The integrity of the container system is crucial to the safety of parenteral products. The presence of glass delamination flakes in product solutions is of particular concern. Glass delamination occurs when the inside surface of the vial begins to degrade and eventually very thin glass flakes begin to spall off of the surface and appear in the liquid product. Glass container stability may be affected by a large number of factors, including the vial manufacturing process, post-formation treatments, depyrogenation and sterilization, and interaction with the drug product. The appearance of flakes in solution indicates that there is significant damage to the vial’s internal surfaces and may lead to recalls and lost revenue. It is crucial to evaluate the compatibility and stability of the product with the glass vials during the initial stages of new drug development. Ideally, accelerated stability samples are tested for the presence of glass delamination as part of the drug development.

Microscopy—using both light microscopes and electron microscopy—is a vital tool in the evaluation of parenteral vials for the presence of glass delamination. At McCrone Associates, evaluation of glass delamination in product vials usually includes three stages. First, light microscopy is utilized to determine if potential glass delamination is present in the product solution. If glass delamination-like flakes are observed, chemical analysis (stage 2) is needed to confirm that the flakes are consistent with glass.
Typically, scanning electron microscopy with energy dispersive X-ray spectrometry (SEM/EDS) is used to confirm that the flakes are chemically consistent with glass delamination. Transmission electron microscopy (TEM) may be used for extremely thin flakes that are difficult or impossible to analyze using SEM/EDS. The third step is to examine the interior surfaces of the vials for evidence of pitting and flaking.

Examining Glass Delamination Flakes
In the first stage of analysis, light microscopy is used to examine intact vials for free floating particles with morphology consistent with glass delamination flakes. Glass delamination flakes are extremely thin and have distinct morphologies in solution and after capture on a filter membrane.

Initially, vials are examined visually using a fiber optic light. The very thin flakes produce a twinkling effect that is characteristic of glass delamination. The vials are then examined using a stereomicroscope to confirm the presence of thin flakes in solution. In order to isolate the flakes, the contents of the vials are filtered using polycarbonate filters. The filters are then examined using a low magnification stereomicroscope using several different lighting conditions including coaxial illumination and oblique illumination, or a combination of both. Coaxial light is also referred to as episcopic or reflected light. This type of lighting is optimal for observation of thin particles, residues and films, as well as surface features. A higher-magnification polarized light microscope (PLM) using episcopic illumination may also be used to examine the filter. (Episcopic illumination is virtually the same as coaxial illumination, but on a PLM microscope.) Glass delamination flakes are extremely thin and thus are usually only visible using coaxial/episcopic illumination. If present, glass delamination is in the form of very thin, sharp-edged, irregular flakes. The flakes range in color from brown for extremely thin flakes to red and blue for thicker flakes. A photomicrograph of typical glass delamination flakes is shown in Figure 1.

Determining Chemical Composition
While glass delamination flakes have a characteristic appearance when viewed using light microscopy (both stereomicroscopy and PLM), it is advisable to determine the chemical composition of the flakes. Thin flake-like materials other than glass can exhibit a similar appearance on polycarbonate filters, so it is important to identify any observed flakes as having chemistries consistent with glass. Scanning electron microscopes (SEM) equipped with energy dispersive X-ray spectrometry (EDS) detectors are used to obtain the elemental profile of small particles. The flakes must be isolated and mounted for analysis in the SEM vacuum chamber. Thicker flakes may be manually removed from the filter and mounted on an appropriate substrate in preparation for SEM/EDS. Very thin flakes can be difficult to remove from the filter and often the flake-like morphology is lost. Alternatively, flakes may be analyzed directly on the filter. A SEM image of glass delamination flakes obtained directly on the filter is shown in Figure 2. This method retains the flake-like morphology, but the EDS spectrum is dominated by the carbon from the filter membrane and peaks arising from the elements in the glass are very small. The minor and trace elements are often not detected. A SEM/EDS spectrum of the flake in Figure 2 is shown in Figure 3. 

Transmission electron microscopy offers new opportunities for analysis of glass delamination samples. In the TEM, a beam of high-energy electrons penetrates the sample, which is typically no more than 100 nanometers thick. The thinness of the sample minimizes scattering of the beam electrons, allowing for high-magnification imaging and high spatial resolution EDS analysis of areas that are a few nanometers or smaller in size. Through-thickness crystallographic information can also be obtained by selected area electron diffraction (SAED), making it relatively straightforward to differentiate crystalline from amorphous specimens and to identify crystalline phases. The TEM is ideally suited for analysis of thin glass delamination flakes, filtered residues and scrapings from glass vials.

TEM presents a major advantage over SEM for samples that are very thin. EDS analysis can be performed on very thin flakes and the resulting spectra have less background and stronger signals for glass elements. Figure 4 is a TEM EDS spectrum of a glass flake on a TEM grid. The spectrum in Figure 4 is much more diagnostic for the confirmation of glass than the SEM/EDS spectrum presented in Figure 3. Isolation methods have been developed to optimize the capture of glass flakes for TEM analysis. This method has potential for detecting glass flake formation in the early stages of delamination and could be very useful in new drug development and testing. Due to potential element-leaching that may have occurred in the delamination process, a comparison of the chemistries between the flakes and the vial glass/vial coating may not produce reliable comparative data, but the elemental composition of the flakes can be used to confirm if they are glass-like material. Also, the interior of the vials may be coated and the composition of flakes from the coating material may not be similar to the original coating chemistry.

Examining Interior Surfaces
The third stage of analysis involves the examination of the vial interior surface for evidence of delamination. The examination of vial interior surfaces may be valuable to determine the location in the vial the delamination is occurring, or if, in fact, the delamination occurred in the product vial. If present, delamination from the interior vial surface will be observed as pitting or surface disruption to the normally smooth interior surfaces. The area near the base of the vial is particularly prone to delamination. At times, lines of demarcation may be noted at the fill line of the liquid product. The interior is best examined directly, not through the walls of the intact vials. In order to access their interior, vials are typically cracked to expose the interior surfaces.

The interior surfaces of the vials may be examined using light microscopy with coaxial or episcopic illumination, differential interference contrast (DIC) microscopic observation, and/or by SEM. The scientist performing the analysis, upon consultation with the client, will determine which visual or instrumental technique(s) will produce the most illustrative data.

Benefitting New Drug Development
Glass delamination has the potential for lost revenue and drug shortages due to product recall. Testing for vial compatibility and stability should be carried out early in the product development process. Light microscopy and chemical analysis using electron microscopy are important tools for identification of glass delamination. TEM potentially can identify thinner flakes at earlier stages in the process, which may be very beneficial for new drug development.