Integrity Testing for Single-Use Bags

Introduction

Compare to many plastic bags used in other industries, single-use bags in biopharmaceutical industry need to be of high quality, provide proper protection to bioprocessing materials, and meet stringent regulatory requirements for biocompatibilities. In particular, they should be able to prevent product loss due to leakage and avert ingress of environmental contaminants such as microorganisms, debris, or reactive gases.

Gross leak in the seams or large holes are visible to the naked eyes, it is a serious issue in GMP production facility and must be mitigated under all circumstances. For examples, a leaking bioreactor would generate a significant financial loss and jeopardize a carefully timed production. In vaccine processes, it might also pose a risk to operator and environment safety. Although tiny microscopic holes may not lead to leak and cannot be seen by naked eyes, they can allow bacteria and other contaminants to enter the bag and contaminant the product, therefore pose risks to product loss and patient safety. For single-use bag systems, the joints and seals between tubing and valves have higher chances of breach of integrity. The ports attached to connectors and tubes are far more likely to allow leakage than the bag film itself. This is especially a concern for complex bag systems with multiple tubes attached or multiple connection points.

 Size of Pinholes and Microbial Ingress

  • Bacterial ingress can occur through pinhole as small as 15 µm.
  • Microbial cut off for pinholes is around 1-10 µM.
  • The microbial penetration through small leaks (<5 µm) is only possible under strong influences such as a very high challenge concentration ( ≥108 cfu/ml), long contact time and high pressure differentials.
  • Pinholes at or below 10 µm present little or no risk of sterility breach.

Regulatory Status

Leak detection and integrity testing is currently the subject of ever-increasing attention by legislative bodies (environmental, health, safety, etc.), resulting in more stringent and definitive standards and specifications. The FDA states that for sterile preparations, sterility testing is not considered sufficient to demonstrate the integrity of pharmaceutical container/closure. An appropriate integrity test should be used, and the sensitivity of the container/closure integrity test should be provided. The approach taken in validating a leak test will be largely dependent on the leak test, the product/package system and the products’ leak rate specification. The FDA has encouraged the use of validated physicochemical leak test methods in lieu of product sterility tests for verifying container closure integrity as a component of product stability studies.

Table 1. Examples of Container Integrity Related Regulatory Articles

Reference Description
ISO 8362-5 Injection containers for injectables and accessories, part 5: Freeze drying closures for injection vials.
FDA For sterile preparations, sterility testing is not considered sufficient to demonstrate the integrity of pharmaceutical container/closures. An appropriate integrity test should be used, and the sensitivity of the container/closure integrity test should be provided.
EU Guide “Manufacture of sterile medicinal products” states that containers closed by fusion, e.g. glass or plastic ampoules should be subject to 100% integrity testing.
EP 6.0, 3.2.9 Rubber closure for containers for aqueous parenteral preparations, for powders and for freeze-derived powders.
USP <381> Elastomeric closure for injections
USP <661> Containers
USP<1207> Container Closure Integrity Testing

Destructive Integrity Testing Methods

Visual inspection are crude methods only used to find large leaks. Snoop®” soap (bubble soap) is an example of using visual method to detect leaks. Microbial ingress method and dye ingress methods are simple, inexpensive methods to determine whether a package/container leaks or not, by watching if the microbials or dye penetrates into the container and contaminate the contents. However, these methods are time consuming, destructive and therefore cannot be a 100% test. 

Non-destructive Testing Method

Several instrumentation-based technologies are currently available to perform container closure testing, such as vaccum/pressure decay, headspace analysis, electrical conductivity, capacitance test (high voltage leak detection), trace gas detection (helium, oxygen, etc), high voltage leak detection, and mass extraction. Many of the technologies have been adopted for on-line 100% inspection. However, each method has its own advantages and limitations, vary in sensitivity and outcome, it is difficult to select one method that fit all application needs, using complementary methods may also help achieve definitive and comprehensive testing conditions. Some factors that need to be considered during method selection are listed below.

  • Accuracy and reproducibility
  • Rest time
  • Handling aspects
  • Condition of the bags after testing
  • Possibility of testing connections
  • Investment costs for equipment
  • Cost per test
  • Footprint of the test equipment
  • Contamination risks
  • IP situation
  • Feasibility of in-place testing (point of use)
  • Compatibility Issues
  • Bag configuration and sizes
  • Severity of Leaks

Pressure Decay Method  Pressure-decay test method is based on the protocol from the American Society for Testing and Materials ASTM F2095-01. Using this method, the bag is first filled with air to a predetermined pressure. It is then left to stabilize for a period of time (e.g., secs to minutes), the pressure is then re-measured. The pressure decreased indicates that some of the air has escaped from the bag, and the precise drop in pressure can be correlated to defect sizes. Using this method, holes of about 250–500 μm can be detected. However, the larger the size of the bag, the less accurate the test becomes as it is difficult to maintain bag shape.

