June 1, 2013 (Vol. 33, No. 11)

Validating the Reproducibility and Reliability of Pressure Decay Methodology

The high value of growth media and the length of time required for a typical cell culture process call for the highest degree of scrutiny when setting up such a run. A leaking bioreactor would generate an important financial loss and jeopardize the carefully timed production schedule in a GMP facility. In vaccine processes, it might also pose a risk to operator safety.

Today, single-use technology is well accepted and the manufacturer’s quality assurance program ensures leak-free single-use bags upon delivery. But what about the risks involved with installation and other handling errors? Training of operators is mandatory, but should it be the only way to mitigate against failures?

A post-installation pre-use test of the entire bioreactor system (including tubing) capable of detecting typical leaks that might have been introduced due to operator handling errors would greatly improve risk-mitigation capabilities in single-use production facilities. A critical example of this situation is the absolute need for proper connection of different tubing elements during bioreactor setup, media preparation, and inoculum transfer because it is at this point that an operator error might immediately result in a leakage.

More than 40 different test methods have been proposed for leak detection based on various technologies such as “sniffers” (for special gases that are used as leak testing agents), thermal imaging, flow measurement, and pressure decay and pressure increase.

Each technology has its own strengths and weaknesses in terms of sensitivity, feasibility, and cost. All these factors have to be carefully considered and balanced when developing a suitable bag-testing device for single-use bioreactors that are designed to make their way into GMP production facilities for vaccine-, monoclonal antibody-, and recombinant-protein-producing companies.

Risk assessment reveals that a predictive leak test for single-use bioreactors has to be performed at the point of use (pre-use but postinstallation). Therefore, testing a single-use bioreactor bag in a separate device as is usually done when applying gas sniffer technology and then installing it into its bag holder would not permit the detection of operator handling errors.

Pressure-decay technology, on the other hand, can be used to test the single-use bioreactor bag after installation in its final configuration directly in its holder. This approach presents a reliable and predictive risk mitigation tool.

Developing a Reliable and Reproducible Methodology

Reliability and reproducibility of a test method have to be established before formally validating it. During the development of a technique based on pressure-decay measurement, researchers discovered early that a leak in a plastic bag is totally masked when pressed against a smooth, hard surface due to the test pressure. Very little of the test gas can escape through the leak, and the reliability of the pressure-decay detection is nil.

The use of specifically designed polyester fleeces acting as a porous spacer between the plastic film of the bioreactor and the holder prevents this masking effect. It delivers reliable and reproducible test values comparable to results obtained with the same test method on bag areas that are not covered by any hard surface.

This approach was qualified by applying the same test method on bags with predefined leaks located on the upper part of the bioreactor bag that is not in contact with the holder, thus not presenting any risk of masking.

Table 1. Minimal Detectable Leak Size

Leak-Test Method General Principles

The objective of the test method is to identify potential damage of installed bioreactor bags (including bag seals, port welds, connections, and bag surfaces), which would result in a loss of the bioreactor content or pose a biosafety risk.

The test method is based on ASTM F2095-01: “Standard Leak Test for Pressure Decay Leak Test for Nonporous Flexible Packages with and without Restraining Plates.” The Sartocheck®4 plus Bag tester (Sartorius Stedim Biotech) is used with a specifically developed bag-tester fleece that prevents masking of any leaks that may have been potentially introduced during installation. It allows point-of-use leak testing of single-use bioreactor bags post-installation and pre-use in its final bag holder.

This pressure-decay leak test can reliably detect defects in the CultiBag STR 50 L of 50 µm and the CultiBag STR 200 L of 100 µm in the bag walls, seals, or connections over the entire flexible bag system, including tubing. The test method is nondestructive and enables the implementation of a reliable and reproducible point-of-use test in bioproduction facilities.

The pressure decay during the test is measured and compared to acceptance criteria established during qualification of the method. The validation approach was separated into two parts: 1) an engineering study to establish the detection limit for different bag volumes and 2) the formal qualification to verify the minimum detectable leak size and establish the test acceptance criteria (maximum allowable pressure decay).

Table 2. Final Test Parameters Established During Engineering Study

Engineering Study

To determine the minimum detectable leak size, a simplified bag (only one bottom port connection for test gas application) was prepared with multiple defined defect patches representing different leak sizes. A defect patch is a circular sheet of film with a laser-drilled and flow-calibrated leak diameter that was welded to the bag surface.

In a second step, one standard bag without a defect patch and one standard bag with a defect patch of the previously determined minimum detectable leak size were used to establish the final test parameters.

The minimal detectable leak size in a CultiBag STR 50 L bag was determined to be 50 µm and at the 200 L scale a leak size of 100 µm could reliably be detected. The results are shown in Table 1.

Based on the results of the engineering study, the final test parameters for the qualification of the method were established (Table 2). These parameters were used to generate an automatic test program for each STR bag volume in the Sartocheck 4 plus Bag tester.

Table 3A. Qualification Study Results (CultiBag STR 50L)

Qualification Study

The purpose of the qualification study was to verify the minimum detectable leak size for the different bag volumes on a statistically significant number of standard bags from different routine production lots.

For the qualification study, standard bags from production without further modification and a standard bag that was prepared with a single defect patch (according to minimum detectable leak size determined in the engineering study) of different lots per bag volume were used. All bags were gamma irradiated. A minimum of 10 test repeats were performed per bag. Tables 3A and 3B summarize the results of qualification.

All tested bags showed expected results, i.e., the nondefect, standard bags passed the test while standard bags prepared with a single defect failed. Hence, the test method using pressure decay combined with the fleeces was successfully qualified and proved to be a robust and predictive method for reliable detection of leaks.

This new single-use bioreactor leak test, which is qualified to be used post-installation but pre-use, now enables the same level of risk mitigation and assurance as previously seen only in conventional bioreactors that could easily be pressure-tested prior to use. The leak test helps to mitigate project delays while also offering a proper risk- mitigation tool, especially in biosafety critical applications typically found in vaccine production.

Table 3B. Qualification Study Results (CultiBag STR 200L)

The authors hold the following positions at Sartorius Stedim Biotech: Magnus Stering, manager, integrity testing solutions, Europe & Asia; Martin Dahlberg, manager, dep. instruments, R&D instrumentation & control; Thorsten Adams, Ph.D., head of product management, single-use bioreactors; Davy De Wilde, director of marketing, fermentation technologies; Europe & Asia; and Christel Fenge, Ph.D., vp, marketing & product management, fermentation technologies.

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