How do water-packs and vial location in packaging affect the freezing of vaccines? This article examines the best practices to avoid freezing. The risk of freezing adds a burden to the already complicated cold chain. The cold chain requires that all vaccines be refrigerated, from manufacturing to administration because heat is damaging to drug potency. Prolonged heat exposure is not the only problem that hinders a successful cold chain. Freezing also can be damaging.

Vaccines must be stored within 2°C to 8°C (36°F to 46°F). Temperature excursions are acceptable when they exceed the range only for a short time. When excursions run for hours or days, however, that’s when they become problematic.  

Manufacturers, handlers, and health personnel can employ many equipment and handling techniques to prevent ruptures or temperature excursions. The use of passive cooling technologies during last-mile transport is one technique. Another is using water-packs that can act as a coolant instead of an electrical connection. A final method is the proper positioning of vaccine vials inside the vaccine carriers.

Using these techniques alone may or may not prevent the risk of freezing and heating. The situation is largely dependent on many factors. Water-packs, for instance, can cool vaccines enough within the acceptable temp range. They also, however, can expose the vaccine to damaging heat and cold if the packs did not undergo a process called conditioning.

Conditioning is allowing the frozen water-packs to warm to 0°C (32°F). Using frozen water packs directly from the freezer could expose vaccines to temperatures below freezing.

If water-packs are only partially frozen, they can only cool the vaccines for a limited time, and are not appropriate for long-distance delivery. In short, the temperature of the water-packs plays a vital part in the stability of the vaccines in transport.  

The location of the vaccines in carriers is also important. The closer they are to the frozen packs, the greater the chance of freezing. The farther away, however, the greater the risk of being exposed to heat (beyond 8°C or 56°F).

Cold Life Passive Cooling Challenges

To better understand factors affecting vaccines, PATH, a Seattle-based nonprofit health NGO, conducted a study on how different temperatures of water-packs can affect vaccines. In the study, PATH measured how changing temperatures of water-packs impacted the cold life (how long vials could maintain temperature before vaccine carriers reach 10°C (50°F). In addition, they examined how the positioning of the vaccine vials inside the carriers – how far or near they are from the water-packs – could expose the vaccines to the risk of freezing.

The study used three 2.5L vaccine carriers (AOV International, AVC-46), as shown in the image below. The carriers were placed in an environmental chamber with an ambient temperature of +43°C (+109°F) and relative humidity of 65% for 24 hours. The study also used 12 0.6L water-packs, filled with tap water to the maximum fill line. The varying temperatures of the water-packs were:

  • Frozen to -25°C (-13°F)
  • Frozen to -10°C (+14°F)
  • Cooled to +5°C (+41°F)
  • Partially frozen to -30°C (-22°F) for two hours
  • Partially frozen to -30°C (-22°F) for four hours
  • Partially frozen to -30°C (-22°F) for six hours
Portable vaccine carrier
Environmental testing chamber

The temperature sensor used was a type-T thermocouple (Omega, OSK2K3671-13), which was monitored using National Instruments (NI) data acquisition units (NI 9211 with chassis cDAQ-9172) and NI SignalExpress software. All vaccine vials, water-packs, and carriers used were equipped with a thermocouple. To prevent the vaccine vials from moving, they were fixed in foam form, with a hole cut in the center.

Vaccine carrier with 4 water packs around the side and vaccine vial in the center

When all the water-packs achieved the targeted temperature, they were placed in the warmed carriers. Upon closing the carrier lid, the logging of the temperatures began. The temperature of the water-packs, vials, inside the carriers, and the environmental chambers were recorded every 30 seconds, until the end of the cold life – when the temperature inside the vaccine vials rose to +10°C (+50°F).

Water-Pack Temperatures Determine the Cold Life

Different temperatures of the water-packs resulted in different cold life results. The table below displays the six kinds of water-packs and how long (hours) they provided an acceptable cold range for the vaccines.

Cold life of different vaccine carriers with water packs of different temperatures

The World Health Organization (WHO) has an established rule for cold life. Their Performance, Quality, and Safety (PQS) requirement for long-range vaccine carriers, as used in this study, is 30 hours of cold life. Both the water-packs frozen at -25°C (-13°F) and -10°C (+14°F) surpassed the PQS cold life requirement. None of the partially frozen water-packs, however, achieved this requirement. This means that to achieve a cold life of 30 hours and beyond, water-packs must be completely frozen to either -10°C (+14°F) or -25°C (-13°F). Partial freezing will only last up to 28 hours, two hours shy from what’s required by WHO PQS.

