of

separation in which the components to be

separated are distributed between 2 immiscible

phases a stationary phase which has a large

surface area and mobile phase which is in constant

motion through the stationary phase.

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Introduction of HPTLC

HPTLC is the improved method of TLC which utilizes theconventional technique of TLC in more optimized way.

HPTLC takes place in highspeed capillary flow range ofthe mobile phase.

There are three main steps HPTLC procedure, they are

1] Sample to analyzed to chromatogram layer, volume precisionand exact position are achieved by use of suitable instrument.

2] Solvent (mobile phase) migrates the planned distance in layer(stationary phase) by capillary action. In this process sampleseparated into its components.

3] Separation tracks are scanned in densitometer with lightbeams in visible or uv region

Steps Involving in HPTLC

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Steps Involving in HPTLC

Sample PreparationSelection of

chromatography layer

Pre-washing

Pre-conditioningApplication of sample

Chromatography development

Detection of spots

Scanning & documentation 57

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Sample preparation

Normal phase chromatography: non polar solvent

Reversed phase chromatography: polar solvent

Selection of chromatography layer

Depends on nature of material to be separatedCommonly used(silica gel, alumina)

58

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Pre-washing

It is purification step

Mainly methanol is usedEssential for quantitative evaluation

Stability testing

59

Pre conditioning

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Pre-conditioning

Also called Chamber Saturation

Low polarity mob. Phase:- no need

High polar mob. Phase:- desirable

For reverse phase saturate chamber with polar

solvent

Sample application

1-5l

Linomat IV Applicator

60

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Linomat lV applicator

61

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Fig. Hptlc instrumentation

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S l ti f HPTLC l t

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Selection of HPTLC plates

Previously hand made plates is used in TLC for both

qualitative and quantitative work. Certain drawbacks with

that is nonuniform layer, formation of thick layer, paved foradvent of precoated plates.

Nowadays precoated plates are available in different formatand thickness by various manufactures. Precaoted plates can

be used for both qualitative and quantitative work in

HPTLC, they are

GLASS PLATES POLY ESTER/POLYETHYLYNE

ALUMINIUM PLATES

Glass Plates:

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Glass Plates:

Offers superior flat and smooth surface.

- fragile

- high weight- higher production cost

Polyester/polyethylene plates:Thickness of plate is 0.2mm.

- It can be produced in roll forms.

- Unbreakable.

- Less packing material is required.

- Development of plate canntbe above temperature1200c loses its shape.

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Aluminium plates:- Thickness of plate is 0.1mm.

- It can be produced in roll forms.

- Unbreakable.

- Less packaging material is required.

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Layer thickness

The layer thickness in HPTLC is around 100-

200cm,in conventional it is 250mm.

Layer prewashing:

- Ascending method

- Dipping method

- Continuous method

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ACTIVATION OF PRECOATED PLATES

The plates are activated by placing in an oven

at 1101200 C for 30 min, this step will removeswater that has been physically absorbed on surfaceat solvent layer.

Freshly opened box of HPTLC plates usuallydoes not require activation.

Activation at higher temp and for longer time isavoided which leads to very active layer and there

is risk of sample being decomposed.

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Solvents used in HPTLC

- Methanol (commonly used)

- Chloroform:methanol:ammonia(90:10:1)

- Chloroform:methanol(1:1)

- Methylene chloride:methanol(1:1)

- Ammonia(1%)solution

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Application of sample and standard

Usual concentration range is 0.1-1g / l,above

this causes poor separation.

Linomat IV (automatic applicator) - nitrogen gas

sprays sample and standard from syringe on TLC

plates as bands.

Band wise application - better separation - high

response to densitometer.

Chromatographic development and drying

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Chromatographic development and drying

After development, remove the plate and

mobile phase is removed from the plate - to avoid

contamination of lab atmosphere.

Dry in vacuum desiccator - avoid hair drier

because essential oil components may evaporate.

Detection and visualization

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Detection and visualization

Detection under UV light is first choice -non destructive.

Spots of fluorescent compounds can be

seen at 254 nm (short wave length) or at 366 nm

(long wave length).

Spots of non fluorescent compounds can beseen - fluorescent stationary phase is used - silicagel GF.

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Non UV absorbing compounds likeethambutol, dicylomine etc - dipping the plates in

0.1% iodine solution.

When individual component does notrespond to UV - derivatisation required for

detection .

