STERILITY PREPARATION

sterility test

This test is aimed at finding out if there are any living microorganisms in pharmaceutical products such as powders and granules for injections that are intended to be sterile. This guarantees that the product contains no contaminating microorganisms prior to administration

Injectable solutions, ophthalmic preparations, sterile powders, implants, and medical devices

types of final dosage form in sterility testing

membrane filtration

A purification process in which liquid is passed through a special membrane filter that retains microorganisms

direct inoculation

A method of sterility testing in which culture media is inoculated with microorganisms and kept under conditions favorable for growth

culture media

Solutions that contain nutrients and are used during the sterility testing to propagate microorganisms.

incubation period

The period of time during which the inoculated culture media are kept in specified conditions in order to allow the organisms to multiply

sterility

The complete absence of viable microorganisms in a products

membrane filtration method

The product is passed through a sterile membrane, and the filter is incubated in a growth medium. Uses Membrane filtration unit, incubators, culture media.

direct inoculation method

The sample is directly inoculated into a growth medium and incubated for microbial growth.

Fluid Thioglycollate Medium and Soybean Casein Digest Medium

Culture Media: Such as ___which allows anaerobic and aerobic microorganisms’ growth, respectivel

sterility test

Statistical Formula: No specific statistical formula, relies on incubation and observation.

Result: No microbial growth observed after incubation.

Standard Value: The set of procedures to perform a sterility test and acceptance criteria are described in the pharmacopoeia such as United States Pharmacopeia (USP), European Pharmacopoeia (EP) and World Health Organization (WHO) guideline

Bacterial Endotoxin Test (BET)

Detects endotoxins from Gram-negative bacteria in pharmaceutical preparations. The test is essential for injectable products and is commonly performed using the LAL test.

endotoxins

Gram-negative bacteria membranes that can lead to fever or shock in humans and contain biologically active amino acids

Gel-Clot Method

BET methods for endotoxins that do not require advanced systems. A simple gel will suffice.

Turbidimetric Method

Used in most quantitative clinical laboratories, it is used for BET to read the result of the reaction between the LAL and the endotoxin containing sample.

Chromogenic Method

chromogenic immunoassays (CIA's) are a promising robust platform for molecular diagnostic application

chromogenic immunoassays (CIA's)

are a promising robust platform for molecular diagnostic application

BET Test

Method: Statistical Formula: the test requires calculation of Endotoxin Maximum Valid Dilution (EMVD), which is the maximum dilution of the sample that can be done, and the endotoxins will still be detectable

MVD = Endotoxin Limit x Sample Concentration over λ

BET test formula

Incubating Device

It holds themreaction mixture at constant temperature which is 37 degrees Celsius plus or minus 1 degreemCelsius for a given amount of time.

Spectrophotometer

Employs in turbidimetric and chromogenic methods where changes in theturbidity or the intensity of the,color are measured

microplate reader

Used for kinetic chromogenic assays and other expansion methods where the absorbance of many samples is measured at the same time.

intravenous medications

Acceptable Result: The acceptable amount of endotoxin largely depends on the type of product and how it is administered. For ____, the limit is generally set at 5 Endotoxin Units (EU) per kilogram of body weight per hour.

intrathecal dosing

More restrictive benchmarks exist for ____ dosing at approximately 0.2 EU/kg/hr

BET Test

Standard Value: The test is based on the guideline and acceptance criteriamspecified in various pharmacopeial standards including United States Pharmacopeia (USP) l, European Pharmacopoeia (Ph. Eur.) , and Japanese Pharmacopoeia (JP); these references explain in detail the methods of study, calculation, and permissible values of contaminants for pharmaceutical preparation

Limulus Amebocyte Lysate (LAL)

Test: A specific method under BET that uses the blood extract of the horseshoe crab (Limulus polyphemus) to detect bacterial endotoxins through coagulation or color change

37°C ± 1°C

LAL test: For the Gel-Clot Method: Water bath or heating block maintained at ___ & Endotoxin-free glass test tube

microplate reader orspectrophotometer

LAL test: For the Chromogenic and Turbidimetric __ capable of measuring at specific wavelengths (e.g., 405-410 nm for chromogenic assays).

37°C ± 1°C

Incubation equipment to maintain

LAL test

Expected/Acceptable Result: Different products and their uses will require different levels of endotoxins. For example, the United States Pharmacopeia states there is an endotoxin limit of 0.25 Endotoxin Units (EU) per milliliter for Water for Injection. A particular product will only be acceptable if the results fall within the stipulated endotoxin limits for that product.

LAL test

Standard Value or System: The acceptance and the examination criteria are stated in USP guidelines, such as the United States Pharmacopeia (USP). In these guidelines, procedures along with sensitivity parameters have been established to carry out the

Pyrogen Test (Rabbit Test or LAL Test)

Evaluates the presence of fever-causing substances (pyrogens) in pharmaceutical products by injecting the test solution into rabbits and monitoring their temperature response

pyrogens

Fever-inducing substances that may be of microbial or chemical origin.

