In the selection of the packaging components for a parenteral drug product, the integrity of the seal between the container and the closure is a primary consideration. Depending upon the nature of the drug product, the packaging must be capable of protecting it from a variety of potential sources of contamination including microbial ingress, moisture and gas exchange. Determining the efficacy of the packaging closure is the purpose of contain closure integrity testing.

Container closure integrity, or CCI, is defined as the ability of a container closure system to provide protection and therefore maintain the efficacy and sterility of a drug product throughout the shelf life. The ability of elastomer components to prevent microbial ingress into parenteral containers is determined through container closure integrity testing (CCIT), which measures the integrity of the seal between closure and container.

The primary seal is formed at the vertical interface of the elastomer seal and the container where the downward force from crimping is applied. Assuming there are no defects in the individual packaging components, this interface represents the principal point of potential packaging failure. Multiple factors must be considered in the selection and application of the appropriate container closure system for a parenteral drug product to assure satisfactory container closure integrity.

Failures in manufacturing, such as improper assembly, inadequate or excessive crimping force, or faulty design can compromise the integrity of the container closure system. Therefore, it is imperative to ensure the correct match stopper/vial dimensions to establish adequate seal integrity. Vacuum loss, gas ingress and exchange, pH adjustments and contaminants can compromise seal integrity which could lead to loss of sterility of the product that subsequently can affect product efficacy and increase the risk to patient safety.

With the increasing trend toward high-value biologics, the requirements for reliable container closure systems have become even more critical. Products such as vaccines, stem cells and proteins are typically sensitive to temperature and the potential for degradation is significant if they are not stored under appropriate conditions. It is not uncommon for some biologics to be stored at temperatures as low as –80°C, and in many cases even lower. It is also common practice for many drug products to be shipped on dry ice, which is approximately –78.5°C, even if long-term storage is only at refrigerator or freezer temperatures. As a result, there is a need to test at or below these temperatures to prove that container closure integrity is maintained under all conditions to which the packaged drug product might be exposed.

A variety of methods can be used to evaluate the CCI of a closed system. Some methods of container closure integrity testing such as dye ingress, microbial ingress or vacuum decay are insufficiently sensitive or simply not suitable under sub-ambient temperature conditions. Helium leak detection is significantly more sensitive than these other methods of CCI testing. In addition to being more sensitive, helium leak detection provides quantitative results that are reproducible and more accurate than qualitative pass/fail results. Although a destructive test, helium leak is frequently the method of choice due to the sensitivity and ability to run samples at sub-ambient temperatures. Once the sample has been prepared, it can be stored and tested while being maintained at the appropriate temperature.

A common physical property of all elastomers is the temperature at which the elastomer loses its elastic properties and changes to a rigid, glass-like state. This is known as the glass transition temperature (Tg). Under ambient temperature conditions, the molecules are in a constant state of thermal motion and constantly change their configuration which provides flexibility and hence the ability to form a seal against another surface. However, at the glass transition temperature the mobility of molecules is significantly reduced and the material becomes brittle and glass-like. The glass transition temperature of common butyl rubbers is -65°C. As a result, there is an understandable concern that these butyl components may not be capable of maintaining closure integrity at –80°C and could potentially compromise the sterility of the drug product.

Experiments were conducted to determine whether a previously established method of helium leak detection at room temperature could be adapted to include testing containers at -80°C. This study was performed over multiple time points including 0, 3, 7, 30 and 90 days. The vials chosen were all 5mL glass. The closures selected for the study were comprised of two formulations of halobutyl rubber stoppers, 20mm in size. All the vials used in this study were sealed and crimped using an in-house pneumatic crimper. All samples were prepared on the same day. Each time point consisted of 20 vials and a positive control for each stopper formulation. In addition to analyzing at different time points, a separate sample set of thirty vials from each of the two stopper formulations were tested at room temperature at time point zero in order to compare the results.

The preparation of each vial consisted of displacing the air in the vial with 100% helium at a volume of ten times the headspace to ensure complete displacement of air from the vial. This procedure is carried out after the container and closure have been assembled and crimped. The procedure consists of puncturing each stopper with two needles, one that supplies the helium and a second needle that acts as a vent through which the air is displaced into a vent trap to prevent over-pressurization of the vial. Once the vial is filled with helium, the puncture sites are covered with epoxy that is then allowed to cure. As this method is specifically designed to test the integrity of the seal at the interface of the glass vial and the elastomeric closure, using epoxy in this manner does not in any way interfere with the analysis. After the epoxy has cured, the set of vials is placed into a freezer at –80°C for at least three hours prior to analysis.

