preparation and evaluation of parenterals

1. PARENTERALS
RINCY RAJAN
1st YEAR M.PHARM
DEPT. OF PHARMACEUTICS
St. JOSEPH’S COLLEGE OF
PHARMACY.

2. Processing of parenteral
preparation
1. Cleaning equipment and containers
2. Sterilization of equipment
3. Compounding the product
4. Filtration
5. Filling procedures
6. Sealing
7. Test for Quality Control
8. Sterilization
9. Packaging
10. Labelling

3. 1. Cleaning equipment and
containers
• Equipment and containers are thoroughly
cleaned with detergent and washing →tap water
→clean distilled water →WFI
• Debris more difficult to remove →hot detergent
• Equipments are mostly disassembled and each
parts are thoroughly scrubbed and cleaned
• Two types of rinsers are used
i. Rotary type rinser
ii. Convey type rinser

4. CLEANING RUBBER AND GLASS BOTTLES:
• Rubber closures and glass bottles are cleaned
in mechanical equipment which is heated
electrically.
• Detergent are mostly used for cleaning the
rubber closures.
• This rubber closures are washed first with
water → autoclave → rinse second time with
preservatives → autoclaved
• Glass containers are cleaned by mechanical
equipment ,which is rotating and the glass
bottles are washed and cleaned.

5. 2. Sterilization of equipment
• Mostly in parenteral equipments are sterilized by using
autoclave before starting of manufacturing or filling
process to remove micro-organism or pyrogen.
• All the equipment including filling equipment,
manufacturing equipments are sterilized before use.
• Methods of Sterilization
i. Steam sterilization
ii. Dry heat sterilization
iii. Filtration sterilization
iv. Gas sterilization
v. Ionizing radiation sterilization

6. 1. Steam sterilization:
• Method: Applying steam under high pressure
• Equipment: Autoclave (steam sterilizer)
• Mechanism of microbial destruction:
denaturation and coagulation of some of
organism's essential protein.
 In presence of moisture, MO are destroyed
at lower temp. than dry heat.
 Temperature- 121-124°C for 15min.

7. 2.Dry Heat Sterilization:
• Method: using heated air
• Equipment: Oven
• Mechanism of microbial destruction: dehydration of
microbial cell after oxidation. Usually conducted at 150
to 170°C for not less than 2 hour.
• Application: Generally employed for substances that
are not effectively sterilized by moist heat such as fixed
oil, glycerol, petrolatum, heat stable powder.

8. Advantages
• Easy to install
• Non toxic and does not harm the environment
• Non-corrosive for metal
Disadvantages
• Time consuming method - Slow heat
penetration
• High temp. are not suitable for most material

9. 3.Sterilization by filtration:
• Method: filtration allows for the exclusion of
organisms based upon size.
Membrane filtration are mainly used method
for system sterilization.
Membrane filtration traps contamination
larger than the pore size on the surface of the
membrane.

10. • Example: Millipore filter which is a thin plastic
membrane of cellulose ester containing uniform
pores constituting up to 80% of membrane's
volume.
• Pore size range from 14 to 0.025μm
• Advantages:
Rapid, inexpensive, effective, large volume
• Disadvantages:
1. cannot use for oily preparation
2. cannot use for moisture sensitive preparations
3. cannot kill spores

11. 4. Gaseous sterilization
• Method: use of gas, such as Ethylene Oxide
• Equipment: special oven, for admission of gas
and humidity
• Application:
Thermolabile powder, plastic (e.g. syringes),
polymers, ophthalmic prep., tubing sets.

12. • Ethylene Oxide (EO)
 Used to sterilize heat- sensitive materials, MO
and spores.
 Used in combination with CO2 to avoid
explosion, >3% EO in air is explosive.
 Mechanism of action of EO is alkylation of
hydroxyl, carbonyl and amino groups of bacterial
enzyme.
• Disadvantages
1. Explosive hazards
2. Toxic
3. Not appropriate for solutions

13. 5.Radiation sterilization
• Equipment: Ultraviolet lamp; for surface
sterilization
-Ionization (Beta rays, Gamma rays, X-rays)
• Application:
Thermolabile drugs (powder)
• Disadvantages:
1. Highly specialized equipment required
2. Effect of irradiation on product and their
containers

14. 3. Compounding the product
• Compounding of product should be
performed under clean environmental
condition.
• It involves two steps
i. Collection of material
ii. Preparation of parental product

15. 4. Filtration
• It is defined as the process in which particles are
separated from a liquid by passing the liquid through a
semi permeable membrane.
• Filtration is frequently the method of choice for
sterilization of solution.
• For filtration of aqueous solutions, semi permeable 0.2
to 2 micron filter papers are used.
• 2 micron filter paper is used as a pre filter, while 0.2
micron paper is used as a last paper before filling.
• For oily solution, cartridge filter paper is used.
• After filtration , the solution must be protected from
environmental contamination until it is sealed in the
final container.

