A High-Resolution Look at Glass Delamination Using Transmission Electron Microscopy (TEM)

Formation of glass delamination flakes and secondary products through reaction of injectable pharmaceutical solutions with glass vial packaging leads to product contamination and costly recalls. Characterization of vials and products by a variety of techniques elucidates delamination mechanisms and drives development of packaging materials and processes to reduce the risk of delamination. Understanding of glass delamination is enhanced by the use of transmission electron microscopy (TEM), a high-resolution characterization technique that provides structural, elemental, and crystallographic information at the micro-level.

Impact of glass delamination on product quality

Glass delamination is characterized by formation and separation of glass flakes from the interior surface of a glass vial containing an injectable drug solution or suspension. It indicates severe extraction of glass by the product, and subsequent reactions can lead to formation of secondary compounds present as particles or residues in the liquid drug product.1,2

The severity of glass delamination depends upon many factors. Among them are the glass composition and vial manufacturing method, presence of glass defects, handling and sterilization techniques, and storage conditions such as time and temperature. The degree of interaction of vial glass with the stored product is also strongly influenced by product characteristics such as pH and presence of buffers, as well as the nature of the active pharmaceutical ingredient (API). This highly undesirable process has the potential to affect large amounts of product, and has been the subject of several recent pharmaceutical recalls.3,4

Multitechnique assessment of glass delamination

Investigation of a glass delamination event typically begins with visual and stereo-microscopic examinations of as-received vials, which may be empty or may contain liquid product. Features such as pitting, striations, or discoloration may be present on the vial glass, and particulate may be observed in the liquid.

Early stages of glass delamination are often observed on the vial interior before glass flakes are evident in the liquid. Samples are prepared for a variety of micro-analyses by 1) filtration of liquid product to isolate flakes and residues, and 2) breaking of vials for examination of interior walls, necks, and bases. The vials are taped before being broken to maintain the spatial relationship of the fragments.5

Figure 1 – TEM image of scraping from vial interior and diffraction pattern from crystalline inclusion. Numbers correspond to EDS spectrum locations.

The glass and isolated materials are further characterized using polarized light microscopy (PLM), scanning electron microscopy with energy dispersive X-ray spectrometry (SEM/EDS), micro-Fourier transform infrared spectroscopy (micro-FTIR), and X-ray photoelectron spectroscopy (XPS). This battery of techniques provides many types of information: images showing morphology of isolated particles, residues and glass defects, elemental spectra and maps that reveal compositional changes such as leaching or migration, and molecular spectra that differentiate drugs from secondary reaction products.

Insights gained from high-resolution TEM

In the TEM, a beam of high-energy electrons penetrates a sample typically no more than 100 nm 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 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. For addressing problems related to glass delamination, the TEM is ideally suited for analysis of thin delamination flakes, filtered residues, and scrapings from glass vials. The results presented here were acquired using the JEOL JEM-3010 TEM and a JEOL JEM-ARM200CF (JEOL Ltd., Tokyo, Japan).

When examined in the TEM, scrapings from an undamaged glass vial will appear featureless when imaged and will yield amorphous electron diffraction patterns. Comparison of EDS spectra acquired from multiple locations will show the glass composition to be quite uniform. In contrast, Figure 1 shows a TEM image of material scraped from the interior base of a vial that had contained a high-pH injectable. Submicrometer inclusions were observed that differed in elemental composition from the surrounding matrix, and were found to be somewhat crystalline. Even such relatively low magnification analyses can reveal significant structural and elemental features that may be inherent in the glass or may be related to delamination.

Figure 2 – TEM images of delamination particles filtered from the same vial; note textural differences.

Glass delamination flakes and secondary products formed during delamination exhibit a variety of microstructures; these are readily apparent in TEM images, but may not be distinguishable using PLM or SEM/EDS. Figure 2 shows high-magnification TEM images of adjacent particles filtered from liquid contained in a severely delaminated vial. The images were acquired at the same magnification, and show the two particles to have distinctly different textures. EDS analysis of several particles of each type showed them to also have different elemental compositions, indicating varying degrees of vial/product interaction.

Figure 3 – TEM image of filtered delamination particle exhibiting a lace-like texture.

The fine dark lines in the image on the left can be interpreted as being either needle-like structures or plate-like structures viewed edge-on. In some cases, such structures have exhibited greater degrees of ordering, forming small bundles or aligning into evenly spaced layers that yielded measurable diffraction patterns. The combination of diffraction results and elemental ratios from EDS analysis has led to more specific phase identification of secondary products exhibiting some degree of crystallinity.

The particle shown in Figure 3 was also filtered from the liquid product in a delaminated vial, and can be seen to have a very different, lace-like texture. As shown in Figure 4, TEM images reveal pits and holes on a much finer scale than might be seen in the SEM, raising the possibility that TEM may be useful for early-stage detection of glass delamination.

Figure 4 – High-magnification TEM image of fine holes and pits in delamination flake.

Figure 5 shows an overlay of element maps acquired from an area around a hole in such a particle. To obtain the element maps, the TEM, which normally employs a static beam of near-parallel illumination, was operated as a scanning transmission electron microscope (STEM). In STEM mode, the beam is converted to a fine, focused probe that can be rastered over an area of the sample as in an SEM. The green component of the overlaid STEM maps shows a concentration of an element present in the drug product around the edges of a hole in the particle. Extensive studies of delamination flakes and secondary products at such high resolution could potentially shed new light on glass delamination mechanisms.

Figure 5 – Overlay of element maps from STEM analysis of delamination flake; note concentration of green component at edges of hole.

Conclusion

Glass delamination is a complex problem, and understanding the mechanisms by which it occurs is crucial to ensuring pharmaceutical product quality and developing more resistant packaging materials. TEM is an ideal technique for analysis of vial glass, thin delamination flakes, and residues, providing a new level of high-resolution morphological, elemental, and crystallographic information. It is best used in conjunction with other techniques commonly applied to glass delamination samples, such as light microscopy, micro-FTIR, SEM/EDS, and XPS.

Pharmaceutical manufacturers can benefit from developing expertise in techniques for sample preparation and analysis applicable to glass delamination events, and many educational institutions can provide suitable training. The Hooke College of Applied Sciences (Westmont, IL; www.hookecollege.com) offers courses in sample preparation, light microscopy, FTIR, SEM, and TEM to aid in development of skills for analysis of glass delamination materials.

References

  1. Iacocca, R.G.; Toltl, N. et al. American Association of Pharmaceutical Scientists, publ. online Aug 26, 2010.
  2. Iacocca, R.G.; Allgeier, M. J. Mater. Sci.2007, 42, 801–11.
  3. Ennis, R.D.; Pritchard, R. et al. Pharm. Devel. Technol.2001, 6(3), 393–405.
  4. Proceedings of the PDA/FDA Glass Quality Conference, Washington, DC, Jun 4–5, 2012.
  5. Diebold, K.J. Mod. Microsc. J. publ. online Oct 20, 2003.

Ms. Schumacher is a Senior Research Scientist, McCrone Associates, Inc., the analytical division of The McCrone Group, 850 Pasquinelli Dr., Westmont, IL 60559, U.S.A.; tel.: 630-887-7100; fax: 630-887-7417; e-mail: eschumacher@mccrone.com. Contributions to this work by Ms. Kristie J. Diebold and Mr. Scott Stoeffler of McCrone Associates, Inc. and Dr. Alan Nicholls of the Research Resources Center at the University of Illinois at Chicago are gratefully acknowledged.

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