Pyrogens and Pyrogen Testing

Pyrogens
Fever producing organic metabolic products arising from microbial
contamination and responsible for many of the febrile reactions in
patients following injection.
Bacterial endotoxins
Causative material: lipopolysaccharide metabolic products from
the outer cell wall of a Gram negative bacteria.
Thermostable
Water soluble

Pyrogens and Pyrogen Testing

USP EU- a bacterial endotoxin limit stated by USP injection
monographs.
Injections are not pyrogen or endotoxin free but are only limited.
Examples from USP32-NF27(12)
Dextrose Injection: Contains not more than 0.5 USP EU per mL for
injections not containing less than 5% dextrose and not more than
10.0 USP EU per mL for injection containing between 5% and 70%
dextrose.
Digoxin Injection: contains not more than 200.0 USP EU per mg of
digoxin.
Gentamicin Injection: contains not more than 0.71 USP EU per mg
of gentamicin.

Pyrogens and Pyrogen Testing

Oxidation
common method used to remove pyrogens.
Done to convert them to easily eliminated gases or nonvolatile
solids which are both separated by fractional distillation.
Potassium Permanganate - oxidizing agent
- efficiency increased by addition of
Barium Hydroxide to impart alkalinity
and make nonvolatile barium salts to
any present acidic compounds.

Pyrogens and Pyrogen Testing

These two reagents + water distilled several times collect under
strict aseptic conditions chemical free distillate with highly pure,
sterile, and pyrogen free water test with official pyrogen test.

Pyrogen test
Uses healthy rabbits that have been properly maintained in terms of
environment and diet before the test. Normal, or control, temperatures
are taken for each animal to be used in the test. These are used as a
base to determine any rise in temperature after injection of sample.
The test uses rabbits whose temperatures do not differ by more
than1 C from each other and whose body temperatures are considered
not to be elevated.

Pyrogens and Pyrogen Testing

Bacterial Endotoxins Test, USP
Uses LAL test (Limulus amebocyte lysate)- developed as a result of the
discovery of an extract from the blood of a horseshoe crab ( limulus polyphemus)
which contains an enzyme and protein system that coagulates in the presence of low
levels of lipopolysaccharides.

More sensitive to endotoxin than the rabbit test

Replacement for the rabbit test
Used for a number of parenteral products

Pyrogens and Pyrogen Testing

Some parenterals cannot be tested with LAL due to interference of the
active ingredient. This products use the pyrogen test.
These are: meperidine HCL, promethazine HCL, oxacillin sodium, sulfisoxazole,
vancomycin HCL, etc.

Since the LAL test is sensitive, interference of an active ingredient can

be overwhelmed by diluting the product with more than twofold of
water.
Products tested in this manner: Diphenhydramine HCL, ephedrine HCL,
meperidine HCL, promethazine HCL, thiamine HCL

Industrial Preparation of Parenteral

Products
In most manufacturing plants, area in which parenterals are made is
maintained bacteria free by:
Use of ultraviolet lights
Filtered air supply
Sterile manufacturing equipment
Sterilized work clothing

Industrial Preparation of Parenteral

Products
Parenteral suspensions - prepared by reducing the drug to a very fine
powder with:
Ball mill
Micronizer
Colloid mill, etc
then suspending the material in a liquid which it is insoluble.
Autoclaving of a parenteral suspension may alter the viscosity of the
product, affecting the suspending ability of the vehicle, or change the
particle size of the suspended particle, altering both pharmaceutical and
therapeutic characteristics.
Autoclaving also destroys parenteral emulsions.

Industrial Preparation of Parenteral

Products
Some injections are packaged as dry powders due to being unstable in
the presence of a liquid.
Method of sterilization of the powder:
Dry heat
Examples of sterile drugs packaged without pharmaceutical additives:
o
o
o
o
o
o
o
o
o

Ampicillin sodium
Ceftizoxime sodium
Ceftazidime sodium
Cefuroxime sodium
Kanamycin sulfate
Nafcillin sodium
Penicillin G Benzathine
Streptomycin sulfate
Tobramycin sulfate

Industrial Preparation of Parenteral

Products

Industrial Preparation of Parenteral

Products
Sterile drugs with pharmaceutical additives and intended to be
reconstituted:
o
o
o
o
o
o
o
o

Cyclophosphamide
Dactinomycin
Erythromycin lactobionate
Hydrocortisone sodium succinate
Mitomycin
Nafcillin sodium
Penicillin G potassium
Vinblastine sulfate

Industrial Preparation of Parenteral

Products

Industrial Preparation of Parenteral

Products
Antibiotics prepared industrially in large fermentation tanks
Sometimes a liquid is packaged along with the dry powder for use at the
time of reconstitution.
Mix-O-Vial
Dry ingredients in the bottom
compartment
Liquid diluent in the top
Separated by a specially
formulated center seal
Offers stability of the product
until it is activated, convenient,
fast, and safe as long as the right
drug and proper diluent are in
correct proportions.
Manufactured by Pfizer

Industrial Preparation of Parenteral

Products
Most commonly employed solvents to reconstitute dry-packaged injections:
Sodium chloride injection
Sterile water for injection
Dry powders packaged in large containers
- caking is prevented upon standing to facilitate dissolution
- uses lyophilization
Treated powders form a honey comb lattice structure that is rapidly
penetrated by liquid.
Solution is rapid due to the exposed powders large surface area.

