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Control of microbial burden of non-sterile pharmaceutical products (EP 2.6.12/2.6.13, USP 61/62)
  • Method suitability test
  • Sample analysis

Drugs contaminated by microbes can cause immediate and long-term harm to patients treated with these products. Microbes may also reduce or even inactivate the therapeutic activity of the product by altering the chemistry of the active pharmaceutical ingredient. Manufacturers have to ensure low bioburden of the finished dosage forms by implementing current guidelines of Good Manufacturing Practice throughout the manufacturing process, storage and distribution of the pharmaceutical preparations. The tests described in the harmonized chapters of E.P. 2.6.12/ 2.6.13 & USP 61/62 allow the quantitative enumeration of mesophilic bacteria and fungi that may grow under aerobic conditions. The ability of the test to detect microorganisms in the presence of the product should be established.

Testing is performed as per the registered specifications which may include: total aerobic microbial count, total yeast and molds count and specified microorganisms such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella species, Bile-tolerant gram-negative bacteria.

Enumeration methods that can be employed include: membrane filtration, plate count method (pour plate or spread method) and most-probable-number (generally considered as the least accurate).


Bacterial endotoxins test (EP 2.6.14, USP 71)
  • Method suitability test
  • Sample analysis

Endotoxins are lipopolysaccharides found in the cell wall of Gram-negative bacteria, which can induce inflammation and fever as an immune response in higher organisms. Reaction to endotoxins can lead to anaphylactic shock and death of patients. The bacterial endotoxin test (BET) aims to detect and/or quantify endotoxins using amoebocyte lysate from the horseshoe crab (Limulus Polyphemus or Tachypleus tridentatus). Any of the six methods described by the harmonized chapters of E.P 2.6.14 & USP 81 can be employed. However, the gel clot limit test is the method of choice in the event of doubt or dispute, unless otherwise described in the monograph. To ensure both the precision and the validity of the test, the label lysate sensitivity test and the test for interfering factors are performed. It must be confirmed that the test solution does not contain any factors that could interfere with the test method. Otherwise, a suitable validated treatment should be employed and the test for interfering factors should be repeated.

Particulate matter analysis (EP 2.9.19, USP 788/789)
  • Method I, Light obscuration method
  • Method II, Microscopy method

Particulate matter consists of mobile, randomly sourced, extraneous substances, other than gas bubbles, that cannot be quantitated by chemical analysis because of the small amount of material they represent and because of their heterogeneous composition. For the determination of particulate contamination in ophthalmic solutions, injections and parenteral solutions two procedures are specified in EP 2.9.19 and USP 788/789. Method I, Light obscuration method is preferably applied; however, it may be necessary to test some articles by the microscopic test to reach a conclusion on conformance to requirements. Any product that is not a pure solution having a clarity and a viscosity approximating those of water may provide erroneous data when analyzed by the light obscuration counting method. Such materials may be analyzed by the microscopic counting method.

Preservative efficacy testing (EP 5.1.3, USP 51)
  • Suitability of the counting method in the presence of the product
  • Sample analysis

In case of pharmaceutical preparations which do not themselves have adequate antimicrobial activity, preservatives may be added to prevent proliferation or to limit microbial contamination. Especially for multidose containers, a probable microbial contamination during the normal conditions of storage and use would present a hazard to the patient. The efficacy of the preservatives in the final container of the product is investigated over the period of validity to ensure that its activity is not impaired by the storage. The test is described under E.P. 5.1.3 and USP 51 and the acceptance criteria are defined by the intended use of the pharmaceutical preparation (e.g., oral solutions, parenteral or eye preparations, ear or nasal preparation etc.). The ability of the procedure to detect challenge organisms in the presence of the product should be established (i.e. suitability of the counting method). 

In vitro genotoxicity test (ICH M7) - AMES test
  • Bacterial reverse mutation test (Ames test) OECD TR 471
  • Ames test Micro Plate Fluctuation assay

The Ames test is an in vitro assay for the detection of the genotoxic potential of various chemical substances including nitrosamines. The assay is performed in compliance with  Good Laboratory Practice (GLP) regulations.

