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Quality control is a basic operation of manufacturing enterprises

Pharmaceutical products are usually produced under strict GMP guidelines. Despite these strict regulations, as a fail-safe measure before batch release, all pharmaceutical products undergo strict sterility testing to determine the possible presence of live microorganisms. It is important to detect pathogenic microorganisms such as bacteria, viruses and fungi before the patient comes into contact with the contaminated product. In a few cases, damaged drugs were put on the market, with devastating consequences for patients and pharmaceutical companies. The sterility test is carried out in accordance with the regulatory requirements defined by the International Pharmacopoeia (USP <71>, EP 2.6.1, JP 4:06). Sterility testing can be done by direct inoculation|transfer or membrane filtration, which is the preferred method. Only when the characteristics of the product do not allow membrane filtration, the sterility test of the product is performed by direct inoculation.

Membrane filtration methods generally rely on closed filtration units containing membranes with a pore size no greater than 0.45 μm, which have been reliably proven to retain microorganisms. Other components of the system include a suitable pressure source (such as a peristaltic pump) that drives the sample through the membrane filter, a suitable membrane rinse solution, and growth|culture media. This closed setup usually meets clean room requirements to eliminate any risk of contamination and subsequent false alarms. After the sample is filtered, the closed system will be incubated, usually for 14 days, and the turbidity will be screened as an indicator of microbial contamination. The Sterisart® tank is a closed system for sterility testing based on membrane filtration methods.

This closed system does not require physical manipulation of the membrane filter, thereby reducing the risk of secondary pollution and false alarms. However, after the prescribed 14-day incubation, when microbial growth makes the growth medium turbid, sample extraction is a prerequisite. If the growth of microorganisms is detected, the identity of the microorganisms and the source of contamination are determined, the sterility test is declared invalid, and the procedure is repeated.

After filtration or during incubation, sterile sampling or addition of sterile enzymes may also be required, such as discontinuing antibiotics that may cause false negatives.

Even before incubation at the specified temperature, precipitation of the filtered test sample, or adverse color changes due to the inherent properties of the compound, can make the growth medium turbid. This complicates the interpretation of sterility testing and the batch release certificate; the batch may require additional testing by taking samples from the tank and subsequent subculture. Sample extraction in traditional sterility testing systems involves piercing or cutting the pipe leading to the tank inlet and then trying to extract the sample carefully without compromising the integrity of the tank or its contents. By cutting the tube to extract the sample, repeated sampling can be avoided. Using other methods for multiple sampling will disrupt the closed system and increase the risk of contamination.

The Sterisart® septum is designed to facilitate repeated sampling during the growth promotion test incubation period. In this report, we show that multiple samples taken through the Sterisart® tank septa-more than 100 times-exceed any possible requirements for aseptic sampling and will not cause system contamination.

1. The culture medium becomes turbid due to the growth of microorganisms, so the microorganisms need to be identified in the root cause analysis.

2. This product makes the medium turbid before culture, and needs to be diluted by subculture.

3. Samples are drawn for microbial contamination testing through rapid detection methods.

4. Reagents against the antimicrobial components of the tested product are added to the sample.

Consumables: Tryptic Soy Broth (TSB) (Gila/BD), Thioglycolic Acid Broth (FTM) (Gila/BD), TSB (Merck), FTM (Merck), Tryptic Soy Agar (TSA) (Merck) ), glass reaction tube, 30 ml (Borosil), needle – 0.90 × 70 mm, 2OG × 2 ¾ (Sterican – B. Braun), syringe – F Luer (Omnifix – B. Braun).

Sterisart® universal pump, incubator (Sartorius Stedim Biotech GmbH), Combisart® 3 branch filter manifold (Sartorius Stedim Biotech GmbH), e.jet pump (Sartorius Stedim Biotech GmbH).

Sterisart® NF Sterility Test System

Ten individual Sterisart® canisters (30 in total) from three septum variants were analyzed in the septum sampling test. One of ten tanks (from each Sterisart® tank type) was used as a negative control (ie, samples were not taken from the tank until the 24th day of the test).

