Abstract
An accelerated lamellae formation (ALF) methodology has been developed to determine the delamination propensity and susceptibility of pharmaceutical glass vials. The ALF process consists of a vial wash and depyrogenation mimic procedure followed by stressing glass vials with 20 mM glycine pH 10.0 solution at 50 °C for 24 h and analyzing the resulting solutions by visual inspection for glass lamellae. ALF results demonstrate that while vial delamination propensity generally correlates with glass hydrolytic resistance, ALF is a more direct test of glass delamination propensity and is not affected by post-production vial washing that can affect results obtained using hydrolytic resistance tests. ALF can potentially be used by pharmaceutical companies to evaluate and screen incoming vial lots to minimize the risk of delamination during the shelf life of parenteral therapeutics, and by glass vial manufacturers to monitor and improve their vial manufacturing processes.
LAY ABSTRACT: Glass flakes can sometimes appear in liquid pharmaceutical drugs contained in glass vials. These glass flakes are a result of several factors related to the glass vial production process, glass vial sterilization procedures, and the formulation of the liquid pharmaceutical drug. Vial testing is routinely done in order to select glass vials that are less likely to form glass flakes. The factors leading to the formation of glass flakes were studied and applied to a method designed to directly screen vials for their propensity to form glass flakes. The washing of vials followed immediately by sterilization at high temperatures was determined to be a critical factor in the formation of glass flakes. As a result, a laboratory mimic of this procedure was incorporated into the newly developed method for screening vials. This mimic procedure as well as robust accelerated incubation conditions and a sensitive visual inspection procedure are key aspects of this vial screening method.
- Glass delamination
- Glass lamellae
- Accelerated lamellae formation
- Visual inspection
- Hydrolytic resistance
- Alkalinity
- Type I borosilicate glass
Introduction
In recent years, glass particles have been observed in injectable protein therapeutics as well as other parenterals contained in glass vials, resulting in a number of product recalls. It has been reported that glass flakes, or lamellae, can be generated by delamination through the interaction between the drug product and glass containers manufactured under suboptimal conditions (1⇓–3). Drug compound (1), high pH, buffer components such as citrate (4), storage temperature, and time all can have a complicated impact on glass dissolution and delamination during a product's shelf life. As the formulation is often constrained due to the stability of the drug product, it is important to select glass containers of the highest quality in order to reduce the likelihood of delamination. Currently, the primary tool for evaluating the suitability of glass vials has been the European Pharmacopeia test for glass surface hydrolytic resistance (alkalinity) (5). However, a correlation between glass vial alkalinity and delamination propensity is not well established and the nature of the alkalinity method is not amenable to a direct determination of vial delamination propensity. More direct methods would be useful to assess and predict the propensity of glass vial delamination and their suitability for use in the pharmaceutical industry. These methods would help to mitigate the risk of potential glass delamination during the shelf life of the product, and the data generated using these methods can assist in the improvement of the glass vial manufacturing process.
The time dependence of delamination as well as its low frequency of occurrence makes the direct study of delamination a challenging endeavor. The potential effect of vial washing and depyrogenation during drug product manufacturing on delamination propensity can also affect the ability to conduct relevant vial screening in a laboratory setting. Many of the reported studies on glass delamination have involved the use of accelerated treatment of glass vials (1⇓⇓–4, 6). While some of these studies have included the visual detection of glass lamellae, most have focused on the detection of soluble extractables in solution and evidence of delamination observed through analysis of the vial surface by scanning electron microscopy (SEM). The ability to accelerate the delamination process is deemed necessary for its efficient study, and it hinges on an understanding of the critical factors in the mechanism of delamination including the impact of washing and depyrogenation and the factors affecting the rate of glass dissolution in aqueous solution. We have studied these factors and have optimized their effects in order to develop an accelerated vial screening method which relies on visual detection of glass lamellae in solution. In this paper, we describe the development and application of accelerated lamellae formation (ALF) for the direct assessment of glass delamination propensity.
