Microbiological testing

Non-sterile Product Testing

Testing is supported in line with the harmonized compendial requirements for the Microbiological Examination Of Non-sterile Products and the Tests for Specified Microorganisms, typically by the Membrane Filtration or Plate-count methods. ALS performs method suitability assessments of the selected test methods, demonstrating recovery of micro-organisms in the presence of product, ahead of routine testing.

ALS routinely handles a broad range of sample matrices, including harsher antibiotics, API’s, disinfectants and preservative-laden aqueous formulations, which can necessitate neutralisation for the removal of antimicrobial activity paralleled to scientific demonstration of absent neutralizer toxicity. The suitability of the counting method must be demonstrated in the presence of product.

All ALS facilities strive to achieve industry best practice inclusive of recommendations set out in USP Chapter <1117> amongst others. While not exhaustive, Compendia acceptance criteria for the microbiological quality of non-sterile dosage forms is principally defined by the route of administration backed by risk-based impact assessments as referenced in USP <1111>. The need for additional controls of objectionable microorganisms should be determined for each product. Alternative criteria is applicable to drug substances, herbal medicines and dietary supplements although in any case, typical requirements include:

Microbial Enumeration Tests

• Total Aerobic Microbial Count (TAMC)
• Total combined yeasts and molds count (TYMC)

Specified Micro-organisms

• Staphylococcus aureus
• Pseudomonas aeruginosa
• Escherichia coli
• Salmonella
• Candida albicans
• Bile-tolerant Gram-negative bacteria (BTGN)
• Clostridia
• Burkholderia cepacia (BCC)

Whilst compendia are harmonized for most non-sterile dosage forms, testing is conducted in accordance with:

• USP ⟨61⟩ Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests
• USP ⟨62⟩ Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
• Ph. Eur. method 2.6.13 Microbiological Examination of Non-sterile Products
• Ph. Eur. method 2.6.12 Microbial Enumeration Tests

ALS can include further specified and objectionable microorganisms into testing regimes, such as isolates from environmental monitoring, where known risks persist.

Other test methods, including rapid microbiological methods, may be used for product testing, but will require validation to demonstrate their suitability and equivalence to the compendial methods. As Technical Projects, ALS can support the development and validation of alternative procedures.

Water Activity Determinations can be supported per USP <922> and Ph. Eur. 2.9.39 methods.

Preservative Efficacy Testing

Antimicrobial Preservative Efficacy (AET), also known as Preservative Efficacy Testing (PET), challenges the effectiveness of the antimicrobial preservative system in nonsterile drugs in preventing the growth of microorganisms that may be introduced into the product during manufacturing or product use. 

Preservative Efficacy Testing is required for all hydrous topical, and oral forms, which contain preservatives, and injections packaged for multiple doses, namely where the product environment (e.g. pH, Water Activity and preservation technique) can be conducive to microbial proliferation.

Suitability testing, as recommend by the compendia, demonstrates the validity of the selected procedure by experimentally demonstrating the recovery of target microorganisms in the presence of product. The preparation procedure should remove residual antimicrobial activity of the product by dilution, filtration or other appropriate inactivators.

ALS has extensive experience in verifying method suitability for a range of formulations and preservative systems and supports Preservative Efficacy Testing by various approaches, including:

• USP <51> Antimicrobial Effectiveness Testing
• Ph. Eur. 5.1.3 - Efficacy of Antimicrobial Preservation
• ISO 11930 – Evaluation of the Antimicrobial Protection of a Cosmetic Product
• Client specific methods and tailored studies

Sterile Product Testing

Bacterial Endotoxin Testing and Sterility testing are routinely supported, the latter under strictly controlled aseptic environments, on sterile waters, drug products and medical devices. 

Endotoxin testing is offered using a number of differing compendia methodologies, such as those prescribed by USP Chapter ⟨85⟩ and Ph. Eur. method 2.6.14, providing options for where one technique in isolation may not be suitable. The presence of endotoxins in a substance or product may be masked or enhanced by factors interfering with the reaction. An initial suitability test, a test for interfering factors, is routinely carried out on three product batches or preparations ahead of routine testing. ALS offers a position of:

• Significant experience with troubleshooting and overcoming interferences
• Routinely handling complex sample matrices including intricate medical devices
• A deep understanding and adoption of compendia and regulatory guidelines
• Fast turnaround times and contingency
• Continued investment into new testing technologies and equipment
• A global support network of experienced microbiologists

Endotoxin testing can be performed in accordance with various compendial and standards:

• USP Chapter ⟨85⟩ Bacterial Endotoxins Test. Current.
• USP Chapter ⟨161⟩ Medical Devices—Bacterial Endotoxin and Pyrogen Tests
• British Pharmacopoeia. (BP) Appendix XIV C. Current.
• European Pharmacopeia. (EP) 2.6.14. Current.
• Japanese Pharmacopoeia (JP) 4.01. Current.
• ANSI/AAMI ST72:2011

Typically going hand in hand with Endotoxin, Sterility testing is supported by compendia methodologies Ph. Eur. method 2.6.1, USP Chapter ⟨71⟩ Sterility Tests as well as ISO requirements for medical devices. Membrane filtration is commonly employed where products are filterable with direct inoculation supporting unfilterable products. Method suitability tests are performed for all new products and whenever experimental conditions change or vary. In support of aseptic manufacturing, ALS also supports media fill simulations as well as Environmental Monitoring (see below).

Container Closure Integrity Testing

Container Closure Integrity Testing (CCIT) is an assay that evaluates the adequacy of container closure systems to maintain a sterile barrier against potential contaminants. Contaminants that could potentially cross a container closure barrier include microorganism, reactive gases, and other substances (USP <1207>). Container closure systems should maintain the sterility and product quality of sterile final pharmaceutical, biological, and vaccine products throughout their shelf-life (Ewan, S. et al., 2015).

Based on the drug formulation (e.g. liquid or lyophilized) and the type of the container (e.g. bottle, vial, ampule and syringe etc.) various methods can be employed to perform the container closure.

Non-destructive

• Electrical Conductivity and Capacitance Test (HVLD)
• Laser-based Gas Headspace Analysis

Destructive

• Blue Dye Ingres Leak Test
• Microbial Immersion Testing

Various CCIT techniques can be supported between ALS and our partners NUVISAN.

Particulate Testing

ALS can support particulate testing alongside microbiological testing of sterile drug products. Particulate contamination of injections and infusions consists of extraneous, mobile undissolved particles unintentionally present in the solutions. It is considered a critical risk with quality control testing commonplace throughout in-process controls and a mandatory testing requirement in release testing specifications. Particulate Contamination is predominately split into two forms: Visible and Sub-Visible. ALS commonly supports the following compendia in connection with the practical application of industry best practices:

• Ph. Eur. 2.9.20 Appendix XIII B. Particulate Contamination: Visible Particles / USP ⟨790⟩ Visible Particulates in Injections
• Ph. Eur. 2.9.19 Method I / USP General Chapter 788: Light Obscuration Particle Count Test
• Ph. Eur. 2.9.19 Method 2 / USP General Chapter 788: Microscopic Particle Count Test

Environmental Monitoring 

Effective microbiological monitoring programmes support overall microbiological control within manufacturing facilities. In the case of aseptic processing, monitoring of cleanrooms RABS (Restricted Access Barrier Systems), and isolators typically includes compressed gases, surfaces, air, and any other materials and equipment that might produce a risk of contamination. Mandatory requirements are set for aseptic processing per USP ⟨1116⟩

Microbiological monitoring and control programs for non-sterile manufacturing are not yet as well defined by compendia or regulatory guidelines. In a similar vein to aseptic, the extent of monitoring depends upon the manufacturing activity although tends be driven by circumstance-specific risk assessments with sampling and testing proportional to supply chain risk. Sound scientific rationale is naturally expected and trend analysis of the generated data helps underpin understanding and effectiveness.

ALS offers a complete range of supportive testing for both Sterile and non-Sterile environmental monitoring programmes, including:

Incubation, enumeration and reporting of:

• Active Air Plates and strips
• Impacted Compressed Gas (supporting ISO 8573 requirements)
• Contact Plates
• Glove Prints (Finger Dabs)
• Settle Plates (Passive Air Monitoring)

Inoculation of blank control plates for Growth Promotion / Fertility testing.

Water Testing (Performance qualification and routine monitoring)

• Potable Feed
• Purified Waters (Bulk and in Containers)
• Product used
• Water used in washing process
• Steam

Surface Monitoring

• Swabs
• Contact Plates
• Drains

In addition to testing, ALS’ offers consultancy services and are proactive in supporting:

• Company-wide education of Environmental Monitoring programmes and their application
• The design and evaluation of Microbiological Monitoring Programmes
• Risk assessments
• Technique Training and Establishment (e.g. culture media selection, incubation condition selection, and sampling best practices)
• The validation of monitoring techniques
• Performance qualification and monitoring thereafter
• Acceptance Criteria Establishment and Limit Setting (Non-Sterile Environments)
• Investigations and root cause analysis