Sterisart® tanks are filled with growth medium in a biological safety cabinet under aseptic conditions. Two Sterisart® tanks are located in the pump bracket, and the Sterisart® piping system passes through the pump head. The outlet of each Sterisart® tank is sealed with a closed wing nut plug. The two sterile ventilation filters are not covered.

The yellow pipe clamp at the outlet of the Y-distributor is opened, and the adjacent white pipe clamp is closed. Insert the double-needle metal needle into the bottle with FTM and turn on the Sterisart® Universal pump. Once the predetermined volume (75 ml) of medium is transferred to the first tank, turn off the pump. The white pipe clamp at the outlet of the Y distributor is opened, and the adjacent yellow pipe clamp is closed. Insert the double-needle metal needle into the bottle containing TSB, and then turn on the Sterisart® Universal pump. Transfer a similar volume (75 ml) of medium to the second tank. Use the two clamps above the tank inlet to seal the pipe and cut off the pipe. Please refer to our Sterisart® NF gamma user manual for an illustration of the described process.

Sterisart® tanks contain TSB, a growth medium recommended for the detection of low-incidence fungi and aerobic bacteria, cultured at 22.5°C for 24 days. The Sterisart® tank containing FTM is a recommended growth medium for culturing aerobic, microaerobe and anaerobic microorganisms at 32.5°C for 24 days.

Under aseptic conditions, extract samples from Sterisart® tanks in a biological safety cabinet. For 17 days, three samples were taken from the top, middle and bottom of the Sterisart® tank each day, each with 100 µl (3 × 2 × 17 = 102 samples). Transfer the extracted sample to a glass reaction tube containing sterile liquid medium, FTM and TSB.

The vial containing TSB was incubated at 22.5°C for 14 days, and the vial containing FTM was incubated at 32.5°C for 14 days. Record the results by photographing each Sterisart® device and the corresponding extracted sample.

Use a black mesh membrane filter placed in a sterile Sartorius Combisart® filter unit and connected to the e.jet pump for final inspection. Fill the funnel with 60 – 70 ml TSB (after the 24-day incubation period of the Sterisart® jar) and filter through the black filter membrane. Use sterile forceps to transfer the filter to the TSA plate and incubate the plate at 36°C for 3-5 days. Then check these boards for microbial contamination.

After 102 diaphragm perforations and repeated sampling, it was determined that all Sterisart® tanks (3×9 containing FTM, 3×9 containing TSB; the 10th tank containing FTM and TSB as respective controls) were sterile and not tested Microbial contamination after 24 days. (Figure 1) Similarly, the extracted samples are also sterile and free of microbial growth, which indicates that the Sterisart® septa facilitates efficient and highly reliable aseptic sampling. Coring may occur when the septum is punctured multiple times or an inappropriate needle type is used. Only after 36 piercings can small particles be observed in some Sterisart® cans. Collect these particles on the black filter membrane after 24 days and monitor their ability to form colonies on the TSA plate. Even after a five-day incubation period, these particles did not show any growth, which indicates that these particles are not biological in nature. Based on their morphology, we conclude that these inert particles are rubber fragments, which are cut from the septum during repeated piercing with a syringe needle. These fragments will not affect the effectiveness of the sterility test and are almost invisible in the growth medium. We recommend taking septa sampling only after unplugging the sterile vent (ie opening the lid) in a controlled environment. Our results show that the Sterisart® tank remains closed and sterile even after continuous sampling of a total of 102 extracts.

All in all, we have proved that the Sterisart® septum is very strong and can maintain a complete sterile environment even after more than one hundred sample extractions. The presence of preservatives and antimicrobial agents in sterility test products has largely hindered the adoption of rapid detection methods that rely on direct inoculation. Membrane filtration and subsequent membrane washing of such products can reduce the risk of false negatives during sterility testing.

In addition, through membrane filtration to facilitate large-volume analysis and sample extraction, we provide users with the ability to combine closed system sterility testing with rapid sterility testing methods.

However, it may be difficult to detect some slow-growing anaerobes using certain rapid aseptic methods. Since septa sampling will not compromise the integrity of the closed system sterility test, we provide customers with the possibility of taking samples for rapid sterility testing, but re-incubating the sterility test within the prescribed 14-day incubation period.

Sartorius' Sterisart® Sterility Test

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