Materials and Methods
Materials
Three milliliter Type 1A—coefficient of expansion of 33—borosilicate glass vials of varying alkalinity were obtained from two different vial vendors (Vendors X and Y). Alkalinity values were supplied by the vial manufacturer. Vials were obtained in an unprocessed state (pre-wash/sterilization). Description of vials in this paper use the letters X or Y to denote the vial vendor followed by the reported alkalinity value for the vial lot (for example, X95%).
Methods
Accelerated Lamellae Formation (ALF) Stress Treatment Development:
Ultrapure water was generated using a MilliQ Synthesis system (Millipore, Billerica, MA). Vials with various volumes of added ultrapure water were placed in a preheated Lindberg Blue M oven (Thermo Scientific, Waltham, MA). After the specified time in the oven, vials were removed from the oven and allowed to cool for approximately 30 min at room temperature. Then 3 cc vials were filled with 1.0 mL of various buffer solutions. Excipients for buffer preparation were obtained from Sigma Aldrich (St. Louis, MO). Filled vials were stoppered with a fluorinated ethylene propylene–coated chlorobutyl stopper, sealed with aluminum crimp caps, and placed in controlled temperature incubators for specified time periods. Experiments comparing the delamination propensity of Type 1A vials of varying alkalinity (Figure 1) used the optimized ALF conditions that included adding 200 μL of ultrapure water, placing vials in Lindberg Blue M oven preheated to 250 °C for 90 min, and allowing to cool for approximately 30 min. These vials were then filled with 1.0 mL of 20 mM glycine pH 10.0 and incubated at 50 °C for 24 h.
Delamination propensity of Type 1A glass vials as a function of vial alkalinity. 3 cc vials of varying alkalinity filled with 200 μL water followed by 90 min in a preheated oven at 250 °C. Vials were then filled with 1 mL 20 mM glycine pH 10 and incubated at 50 °C for 24 h followed by visual inspection using APK unit. n = 140 vials tested for each lot.
Visual Inspection:
Vials were inspected visually for the presence of glass lamellae with the aid of an Ampoule Particle Kensa (APK) unit (Eisai Machinery Co. Ltd., Tokyo, Japan), which illuminates the vial from below and includes a mechanism for spinning the vial prior to observation. After application of a pulsed spin for approximately 1 s, vials were inspected against a black background on the APK unit for the presence of lamellae. Glass lamellae are differentiated from other types of particles in solution by their unique morphology and their ability to reflect light. In contrast to other particles, glass lamellae will appear to sparkle as they rotate in solution in the presence of proper lighting and background. Data was recorded for each vial inspected as containing or not containing visible lamellae. The data for each lot of vials is expressed as the percentage of vials tested that contain lamellae.
Results
Development of Accelerated Incubation Conditions
Accelerated incubation conditions were determined by filling washed and depyrogenated vials with various buffers followed by incubation at a range of temperatures and durations. At various timepoints, vials were removed from the incubators and visually inspected for the presence of glass lamellae. During this experiment, it was observed that vials containing lamellae at a particular timepoint would not always contain visible lamellae at subsequent timepoints (Table I). A likely explanation for this phenomenon is the dissolution of the lamellae in solution over time. Higher temperatures, while accelerating the initial appearance of lamellae, caused a proportional increase in the rate of lamellae dissolution. Because of this observation that glass lamellae are apparently unstable in solution due to dissolution, accelerated incubation conditions were optimized to increase the robustness of lamellae formation and detection in the vial screening method. To optimize the incubation conditions for use in the ALF method, variations in glycine concentration, buffer solution pH, incubation temperature, and incubation time were studied for their effect on lamellae formation and detection. The optimized accelerated method conditions, 20 mM glycine pH 10 at 50 °C for 24 h, represent a balance between the time to lamellae formation and the stability of lamellae in solution. Using these conditions, the formation of lamellae in susceptible vials appears to be robust and was not significantly affected by minor variations in buffer or incubation parameters.