Fever Threshold

It is the range of temperature increase in rabbits that makes a sample potentially pyrogenic.

Coagulation Reaction

In the LAL test, endotoxins have the ability to gel andmself-clump into a solid clot. This is normally a sign of contamination

thermometers, animal keeping facilities, temperature surveillance systems

Equipment: Rabbit Test:

Microplate reader, LAL reagent, incubation system

LAL Test

(>0.5°C for individual rabbits or >1.4°C in three rabbits combined)

Result: No significant increase in rabbit body temperature __

pyrogen test

Pharmacopeial Standards:United States Pharmacopeia (USP) <151> Pyrogen Test

USP Bacterial Endotoxins Test European Pharmacopeia (EP) anJapanese Pharmacopeia (JP) guidelines

leaker’s test

is a quality control procedure used to detect defective or compromised containers in pharmaceutical packaging, particularly ampules and vials. The test ensures that these containers are properly sealed and can maintain sterility and integrity throughout their shelf life

vacuum pressure

A pressure level lower than the surrounding atmospheric pressure, used to force dye into leaks.

dye penetration

A method where a colored solution is used to detect leaks by entering compromised areas of a containers

vacuum pressure method

This method checks for leaks by placing containers in a vacuum chamber and reducing external pressure. If a container has a defect, air or liquid enters or escapes, revealing the leak.

Dye Penetration Test

involves submerging containers in a dye solution (e.g., methylene blue) under vacuum. The vacuum forces the dye into any cracks or leaks.

leaker’s test

Specific equipment used: Vacuum chamber, Dye solution (commonly methylene blue), magnifying lenses/automated inspection system

leaker’s test

Expected/Acceptable result: The absence of dye penetration in the container, indicating no leaks or defects. Containers with visible dye inside are rejected as defective.

leaker’s test

Pharmacopeial Standards:

• United States Pharmacopeia (USP) <381> Elastomeric Closure Integrity

• USP <1207> Package Integrity Evaluation

• European Pharmacopeia (EP) guideline

Particulate Matter Test

is a quality control procedure used for removing, counting, and sizing undissolved particles in pharmaceutical products in injectables and parenteral solutions.

Particulate Matter Test

is required on all pharmaceutical products that come in contact with human blood or tissue

microscopy

A technique used to visualize and analyze the particulate matter under magnification.

Light Obscuration

A method to detect and count particles based on the interruption of light (usually a laser light source) by particles in a liquid

Light Obscuration Method

The solution is passed through a particle counter, where light interruption is used to detect and count particles.

laser diode

The light source is a ___, which illuminates individual particles in the stream to generate a shadow or blockage of light on the detector.

Particulate Matter Test

Statistical formula: uses statistical techniques such as; the Mean Particle Count per Volume

Particulate Matter Test

Specific Equipment used: HIAC, PAMAS (Light obscuration particle counters), and Computer Software

≤100 mL

PMT: Small-Volume Parenterals (SVPs) (__):

≤6000 particles per container

PMT small volume: Particles ≥10 µm: __

≤600 particles per container

PMT small volume: Particles ≥25 µm:

>100mL

Large-Volume Parenterals (LVPs) (__):

≤25 particles per mL

PMT large volume: Particles ≥10 µm:

≤3 particles per mL

PMT large volume: Particles ≥25 µm:

clarity test

This test assesses the transparency and absence of particulate matter/impurities in liquid pharmaceutical products. A clear solution indicates the absence of visible impurities, ensuring the product’s quality and safety.

turbidity

The cloudiness or haziness in a liquid caused by large numbers of individual particles

particulate matter

Undissolved particles, other than gas bubbles, present in the liquiid

clarity test

USP <790> Visible Particulates in Injections

USP <788> Particulate Matter in Injections

European Pharmacopeia (EP) 2.9.20

Clarity and Degree of Opalescence

safety test

This test evaluates whether a pharmaceutical product causes any toxic reactions when administered. It involves administering the product to test animals and observing for adverse effects, ensuring the product’s safety for human use.

Abnormal Toxicity Test

Safety Test is also known as the

toxicity

The degree to which a substance can harm humans or animals.

LD50 (Lethal Dose 50)

The dose of a substance that kills 50% of the test population.

safety test

Statistical Formula: The Safety Test involves observation of adverse effects, statistical analysis may be applied to interpret the data, especially in determining metrics like LD50.

safety test: Selection of Test Animals: Typically healthy ___are used.

intraperitoneal route

Administration: Inject a specified dose of the product into the animals, usually via the

safety test

Expected/Acceptable Result: No test animals should exhibit signs of toxicity or abnormal reactions during the observation period

safety test

Pharmacopeial Standards:

USP <88> Biological Reactivity Tests, In Vivo

USP <87> Biological Reactivity Tests, In Vitro

European Pharmacopeia (EP) 2.6.9

Abnormal Toxicity Test (historically used but now largely replaced) ICH Guidelines (International Council for Harmonization) for Toxicity Testin