Industrial Preparation of Parenteral

Products
Hospira ADD-Vantage System

Ready to mix sterile IV product

Designed for intermittent administration of

potent drugs that do not have long term
stability in solution

Antibiotics and other drugs do not have to

be mixed until prior to administration

Consists of two components:

Flexible plastic IV container
Partially filled with diluent
Glass vial of powdered or liquid drug

Industrial Preparation of Parenteral

Products
Monovial Safety Guard

Manufactured by Becton Dickinson Pharmaceutical

Systems

IV infusion system for use in preparing

extemporaneous small-volume infusions using plastic
minibags

More favorable, less time consuming, costs less than

traditional:
Transfer needle and vial (TFN) method
Syringe and vial (SYR) methods

Integrated drug transfer mechanism with a protective

shield surrounding the attached transfer needle

Reconstitution and transfer of the drug into an

infusion bag are accomplished safely, quickly, and with
few materials.

Packaging, Labeling, and Storage of

Injections
Containers for injections must not interact physically or chemically with
the preparation
If container is made of glass, it must be clear and colorless, or light amber
to permit inspection of its contents
Injections are placed either in:
Single dose container- hermetic container intended for single dose of administration
wherein once opened, it cannot be resealed; may be in ampules or single dose vials
Multiple dose container- hermetic container that permits successive withdrawal of its
contents without changing the strength, quality, or purity of the remaining portion

Some products are packaged in pre-filled syringes, with or w/out special

administration devices

Packaging, Labeling, and Storage of

Injections
CONSTITUTION OF OFFICIAL GLASS TYPES
TYPE

GENERAL DESCRIPTION

Highly resistant borosilicate glass

II

Treated soda-lime glass

III

Soda-lime glass

NP

General purpose soda-lime glass

Type I, II, III are suitable for parenterals with type I being the most
resistant to chemical deterioration.

Packaging, Labeling, and Storage of

Injections
Clarity- prime prerequisite; clear and free of all particulate matter
Precautions to be taken during manufacture, storage, and use of product:
Filter parental solution before transferring into the container
Containers must be chemically resistant and of highest available quality
-prevents leaching
After selection, container must be carefully cleaned
Extreme care must be exercised during container filling
Personnel must wear monofilament fabric uniform, face hoods, caps, gloves, and
disposable shoe covers
After filling of container, it must be visually or automatically inspected
- Done by passing through a light source with black background
- Particles 50 um are detected

Packaging, Labeling, and Storage of

Injections
Methods to detect smaller particulate matter:
Microscopic examination
Coulter Counter electronically counts particles in samples

Particulate matter has the potential to induce thrombi and vessel

blockage.
Depending on chemical composition, it has potential to introduce to the
patient agents that are undesired and toxic.
Some single dose preparations are to be administered rapidly in small
volumes, but others infuse slowly into the circulatory system over hours.

Packaging, Labeling, and Storage of

Injections
Small volume parenterals formulated so that a convenient amount of
solution contains the usual dose of the drug.
Larger volumes of more diluted solutions are administered intravenously
and intramuscularly.
Large- volume single-dose preparations used to expand the blood
volume or to replenish nutrients or electrolytes
-given by slow IV infusion
-does not permit withdrawal of more than 1,000 mL
Intraspinal, intracisternal, or peridural administration must be packaged
only in single-dose containers.

Packaging, Labeling, and Storage of

Injections
Multiple dose containers
Affixed with rubber closures to permit penetration of a hypodermic needle
without removal or destruction of the closure.
As long as the needle at the time of entry is sterile, the injection will remain
sterile
Required to contain antibacterial preservatives
Not permitted to allow withdrawal of more than 30mL

Usual container contains about 10 usual doses of the injection

Packaging, Labeling, and Storage of

Injections
For labeling, revised injectable product nomenclature became official in
USP 23 on January 1,1995.
Center for Drug Evaluation and Research encouraged manufacturers to
place a flag, or reminder statement, on the labels of the product for 6
months, alerting practitioners to the changes.