Vacuum Decay Test Method. Vacuum decay leak testing is a variation of pressure/vacuum change leak tests.  The method consists of placing the test container in a chamber, sealing and then evacuating the chamber to a predetermined vacuum level, isolating the vacuum source and then monitoring the rise in pressure (vacuum decay) inside the chamber resulting from container leakage. Vacuum decay leak test method sensitivity is dependent on several factors, including, test chamber design, pressure transducer sensitivity, test vacuum level, test system dead space volume, and total test time. Thus, the capabilities of any given vacuumed decay leak test method are specific to both the leak test instrument and its manufacturer. Also reference ASTM F2338-09 for detailed test method parameter selection.

Tracer Gas Testing (Helium Mass Spectrometry)  Helium leak detection technology is a highly sensitive and accurate method to confirm the presence or absence of channels and/or holes in containers.  During the testing, the bag is filled with helium instead of air and placed inside a sealed rigid container. Vacuum is applied to the rigid container to ensure that if there are any small defects in the bag, helium will escape from the bag into the container. A mass spectrometer is used to detect helium and quantify it, and this can be correlated to the defect size. This technique allows detection of holes as small as 10 μm.

Mass or Volumetric Flow Measurement   Mass or volumetric flow measurement uses mass or volume flow meter to determine the rate gas flowing, it requires a very precise pressure control throughout the test. Slightly overshooting or undershooting the initial test pressure would directly affect the measured flow and result in false estimation of the test value. This methodology cannot be applied to large bags, because it is more difficult to accurately adjust the pressure in large containers.

High voltage leak detection (HVLD)  High voltage leak detection (HVLD) applies high voltage current to liquid filled glass or plastic containers. The current is applied to the non-conductive container and looks for a voltage differential, which would represent an integrity breech. The testing requires no sample preparation, produces rapid results, and possible to detect 5-10 µm defects. However, several factors should be carefully tested and measured to define operating parameters such as voltage and gain. The users should also consider other factors such as wall thickness, content, temperature, humidity, speed of the conveyor belt, etc. 

Headspace Analysis  Headspace analysis is also highly sensitive, it uses frequency modulation spectroscopy as an optical measurement method for transparent containers. In this method, the light from the near-infrared laser light passes through the containers and measures the amount of light or frequencies absorbed, which is proportional to the pressure inside the container. The test may be performed overtime to provide quantitative measures of a sample’s leak rate.

Method Selection

Most single-use bags used in biopharmaceutical manufacturing feature different design components, such as tubing, filters, fittings and connectors. The integrity testing strategy should be based on thorough understanding of the system construction, design and manufacturing process. application conditions and requirements. A risk-based approach can be used (e.g., failure modes and effects) to determine the testing frequency and testing methods. At initial evaluation, users need to collect all the knowledge of the existing methods, for example, supplier testing methods and results, gap analysis, supplier recommended testing, and literature documented results. In next step, the team design the testing based on the knowledge of the methods and material and determining testing parameters, methods, and appropriate acceptance criteria. The designed methods can be further optimized and acceptance criteria should be established. The method effectiveness can also be observed during the design, testing and optimization stages. Once the method is tested and optimized, the method can be further validated to establish method characteristics, such as detection limit, range, accuracy, precision and robustness. Once the testing method is finalized and fully validated, the method can be implemented for routine testing.

Figure 1.  Examples of container integrity testing methods selection process. It is recommended that user spend more efforts in the testing design and optimization stages to understand method limitation, effectiveness, and robustness.

Conclusion

Single-use bags may contain most precious pharmaceutical products and it should provide complete integrity during the time of use to ensure the quality and safety of the products. Batch failure due to leakage or sterility breach represents an unacceptable financial loss, so users need to evaluate different methods to ensure the integrity of their single-use systems is absolute. Although several destructive and non-destructive techniques already exist in pharmaceutical industry, the selection of non-destructive method is still challenging, as it requires end-user’s thorough understanding of the technology and careful evaluation of the instrument before implementation.

Reference