Pharmaceutical and Biologics

 

Sterility test: USP <71>, EP 2.6.1


Nelson Labs Europe performs sterility tests under the most stringent test conditions in a sterility isolator. All materials and test items enter the isolator through a transfer hatch. Before entering the manipulation area the outer surfaces of all materials are decontaminated using Vaporised Hydrogen Peroxide (VHP). This prevents the manipulation area from being contaminated and assures an absolute sterile working environment.
When performing sterility tests in a well validated isolator, the risk of a false positive sterility test is virtually down to zero. When the test is performed in a sterility isolator, the influence and the efficacy of the surface decontamination should be validated. This way, false negative results can be excluded.
 

In order to validate the possible penetration of VHP through the packaging material, the test items are inoculated with a small number of S. aureus. Subsequently, the outer surface of the test items are decontaminated in the transfer hatch of the isolator and the viability of the micro-organism is determined.

 

Bacterial Endotoxins (LAL): USP 85, EP 2.6.14


Pyrogens are a group of substances capable of eliciting a fever response upon injection or infection. Endotoxin is a subset of pyrogens that is strictly of gram-negative bacterial origin. Dozens of microbiological compounds have been found to induce fever, but many do so only weakly or only at very high doses.
Most reactive (by far), most occurring and most resistant to extreme conditions of all possible pyrogens are gram-negative bacterial endotoxins. This is why the focus of pyrogens is mainly on endotoxins.
LAL-activation is considered analogous to the response that is considered to be pyrogenic but is more specific for bacterial endotoxin and at much lower levels of detection.
Endotoxins or Lipopolysaccharides (LPS) are a part of the outer cell wall of gram-negative bacteria.


LAL chromogenic kinetic assay


Endotoxins catalyse the activation of a proenzyme in the Limulus Amebocyte Lysate (LAL). The rate of activation is determined by the concentration of endotoxin present. The activated enzyme induces the formation of colour which is measured photometrically continuously throughout the incubation period. The concentration of endotoxin present is characterised by the time needed to increase the optical density by 0.2 units. A calibration curve is developed from an E. coli endotoxin standard from which reaction times of unknowns can be translated into Endotoxin concentration.
The kinetic approach for the LAL test allows for a cost-effective, automatable and quantifiable method to detect gram-negative bacterial pyrogens. It is also one of the few existing methods with a theoretical detection limit of 0.001 EU/mL.


Inhibition & Enhancement


The constitution of the test liquid (or extract) may influence the proper functioning of the enzyme and thus result in an over- or underestimation of the endotoxin concentration. To exclude this inhibition or enhancement of the enzyme, a positive product control (PPC) for each sample is included in the assay.

 

Microbiological Examination of Non-Sterile Products: Microbial Enumeration Tests: USP <61>, EP 2.6.12


These tests will allow quantitative enumeration of mesophilic bacteria and fungi that may grow under aerobic conditions.
The tests are designed primarily to determine whether a substance or preparation complies with an established specification for microbiological quality.
Validation: Suitability of the counting method in the presence of the product.
For each type of product where bioburden is to be determined, the applied microbial recovery method should be determined during a validation study prior to the routine application of the desired method.
An artificial bioburden of less than 100 CFU of one of the following organisms will be applied to the test item: Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Candida albicans or Aspergillus niger. Subsequently, the bioburden of the test items will be determined using the selected method. A count of any test organism not differing by a factor greater than 2 from the value of the control in the absence of the product must be obtained.

If growth inhibition is noticed, the use of a suitable neutralizer should be validated.


Total Aerobic Count
 

2 x 10 mL of the product will be membrane filtered and the membranes transferred once to Trypticase Soy Agar (TSA) plates and once to Potato Dextrose Agar (PDA) plates. TSA plates will be incubated for 3-5 days at 30-35°C. PDA plates will be incubated for 5-7 days at 20-25°C. 
The results are expressed as CFU/g.
Acceptance criterions can be found in specific monographs of the pharmacopeia.

