Quality attributes and evaluation

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

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.

Pyrogens

1. Endotoxins, exotoxins, and pyrogens

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.

2. Endotoxin components and tolerance limits

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.

3. Sources

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.

4. Depyrogenation

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.

5. Detection

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.

Particulate matters

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.