Wednesday, May 27, 2009

Sterility testing: broth validation



bioMérieux has carried out rigorous testing on the packaging and the media growth performance of its new cleanroom range of sterility testing bottles

BioMerieux has developed a new range of sterility testing bottles, double wrapped and ethylene oxide decontaminated, filled with high performance culture media BioMerieux has developed a new range of sterility testing bottles, double wrapped and ethylene oxide decontaminated, filled with high performance culture media

BioMérieux has developed a new cleanroom range of sterility testing bottles, double wrapped and ethylene oxide decontaminated, filled with high performance culture media. A range of tests were carried out to show the suitability of the new packaging and to illustrate the growth performance of the media.

Sterility testing is critical in the pharmaceutical quality control process. It is a very precise procedure, where asepsis of the environment must be mastered for a correct interpretation of results. The test for sterility is carried out under aseptic conditions, usually in a cleanroom or an isolator. Pharmacopoeias (USP, EP or JP) define strict procedures to ensure that no contaminating micro-organism has been found in sterile products.

Pharmaceutical companies perform cleaning procedures to eradicate all viable and non-viable contamination. This work shows that the new bioMérieux Clean Room range, which is ethylene oxide (EO) treated and packed in plastic box and double wrapped, brings a well adapted solution for an easy and safe use in an isolator, while maintaining high growth performances.

Materials and methods

The following bioMérieux products were tested in this study:

• Trypcase Soy Broth Clean Room (TSB-CR) (bioMérieux reference 42633)
• Clear Thioglycollate Clean Room (CT-CR) (bioMérieux reference 42634)
• Standard Trypcase Soy Broth (TSB) (bioMérieux reference 44011)
• Standard Clear Thioglycollate (CT) (bioMérieux reference 44021).

To control the biological indicator growth, the following culture media were used: Trypcase Soy Agar (TSA) plate (bioMérieux, reference 43011) and Trypcase Soy Broth (TSB) tube (bioMérieux reference 42100).

According to the ISO 11138-2 standard requirements,1 Biological Indicators (BI) of endospores of Bacillus atrophaeus NRRL B 4418 in a strip (106 to 107 endospores/strip) were used to assess the performance of the sterilisation process employing EO gas.

BI control

BI strips were tested before use for enumeration and purity. BI growth: BI endospores were extracted from a strip in 10ml of physiological sterile saline solution with sterile glass beads by a vortex. Dilutions were used to perform the inoculation of TSA plates that were incubated in aerobic conditions for 48 hours at 30-35°C. A final count was made. One hundred microlitres of the 10-1 dilution of the extraction was used to inoculate TSB tubes. Tubes were incubated in aerobic conditions for seven days at 30-35°C and the growth in the medium was checked for a minimum of 4 McFarland (McF) turbidity.

Gas chromatography studies: The measure of EO, ethylene glycol (EG) and ethylene chloridrin (EC) residue concentrations was performed using a gas chromatography, headspace method. For EO residues, the intact bottles were put in 300ml of deionised water; the box was cut off and put in 500ml of deionised water. Extraction solutions with bottles or box were incubated 24h at 20-25°C. Two millilitres were heated at 80°C for 1hr before injection of an aliquot in the gas chromatograph.

For measures of EG and EC, samples were prepared and incubated in the same way as described for EO. For the septum analyses, residues were extracted in 50ml of deionised water from only one septum. The extraction solution was directly injected in the equipment without the heating phase.

Culture media growth promotion

TSB-CR media growth studies were performed on 49 aerobic strains. CT-CR media growth studies were performed on 28 aerobic and 22 anaerobic strains. These different strains came from ATCC‚2 or were randomly isolated from cleanroom environments. An inoculum containing 10 to 100 CFU was introduced in the Clean Room bottles and in TSB and CT reference culture media. Bottles were incubated up to 7 days at 30-35°C for anaerobic growth and at 20-25°C for aerobic growth. The micro-organisms’ growth was checked after 2, 5 and 7 days of incubation; the reading was made according to an internal turbidity scale (from 0 McF = no growth up to 5 McF = growth > 109 CFU).

The study describes the EO decontamination process on new Clean Room bottles and the validations made to ensure the perfect safety and efficiency of the product.

New packaging

BioMérieux’s new Clean Room Range (TSB-CR and CT-CR) consists of six 100ml culture media bottles that are packed in an Akylux 2 box to reduce the level of bioburden particles compared with classical cardboard packaging. The box is double wrapped in sterilisation bags with a sterilisation indicator. Each bag is individually sealed to protect the product from contamination and allows isolator sterilisation.

The validation report of EO decontamination3 describes the location of 50 BI used to map the decontamination efficiency:

• 10 BI were used to validate the EO decontamination homogeneity of this specific load;

• 40 BI were positioned in different TSB-CR and CT-CR boxes all around the pallet according to the ISO 11135-1 standard4 in order to follow the efficiency of the EO sterilisation.

Different configuration variables could be changed in the decontamination protocol; therefore, four decontamination conditions (A, B, C, D) were tested (see Figure 1).