 

Microbiological Examination of Non-Sterile Products: Tests for Specified Microorganisms: USP <62> and EP 2.6.13


These tests will allow determination of the absence of, or limited occurrence of, specified microorganisms that may be detected under the conditions described. The tests are designed primarily to determine whether a substance or preparation complies with an established specification for microbiological quality.
Validation: Suitability of the counting method in the presence of product
For each type of product where bioburden is to be determined, the applied microbial recovery method should be determined during a validation study prior to the routine application of the desired method.
An artificial bioburden of less than 100 CFU of a specific micro-organism will be applied to the test item. Subsequently, the bioburden of the test items will be determined using the appropriate method.
A count of the test organism not differing by a factor greater than 2 from the value of the control in the absence of the product must be obtained.
If growth inhibition is noticed, the use of a suitable neutralizer should be validated.


Test for Specific Micro-organisms


The product will be membrane filtered and the membranes transferred to selective growth media promoting only growth of the specific micro-organisms. The plates will be incubated for a specified time at the appropriate temperature.
The results are expressed as CFU/g.
Acceptance criterions can be found in specific monographs of the pharmacopeia.

 

Mutagenicity Assays (AMES): OECD 471


The Ames test is a biological assay to assess the mutagenic potential of chemical compounds. As cancer is often linked to DNA damage, the test also serves as a quick assay to estimate the carcinogenic potential of a compound since the standard tests for carcinogenicity done on rodents take years to complete and are expensive to do.
The test uses several strains of the bacterium Salmonella typhimurium and/or Escherichia coli that carry mutations in genes involved in histidin or tryptophan synthesis, so that they require histidin or Tryptophan for growth. The mutagen's ability to cause a reversion that will allow the strain to grow on a histidin/tryptophan-free medium is tested. The tester strains are specially constructed to have both frame shift and point mutations in the genes required to synthesize histidin/tryptophan, which allows for the detection of mutagens acting via different mechanisms. Some compounds are quite specific, causing reversions in just one or two strains. The tester strains also carry mutations in the genes responsible for Lipopolysaccharide synthesis, making the cell wall of the bacteria more permeable, and in the DNA repair system to make the test more sensitive. Rat liver extract is added to simulate the effect of metabolism, as some compounds, like benzopyrene, are not mutagenic themselves but their metabolic products are.

 

Antibiotic Assay USP <81>


The activity (Potency) of antibiotics may be demonstrated under suitable conditions by their inhibitory effect on microorganisms. A reduction in antimicrobial activity also will reveal subtle changes not demonstrable by chemical methods. Accordingly, microbial or biological assays remain generally the standard for resolving doubt with respect to possible loss of activity. Two methods are employed, the cylinder-plate or turbidimetric assay.


The cylinder plate assay depends upon diffusion of the antibiotic from a vertical cylinder through a solidified agar layer in a Petri dish or plate to an extent such that growth of the added microorganisms is prevented entirely in a circular area or “zone” around the cylinder containing a solution of the antibiotic.
A standard dilution series of a reference antibiotic is prepared. The size of the inhibition zone of the test sample is measured and compared to the size of the zone of inhibitions of the standards, to determine the potency of the test sample.
 


Example of the zone of inhibition assay. The size of the zone is relative to the potency of the antibiotic.

 

The turbidimetric method depends upon the inhibition of growth of a microbial culture in a uniform solution of the antibiotic in a fluid medium that is favourable to its rapid growth in the absence of the antibiotic.
The turbidity of the test sample is measured photospectrometically and compared to a dilution series of a reference antibiotic to determine the potency of the test sample.

Contact:

 

Nelson Labs Europe
Romeinsestraat 12
B-3001 Leuven
Belgium
+32.16.400484
InfoEurope@nelsonlabs.com

Nelson Labs Europe | Pharmaceutical and Biologics