Dissolution of Lamellae under Accelerated Incubation Conditions
Development of Wash/Depyrogenation Mimic
It was initially determined that only vials that had been washed and depyrogenated would form visible lamellae upon incubation under the accelerated conditions studied. No glass lamellae were detected in unprocessed vials that had not been previously washed and depyrogenated under the incubation conditions studied (Table II). The apparent requirement that vials be pretreated by washing and depyrogenation for delamination to occur upon subsequent incubation presents a challenge for the development of a method to screen unprocessed vials. For this reason, a lab-scale wash and depyrogenation mimic procedure was developed to allow for an assessment of the delamination propensity of unprocessed vials.
Effect of Vial Processing on Lamellae Occurrence under Accelerated Conditions
The effects of washing and depyrogenation parameters on glass vial delamination were studied using laboratory equipment to mimic the conditions encountered in a manufacturing setting. Exposure of vials to high temperature, with and without residual moisture, was investigated. Following exposure to these pretreatment conditions, vials were filled with 1 mL 20 mM glycine pH 10, incubated at 50 °C for 24 h, and visually inspected for lamellae. Conclusions made from these experiments on the wash depyrogenation mimic effects may be somewhat specific to the subsequent incubation conditions studied, with the results indicating the combined effect of the two phases of vial stress treatment. Heating dry vials at 250 °C and washing vials with 80 °C water without subsequent heating at 250 °C were insufficient pretreatments, and no lamellae were observed in vials exposed to these conditions. However, placing vials containing a small volume of added water in an oven at 250 °C was a sufficient pretreatment to induce lamellae formation upon subsequent incubation at 50 °C for 24 h after filling with 1 mL of 20 mM glycine pH 10 solution.
In order to optimize the volume of water added to the vials, varying amounts of water were added to the vials followed by exposure to 250 °C for 90 min. Results in Table III indicate that addition of 200 μL water to the vials appears optimum for an effective pretreatment, resulting in delamination of susceptible vials. The addition of water volumes greater than 400 μL prior to 250 °C exposure eliminated the propensity of vials to delaminate.
Effect of Water Fill volume of Wash/Depyrogenation Mimic on Vial Delamination
The temperature of the preheated oven was varied to determine the effect of this temperature on the delamination mechanism. It appears from Table IV that there is a critical minimum temperature between 175 and 200 °C that is required to prime vials for delamination.
Oven Temperature
Experiments were performed to assess the impact of time at 250 °C on the extent of water drying and the impact on vial delamination. Approximately 4 min in the preheated oven at 250 °C is required to dry 200 μL of water in a 3 cc glass vial. Vials heated for 3 min contain 25–50 μL of residual water from the added 200 μL, and after 4 min the vial interior appears dry. Table V shows that this drying process at 250 °C is sufficient pretreatment to prime susceptible vials for delamination.
Time in Preheated Oven (250 °C) for Vials Containing 200 μL Water
Based on these results, an optimized lab-scale wash/depyrogenation mimic procedure consisting of placing 3 cc vials containing 200 μL of water in a preheated oven at 250 °C for 90 min. Table VI shows that vial pretreatment using the optimized wash/deprogenation mimic is comparable to the effect of washing and depyrogenation on vial delamination propensity.
Effect of Depyrogenation Mimic Procedure and the Role of Water
Results of Accelerated Stress Treatment on Vial Lots of Varying Alkalinity
Figure 1 shows the results of the application of the ALF stress treatment and visual inspection on seven vial lots. The results indicate a general trend towards higher delamination propensity with increasing vial lot alkalinity. However, some deviations from this trend are obvious due to several outliers. In particular, the three vial lots with alkalinity values of 58%, 60%, and 65% vary significantly in their propensity to delaminate. These vial lots, while similar in alkalinity, were produced by different vendors and by varying manufacturing processes within a single vendor. It appears that these different manufacturing processes could lead to vials with comparable alkalinity but with greatly varying delamination propensities. Lot Y58% in particular differs from lot Y60% in that it was treated with a postproduction water wash in the course of its manufacture. This treatment apparently lowered the measured alkalinity of the lot without correcting the root cause of delamination. This data illustrates the value of this direct stress approach to vial screening, as reliance on alkalinity alone would have been a poor assessment of delamination propensity for some of the vial lots tested in this experiment.