The helium leak instrument employed in this study is a mass spectrometer that is tuned to detect only helium. Prior to analyzing samples, the instrument is calibrated against NIST traceable leak standards. Immediately upon removal from the –80°C freezer, the vials are placed on dry ice to maintain the samples at the desired temperature. A chiller capable of maintaining a constant temperature of –80°C is connected to the instrument and vials are individually analyzed. The instrument places the sample under high vacuum and measures the helium as it escapes from the sample vial. The helium partial pressure present in the leak detector is measured by the mass spectrometer and displays this “leak” from the sample vial as a leak rate, which is measured quantitatively. After the leak rate has been determined, the vials are allowed to warm to room temperature. At this point, the helium concentration inside the vial is measured with a calibrated headspace analyzer probe to determine the amount of helium remaining in the vial. The helium concentration and the measured helium leak rate are then used to calculate the actual helium leak rate of the vials, which has units of standard cc/sec. This leak rate is proportional to the size of the leak through which the helium escapes.

The results from this study illustrated that both stopper formulations had similar leak rates throughout the different time points. On average, the leak rates at –80°C were in the low 10-8 cc/sec range. Comparatively, the leak rates at room temperature were in the low 10-7 range. The leak rate displayed a decrease of approximately an order of magnitude when comparing room temperature samples to samples analyzed at –80°C.

The critical point for leak rate was based on findings by Dr. Lee Kirsch¹ whose studies illustrate a correlation of leak rate to microbial ingress. It is important to note that the experiments performed by Dr. Kirsch and his associates were tested at room temperature. Our methodology used his critical point for leak rate of 6.0E-6 std cc/sec as a reference for determining failing leak rates for our vials. The results of our study are significantly below this specified limit of failure. The average leak rate for the positive controls was 7.7E-3 std cc/sec, which clearly demonstrates that even at –80°C, the instrument will detect a breach in the integrity of a sealed container.

Because this study was performed at conditions below room temperature, there were questions concerning the critical leak rate. As the testing was performed at a significantly reduced temperature, it was anticipated that the critical leak rate would be lower. The helium fill of the samples was necessarily performed at room temperature. As a result the pressure of helium in the vial would be significantly reduced at such a low temperature and therefore the critical leak rate needed to be adjusted to account for this pressure reduction. In order to accomplish this, the internal pressure was determined using the ideal gas law. An assumption was made that the critical leak rate would be reduced in direct proportion to the reduction in internal vial pressure. Based on this assumption, a critical leak rate of 3.9E-6 std cc/sec was calculated for samples tested at –80°C. Since all the results, with the exception of the positive controls, were in the 10-8 std cc/sec range, it was demonstrated that container closure integrity was maintained throughout the study.

Helium leak detection is an ideal way of performing container closure integrity, even at temperatures as low as –80°C. While there are other techniques for monitoring CCI available, many of these are unsuitable for use at –80°C temperature. The highly sensitive nature of the instrument allows for leaks as low as a 2µm hole size to be detected as a failure. The quantitative results provide the analyst with a numerical value that allows for better data trending and higher confidence in the efficacy of the packaging system than is possible with qualitative methods.

Although this work has provided valuable data, there is a need for further studies into even lower temperature regions and other packaging systems. There is already an emerging need to extend container closure integrity testing into the cryogenic temperature range. If pharmaceuticals are to be stored at cryogenic temperatures, then container closure integrity will have to be established under those conditions. As has been clearly demonstrated by this study, helium leak testing is uniquely suited to address this need. Also, newer packaging systems such as pre-filled syringes and complex self-dosing devices are being increasingly utilized for these types of drug products and CCI will have to be proven for these types of packaging as well. 

References

1. L.E. Kirsch, L Nguyen, C.S. Moeckly, R. Gerth, “Pharmaceutical Container/Closure Integrity II: The Relationship Between Microbial Ingress and Helium Leak Rates in Rubber-Stoppered Vials”, PDA Journal of Pharmaceutical Science and Technology”, 51(5):195 (1997)