It is never convenient when the test result for a product, that has historically been within specification, comes back with an Out of Specification (OOS) result. It is however, critically important for safety, regulatory, and economic reasons, to determine what is causing an OOS result. The time and resources required to determine the root cause for a failure can return valuable, and sometimes surprising results. “The Mystery of the Disappearing Fentanyl” is one of those situations. What was originally thought to be a testing issue, turned out to be something entirely different, and enlightening.
This case study summarizes an investigation that was conducted to determine the root cause of an unexpected low and OOS result for a 50mcg/mL Fentanyl injectable drug. The container closure system for this product was comprised of a 2mL amber glass vial, a grey bromobutyl rubber stopper, and an aluminum crimp seal. The fill volume was 2mL. Historically, the potency result for this specific medication averaged 95% (i.e. 47.5mcg/mL) of label value. On one occasion, the result was 80% (i.e. 40mcg/mL) of label value. Nothing obvious turned up in the preliminary laboratory and production investigations that pointed to a testing or compounding error. However, because of the consistency of the historical results, a decision was made to repeat the test just in case a less obvious error in testing had occurred. Upon retest, the potency value decreased to 70% (i.e. 35mg/mL). Based on these results, a full-scale investigation was initiated.
It was theorized that the container closure system may be contributing to the low result, so the following experiment was designed to determine if the CCS was the root cause of the issue. The medication was produced in a glass container using the standard formula and compounding process. A portion of the bulk was filled into the standard container closure system, as either a 2mL or 0.2mL fill (6 vials for each fill volume). Half of the vials containing the 2mL and the 0.2mL fills were inverted so that the medication was in contact with the stopper (see Illustration 1). The solution was not in contact with the stopper when stored in the upright condition. The container holding the remaining bulk solution was sealed and stored at room temperature (RT) for 24 hours (Control Condition). All vails, 2mL and 0.2mL fills upright and inverted, were also stored at RT for 24 hours (Test Conditions). After 24 hours of storage, all the vials, as well as the bulk solution, were tested for potency using the standard potency testing procedure against a USP Reference Standard. The results may be found in Table 1.
TABLE 1: Potency Result vs Fill Volume and Storage Orientation
| Condition | Vial 1 | Vial 2 | Vial 3 | RSD | % of Control |
| Control Bulk | 99.1% | 100.2% | 100.8% | 0.86% | 100.0% |
| Test 1 – 2mL Upright | 99.9% | 99.1% | 98.8% | 0.59% | 99.2% |
| Test 2 – 2mL Inverted | 95.4% | 94.3% | 97.4% | 1.64% | 95.7% |
| Test 3 – 0.2mL Upright | 93.7% | 94.8% | 96.5% | 1.50% | 95.0% |
| Test 4 – 0.2mL Inverted | 63.8% | 57.9% | 61.8% | 4.94% | 61.1% |
Based on the results of this experiment, we can conclude that the low potency result had a least two contributing factors.
- The first factor is non-specific binding to the container closure, with the stopper having the more significant impact. It is typical for plastic and rubber to have significantly higher numbers of non-specific binding sites than glass. Evidence of this can be see in the potency results for the inverted vials, where the solution was in direct and continuous contact with the stopper was 5 to 8 times lower than the upright vials. The difference in potency results for the inverted vials (i.e. Test 2 and 4), was 4.3% and 38.9% lower than the control condition, as compared to the upright vials (i.e. Test 1 and 3) which were 0.8% and 5.0% lower than the control.
- The second factor is the fill volume. A lower fill volume means that there are fewer total molecules to bind to the non-specific sites. However, there are still the same number of non-specific sites, which will result in a more pronounced drop in potency for low volume, as compared to higher volume solutions. The results for the vials containing the 0.2mL fill were significantly lower than the 2mL fill counterpart. The potency of the 0.2mL upright fill was 4.2% lower than its 2mL upright counterpart, and the 0.2mL inverted was 34.6% lower than the 2mL inverted counterpart.
In the experiment described above, the relatively low concentration for fentanyl, combined with the low volume and the higher number of non-specific binding sites on the stopper, resulted in significantly lower potency results when the vials were stored in the inverted orientation.
This information can be used to explain a decrease in potency, however it does not explain why the result went from a historical average potency result of 95%, to a potency result of 80% on the batch in question, and then 70% on the retest. To explain this, we had to look beyond the production process, test procedure, container closure system and storage conditions, and at other factors.
After doing some additional research, we discovered that the sampling process for the batch in question had changed. For all previous batches, the laboratory had been sent sufficient vials to perform the all the tests using different vials. With the batch in question, only one vial was sent to perform all the testing. In situations where only one container is sent, the container is sent first to the microbiology lab, where half of the contents (i.e. 1mL in this case) of the container is sampled under aseptic conditions to perform the sterility test. The remaining contents of the container (i.e. 1mL) is sent to the chemistry laboratory for potency testing. Half the contents (i.e. 0.5mL) is used to perform the original potency test, leaving 0.5mL for the retest.
As a result of having only one vial to perform all the tests, the process had to be extended to multiple days. When multiple vials are used, all tests are performed in parallel fashion and performed on the same day. When only one vial is used, the tests need to be performed sequentially, which adds time to the process. On this occasion the original potency test was not performed until the day after the sterility test was started, and the retest was not performed until one day after the original potency test.
Based on this new information, we determined that the original potency result (i.e. 80%) for the batch of Fentanyl in question, was due the vials containing the lower volume (i.e. 1mL), being stored for 24 hours on their side. The retest potency result of 70% was just a continuation of that trend, given an even lower volume (i.e. 0.5mL) and the additional storage time. This also explains the historical average result of 95%. The orientation of the standard product (i.e. 2mL fill vials) during the handling process was never controlled, and therefore likely on its’ side (i.e similar to Test Condition 2), resulting in approximately 5% of the fentanyl molecules binding to the container closure system (i.e. mainly the rubber stopper).
There are numerous approaches that can be used to correct for situations like the one in this case study. The easiest and quickest way is to simply adjust the formula to compensate for the active ingredient that binds to the container closure system. More complicated and costly approaches are to change the container closure system to one that has lower binding characteristics, or to add ingredients to the formula that block the non-specific binding sites. In both these scenarios, additional validation will be required.
This case study is an excellent example of why it is important to perform investigations, and to keep an open mind, because the root cause of a problem is not always obvious. Had we not performed this investigation, we may never have discovered the non-specific binding issue, nor the downstream impact of something as simple and seemingly innocuous as a sampling procedure. Now that we have this new information, we can apply it to other situations, thereby minimizing the likelihood of similar issues in the future, resulting in significant cost savings and better service.