Packaging, Labeling, and Storage of

Injections
Labels on containers of parenteral products must state the ff:
Name of preparation
For liquid preparation, the percentage content of drug or the amount of drug in a
specified volume
For a dry preparation, the amount of active ingredient present and the volume of liquid
to be added to prepare a solution or suspension
Route of administration
Statement of storage conditions and expiration date
Name of manufacturer and distributor
Identifying lot number capable of yielding complete manufacture history

Packaging, Labeling, and Storage of

Injections
Preparations for dialysis, hemofiltration, or irrigation solutions should bear
a statement indicating that the solution is not intended for IV infusion.
Containers have an area free of label to permit inspection of the contents.
If inspection reveals presence of particulate matter, preparation should be
discarded.
Each individual monograph for the official injection states:

Type of container
Type of glass preferred
Exemptions if any to usual package size limitations
Special storage instructions

Packaging, Labeling, and Storage of

Injections
Injections prepared from chemically pure medicianal agents are:
Stable at room temperature
Stored without special concern or conditions

Biologic products such as:

Insulin injection
Various vaccines
toxoids
Toxins

Should be stored under refrigeration.

Environmental issues
Hospira, Baxter Healthcare, B. Braun, are shipping containers free from
PVC and di-2-ethylhexylpthalate (DEHP).
PVC or polyvinyl chloride- popular thermoplastic that contains high levels
of chlorine ; referred to as the "Poison Plastic" due to the toxic pollutants
it might release.
There is a huge concern regarding the effects of DEHP on male neonate
patients.

PVC bags can leach DEHP into the fluid of the container.
Another concern is the amount of plastic waste.
In response, Hospira launched its VISIV line of IV containers.

Environmental issues
VISIV containers:

Are PVC/DEHP free

Do not contain any overwrap
Has a sterile port
During emergency, does not have to sterilize the port prior to administration
Provides thermal stability
Moisture barrier properties
Inertness
Avoids leaching

Problems associated with PVC/DEHP:

IV admixtures still need preparation in glass containers
Costly to manufacture than non-PVC/DEHP plastic bags

Quality Assurance for PharmacyPrepared Sterile Products USP <797>

June 1, 2008 revised USP Chapter <797>, Pharmaceutical Compounding
Sterile Preparations became official.
USP <797> - provides the minimum practice and quality standards for
compounded sterile preparations (CPSs).
- Applies at all times to those who compound sterile
preparations
Three risk levels of (CPCs):
Low risk level
Medium risk level
High risk level

Quality Assurance for PharmacyPrepared Sterile Products USP <797>

Risk levels based on the potential of contaminating a low risk level or
medium risk level CSP or failure to sterilize a high risk level CSP which can
harm the patient and lead to death.
Low risk level and medium risk level CSPs sterile ingredients and devices
are used and sterility is maintained
High risk level CSPs must be sterilized before administration
Any CSP with a nonsterile component is always high risk level.

Quality Assurance for PharmacyPrepared Sterile Products USP <797>

Immediate use CSPs section only for emergency or situation wherein
the patient immediately needs to receive a low risk level CSP
- May occur in a:

Respiratory or cardiac arrest

Emergency room
Operating room
Combat zone
With the preparation of a diagnostic agent

Quality Assurance for PharmacyPrepared Sterile Products USP <797>

Compounding process cannot exceed one hour.
Compounding under immediate use conditions increases the likelihood
of microbial contamination and potential patient harm.

Facilities for compounding CSPs are designed and environmentally

controlled to minimize airborne contaminations.
PECS or sources of ISO class 5 air quality:

Laminar airflow workbenches

Compounding aseptic isolators
Compounding aseptic containment isolators (CACI)
Biological safety cabinets (BSC)

Quality Assurance for PharmacyPrepared Sterile Products USP <797>

PECs should be recertified at least every 6 months or whenever the
device or room is relocated or altered or a major service to the facility is
performed.
Exposure to hazardous drugs may lead to adverse events which may
range from skin rashes to effects on the respiratory system and cancer.
Preparation of such drug shall occur in an ISO Class 5 environment.
Closed system transfer devices (CSTDs) that prevent venting or exposure
of the drug to the environment shall be used within the ISO Class 5
environment.

Quality Assurance for PharmacyPrepared Sterile Products USP <797>

For institutions that compound a low number of hazardous drugs, a
CSTD used within a BSC or CACI in a nonnegative pressure room is
acceptable.
Key components in minimizing the risk to patients are:
Proper hand hygiene and garbing practices
Personnel aseptic technique
Disinfection of compounding practices

Quality Assurance for PharmacyPrepared Sterile Products USP <797>

Compounding personnel must be :
completely trained in the theoretical and practical aspects of aseptic
manipulation
Must pass written examinations and media fill testing before being allowed to
prepare CSPs for patients
Must take gloved fingertip sampling which assesses competency in performing
hand hygiene and garbing

USP <797> also includes sections on:

Establishing beyond use dates

Compounding radiopharmaceuticals and allergen extracts
Characteristics of a quality assurance program
Verification of compounding accuracy and sterility
Finished preparation release checks and test
Elements of quality control