Among the four different conditions tested, only condition D shows a 6-log killing of all BI. Condition D was tested successfully on three different batches to validate the reproducibility and in two different decontamination chambers. These conditions were optimised to ensure complete decontamination of the product.

Validation of the aeration protocol after EO decontamination

EO is a highly reactive molecule that generates two major derivative compounds: EG and EC. EO reacts with water to produce EG. EC is synthesised from EO and hypochlorous acid (and generally in the presence of chlorine).

According to the ISO 10993-7 standard,5 EG and EC residues were checked on all products that were decontaminated using EO. In the case of the new Clean Room range of bottles, product exposure to EO is for less than 24h, meaning it was classified in the “limited exposure” category.

The aeration validation protocol6 was created using a gas chromatography method. The maximum EO rate allowed is 20mg/article (sterilisation bags are not tested as they do not fix EO). Two other chemical compounds derived from EO were also checked on products. The highest yields of EG occur at acidic or neutral pH with a large excess of water. According to the ISO standard, there is no threshold for EG residues. However, the maximum rate for EC is 12mg/article following the ISO10993-7.

Results of the gas chromatography analysis on decontaminated and non-decontaminated products are shown in Table 1. This table demonstrates that after one day of aeration, the level of EO residues on a six bottle pack is the same as the level observed without decontamination, with an average of less than 0.55mg/article, that is clearly under the maximum rate of 20mg/article allowed in the standard.

The EG and EC measures were made only after three days because these chemical compounds were produced from EO residues after aeration. The level of EC observed was the same as that for the non-decontaminated product. In the worst case, the EC level on a product was less than 2.46mg/article, that is clearly under the maximum rate of 12mg/article. Although no threshold was described in the ISO standard for EG residues, results compared with non-decontaminated products showed that the sterilisation process did not produce EG.

These results demonstrate the excellent aeration protocol efficiency. They allowed validation of the aeration kinetic: 1 day minimum / 3 days maximum aeration time.

Airtightness of bottle

The decontamination cycle subjected the Clean Room bottles to extreme temperature and pressure conditions. Detection of any EO, EG and EC residues inside the bottles (broth and air) was by gas chromatography analysis (as described in the ISO 10993-7 standard). The results are shown in Table 2. Control bottles were not treated with the EO sterilisation cycle.

All results were below the quantification threshold of the equipment. This clearly shows that EO did not get into the Clean Room bottles.

TSB-CR and CT-CR broths produce an interfering peak in the chromatography analysis. Therefore, it was decided to substitute the media with deionised water for the EG and EC analysis. No EC residue was found in the bottle and the result was comparable to the negative decontamination control. Furthermore, a cleanroom bottle septum contains chlorobutyl so the decontamination process could have produced an EC compound inside the bottle.

For this reason, the test for EC residues was also carried out on the septum (see Table 3). Compared with the non-decontaminated bottles, the level of EC residues increased on the septum. However, it remained at a very low concentration, much lower than that authorised by the ISO standard (i.e. 12mg/article).

Together, these tables show the excellent resistance of the new bottles to EO decontamination action. No toxic residues were found inside the TSB-CR or CT-CR bottles that could have an impact on the media.

Culture media growth promotion: The Clean Room bottle culture media are exposed during the sterilisation process to conditions that could damage their properties. To verify the growth performance of TSB-CR, 49 aerobic strains were tested in parallel in TSB and TSB-CR culture media. The broth turbidity was measured for each strain and all results were summed for each culture medium (see Figure 2).

The results presented in Figure 2 show that the performance of TSB-CR medium is equivalent to TSB reference medium.

CT-CR growth promotion tests were also carried out using 28 aerobic micro-organisms and 22 anaerobic micro-organisms. CT and CT-CR culture media were inoculated with these different strains. The broth turbidity for each strain was measured for aerobes and anaerobes and the sum of the results was calculated for each culture medium. As Figure 3 shows, growth of micro-organisms was similar for CT-CR and CT culture media.

All data obtained during the growth promotion test support the fact that the decontamination process doesn’t have any effect on the performance of the different culture media.

The sterility test aims to check the sterility of the final product. Considering the economic impact of false positive or negative results for pharmaceutical companies, such a test must be performed in a very strictly monitored environment to avoid external contamination. In this case, it was shown that the Clean Room range of bottles, double wrapped and EO decontaminated, were developed to reduce the risk of potential contamination during the sterility test and so to decrease the potential risk of false positives.

The EO sterilisation process was optimised to ensure complete asepsis of the Clean Room bottles, and the double wrapped packaging allows for a microbe-free insertion in cleanrooms or in isolators.

Furthermore, the user safety and the product performances are guaranteed by the absence of chemical residues outside and inside the bottles following sterilisation. Despite the hard decontamination process conditions, TSB-CR and CT-CR kept excellent growth performance that was comparable to culture media not sterilised by EO.

In conclusion, this study confirms that bioMérieux new Clean Room bottles are suitable for sterility testing use and they bring to pharmaceutical industries a well-adapted solution for their constraints.

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