HPTLC

00

TLC

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100m

High due to smaller particle size

generated

3 - 5 cm

Shorter migration distance and the

analysis time is greatly reduced

Wide choice of stationary phases

like silica gel for normal phase and

C8 , C18 for reversed phase modes

New type that require less amount

of mobile phase

Auto sampler

Use of UV/ Visible/ Fluorescence

scanner scans the entire

chromatogram qualitatively andquantitatively and the scanner is

an advanced type of densitometer

250m

Less

10 - 15 cm

Slower

Silica gel , Alumina &

Kiesulguhr

More amount

Manual spotting

Not possible

PP

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APPLICATIONS

Pharmaceutical Researches

Biomedical Analysis

Clinical Analysis

Environmental Analysis

Food Industry

Therapeutic drug monitoring to determine concentration of drugand its metabolite in blood, urine etc

Analysis of environmental pollutions levels

Quantitative determination of prostaglandins and thromboxanes

in plasma

Determination of mercury in water

Analysis of nitrosoamines in food and body fluids Determination of sorbic acid in wine

Characterization of hazards in industrial waste

REFERENCES

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REFERENCES1. Principles of instrumental analysis skoog, Holler,

Nieman

2. Instrumental methods of analysis Willard

,Merrit, Dean

3. Pharmaceutical analysis Munson

4. Sharma J.friedB.Handbook of TLC

5. Kasture A.V A text book of Pharmaceutical Analysis(Instrumental methods), 14thedition, page no.28-30

6. Elke Hahn Deinstrop Applied TLC, 2nd

edition7. Sitewww.Google.com (Google images)

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THANK YOU

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Sterility testing

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DEFINITION: Sterility Testing: It is a procedure carried out to detect and

conform absence of any viable form of microbes in or on pharmacopeia

preparation or product.

PRINCIPLE : Sterility testing only shows that organisms capable of growing

in selected conditions are absent from the fraction of batch that has been

tested. If the microorganism are present in the product can be indicated by

a turbidity in the clear medium.

OBJECTIVE OF STERILITY TESTING:

For validation of sterilization process.

To check presence of microorganisms in preparation which are sterile.

To prevent issue of contaminated product in market.K.I.P.M. GIDA, GORAKHPUR, U.P.

STEPS INVOLVED IN STERILITY TE TESTING

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1) Sampling

2) Selection of the quantity of the product to be used

3) Method of sterility testing

i ) METHOD 1 Membrane filtration method

ii) METHOD 2 Direct inoculation method

4) Observation and interpretation Must be carried out under

aseptic condition.


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Selection of the quantity of the product to be used

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Selection of the quantity of the product to be used for sterility testing

depends mainly on the volume or weight in the container.


Method of sterility testing

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Membrane filtration method (METHOD 1):

Membrane filtration Appropriate for : (advantage)

Filterable aqueous preparations

Alcoholic preparations

Oily preparations

Preparations miscible with or soluble in aqueous or oily (solvents

with no antimicrobial effect)

All steps of this procedure are performed aseptically in a Class 100

Laminar Flow Hood


Membrane filter 0.45porosity

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Filter the test solution

After filtration remove the filter

Cut the filter in to two halves

First halves (For Bacteria) Second halves (For Fungi)


(Fluid Thioglycollate medium)



(Soyabeans-Casein Digest medium)


Observe the growth in the media

Observe the growth in the media


Direct inoculation method (METHOD 2):

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Suitable for samples with small

volumes

volume of the product is not morethan 10% of the volume of the

medium

suitable method for aqueous

solutions, oily liquids, ointments ancreams

Direct inoculation of the culture

medium suitable quantity of the

preparation to be examined is

transferred directly into the

appropriate culture medium &

incubate for not less than 14 days.


Observation and results

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Culture media is examined during and after at the end of incubation.

The following observations are possible:

1) No evidence of growth Pass the test for sterility.

2) There is evidence of growth Re-testing is performedsame no.

of sample, volume & media as in original test No evidence of

growth Pass the test for sterility.

3) There is evidence of growth isolate & identify the organism.

Re-testing is performed with twice no. of sample if:

No evidence of growth Pass the test for sterility.

There is evidence of growth Fail the test for sterilityK.I.P.M. GIDA, GORAKHPUR, U.P.

Particulate matter monitoringP ti l t tt i d fi d t d bil i l bl tt

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Particulate matter is defined as unwanted mobile insoluble matterother than gas bubble present in the product.

If the particle size of foreign matter is larger than the size of R.B.C.. It

can block the blood vessel.

