Category: Manufacturing and Bioprocessing
Purpose: Many aluminum-adjuvanted vaccines are freeze-sensitive products that require attentive cold chain adherence. Vaccine freezing may reduce the immunogenicity of the vaccine and should be prevented. In the US, vaccines that have experienced subzero temperatures are set aside or discarded. The World Health Organization uses the “Shake Test” to compare one vial in a batch that is suspected to have experienced subzero temperatures with a vial that has been intentionally frozen. The shake test relies upon visually apparent changes in a vaccine vial that has been frozen. This qualitative test of a single vial in a batch determines the fate of the entire batch, takes 5 to 45 minutes to complete, and requires overnight freezing of a control vial. A relatively rapid, new quantitative method for measuring vaccine freezing was explored in this project.
Methods: Three vaccines containing three different aluminum adjuvants were purchased from the U.S. market. The vaccines contained Al3+ in quantities of 0.33 mg per dose/vial (0.66 mg/mL) for Daptacel® as aluminum phosphate, 0.5 mg per dose/vial (0.5 mg/mL) for Engerix-B® as aluminum hydroxide, and 0.45 mg per dose/vial (0.45 mg/mL) for VAQTA® as amorphous aluminum hydroxyphosphate sulfate. Each vaccine carton contained 10 vials. For each vaccine, 5 vials were maintained at proper storage conditions (2-8°C) and 5 vials were exposed to subzero (-18°C) temperatures overnight. Exposure to subzero temperatures and thawing at 4°C was repeated three separate times. Visual inspection was performed upon removal from subzero temperature exposure. Any frozen vials were thawed at 4°C and maintained at 2-8°C for benchtop NMR relaxometry experiments. The water proton (1H2O) transverse relaxation time, T2, for each intact vaccine vial was obtained by a single exponential fitting of an echo signal decay, collected from the Carl-Purcell-Meiboom-Gill (CPMG) pulse sequence. The relaxation rates, R2, for each vaccine vial were calculated (T2 = 1/R2).
Results: Vaccine vials were initially monitored over six non-consecutive days. The average R2(1H2O) for the 6-day monitoring phase prior to freeze/thaw experiments, with standard deviation of 6 measurements (from 6 different days) is: Daptacel® 2.541 ± 0.016 s-1, Engerix-B® 1.239 ± 0.002 s-1, and VAQTA® 3.238 ± 0.011 s-1. After each overnight exposure to subzero temperatures, the vials that were visually noted as frozen had a significantly lower R2(1H2O) than the control vials that did not experience subzero temperatures. Changes in the product due to freezing were detected by R2(1H2O) measurements. Visual observation also revealed that within one carton of vaccines, of vials exposed to the same subzero temperature conditions, some froze and others did not. This vial-to-vial variability was apparent in the R2(1H2O) measurement. Vaccine vials that were exposed to subzero temperatures overnight but did not freeze had a R2(1H2O) that matched the control vaccine vials that were never exposed to subzero temperatures.
Conclusion: The R2(1H2O) detects prior freezing in thawed vaccine vials non-invasively. Freezing varies between vaccine products exposed to the same subzero temperatures, and also varies within a product carton. Vaccine vials exposed to subzero temperatures that did not freeze have a significantly different R2(1H2O) from vaccine vials that froze. These results indicate that R2(1H2O) may be a useful tool in determining whether a vaccine vial has experienced freezing during transportation or storage.