Biological Testing

USP <81> Antibiotics – Microbial Assays

Microbial assay is the gold standard method to assess the activity (potency) of antibiotics. The antimicrobial activity of the antibiotic is assessed by measuring the “zones of inhibition” on lawns of specific microorganisms plated at specific concentrations so that the zones will be sized appropriately for the concentrations of antibiotics defined in the chapter. This approach is called the peni-cylinder, or cylinder-plate method.

In addition to the cylinder-plate method, USP also describes the turbidimetric method where the potency of the antibiotic is assessed by looking at concentration-specific reduction in microbial concentrations that are quantified using a spectrophotometer. This method depends upon the inhibition of growth of a microbial culture in a uniform solution of the antibiotic in a fluid medium that is favorable to its rapid growth in the absence of the antibiotic.

USP <85> Bacterial Endotoxin Tests

Endotoxin is ubiquitous substance within a gram-negative bacterial cell wall and released when the bacteria ruptures or disintegrates. Endotoxin consists of lipopolysaccharide (LPS) and lipoprotein. Endotoxin is a pyrogen often producing

fever due to the immune response it generates in the host.

The Bacterial Endotoxins Test (BET) is a test to detect or quantify endotoxins from using amoebocyte lysate from the horseshoe crab.

There are three techniques for this test: the gel-clot technique, which is based on gel formation; the turbidimetric technique, based on the development of turbidity after cleavage of an endogenous substrate; and the chromogenic technique, based on the development of color after cleavage of a synthetic peptide-chromogen complex.

Pharmetric predominantly uses Charles River MCS technology, but also utilizes Associate’s of Cape Cod’s (ACC’s) Pyros Kinetic Flex technology. The Charles River MCS utilizes chromogenic technology, while the Pyros Kinetic Flex utilizes chromogenic or turbidometric technology.

Dry Heat Oven Validation / Verification

Endotoxin Challenge Vials are commercially available vials containing nominal values of endotoxin. These vials can be used like the BIs for steam sterilization during dry heat oven cycles. The vials can also be used for annual verification that the dry heat oven is working properly, or it can be used in conjunction with other tests (such as testing the actual materials in a full load cycle) in an initial oven validation. The counts are log₁₀-transformed, and log-reductions determined. The oven cycles should meet at least 3-log reduction in endotoxin levels when compared to control, unbaked vial(s).