The permit limits of particulate matter as per I.P.are follows:

Source of particulate matter:

1. Intrinsic contamination: The material which are originallypresent in the parenteral solution e.g. Barium ions leach inparenteral & react with sulphur ions in the product to formbarium sulphate crystals.

2. Extrinsic contamination: The material which comes from theenvironment e.g. Shedding of material from cloth, body, &

cotton, paper, rubber, tissue etc.K.I.P.M. GIDA, GORAKHPUR, U.P.

Methods for monitoring particulate matter contamination:

1) Vi l th d

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1) Visual method

2) Coulter counter method

3) Filtration method4) Light blockage method

Identification of particulate matter:

1) Microscopy

2) X-ray powder diffraction

3) Mass spectroscopy

4) Polarized light spectroscopy

5) Scanning electron microscopy (SEM)


Faculty seal packaging / leaking testing

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The sealed ampoules are subjected to small cracks which occur due

to rapid temperature changes or due to mechanical shocks.

Vials & bottles are not suitable for this test because the sealing

material used is not rigid.

Filled & sealed ampoules

Dipped in 1% Methylene blue solution

Under negative pressure in vacuum chamber

Vacuum released colored solution enter into the ampoule

Defective sealing


Pyrogen Testing

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Pyrogen = Pyro(Greek = Fire) + gen(Greek = beginning).

Fever producing, metabolic by-products of microbial growth and

death. Bacterial pyrogens are called Endotoxins. Gram negative bacteria

produce more potent endotoxins than gram + bacteria and fungi.

Endotoxins are heat stable lipopolysaccharides (LPS) present in

bacterial cell walls, not present in cell-free bacterial filtrates Stable to at least 175oC; steam sterilization ineffective

Water soluble; monomer unit of LPS can be 10,000 Daltons (1.8 nm)

so endotoxins can easily pass through 0.22mfilters

Sources: Water (main), raw materials, equipment, processenvironment, people, and protein expression systems if using gram

negative bacteria.



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Pyrogen elevate the circulatory levels of inflammatory cytokines

which may be followed by fever, blood coagulation, hypotension

Low doses of Pyrogen: asymptomatic inflammation reaction

Moderate doses: fever & changes in plasma composition

High doses: cardiovascular dysfunction, vasodilation,

vasoconstriction, endothelium dysfunction, multiple organ failure

& finally death.

Sources of pyrogen1) Equipment

2) Containers (Glass , plastic , metal)

3) Solvent

4) Solute K.I.P.M. GIDA, GORAKHPUR, U.P.

Elimination of pyrogens

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Dry heat sterilization : For glass wares, metal equipments,

powders, waxes, oils, heat stable drugs

650 oC temp - 1 min


180o

C temp - 240 min

Ultra filtration

Reverse osmosis : RO membrane is composed of cellulose acetate

phthalate/ polyamide

Distillation

Adsorption method


Principal:

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Rabbits are used to perform this test because their body temp

increases when pyrogen are introduced into their bodies by

parenteral route 3 healthy adult rabbits of either sex, each weighing NLT 1.5 kg are

selected

Do not use any rabbit

having a temp higher than 39.8 oCShowing temp variation >0.2 oC between two successive reading

in the determination of initial temp

Sham test is performed within 7 days of actual test

Animal showing temp increase over 0.6 oC should be removed frompyrogen testing


Method :

Dissolve the subs being examined in, or dilute it with a pyrogen free

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Dissolve the subs being examined in, or dilute it with a pyrogen freesaline solution

Warm the liquid being examined to approx. 38.5o C temp before

injection The volume of injection is NLT 0.5ml/kg & NMT 10ml/kg of body

weight

Withhold water during test

Clinical thermometer is inserted into the rectum of rabbit to recordbody temp

2 normal reading of rectal temp are should be taken prior to the testinjection at an interval of half an hr & its mean is calculated- initialtemp

The solution under test is injected through an ear vein

Record the temp of each rabbit in an interval of 30 min for 3 hrs

The difference between initial temp & maximum temp is recorded-taken as response


Interpretation of results

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d f d f b l

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To detect or quantify endotoxins of gram-ve bacterial origin

Reagent: amoebocyte lysate from horseshoe crab (Limulus

polyphemus or Tachypleus tridentatus).