Discussion
Accelerated conditions for lamellae formation are based on the understanding that the glass delamination phenomenon is dependent upon the structural impact of vial forming and processing conditions as well as chemical dissolution of the glass matrix. Vial forming is a critical determinant of delamination propensity, and the inherent variability of this process is the purpose of vial screening method implementation. Based on our experiments, vial processing by washing and depyrogenation can be a significant contributor towards realizing the delamination potential of a particular vial, and a mimic of its effect has been incorporated into our methodology. It appears that heating moisture-containing vials to dryness at high temperature is a sufficient pretreatment for achieving this effect. Water is known to penetrate the surface of glass via several mechanisms including the diffusion of molecular water, ion exchange involving the interdiffusion of hydronium and sodium ions, or through the hydrolysis of silicon-oxygen bonds (7, 8). All of these mechanisms lead to corrosion of the glass surface and increase the likelihood of glass delamination. As would be expected, exposure of water-containing vials to temperatures ≥200 °C appears to accelerate this corrosion and facilitates delamination. Based on data in Tables III–V, there may be additional factors related to the water level in a vial and the effect of rapid dessication of a hydrated vial that require further study to understand their effect.
The final step in the process of lamellae formation is the dissolution of susceptible areas of the vial by aqueous solution and the release of lamellae from the surface. Based on our understanding of glass dissolution chemistry, we have incorporated high temperature and high pH into the ALF methodology. While glass dissolution is a component factor in the glass delamination mechanism, we have found that a fine balance exists between the acceleration of lamellae formation and excessive dissolution that prevents lamellae detection. In fact, conditions reported to favor delamination, such as citrate containing buffers at high temperature, often show no evidence of glass lamellae as a result of dissolution. We find that 20 mM glycine pH 10 at 50 °C for 24 h represents optimized conditions for the purpose of accelerating the delamination phenomenon while allowing robust detection of formed lamellae. The application of a direct approach to the study of glass vial delamination allowed for us to correlate the glass dissolution events leading to the presence of leachable ions in solution (alkalinity) to the appearance of glass lamellae in solution. Previous reports have stated that the appearance of lamellae was a lagging event and not a reliable indicator of delamination (3). While this may be true, given that the actual appearance of the lamellae is the most important event with regards to the effect of delamination on the safety and quality of parenteral pharmaceuticals, the use of an accelerated method for the reliable generation and detection of these lamellae is critical for the study and assessment of glass vial quality. The ALF method thus relies heavily on the visual inspection method for detection of lamellae in solution.
The use of the APK unit to aid in this visual inspection provided several advantages. The lighting provided by the unit appears to enhance the ability to observe the lamellae based on their reflective properties. Also, while in most cases the appearance of lamellae using ALF did not require the application of any mechanical force to dislodge the flakes and allow detection in solution, the use of the spinning mechanism associated with the APK unit increases the likelihood of lamellae detection for vials with lamellae remaining weakly bound to the vial surface. Despite the use of a percentage scoring system for comparing vial lot lamellae formation frequency, manual visual inspection remains a poorly quantitative technique. The possibility exists for the development of automated detection methods using video imaging analysis or subvisible particle methods such as microflow imaging. These methods could improve the sensitivity, consistency, and quantitative nature of lamellae detection.
In addition to inspecting vials for lamellae, it is possible to examine the inner surface of the vial to detect evidence of delamination following a stress treatment such as ALF. Previously, this was a cumbersome process requiring destructive and time-consuming vial preparation and the use of a scanning electron microscope. Due to the nature of the SEM method, screening a statistically significant sample size from a particular vial lot would be difficult. A recent report details the use of dynamic interference contrast (DIC) microscopy for the purpose of examining the inner surface of glass vials for evidence of delamination (9). The DIC method possesses advantages over SEM related to sample throughput, and the use of DIC in conjunction with ALF could increase the level of sensitivity in an evaluation of glass vial quality.
