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.

Rapid Sterility Testing 

ALS offers rapid sterility testing in our GMP-certified laboratory using calScreener+™ technology. This innovative method reduces analysis time from the traditional 14 days to just 3 days, enabling faster product release without compromising safety or accuracy.

The process 

• Two testing units: one maintained at 20–25°C and another at 30–35°C, both equipped with internal and external thermostats and multi-calorimeter systems
• CalView software is used for instrument control and data collection
• High sensitivity with an LOD of <5 CFU, ensuring exceptional sterility assurance and minimal risk of undetected microbial growth. 

Advantages of calScreener+™

• Faster results compared to traditional sterility methods (3 days vs. 14 days)
• Fully validated according to international standards: USP <1223> and <1071>, EP 5.1.6, EP 2.6.27, and PDA TR 33
• Comparable to traditional methods and compliant with regulatory requirements
• Ideal for routine quality control of pharmaceuticals and medical devices

Bacterial Endotoxin Testing 

At ALS in Sollentuna, we provide bacterial endotoxin testing (LAL testing) to ensure the safety of injectable pharmaceuticals and medical devices.

Bacterial endotoxins are lipopolysaccharides (LPS), components of the outer membrane of Gram-negative bacteria, known to cause fever and illness when introduced into the bloodstream. Endotoxins are heat-stable, and their toxicity is independent of the presence of viable bacterial cells, making accurate detection essential for patient safety.

Gel-Clot Method 

At ALS Sollentuna, we offer endotoxin testing using the Gel-Clot method, available in both qualitative and semi-quantitative formats. This test uses lysate reagents with a defined sensitivity (for example, 0.03 EU/ml). When the endotoxin level meets or exceeds the sensitivity limit, a gel forms as a visible result of a cascade reaction.

In semi-quantitative analysis, serial dilutions are performed to determine the exact endpoint for gel formation. Each sample is tested in duplicate and spiked in duplicate, and a standard curve is prepared for every analysis to ensure reliability and precision.

Kinetic Chromogenic Method (Endosafe® MCS)

For quantitative endotoxin analysis, ALS Sollentuna utilizes the Endosafe® MCS system, an advanced endotoxin detection platform that provides accurate results in approximately 15 minutes. The system uses FDA-licensed Endosafe® PTS cartridges and Endoscan-V® software, which is 21 CFR Part 11 compliant, ensuring full traceability for pharmaceutical and medical device testing.

The MCS system applies the kinetic chromogenic LAL method, measuring color intensity as a function of endotoxin concentration. Each single-use cartridge contains pre-measured LAL reagent, chromogenic substrate, and control standard endotoxin (CSE) to ensure high accuracy and reproducibility.

With long-standing experience in microbiological testing, ALS Sollentuna offers dependable, regulatory-compliant endotoxin analysis with fast turnaround times and expert local supported by the global ALS network.

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

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

Mycoplasma Testing 

ALS can identify mycoplasma contamination in pharmaceuticals within a single working day. The speed and precision offered by qPCR technology (quantitative PCR) make our MycoSEQ™ Plus Mycoplasma Detection Kit a more efficient alternative compared to traditional culture-based methods.

Mycoplasma testing is a regulatory requirement from global authorities such as the FDA and EMA. In cell-based pharmaceutical production, mycoplasma can pose a quality and safety risk by affecting the cell’s physicochemical properties and may also reduce production yield and process efficiency.

Expertise-Driven Testing in Sollentuna

• Advanced technology: We use the latest qPCR technology to ensure speed and accuracy
• Flexibility: We offer tailored solutions to meet specific customer needs 
• Quality assurance: Our methods are fully compliant with international standards and validated for regulatory adherence