QUALITY CONTROL OF

CEFEPIME INJECTABLE DRUG

Name: Namita Dhiman
Class: M.Sc. Final Year
Roll no. : 2023037026
Content…
• Industry profile
• Introduction
• Structure
• Quality control
1. Importance
2. Sterility testing
3. Endotoxin test
4. Environmental monitoring
• Conclusion
• References
Industry profile
• GMH Organics, an Indian
pharmaceutical company renowned
for its expertise in Sterile
Antibiotics, was established in 2005.
• Since its inception, GMH Organics
has evolved into one of India’s
foremost producers of small volume
parenteral, specifically in the dry
powder Injectable beta lactam
group.(https://gmh.co.in/)
Introduction…
Cefepime is a fourth-generation cephalosporin antibiotic with broad-
spectrum activity against gram-positive and gram-negative bacteria,
including multidrug-resistant strains.
It works by inhibiting bacterial cell wall synthesis, making it effective
for severe infections such as pneumonia, urinary tract infections, skin
infections, and septicemia.
• Cefepime is commonly administered via intravenous or intramuscular
injection. Its extended spectrum of activity and resistance to beta-
lactamases make it a preferred choice for hospital-acquired infections.
(Chapman TM, Perry CM. Cefepime: a review of its use in the
management of hospitalized patients with pneumonia. Am J Respir
Med. 2003;2(1):75-107. doi: 10.1007/BF03256641. PMID: 14720024.)
Structure…
• Its structure is based on the typical cephalosporins bicyclic system
that combines two rings in a fused structure.
• Its molecular formula is C19H24N6O5S2
• Structure:
1. The B-lactam ring:
2. The Dihydrothiazine ring:
Fig. 1 Structure of Cefepime
 Quality control…
IMPORTANCE
• It directly impact patient safety by ensuring that injectable drugs are
sterile, free from contaminats and minimising the risk of adverse
reaction due to contamination.
1. Patient safety
2. Sterility assurance
3. Purity
4. Consistency
5. Compliance with regulatory standards
STERILITY testing
Introduction:
• The sterility of a product is defined by the absence of any viable And
actively multiplying microorganisms when tested in specified media.
• The test is applied to substance, preparations or articles which,
according to the Pharmacopoeia, are required to be sterile.
• Turbidity in the broth media usually indicates contamination
• Test is performed on the end –product and is one of the quality
control tests specified for release of a batch of sterile product.(Indian
Pharmacopoeia,2010, Vol-I,p:56-63,36-37,28-33,196-198)
Media used
1. Fluid Thioglycollate Medium(FTM):
• It is used with clear fluid products.
• FTM is primarily intended for the culture of anaerobic bacteria;
however it will also detect aerobic bacteria.
2. Soyabean Casein Digest Medium(SCDM):
• Used for turbid or viscid product.
• For both fungi and aerobic bacteria Medium 1 are adjusted to pH 7.
• Medium 2 is adjusted to 7.3+0.2 Autoclave at 121°C for 20mins
Wherever culture media are employed in the manufacturing of
antibiotics they need to contain inactivating substances to neutralize
efficiently these antibiotics to ensure that any sensitive contaminant
microorganisms maintain their ability to grow.
• In manufacturing facilities where antibiotics are produced, β-
lactamase enzymes such as penicillinase (penase) and
cephalospopinase (Cephase) are contained in the monitoring media
to inactivate β-Lactam antibiotics.
• Produced antibiotics must also be inactivated prior to sterility testing
to allow for the detection of potential contaminants which are
sensitive to the antibiotic. (Indian Pharmacopoeia,2010, Vol-
I,p:56-63,36-37,28-33,196-198)
Test methods