16. 5.Filling Procedures
FILLING EQUIPMENT FOR LIQUIDS:
• Here, the measured amount of liquids deliver from the
small orifice into the container.
• The size of the delivery tube is governed by opening in
the container to be used, the viscosity and density of
the liquid and the speed of delivery desired.
• The tube must free enter the neck of the container and
deliver the liquid deep enough to permit air to escape
without sweeping the entering liquid into the neck or
out of the container.
• Filling machine parts should be constructed of
nonreactive materials such as borosilicate glass or
stainless steel.

17. • The solution are usually filled in the bottle by
gravity, pressure or vacuum filling device.
• Emulsion and suspension required specially
designed filling equipment because of their
high viscosity.
• Powders such as antibiotics, are more difficult
to subdivide accurately and precisely into
individual dose containers than are liquid.

18. 6.Sealing
• Container should be sealed in the aseptic area
in immediately adjacent to the filling machine.
• It is obvious that a sterile container that has
been opened can no longer be considered to
be sterile. Therefore, temperature proof
sealing is essential.

19. • SEALING AMPOULES:
• Ampoules may be closed by melting a portion
of the glass of neck to form either tip-seals or pull
seals.
• Tip-seals are made by melting sufficient glass at
the tip of the ampoule neck to form a bead of
glass and close the opening.
• Pull-seals are made by heating the neck a
rotating ampoule below the tip, then pulling the
tip away to form a small, twisted capillary just
prior to being melted closed.
• Excessive heating of air and gasses in the neck
causes expansion against the soft glass with the
formation of fragile bubbles at the point of seal.

20. SEALING OF VIALS,PLASTIC BOTTLE:
• When closures are to be inserted by
machines, the surface of the closures is
usually halogenated or coated with silicone to
reduce the friction.
• Aluminum caps are used to hold rubber
closures in place

21. 7. Quality control test
• Quality control involves operational
techniques and activities that are used to
assure product compliance to specifications.
• There are 3 general areas of QC.
• They are - incoming stock
- manufacturing &
- the finished product.

22. Quality control are classified into:-
1. Sterility Test
2. Clarity Test/ Particulate matter monitoring
3. Leakage Test
4. Pyrogen Test

23. • 1. STERILITY TEST:
• Most reliable step of QC.
• To detect the presence of viable forms of
microbes in pharmacopeial final preparations.
• All the final preparations must confirm to the test
for sterility as prescribed in pharmacopoeia.
• The test for sterility is usually carried out under
aseptic conditions.

24. • PRINCIPLE:
If viable microbes are placed in a
nutritive medium, kept at favourable
temperature, the organisms will grow & their
presence can be identified by turbidity in the
clear medium.
Sterility test is mainly carried out by 2 methods.
A. Membrane filtration method
B. Direct inoculation method
To carry out these tests, a sterility test media is
essential.

25. • Sterility test media:
• It should be such that it supports the
growth of aerobes, anaerobes and fungi.
• If the final product is clear, then fluid thioglycollate medium can
be employed.
• If the product is turbid & viscid, fluid thioglycollate medium
devoid of agar is used.
Fluid thioglycollate medium Anaerobes & aerobes
Soya bean-casein digest
medium
Fungi & aerobic bacteria

26. • Membrane filtration method:
• This method is employed in the following
cases:
1. oil & oily preparations
2. alcoholic preparations
3. for preparations miscible with or soluble in
aqueous or oily solvents.
This methods can be explained through various
simple steps.

27. • Step 1: Selection of
membrane:
• Membrane selected should have a
nominal pore size not greater than
0.45µm and diameter
approximately 47mm.
• Should have effectiveness to retain
micro-organisms to be established.
• Eg: cellulose nitrate filters
cellulose acetate filters, etc.
For products containing antibiotics,
specially adapted membrane filters
are used.

28. • Step 2: Washing the membrane
• Before the start of sterility test, the membrane is
rinsed with diluting or rinsing fluid.
• Fluid A- Sterile solution- 1gm peptic digest of
animal tissue in 1lt of water.
• Fluid D- Contains suitable emulsifying agent like
polysorbate 80 at a conc of 10gm/lt.
Fluid A Aqueous solutions or soluble solids
Fluid D (3 TIMES) Oil, oily solutions, w/o emulsions

29. • Step 3:Transfer of product to the membrane
• After rinsing, the contents in the product container is
transferred to the rinsed membrane.
• Aqueous & oily solutions of low viscosity may be
filtered without prior dilution.
• Viscous oils, w/o emulsions, etc may be suitably diluted
with sterile diluents like isopropyl myristate before
passing through the membrane.
• Pre-filled syringes(without attached needles), the
contents of syringes are expelled into separate pooling
vessels prior to transfer whereas if separate needle is
attached, the contents are directly expelled.

