Sterile Water Tank Testing

Sterile water tank inspection mainly conducts scientific testing and analysis on the microbial limit testing, endotoxin testing, insoluble particulate matter testing, physical and chemical index testing, sealing and integrity testing and surface cleanliness and biofilm risk testing, objectively assessing the product quality grade and issuing a detailed test report, providing a basis for manufacturers' quality control and downstream users' product acceptance.

Testing Items

Microbial limit testing: This is the core of the sterile water tank inspection, aiming to confirm whether the microbial pollution such as the bacteria, moulds and yeasts exists in the tank inner walls and stored water. Usually adopting the plate count method, after the water samples or contact plate samplings are taken, the culture is carried out under the specified conditions, calculating the colony forming units (CFU). The standards require that within the testing cycles of the sterile water tanks, no viable bacteria should be detected in their inner surfaces and water samples, to ensure that the water quality reaches the sterile level.

Endotoxin testing: The endotoxins are the lipopolysaccharide components of the gram-negative bacterial cell walls, with the strong pyrogenicity. Even if the microorganisms are killed, the residual endotoxins may still cause the adverse reactions. The testing usually uses the Limulus amebocyte lysate (LAL) reagent method, quantitatively determining the endotoxin contents in the water through the gel method or dynamic turbidimetric method, etc. For the sterile water tanks of the high-purity water or pharmaceutical water systems, the endotoxin levels need to be strictly controlled below the limits specified in the corresponding pharmacopoeias (such as USP and ChP).

Insoluble particulate matter testing: Whether the particles invisible to the naked eyes (such as the metal chips, rubber particles and fibers) exist in the water tanks and delivery systems is tested. Usually, the light obscuration method or microscopic counting method particle counters are used to analyse the water samples. According to the pharmacopoeia standards (such as USP <788>), the quantities of the particles with the particle sizes ≥10μm and ≥25μm are classified and controlled, preventing the particles from affecting the subsequent processes or product qualities.

Physical and chemical index testing: To ensure the chemical purity of the water quality, a series of physical and chemical parameter testing needs to be carried out on the water in the tanks. The key items include the conductivity (reflecting the total ion contents), total organic carbon (TOC, reflecting the organic pollutant levels), pH values, nitrates and heavy metals, etc. These indicators should continuously meet the purified water or water for injection standards of the Chinese Pharmacopoeia, supporting the chemical basis of the "sterile" state.

Sealing and integrity testing: The sealing properties of the tank bodies, interfaces, manholes and breather filters (if installed) are tested, preventing the intrusion of the external microorganisms and particles. The methods include the pressure holding testing, vacuum decay testing, or the online integrity testing with the particle counters (for the breather filters). Ensuring that the system can maintain the closed sterile environments under the positive or negative pressure fluctuations.

Surface cleanliness and biofilm risk testing: Through the surface samplings (such as the contact plates and cotton swab wiping methods), the microbial testing is carried out on the key parts such as the tank inner walls, spray balls and level gauges, evaluating the cleaning and disinfection effects. At the same time, the risks of the biofilm formation need to be paid attention to. The ATP bioluminescence testing can be used for the fast screening, or the deeper surface analysis can be regularly carried out.

Testing Scope

All the internal surfaces of the tank bodies: The testing covers all the surfaces in contact with the water such as the top cover inner walls, side walls, bottoms, welds and internal support structures of the tanks. These areas are the potential risk points of the microbial attachment and biofilm formation, especially the parts difficult to clean such as the dead corners and welds, which need to be the key sampling areas.

The stored media (water) bodies: The sampling testing is carried out on the purified water, water for injection or other process water stored in the tanks. The sampling points should be representative, usually including the return water ports, outlet ports and tank middles. The testing contents cover the microorganisms, endotoxins and all the key physical and chemical indicators.

Ancillary equipment and interfaces: The testing scope extends to the equipment directly connected to the tanks, such as the circulation pumps, spray balls, level sensors, temperature sensor sleeves, breather filters (hydrophobic filter elements), sampling valves and drain valves. These components are the weak links of the system integrity and must be included in the testing and verification systems.

Point of use (POU) water quality: Although the testing subject is the sterile water tank, its ultimate purpose is to guarantee the water quality at the points of use. Therefore, the testing scope should extend downstream, verifying whether the distribution pipelines from the tanks to the various points of use can maintain the sterile and chemical qualities of the water, ensuring the safety of the "last kilometre".

Environment and air: For the installation environments that are not completely sealed, the airborne particle and microbial monitoring (such as the settling bacteria and airborne bacteria) needs to be carried out on the air in the rooms where the tanks are located. At the same time, the intake air qualities of the breather filters are checked, preventing the environmental air from becoming the pollution sources.

Cleaning and disinfection effects: The testing scope includes verifying the effectiveness of the cleaning and disinfection procedures. That is, after the execution of the specified CIP (cleaning in place) or SIP (sterilization in place) procedures, all the above scopes are immediately tested, proving that the procedures can reduce the pollution below the acceptable standards.

