Injectable products are usually required to be tested for the following characteristics:
Quality attributes
and evaluation
In
addition to meeting the physical and chemical stability attributes of the
dosage form being formulated, all parenteral products must be sterile,
nonpyrogenic, and free from extraneous insoluble materials. Injectable products
are usually required to be tested for the following characteristics:
Sterility
testing is carried out by incubating the drug product in a conducive
environment for microbial growth. Such conducive environment includes
appropriate temperature, humidity, and nutrient media. Microbial growth is
monitored after a given period of time, determined by standard protocols for
each type of microbes.
There
are two methods of sterility testing:
·
Direct inoculation: The drug product
is added to the nutrient media and
incubated, followed by observation for any microbial growth.
·
Membrane filtration: Whenever the
nature of the drug product is likely
to hinder the detectability of microbial growth, the product is filtered
through a membrane and the membrane is incubated in nutri-ent media for
observation of microbial growth.
Typically,
two culture media are used: (1) trypticase soy broth and (2) fluid
thioglycollate medium. The sterility of each sterilized batch of medium is
confirmed by incubating a portion of the batch at 20°C–25°C when tryp-ticase
soy broth is used as a culture medium, but at 30°C–35°C when fluid
thioglycollate medium is used.
Bacterial
toxins could be endotoxins or exotoxins. Endotoxins are the struc-tural
molecules of certain gram-negative bacteria that are recognized by the human
immune system, resulting in fever and immune reaction. Exotoxins, on the other
hand, are the toxins secreted by microorganisms, such as bacteria, fungi, and
algae. When injected, both endotoxins and exotoxins can induce fever, that is,
pyrogenic. Such substances are termed as pyrogens. Some of the effects caused
by pyrogens in the body are an increase in body temperature, chills, cutaneous
vasoconstriction, a decrease in respiration, an increase in arterial blood
pressure, nausea and malaise, and severe diar-rhea. When compounding a sterile
injectable product from nonsterile com-ponents, there is always a concern about
endotoxin contamination.
The
endotoxins might originate from the microbes that get destroyed dur-ing
sterilization. The lipopolysaccharide (LPS) portion of the cell wall that gets
released during cell lysis, is the principal constituent of the endogens that
cause the pyrogenic response. The LPS can be sloughed off the bacteria, which
do not have to be living for the LPS to be pyrogenic. Gram-negative bacteria
produce more potent endotoxins than gram-positive bacteria and fungi.
Endotoxin
levels higher than 5 endotoxin units (EU)/kg/h can elicit pyrogenic response on
IV injection. The maximum permissible levels in the United States (mandated by
the U.S. FDA) are 0.2 EU/kg products for intrathecal, 5 EU/kg products for
nonintrathecal injectable, and 0.25–0.5 EU/mL for sterile water. Intrathecal
injections of 0.2 EU/kg can cause pyro-genic response. One EU is approximately
100 pg (picogram, i.e., 10−12 g) of Escherichia coli LPS, present in approximately 105 bacteria.
Water
is the main source of pyrogens. This is because Pseudomonas, a gram-negative bacterium, grows readily in water.
Other sources of endo-toxins or pyrogens are raw material, processing
equipment, and human contamination.
Endotoxins
are not completely removed by filtration and steam steriliza-tion. Endotoxins
can be destroyed by dry heat. Thus, when compounding a sterile product from
nonsterile starting material that can withstand the heat of 200°C, it should be
depyrogenated. If a particle is depyrogenated, it is also sterile.
A
preferred method for the detection of pyrogens is the limulus amebocyte lysate
(LAL) test. A test sample is incubated with amebocyte lysate from the blood of
the horseshoe crab, Limulus polyphemus.
A pyrogenic substance causes gelling.
Parenteral
solutions are carefully inspected for the presence of any foreign particles,
such as glass, fibers, precipitates, and any floating material by microscopy,
video imaging, visual inspection, and/or particle counters. Sources of
particulate matter include the raw materials, processing and filling equipment,
the container, and environmental contamination. Any parenteral product samples
found containing particulate matter are dis-carded. If the quantity and the
type of discard exceed a predetermined quality threshold, an investigation is
initiated to determine and remediate the cause of the particulate.
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