The name of the test is also Limulus amebocyte lysate (LAL) test

Mechanism of LAL Test:The test is based on the primitive blood-clotting mechanism of

the horseshoe crab

enzymes located with the crab's amebocyte blood cells endotoxin

Initiation of an enzymatic coagulation cascade

proteinaceous gelK.I.P.M. GIDA, GORAKHPUR, U.P.


d d

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Avoid endotoxin contamination

Before the test:

interfering factors should not be present

equipment should be depyrogenated the sensitivity of the lysate

should be known

Test:

equal Volume of LAL reagent and test solution (usually 0.1 ml of

each) are mixed in a depyrogenated test-tube Incubation at 37C, 1 hour

remove the tube - invert at (180) observe the result

pass-fail test K.I.P.M. GIDA, GORAKHPUR, U.P.

LAL test


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1. The gel-clot technique - gel formation

2. The turbidimetric technique - the development of Turbidity aftercleavage of an endogenous substrate

3. The chromogenic technique - the development of color after

cleavage of a synthetic peptide- chromogen complex

Advantages of LAL testFast - 60 minutes vs. 180 minutes

Greater Sensitivity ,Less Variability

Much Less False, Positives ,Much Less Expensive

Alternative to Animal Model, cheaper,particularly useful for:

Radiopharmaceuticals and cytotoxic agents

Blood products

Water for injectionK.I.P.M. GIDA, GORAKHPUR, U.P.


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The production area where the parenteral preparation are

manufactured can be divided into five sections:

Clean-up area

Preparation area

Aseptic area

Quarantine area

Finishing & packaging area


Clean-up area:

It is not aseptic area

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It is not aseptic area.

All the parenteral products must be free from foreign particles &

microorganism.

Clean-up area should be withstand moisture, dust & detergent.

This area should be kept clean so that contaminants may not be carried out

into aseptic area.

Preparation area:

In this area the ingredients of the parenteral preparation are mixed &preparation is made for filling operation.

It is not essentially aseptic area but strict precautions are required to prevent

any contamination from outside.


Aseptic area:

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The parenteral preparations are filtered, filled into final container

& sealed should be in aseptic area.

The entry of personnel into aseptic area should be limited &

through an air lock.

Ceiling, wall & floor of that area should be sealed & painted.

The air in the aseptic area should be free from fibers, dust and

microorganism.

The High efficiency particulate air filters (HEPA) is used for air.

UV lamps are fitted in order to maintain sterility.


Quarantine area:

After filling sealing & sterilization the parenteral product are held

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After filling, sealing & sterilization the parenteral product are held

up in quarantine area.

Randomly samples were kept foe evaluation.

The batch or product pass the evaluation tests are transfer in to

finishing or packaging area.

Finishing & packaging area:

Parenteral products are properly labelled and packed.

Properly packing is essential to provide protection against

physical damage.

The labelled container should be packed in cardboard or plastic

container.

Ampoules should be packed in partitioned boxes



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Lyophilization or freeze drying is a process in which water is removed

from a product after it is frozen and placed under a vacuum, allowingthe ice to change directly from solid to vapor without passing through

a liquid phase.

The process consists of three separate, unique, and interdependent

processes;

Freezing,

Primary drying (sublimation), and

Secondary drying (desorption).


The advantages of Lyophilization include:

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Ease of processing a liquid, which simplifies aseptic handling

Enhanced stability of a dry powder

Removal of water without excessive heating of the product

Enhanced product stability in a dry state

Rapid and easy dissolution of reconstituted product

Disadvantages of Lyophilization include:

Increased handling and processing time

Need for sterile diluent upon reconstitution

Cost and complexity of equipment


The Lyophilization process generally includes the following steps:

Dissolving the drug and excipients in a suitable solvent generally

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Dissolving the drug and excipients in a suitable solvent, generally

water for injection (WFI).

Sterilizing the bulk solution by passing it through a 0.22 micronbacteria-retentive filter.

Filling into individual sterile containers and partially stoppering

the containers under aseptic conditions.

Transporting the partially stoppered containers to the lyophilizerand loading into the chamber under aseptic conditions.

Freezing the solution by placing the partially stoppered containers

on cooled shelves in a freeze-drying chamber or pre-freezing in

another chamber.

Applying a vacuum to the chamber and heating the shelves in

order to evaporate the water from the frozen state.

Complete stoppering of the vials usually by hydraulic or screw rod

stoppering mechanisms installed in the lyophilizers.K.I.P.M. GIDA, GORAKHPUR, U.P.

There are many new parenteral products, including anti-infectives,

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biotechnology derived products, and in-vitro diagnostics which are

manufactured as lyophilized products.

Additionally, inspections have disclosed potency, sterility and stability

problems associated with the manufacture and control of lyophilized

products.


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