Testing Methods

Microbial culture method: This is the classic qualitative and quantitative method. For the water samples, the membrane filtration method is adopted. The quantified water passes through the 0.45μm or 0.22μm filter membranes to intercept the microorganisms, and then the filter membranes are placed on the appropriate agar culture media (such as the R2A media) for the culture and counting. For the surfaces, the contact plates or cotton swab wiping are used and then inoculated for the culture. The culture temperatures and times need to be specified according to the microorganism types (such as the mesophilic bacteria and moulds).

Limulus amebocyte lysate method (LAL): Used for the quantitative endotoxin testing. The gel method judges the endotoxin limits by observing the gel formation after the samples are mixed with the LAL reagent; the photometric methods include the dynamic turbidimetric method and chromogenic substrate method, precisely calculating the endotoxin contents (EU/mL) by measuring the absorbance changes with the instruments. This method is fast and sensitive, and is the mandatory testing item of the pharmaceutical water systems.

Online and offline instrumental analysis method: The physical and chemical indicators mostly adopt the combination of the online instrument monitoring and offline laboratory analysis. The conductivity and TOC usually install the online sensors for the continuous monitoring and data recording. The pH values, nitrates and heavy metals are regularly sampled, using the laboratory equipment such as the pH meters, ion chromatographs and atomic absorption spectrometers for the precise analysis.

Pressure holding/vacuum decay testing method: Used to verify the system sealing. The clean compressed air is filled into the sealed water tanks or pipeline systems to a certain positive pressure (or pumped to a certain vacuum), and after the pressure holding for a period of time, whether the pressure (or vacuum) drop is within the allowable ranges is observed. This method can effectively find the macro leakage points.

Particle counting method: Used for the insoluble particulate matter testing. In the offline testing, the dedicated liquid particle counters are used to measure the water samples in the sampling bottles. In the online testing, the sensors can be installed in the bypass of the circulation pipelines to monitor the system particle levels in real time. The testing needs to be carried out under the laminar flow clean benches, avoiding the environmental interference.

Rapid microbiological testing method: Such as the ATP bioluminescence testing, which quickly evaluates the residual contamination levels of the surfaces after the cleaning and disinfection by detecting the adenosine triphosphate in the microorganisms, and can obtain the results within minutes. Suitable for the fast feedback of the daily monitoring and cleaning verification, but usually as the auxiliary and early warning means, and cannot replace the standard culture methods.

Testing Standards

Domestic Standards:

1. Pharmacopoeia of the People's Republic of China (ChP) — Purified water and water for injection monographs

2. GB/T 16293, Test method for airborne microbe in clean room (area) of the pharmaceutical industry

3. GB/T 16294, Test method for settling microbe in clean room (area) of the pharmaceutical industry

4. GB/T 25915, Cleanrooms and associated controlled environments

International Standards:

1. USP <788>, Particulate matter in injections

2. USP <643>, Total organic carbon

3. ISO 14644-1, Cleanrooms and associated controlled environments — Part 1: Classification of air cleanliness by particle concentration

4. European Pharmacopoeia (EP) — Purified water and water for injection monographs

Note: The applicable standard can be selected according to the product use and customer trade requirements; standards agreed in the contract take precedence over general standards.

Testing Instruments and Equipment

Microbial testing equipment: The core equipment includes the autoclaves (for the sterilization of the culture media and utensils), constant-temperature incubators (for the multi-temperature culture, such as 20-25°C for the mould culture and 30-35°C for the bacterial culture), biological safety cabinets or clean benches (providing the sterile operation environments), membrane filtration devices, colony counters or automatic colony counting instruments. In addition, the standard culture media and dilution liquids need to be equipped.

Endotoxin testing equipment: The main equipment is the LAL reagent reaction instruments, such as the constant-temperature metal baths or test tube heaters for the gel method observation, as well as the dedicated microplate readers or dynamic test tube detectors for the photometric method. The supporting equipment includes the pyrogen-free pipettes, pyrogen-free tips and pyrogen-free reaction test tubes, and the whole operations need to be carried out in the pyrogen-free environments.

Physical and chemical index analysis instruments: The online monitoring needs to install the calibrated conductivity meters and TOC analysers. The offline laboratory analysis needs the high-precision pH meters, ion chromatographs (for the anion analysis), atomic absorption spectrophotometers or inductively coupled plasma mass spectrometers (for the heavy metal analysis), UV-visible spectrophotometers, etc. All the instruments need to be regularly calibrated.

Particle testing equipment: The liquid particle counters are the core equipment, with the principle of the light obscuration method. The equipment needs to accurately distinguish and count the particle quantities of the different particle size channels (such as ≥2μm, ≥5μm, ≥10μm and ≥25μm). At the same time, the clean sampling bottles, ultrasonic cleaning machines (for cleaning the sampling bottles) and online sampling adapters need to be equipped.

Integrity testers: Used to test the integrity of the tank breather filters, usually adopting the dedicated instruments based on the diffusion flow testing or water intrusion testing principles. The equipment can automatically carry out the testing, calculate and judge whether the filter elements are qualified, and is the key equipment of ensuring the effectiveness of the sterile barriers.

Sampling and auxiliary equipment: Including the sterile sampling bottles, sterile syringes, surface contact plates, sterile cotton swabs, alcohol lamps, pressure gauges, vacuum gauges and temperature recorders. For the verification testing, the biological indicators (such as the Geobacillus stearothermophilus) and chemical indicators may also need to be used to verify the effects of the SIP (steam sterilization).