30. Step 4: Culturing in broth
• After filtration of contents, the membrane is
aseptically cut into 2 halves.
Each half transferred to both the medium.
Both incubated for not less than 14 days.
Fluid thioglycollate Incubated at 32.5±2.5 ◦c
Soya bean-casein Incubated at 22.52±1◦c

31. • Step 5: Interpretation of result
• The media is examined for microbial growth at intervals during
the incubation period.
The test may become invalid if
1. The data of microbiological monitoring or testing facility or the
testing procedure used reveals a fault.
or
2. Negative control that shows microbial growth. When a test is
declared to be invalid, it is repeated same as original
If no evidence of microbial
growth
Product complies the test for
sterility
If evidence of microbial growth
(bacterial colonies)
Product does not complies the
test for sterility

32. • Direct inoculation method
• Implemented when membrane filtration method
appears to be difficult.
• In here, the quantity of preparation to be tested is
directly transferred directly into the culture medium or
vice versa, so that the volume of product is not more
than 10% of the volume of the medium.
• If the product to be tested contain any anti-microbial
agent, using suitable reagent it should be neutralised
before the test.
• For large volume products, we use conc. culture
medium.

33. • The inoculated medium is then incubated for not less
than 14 days.
• Observe the culture during incubation period.
• If the medium render to be turbid, result interpretation
will be impossible. In such cases, after 14 days of
incubation, transfer the contents to a fresh vessel of
the same medium. Then incubate both the fresh and
the original for 4 more days.
• At last, the result interpretation is done as same as for
membrane filtration method.

34. • If no evidence of microbial growth
- Product complies the test for sterility
• If evidence of microbial growth (bacterial colonies)
-Product does not complies the test for sterility
The test may become invalid if
1. The data of microbiological monitoring or testing
facility or the testing procedure used reveals a
fault.
or
2. Negative control that shows microbial growth.
When a test is declared to be invalid, it is repeated
same as original

35. • Scheme for sterility test by direct inoculation method

36. 2. CLARITY TEST
• This test is also referred as particulate matter monitoring.
• Particulate matter refers to unwanted mobile insoluble
foreign matter other than gas bubbles present in the
product.
• It is to be noted that there occur a very dangerous
situation when the particle size is greater than RBC because
they may block the blood vessels.
• The QC department detect and discard individual
containers of a product that is considered to be unclean.

37. • For particulate matter, the pharmacopoeia has
established certain limits such as
50 particles of 10µm & larger
5 particles of 25µm & larger
Mainly we discuss here 2 methods
i. Visual method
ii. Coulter counter method

38. • Visual method:
It is the most used and relevant method for clarity
testing.
It is an old method.
According to this method, the filled containers are
examined against a strong illuminating background
with a swirling motion since moving particles are
easier to identify than stationary particles.
Inversion of containers can be done to view heavier
particles.
Dark background- Light particles
Light background- Dark particles

39. This is a model for visual inspection
machine.

40. • Coulter counter method:
• This is the test that is based on the principle
that there will be an increase in the resistance
as a particle approaches and passes through
the orifice (2 electrodes).
• This method require destruction of the product
unit since an electrolyte is added to the
preparation before its evaluation.

41. • Some other methods of clarity testing can be
listed as Filtration method, Light scattering
method, Light absorption, Light blockage
methods, etc...
• Once the particles are detected, then they are
identified by various methods like microscopy,
X-ray powder diffraction, mass microscopy,
micro-chemical tests, polarised light
microscopy and scanning electron microscopy.

42. • 3. Leakage test
• Ampoules are intended to provide hermetically
sealed container for a single dose.
• If any capillary pore tiny cracks are present
microbes or any other contaminants may enter
the ampoule or the contents may leak out and
spoil the product.
• The leak test is intended to detect any
incompletely sealed ampoules, presence of
cracks, openings, etc so that they may be
discarded.

43. • Leak test is usually done by
• producing a negative pressure
• within an incompletely sealed ampoule,
• usually in a vacuum chamber,
• while the ampoule is entirely submerged
• in a deeply coloured dye solution.
• The atmospheric pressure then causes the dye to
penetrate if any opening is present. Only a tiny drop of
dye may penetrate a small opening.
• Dye solution- 0.5-1.0% methylene blue.
• The presence of dye colour confirms the leakage &
hence the product is rejected.