The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs.  Following the OECD 471 Guideline for the Testing of Chemicals, four S. typhimurium strains, TA98, TA100, TA1535, and TA1537, and one E. coli strain, WP2 uvrA (pKM101), are employed in the presence and the absence of an exogenous metabolic activation system. The test is performed in triplicates using at least five concentrations of the test item, up to the lowest cytotoxic concentration, but not exceeding 5 mg/plate as determined in the frame of the dose range finder experiment.

As per the requirements of EMA Q&A, Appendix 3: Enhanced Ames test Conditions for N-nitrosamines (EMA/451666/2023), the pre-incubation method should be used and the recommended preincubation time is 30 minutes. Ames assays should be conducted in the absence of a post-mitochondrial fraction (S9), and also in the presence of 30% rat liver S9, as well as 30% hamster liver S9. The rat and hamster post-mitochondrial fractions (S9s) should be prepared from rodents treated with inducers of cytochrome P450 enzymes (e.g., a combination of phenobarbital and β-naphthoflavone). Additionally, two class-related positive controls for nitrosamines are incorporated in the assay, namely; N-Nitrosodimethylamine (NDMA), CAS number 62-75-9, 1-Cyclopentyl-4-nitrosopiperazine (CNP), CAS number 61379-66-6 and a representative NDSRI.

Biological assays
  • Microbiological assay of antibiotics - EP 2.7.2, USP 81

The potency of an antibiotic pharmaceutical preparation can be demonstrated by the inhibitory effect on a reference microorganism under suitable conditions. A reduction in antimicrobial activity may not be demonstrated by chemical methods. The assay may be performed with two general techniques: diffusion agar assay (or cylinder plate assay) and the turbidimetric assay. The suitable design should be selected and verified. In case of the diffusion method, the inhibition zones of the sample are compared with the inhibition zones of the equipotent concentrations of the reference standards, while in turbidimetric assays the absorbance or transmittance is evaluated. Appropriate statistical methods are employed for the calculation of the potency.


  • Heparin biological activity

The anticoagulant activity of heparin is determined in vitro by its ability to accelerate the inhibition of thrombin (i.e. factor IIa) by antithrombin. The anti-IIa and anti-Xa assays can be end-point assays (determination of absorbance) or kinetic assays (change of absorbance per minute). Appropriate statistical methods are employed for the calculation of the potency of the substances. The method is described in E.P. 2.7.5 while similar methods are applicable for low-molecular mass heparins as well. Method development, validation and routine testing is performed in house. 

Filter validation studies (PDA TR 26, ISO 13408-2:2018)

Sterilizing filtration is the process of removing microorganisms from a fluid stream without adversely affecting product quality. To this end, most filter manufacturers provide results of tests performed according to applicable compendial methods to qualify their filters as suitable for pharmaceutical applications. However, this qualification documentation supports but does not replace, performance qualification as part of process validation conducted by the filter user. Qualimetrix facilitates this task, by undertaking the following tests according to PDA technical report No 26/2008, for which a short description is given:

  • Filterability test: The scope of the filterability test is to evaluate the potential clogging of the membrane by measuring the rate of the filtration (filtered weight against time), under constant pressure. This test also serves as a means of predicting manufacturing-scale performance by using flux and pressure conditions representative of the actual process.
  • Filter extractables (and potential leachables): It is a crucial part of filter validation studies to ensure that the filter does not adversely affect the process stream. To this end, filter extractable and possibly filter leachable species should be assessed. Extractables are chemical compounds that can be extracted from product contacting surfaces when exposed to an appropriate solvent under exaggerated conditions (i.e. time and temperature). Leachables are chemical compounds that migrate from a contact surface into the drug product or process fluid during storage or normal use conditions. The first step of the approach followed is to perform an extraction study with a suitable model solvent simulating worst-case conditions. Non-specific methods, such as non-volatile residue (NVR) and Fourier-transform infrared spectroscopy (FTIR) are employed to provide general quantitation and qualification of potential leachables. This represents the essential and simplified approach. nvr
  • Alternatively, and upon client’s request, an extensive extractable species profile assessment can also be implemented, by using various extraction media, to cover and extract the total pool of potential leachables, and by employing specific orthogonal techniques (LC/UV/MS, GC/MS, ICP/MS) to address their chemical diversity and provide information with respect to both their identity and concentration levels. The second step is to evaluate the levels of extractables following a risk-based methodology in order to determine the need for conducting a leachable species study. Both extractables and leachables testing for filters and other production-related materials (e.g., tubes) are an integral part of our services portfolio.
  • Compatibility: It is well known that numerous chemical interaction possibilities exist in a filter system. The effects of these interactions should be adequately characterized prior to filter selection and in most cases a simple chemical compatibility chart will not often provide enough information for predicting filter system compatibility, thereby requiring additional testing. Integrity testing is a non-destructive test that relates to microbial retention and is a determinant of compatibility. This test can be performed by means of either a diffusive / forward flow or bubble-point test. It is worth noting that both extractables and integrity testing constitute a combination of tests that serve to establish compatibility and detect subtle incompatibilities that a single test is usually not able to reveal.
  • "Product wetted" integrity test (Bubble point & Forward flow): In case the integrity test of the process-scale filter is performed online using the actual solution for the wetting procedure, "product-wetted" specifications for the integrity testing parameters should be calculated. This study determines the forward flow and bubble point test limits for a membrane filter that is wetted with process fluid.
  • Viability study: The aim of bacterial retention validation studies is to have documented evidence demonstrating that the filtration process will consistently remove high levels of a standard bacterium or relevant bioburden isolate, suspended within the product or product simulant, under simulated process conditions. Hence, the first and most crucial step is to demonstrate that the challenge organism (e.g. Brevundimonas diminuta, ATCC 19146) is viable after a specific contact period of time with the product. The population (survivals) of the test microorganism is compared to the microorganisms spiked in a control solution over the defined process time. The product is characterized based on the log reduction of the population of the challenge microorganism according to the approach described below:
    • Non-Bactericidal: A decrease in the viability of the micro-organism of less than one log over the process time.
    • Moderately Bactericidal: A decrease in the viability of the micro-organism of less than one log over 60 minutes and of 1 log or more over the process time.
    • Bactericidal: A decrease in the viability of the micro-organism of greater than one log over a minimum of 60 minutes duration.
  • Flush studies & Viability test for product simulant. If B. diminuta is not viable in the liquid (bactericidal product solution), the product fluid can be modified to ensure the viability of the challenge organism, the exposure time can be reduced to ensure that the challenge organism remains viable or a product simulant can be used instead. The ideal simulant solution would minimize adsorptive retention, ensuring that the sterilizing action of the filter under consideration is the consequence of sieve-retention. Moreover, it should match the product as closely as possible in terms of its physical and chemical characteristics (pH, osmolality, surface tension, viscosity), without adversely affecting the challenge micro-organism. The viability of the challenge microorganism in the product simulant should be verified. Moreover, the flush studies should be conducted. The filter flush studies are performed in order to validate the appropriate flushing volumes to be applied during the bacterial challenge test, in order to rinse any bactericidal residues from the filters, prior to challenging the altered product or simulant fluid with B. diminuta. The flush study consists of three distinct phases, the flushing of the test filter membranes, the collection of the final effluent, passed through the test filter membranes and the flushing of the recovery membranes, that are assembled downstream of the test filter membranes, in order to recover any microorganism that might penetrate the filter membranes.
  • Bacterial challenge: The bacterial challenge test validates filter membrane classification and demonstrates complete microbial removal from the product. The challenge organism is inoculated into the product (or surrogate) to deliver a minimum challenge level of 107 cfu/cm2 of filter surface area. The bacterial retention validation study should demonstrate that the filtration process of the product with the use of the specified filter, achieves the consistent removal of the high levels of the standard bacterium Brevundimonas diminuta (ATCC 19146) during the simulated processing conditions.

Optionally and upon request, an adsorption test can also be performed, in order to assess potential product binding to the filter membrane that could affect product composition and concentration.


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