Methods

Membrane
Direct
Filtration
Inoculation
Method
1. Membrane filtration method:
• Membrane has a nominal pore size not greater than 0.45 micron and
diameter of approximately 50mm.
• Flow rate: 55-75ml/min at pressure 70mm Hg.
• Cellulose nitrate filters are used.
• This method basically involves filtration of sample through membrane
filters.
• The filtration is assisted under Vacuum after filtration completion the
membrane is placed in plates/test tube containing FTM, SCDM
medium.
• For bacteria 20-25°C, for fungi 30-35°C.
• Incubate the media for not less than 14 days.
Fig. 2 Vaccum pump
• Used for:
• An oil or oily preparation.
• Ointments that can be put into solutions.
• Soluble powder.
• Liquid products where volume in a container is 100ml or more.
• Non bacteriostatic solid not readily soluble in culture media.
• Products ,volume & quantities, media preparation are given in the
above tables.
2. Direct inoculation method:
• It involves a direct inoculation of required volume of a sample in two
test tubes containing a culture medium that is FTM, SCDM.
• Volume of the preparation under examination is not more than 10%
of the volume of the medium.
• Incubate the inoculated media for not less than 14 days.
• Products, volume & quantities, media preparation are given in the
above tables.
Observation & Result
• At intervals during the incubation period and at its conclusion,
examine the media for macroscopic evidence of microbial growth.
• If the material being tested renders the medium turbid so that the
presence or absence of microbial growth cannot be readily
determined by visual examination, 14 days after the beginning of
incubation transfer portions (each not less than 1 mL) of the medium
to fresh vessels of the same medium, and then incubate the original
and transfer vessels for not less than 4 days.
If no evidence of microbial growth is found, the product to be examined
complies with the test for sterility. If evidence of microbial growth is
found, the product to be examined does not comply with the test for
sterility, unless it can be clearly demonstrated that the test was invalid for
causes unrelated to the product to be examined. The test may be
considered invalid only if one or more of the following conditions are
fulfilled:
a. The data of the microbiological monitoring of the sterility testing
facility show a fault.
b. A review of the testing procedure used during the test in question
reveals a fault.
• c. Microbial growth is found in the negative controls.
• d. After determination of the identity of the microorganisms isolated
from the test, the growth of this species (or these species) may be
ascribed unequivocally to faults with respect to the material and or the
technique used in conducting the sterility test procedure.
Fig.3 SCDA plate Fig.4 FTM tube
Endotoxin test
• Endotoxin is a pyrogenic substance that is found in the cell wall of
Gram negative bacteria.
• Pyrogenic substance can induce fever when injected into the blood or
cerebo spinal fluid.
• It is associated with injectable products.
• Sterile production procedures are needed.
• Sterilization does not remove the toxin.
• It is heat stable.
Fig. 5 cell wall of Gram negative bacteria
Methods

