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June 15, 2017 (Vol. 37, No. 12)

Validating a Point-of-Use Leak Test

Description of a Six-Sigma Procedure for a Flexsafe STR Single-Use Bioreactor

  • Single-use bioreactors have been successfully implemented by the biopharmaceutical industry up to the 2,000-L scale. The performance of single-use bioreactors is comparable if not superior to stainless-steel alternatives, even in the most demanding applications such as continuous cell culture (Sherman et al., 2016).

    The consequences of a loss of bioreactor integrity, whether single use or stainless steel, are significant and costly. It could lead to greater risk of contaminations, disruption to production schedules, and the need to discard inoculums prepared in the seed train and expensive cell culture media used to fill the bioreactor.

    Leaks from bioreactors can be a biosafety hazard, as is the case during the production of viral vaccines. A bioreactor leak may be present for a number of days before it is detected, contaminating the production environment and exposing operators to potentially hazardous materials.

    A variety of methods are available to detect leaks in single-use bags. However, they require the assembly of the bag in a separate device for testing before installation of the bag into the bioreactor bag holder. This leads to an additional risk of the bag being damaged following the test.

    A point-of-use leak test after installation and directly before use presents the best solution to prevent financial loss by saving high-value growth media and production time. A point-of-use leak test method based upon pressure decay has been reported previously (Stering et al., 2013)

  • Pressure Decay Method

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    Figure 1. Installed fleece in an STR® 2,000 bag holder.

    The defect size that the pressure decay method can detect is appropriate for the typical range of defects being introduced by transportation or handling. The test time is short compared to the long cultivation times, and is completely automated, with the bag already installed in the bioreactor bag holder. The pressure decay test therefore allows for easy and efficient operation under GMP cleanroom conditions, and presents a robust and reliable test method with a strong validation to increase safety with low additional effort.

    Sartorius Stedim Biotech has launched the Flexsafe® film to address the industry’s needs for bioreactor bags that provide consistent cell-growth and extractables profiles, superior robustness and ease-of-use, and assurance of supply and suitability for application across the entire process train. Use of the new film has been demonstrated in cell-culture applications (Reglin et al., 2014). This paper describes the validation of the point-of-use test for single-use bioreactor bags constructed from the Flexsafe film.

    The leak test is performed after the installation of the bag into its holder. To ensure 100% coverage of the bag surface, a fleece is installed into the holder prior to the bag’s installation (Figure 1). This acts as a porous spacer between the bag and the stainless-steel surface, avoiding masking effects by allowing air to flow through potential defects in the bag film.

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    Figure 2. Validation approach

    The bioreactor bag is inflated with compressed air to a defined test pressure of 50 mbar to perform the test. The pressure drop is measured and recorded for 20 minutes after a given stabilization time. This stabilization period eliminates temperature-dependent pressure changes and volume expansion due to the flexibility of the bag film. A complete test takes between 45 and 160 minutes depending on the volume of the bioreactor and allows for detection of defect sizes between 50 µm and 600 µm.

    We apply a one-for-all validation procedure to ensure validation robustness with a Six-Sigma confidence interval, independent of the test method or whether this method is used in our manufacturing sites as a quality release test or at customer sites as a point-of-use leak test. Based on a statistically relevant number of tests, the optimum parameter sets were determined and verified either for individual bags and bag assemblies, or for complete product families.

    The validation is done in a three-step approach (Figure 2), using nondefective samples as well as samples prepared with calibrated defects deliberately introduced.

  • Achievable Sensitivity

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    Figure 3. Test results of a parameter study for Flexsafe STR 2,000 L.

    Defect Definition and Conformity Check

    The defect size range used for validation is defined by taking into account the market needs, technical feasibility, and experience in capabilities shown in previous validations. It can either be a fixed defect size or a range in order to find the best achievable sensitivity.

    Flexsafe STR bags with defects of the chosen size or sizes are then manufactured. The defects can be introduced using microcapillaries or film patches with laser-drilled holes. To avoid tests being performed under the wrong conditions, each defect is flow calibrated during manufacturing and checked before, in between, and after its use for conformity. Non-conforming defects could occur if defects were blocked by dust, for example.

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    Table 1. Preliminary Test Parameters Resulting from Parameter Study

    Parameter Study

    A preliminary parameter study is performed to establish a set of parameters to be used in the final validation study. The matrix of test parameters can lead to a high number of combinations. For the pressure decay measurement, the matrix consists of filling time, stabilization time, test time, test volume, and defect size.

    The final test parameter sets from a previous validation study on the Cultibag® product family (Stering et al., 2013) were used as preliminary sets for the validation on most of the bags sizes. However, a new parameter study was performed for the STR® 2,000-L bags, which had not formed part of the previous study.

    In this study on 2,000-L bags, a defect size range of 600 µm–1,000 µm was chosen based on experience from previous validations. The same test pressure, stabilization time, and test time were used as for the Cultibag family for consistancy. Testing showed that samples with 800 µm defects could be clearly differentiated from nondefective samples. Samples with defects of 600 µm were then tested and found to be detectable (Figure 3). Table 1 shows the complete set of preliminary test parameters that was subsequently used for the validation study.

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    Table 2. Final Test Parameters Resulting from Validation Study Conclusion

    Validation Study

    Six hundred fifty tests were performed on 11 bags within the volume range to confirm the preliminary parameters that had been set. Batch variation was also taken into account in order to show batch to batch consistency. The acceptance criteria were refined using the increased number of tests and greater statistical validity.

    Using an analysis including a high Six-Sigma confidence interval led to a robust test with the parameter shown in Table 2. Those parameters are to be used to program a test program in the Sartocheck® bag tester. 

  • Conclusion

    In this study, we have extended our well-established leak test from our Cultibag STR product family (Stering et al., 2013) to our new Flexsafe STR product line. We validated that same operating procedures apply for this point-of-use leak test, using the Sartocheck bag tester and a porous spacer, allowing for a smooth transition from Cultibag to Flexsafe applications.

    After thorough validation studies, the previously used minimum detectable leak sizes and allowed maximum pressure drops over the 20 minute measuring time were adjusted for the STR 50 L and 200 L bags. This occurred because of the improved characteristics of the Flexsafe bag, such as increased flexibility of the film. Furthermore, detectable leak sizes and allowed maximum pressure drops for STR 2,000-L bags were established.

    This new validation of a single-use bioreactor leak test is qualified to be used post-installation, but used prior to installation in order to reduce financial risk caused by the potential waste of expensive cell-culture media and preparation effort. It helps to mitigate project delays and offers a proper risk-mitigation tool, especially in biosafety critical applications typically found in vaccine production.

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