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Quantitative methylene blue staining of pharmaceutical glass vials

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This presentation provides a summary of recent work by my research group on the use of methylene blue staining as a method for characterizing pharmaceutical glass vials.

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Quantitative methylene blue staining of pharmaceutical glass vials

  1. 1. Quantitative methylene blue staining of pharmaceutical glass vials Matthew M. Hall, Associate Professor of Biomaterials & Glass Science Kazuo Inamori School of Engineering at Alfred University E-mail: hallmm@alfred.edu
  2. 2. Methylene Blue (MB) staining can been used to qualitatively evaluate surface defects in glass vials Sulfur-treated Type IA vials autoclaved and then stained with MB solution SEM image of corroded surface in heel region after autoclaving Images taken from presentation by Daniele Zuccato (Stevanato Group) to PDA Metro Chapter Available at http://www.pda.org/docs/default-source/website-document-library/chapters/presentations/metro/ glass-delamination-risks-reality-and-regulatories.pdf?sfvrsn=6
  3. 3. Methylene Blue (MB) staining can been used to qualitatively evaluate surface defects in glass vials • There is no standard procedure for performing MB staining of glass vials • Typical methods use concentrated MB solution (1% (w/v) is common) with staining times ranging from 15 minutes to 24 hours • Can identify regions of selective staining within a single vial (e.g., heel versus body) and perhaps make observations about relative staining behavior among different vials • Qualitative information may be helpful, but does not necessarily produce data that can be readily documented and acted upon
  4. 4. Quantitative results may also be achieved with MB staining under appropriate conditions 5 4 3 2 1 0 400 450 500 550 600 650 700 Optical Absorption (cm-1) Wavelength (nm) 1) 1:50,000 2) 1:100,000 3) 1:150,000 4) 1:200,000 5) 1:300,000 6) 1:400,000 1 g MB/100 mL WFI stock solution 1:400,000 dilution of stock with WFI • UV-Vis spectroscopy was used to measure the optical absorption spectra for a series of MB solutions prepared by serial dilution of a 1% (w/v) stock solution • 1:200,000 MB solution was found to be suitable for staining; avoids noisy spectrum (see spectrum #1 above) and provides adequate detection capabilities when used to stain vials in the 8 to 10 mL size range
  5. 5. Quantitative results may also be achieved with MB staining under appropriate conditions • An experiment was designed to evaluate if MB staining could discriminate between acceptable and defective glass vials • Type 1 glass vials provided by supplier on condition of anonymity • Two lots of vials – one passed a standard hydrolytic resistance test (“Pass” vials); the other lot failed the same test (“Fail” vials) • Chemical stability behavior of same vials previously reported in: Kucko et al. (2013). Fill volume as an indicator of surface heterogeneity in glass vials for parenteral packaging. Journal of Pharmaceutical Sciences, 102: 1690-1695. • Pass and Fail vials were filled to 90% maximum capacity with MB solution prepared by 1:200,000 volumetric dilution of a 1% (w/v) stock solution • Vials exposed in triplicate to dilute MB solution for time periods ranging up to 24 hours • Optical absorption of dilute MB solution measured after each incubation period
  6. 6. Quantitative results may also be achieved with MB staining under appropriate conditions 1.20 1.15 1.10 1.05 1.00 0.95 0.90 0.85 0.80 0.75 0.70 Decrease in optical absorption occurs when vial is stained by MB solution. Error bars represent 2 standard deviation of result measured in triplicate and are smaller than symbol when not visible. 0 3 6 9 12 15 18 21 24 Optical Absorption of Post-Staining 1:200,000 MB Solution (cm-1) Time (hr) Pass Fail • Both vial types exhibited detectable staining by dilute MB solution • Staining was visible to the unaided eye in the heel region of the vials • Staining behavior is time dependent and unique to vial type • Staining is similar within 1st hour • Optical absorption behavior reaches a local minimum at short times, followed by an increase and then gradual decline • Behavior suggests that interior surface of vial is being altered while exposed to MB solution, thereby influencing extent of MB absorption/adsorption • Fail vials always stained more than Pass vials after 1 hour of exposure • Quantitative MB staining can be used as a simple method for identifying drifts in manufacturing process
  7. 7. Quantitative results may also be achieved with MB staining under appropriate conditions Electron microscopy was used to probe origins of different MB staining behavior in the heel region of Pass and Fail vials Pass vial, As received (15,000) Pass vial, 24 hr stain (15,000) Pass vial, 24 hr stain (~107,000) • Pass vials show circular pitting in heel region after 24 hr exposure to dilute MB solution • Circular pitting is characteristic feature of many vials produced from converted tubing
  8. 8. Quantitative results may also be achieved with MB staining under appropriate conditions Electron microscopy was used to probe origins of different MB staining behavior in the heel region of Pass and Fail vials Fail vial, As received (15,000) Fail vial, 24 hr stain (9,900) Fail vial, 24 hr stain (30,000) • Fail vials also showed circular pitting although larger structures were apparent, including semi-circular cracks
  9. 9. Quantitative results may also be achieved with MB staining under appropriate conditions Combination of FIB and STEM used to further examine heel region of Fail vials Platinum layers associated with sample preparation procedure Foamed glass shell of blister Void within blister Bulk glass below blister • FIB used to generate cross-section that was subsequently imaged by STEM • Cross-section shows blister consisting of void surrounded by thin shell of foamed glass, perhaps due to overheating during conversion process; shell was rich in alkali, which explains poor corrosion resistance • Outer perimeter of blister faintly visible by FESEM (see middle image of last slide); cracking occurred within shell • Combination of void and/or high surface blisters presumably contributed to increased MB staining
  10. 10. Summary • Quantitative MB staining may be achieved using appropriately diluted MB solutions • In this case, acceptable and defective glass vials were clearly detectable by differences in quantitative staining behavior • The degree to which these vials were acceptable or defective is not known. Further method development is needed to determine if vials that are barely acceptable or defective by standard measures can be resolved • If found to be sufficiently sensitive, quantitative MB staining could provide a simple and inexpensive alternative method for detecting defective vials and monitoring the vial manufacturing process
  11. 11. Acknowledgements • UV-Vis results and lower magnification SEM results were generated by undergraduate and graduate students (Tim Keenan, Kelsey Gloss, and Simon Chon) working in my research group at Alfred University • High magnification imaging of blister cross-sections were provided in collaboration with Jeff Shallenberger at Evans Analytical Group Contact information • Office phone: 607-871-3143 • Assistant’s phone: 607-871-2486 • Email: hallmm@alfred.edu

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