Methods

Gel Clot Turbidimetric

Method method
1. Gel clot method:
• In this method equal amounts of a test sample, for instance an
injectable chemotherapy solution, and the gel clot LAL/TAL are mixed
in a test tube.
• The combined solution is incubated at 37 °C for 60 minutes. After the
incubation, the tube is inverted.
• If sufficient endotoxin is present in the test sample, the solution
would have clotted during the hour incubation and a gel will remain in
the bottom of the inverted tube.
• If the sample does not contain detectable endotoxin, no clot will
form and liquid will run down the side of the inverted tube.
• The gel clot method is still widely used throughout the world.
• Bacterial endotoxin test is done to detect or quantify endotoxin of
Gram negative bacterial origin using amoebocyte lysate from horse
shoe crab.
• The blood of the horseshoe crab is blue due to the copper-based
oxygen carrying protein hemocyanin . The amebocytes can be seen
as the white pellet at the bottom of the blue liquid in the bottles.
• The blood clotting system contains a cascade of three inactive
enzymes and a clottable protein, coagulogen. In the presence of
endotoxin, the first enzyme, Factor C is activated which activates
Factor B. Factor B activates the clotting enzyme which cleaves
coagulogen into coagulin.
• Coagulin molecules stick together to form a clot to protect the
horseshoe crab from an exposure to Gram-negative bacteria or
endotoxin.
• Frederik Bang and Jack Levin’s test uses the blood clotting system to
form a gel clot in a test tube when endotoxoin is present in the test
sample.
Fig. 6 Clot formation process
2. Turbidimetric method
• The turbidimetric method is a widely used technique for the
detection and quantitative analysis of endotoxin concentrations.
• Endotoxins are lipopolysaccharides (LPS) found in the outer
membrane of gram-negative bacteria and can cause significant
immune responses and other adverse effects in humans.
Observation & Result
Environmental monitoring
• Microbial Monitoring is a program designed to demonstrate the
control of viable (living microorganisms) and non-viable particles in
critical areas.
• These areas include clean-rooms for drug fill/finish, formulation tank
rooms, laminar flow hoods, biological safety hoods, isolators,
Intravenous (IV) compounding areas and sterile packaging.
• It is done to check the quality of air.
1. Settle Plate Method:
• The 90mm Petri plates are being prepared with 20ml-25ml of SCDA
(Soybean casein digest agar), after the pre-incubation of 24 to 48
hours the Petri plates are allowed to be exposed for the 4 hours in the
manufacturing area and then it is incubated at 20°C-25°C for 72 hours
and observation is being recorded. Later the same plate is then
incubated at 30°C-35°C for 48 hours and later the observation is being
recorded.
2. Active Air Sampling
• It is performed in the pharmaceutical manufacturing plants. The
purpose here is to monitor the contamination present in the
environment which ultimately causes contaminate in the medicines
i.e. being manufactured in specified areas.
• The contaminants can be non-viable particles and viable particles as
well.The 90mm Petri plates are being prepared with 20ml-25ml of
SCDA (Soybean casein digest agar), after the pre-incubation of 24
hours the Petri plates are allowed for sampling and the Active Air
Sampling procedure is to be started and to be continued up to 10
minutes or as per the time validated by each industry and then it is
incubated at 20°C-25°C for 72 hours and observation is being
recorded. Later the same plate is then incubated at 30°C-35°C for 48
hours and later the observation is being recorded.
3. Surface Monitoring methods:
• The surface monitoring procedure is performed to monitor the
contaminants which stick to the surface of the working machine,
laminar air flows, conveyor belts, door surface, floor surface, window
glass wall surface. Then it is incubated at 20°C-25°C for 72 hours and
observation is being recorded. Later the same plate is then incubated
at 30°C-35°C for 48 hours and later the observation is being recorded.
Surface Monitoring is performed by the following two ways:
(1). Contact Plate Method:
• The 55mm RODAC Petri plates are being prepared, after the pre-
incubation of 24 – 48 hours, the Petri plates are allowed to get in
contact with the working machine, laminar air flows, conveyor belts,
door surface, floor surface, window glass wall surface, etc. Then it is
incubated at 20°C-25°C for 72 hours and observation is being
recorded. Later the same plate is then incubated at 30°C-35°C for 48
hours and later the observation is being recorded.
(2). Swab Test Method:
• Normal saline is used for this purpose. Swab sticks are dipped in
normal saline in the swab collection tubes and this configuration is
used for surface monitoring by swab test. All those locations where
sample collection by the contact plate is not possible, swab test are
then performed for all those locations, like a hopper, LAF corners,
door handle wall corners, etc. The collected samples are then is being
solidified by SCDA (Soybean casein digest agar) via pour plate
techniques. Then it is incubated at 20°C-25°C for 72 hours and
observation is being recorded. Later the same plate is then incubated
at 30°C-35°C for 48-hour sand later the observation is being recorded.
• CLEAN ROOM AND ITS CLASSIFICATION
• A cleanroom is a facility ordinarily utilized as a part of specialized
industrial production or scientific research, including the manufacture
of pharmaceutical items and microprocessors. Cleanrooms are
designed to maintain extremely low levels of particulates, such as
dust, airborne organisms, or vaporized particles. Cleanrooms typically
have a cleanliness level quantified by the number of particles per
cubic meter at a predetermined molecule measure. The atmosphere
outdoor air in a typical urban area contains 35,000,000 particles for
each cubic meter in the size range 0.5 μm and bigger in
measurement, equivalent to an ISO 9 cleanroom, while by
comparison an ISO 1 cleanroom permits no particles in that size range
and just 12 particles for each cubic meter of 0.3 μm and smaller.
• Temperature:
• Temperature and relative humidity or RH is critically controlled in the
cleanrooms for human comfort & for the stability of the products.
• The general limit for temperature is 20 to 25 °C & relative humidity is
maintained at 55±5%.
• The humidity & temperature for moisture & heat sensitive products can
be adjusted as per requirement.
• Differential Pressure In Cleanroom:
• Difference of atmospheric pressure between one area and adjacent
areas is known as differential pressure.
• As per WHO guidelines of HVAC 10 to 15 Pascal differential pressure
must be maintained.
References
• https://gmh.co.in
• Chapman TM, Perry CM. Cefepime: a review of its use in the
management of hospitalized patients with pneumonia. Am J Respir
Med. 2003;2(1):75-107. doi: 10.1007/BF03256641. PMID: 14720024.
• Indian Pharmacopoeia,2010, Vol-I,p:56-63,36-37,28-
33,196-198
Thank you