Borosilicate glass, from which pharmaceutical vials are produced, is primarily a tetrahedral network of covalent bonds of silicon oxygen and boron oxygen. In the process of converting the glass tubing into vials, however, excessive heat can cause the migration of sodium towards the inner surface of the vial (1). We have shown that glass vial alkalinity values show a rough correlation with delamination propensity as determined using ALF. This is due to the fact that the increased levels of sodium at the inner glass surface, seen as a result of excessive heat during vial forming, will result in higher alkalinity as well as increased delamination propensity. However, as we have shown, manufacturing variations such as water rinsing of vials can greatly affect the alkalinity values obtained without correcting structural defects on the glass surface, thus negatively affecting the correlation between alkalinity and delamination propensity as assessed by ALF. Despite this, the information on the hydrolytic resistance of vials remains valuable for a complete assessment of the factors affecting vial quality.
For studying the effect of a sterilization process on delamination, a method such as ALF using glass lamellae detection is essential. Based on the data presented here, the effect of vial processing by washing and depyrogenation is indicated to be especially critical in the delamination of glass vials. The combination of residual moisture from vial washing and heat during the depyrogenation process appears to be important in the priming of vials for subsequent delamination.
The ALF conditions were designed to screen vials for use with aggressive citrate-containing, neutral pH formulations which are known to increase the risk of glass delamination. Screening vials for use with lower or higher delamination risk drug product formulations can use modified ALF conditions to more appropriately reflect the necessary level of delamination resistance.
Despite advancements in vial testing methods, it remains impossible to nondestructively screen every vial prior to filling. Vials of high delamination propensity may remain in high-quality vial lots due to lot heterogeneity and inconsistency in the manufacturing process. Thus, in order to reduce the risk of delamination further, vial processing changes could be made in addition to implementing improved vial screening procedures. It appears that optimized vial drying procedures designed to better eliminate excess moisture prior to depyrogenation would constitute a favorable process improvement that would significantly reduce the risk of delamination.
Conclusions
A method has been developed and optimized for the purpose of assessing the delamination propensity of Type 1A glass vials. This method significantly increases the confidence in glass vial quality testing due to its direct and sensitive nature. While the method has only been shown to be effective for Type 1A vials, a similar type of method would likely be useful in the evaluation of the relative delamination propensity of Type 1B and other types of glass vials, including those with surface modifications or coatings. Using this method, the effects of vial manufacturing and processing conditions on vial delamination propensity can be directly assessed. Washing and depyrogenation appear to be critical factors in the delamination propensity of glass vials, with residual moisture and heat playing a significant role. While washing and depyrogenation may not be a prerequisite to delamination in every instance, it appears to significantly increase the likelihood of lamellae detection in susceptible vials within the timeframe of the accelerated conditions studied. While there is a positive correlation between the vial alkalinity and the measured delamination propensity, the ALF method could be superior to alkalinity in determining the true delamination propensity of glass vials. Delamination of glass vials appears to be influenced by glass vial quality, washing and depyrogenation parameters, drug product formulation, and drug product handling and storage conditions. Accelerated stress testing can aid in the evaluation of these variables and their impact on drug product delamination propensity. Glass vial manufacturers can use ALF to monitor and improve their glass manufacturing process to make vials more suitable for parenteral drug products. Pharmaceutical companies can use ALF to objectively and quantitatively evaluate and qualify vial suppliers, screen incoming vial lots, assess new vial types, facilitate formulation and product manufacturing development, and to investigate product and process deviations should they occur.
Conflict of Interest Declaration
The authors declare that they have no competing interests.
Acknowledgements
The authors wish to thank Zai-Qing Wen, Yasser Nashed-Samuel, Gianni Torraca, Peter Masatani, Shawn Cao, Nancy Jiao, Margaret Ricci, Monica Goss, and Sekhar Kanapuram for their assistance and helpful discussions.
- © PDA, Inc. 2013
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