44. Representation of leakage test machine

45. • Vials and bottles are subjected to such leak tests
because the rubber closures are not rigid.
• For such cases, a spark tester probe is applied to
the outside of the bottle moving from the liquid
layer to the air space. A blue spark discharge
occurs if the air space is identified.
• In case of bottles, a vacuum remains during its
shelf-life. The presence of vacuum is detected by
striking the base of bottle with the heel of hand
to produce a typical water hammer sound.

46. 4. Pyrogen test
PYROGENS:
These are the metabolic products of microbes.
Most bacteria, moulds and viruses produce
Pyrogen.
Most potent pyrogenic substance called endotoxins
are produced by gram negative bacteria .
Pyrogens when injected into a human, shows
marked rise in the temperature , chills, body
aches, cutaneous vasoconstriction and increased
arterial blood pressure.
The most likely source of pyrogens are water,
contaminated solutes and containers.

47. • Rabbit test

48. Rabbit Pyrogen Test
• Rabbits must be healthy and mature
• New Zealand or Belgian Whites used
• Either sex may be used
• Must be individually housed between 20 and 23°C
Rabbit test
• Selection of animals - healthy, adult, not less than 1.5kg.
• Housing of animals - environmental problems: presence of
strangers (unknown place), noise.
• Equipment and material used in test - glassware, syringes,
needles.
• Retaining boxes - comfortable for rabbits as possible.
• Thermometers - standardized position in rectum, precision
of 0.1°C.

49. Preliminary test (ShamTest)
• Intravenous injection of sterile pyrogen-free
saline solution
• To exclude any animal showing an unusual
response to the trauma (shock) of injection
• Any animal showing a temperature variation
greater than 0.50 C is not used in them a in
test
• All glassware, syringes and needles must be
pyrogen free by heating at 2500 C for not less
than 30min.

50. Main test:
• Group of 3 rabbits
• Preparation and injection of the product:
• Warming the product to 37±2°C
• Dissolving or dilution
• Injection site: ear vein
• The injected volume: about 10ml per kg of
body weight over 10min. duration
• Record temperature at 30min. Interval for
3 hours

51. • Interpretation of the results:
• The test is carried out on the first group of 3
rabbits; if necessary on further groups of 3
rabbits to a total of 4 groups, depending on
the results obtained.
• Intervals of passing or failing of products are
on the basis of summed temperature
response.

52. If the difference is negative, the result is counted as zero response.
• Result interpretation:
• Later with the 2nd case, if not more than 3 of 8 rabbits show
individual response of 0.6 or more and if the sum of the
response of 8 rabbits does not exceed 3.7 , the preparation
passes the test for pyrogen.
Sum of response of
3 rabbits
Individual response
of a rabbit
Result
Does not exceed
1.4◦c
Less than 0.6◦c Product pass the
test
Is or Exceed 1.4◦c Is or is more than
0.6◦c
Continue the test
with 5 more rabbits

53. Limulus Amebocyte Lysate {LAL} Test
• Recently added method.
• Invitro test.
• Rapid.
• 5 to 10 min more sensitive than rabbit test.
• Semi-quantitative.
• PRINCIPLE: The endotoxins of gram negative bacteria
forms a firm gel within 60 mins in the presence of
lysate of amebocytes of limulus polyphemus
{horseshoe crab} when incubated at 37◦c.
• Hence the test is only effective with gram negative
bacteria which constitute the majority and the most
potent of the pyrogens.

54. • The test also has been
automated.
• This test can also be called as
Bacterial Endotoxins Test {BET}.
• To provide standardization for the
test, the USP has established a
reference endotoxins against
which lots of lysate are
standardized.
• Therefore, the sensitivity of the
lysate is given in terms of
Endotoxin Unit {EU}.
HORSESHOE CRAB

55. Enzyme +
protein
Endotoxins
Coagulation - Formation of gel
~ 30 mins
Extract from blood
cells (Amoebocyte
lysate)
Horseshoe crab
(Limulus
polyphemus)

56. 10. Packaging and labeling:
• The packaging should be neat ,clean and
attractive in appearance if it is to convey to the
user .
• The quality, purity and reliability that should be
conformed from the carton or label.
• The package must be accurately and completely
provide the user information necessary for its
use.
• The labeling should be identified and shows the
required things on label.
• It should be stored as per its necessary.

57. Reference :
1. Pharmaceutical dosage forms: parenteral
medications of industrial pharmacy ,
Lachmann/ Lieberman's, 4th edition, pg.no.
864-871.
2. Dispensing pharmacy by R M Mehta, pg.no.
303-311.
3. The science & practice of pharmacy,
Remington, 21st edition, pg.no. 1367-1374.