Since the PQS specification does not include a cold life requirement for vaccine carriers, and only for water-packs, the study focused on the temperatures of the vials. When the temperature of the vaccine carriers reached +20℃ (+68°F), that’s when the temperature of the vaccine vials reached the same temperature, thus still adhering to the PQS requirement of +20℃ (+68°F) for the cold life.

In the figures below, the temperatures of all vials, carriers, water-packs, and environmental chambers are displayed all together. It shows how the temperature of water-packs affected everything, especially the vaccines. Two studies were conducted for the -25°C (-13°F) water-packs. Both of these studies state that -25°C (-13°F) frozen water-packs can freeze the vaccines.

Temperatures recorded during –25°C frozen water-pack cold life test using carriers
Temperatures recorded during frozen –25°C water-pack cold life test using carrier 2 (detail).
  • Abbreviations used:
  • Chamber: environmental chamber;
  • C2:  carrier 2
  • V2: vial in carrier 2;
  • C2-IP#: carrier 2 water-pack number 1, 2, 3, 4

On the other hand, the use of -10°C (+14°F) frozen water-packs and -30°C (-22°F) partially frozen water-packs (six hours) respectively exhibited no vaccine freezing. The temperature inside the vial remained in the acceptable temperature range and did not drop to 0°C (+32°F), in contrast to figures 5 and 6 where accidental freezing occurred.

Graph shows that the vaccine vial never froze when water-packs with a temperature of -10°C were used.
Graph shows that the vaccines never froze when partially frozen water-packs were used

In figures 7 and 8, all vaccines did not freeze. The use of water-packs frozen at -10°C (+14°F) and partially frozen at -30°C (-22°F) contributed a lot to these results. But the absence of freezing in both cases raised the question of whether all locations inside the carriers were free of freezing risk. To answer this question, the cold life test of -10°C (+14°F) was repeated, but this time, two vaccine vials were placed in different locations, one in the center, the other at the corner closest to the frozen water-packs.

Can the vial location affect freezing risk?

The study used six vaccine carriers, specifically, the Nilkamal, BCVC 44-A. These were warmed in an environmental chamber at +43℃ (+109.4°F), with a relative humidity of 65% for 24 hours. The study used 24 water-packs filled with 5ml of water, cooled to +5℃ (+41°F) overnight. The same temperature sensors and acquisition software and hardware were used for cold life monitoring.

In performing the cold life test in triplicate, the study yielded nine different data sets for each water-pack freezing temperature. The temperature of the vials, carriers, and the water-packs were very similar to the temperature of the given water-packs. The graph below shows the data for two different water-packs -25°C (-13°F) and -10°C (+14°F).

Graph shows the central and corner vial temperature reading based on a water-pack frozen at -25°C

Both center and corner vials in the -25°C (-13°F) frozen water-packs cooled to temperatures at or below 0°C (+32°F). In eight out of nine tests, the corner vial froze below 0°C (+32°F), then warmed back up to 0°C (+32°F). In six out of nine tests, the center vial had the same reaction as most of the corner vials. In the other three tests, the center vials cooled and warmed up to the same temperature as the water-packs, from 0°C (+32°F), then warmed back up.

Graph shows the central and corner vial temperature reading based on a water-pack frozen at -10°C

In using the -10°C (+14°F) water-packs, only the corner vials cooled at or below 0°C (+32°F). The center vial did not freeze, only cooled between 0°C (+32°F) and +10°C (+50°F).


The PQS cold life requirement for long-range vaccine carriers is 30 hours. To achieve that, water-packs must be frozen at either -25°C (-13°F) or -10°C (+14°F). The other methods used in this study such as the partial freezing never came close to a 30-hour cold life threshold.

Conditioning is still a vital part of the process. It should be noted that conditioning is different from the partial freezing method used in this study. In conditioning, the ice mass in the middle is large enough to still cool the vaccines, while partial freezing may appear frozen because most water-packs freeze from the outside to the inside.

Cooled packs (+5°C or 41°F), on the other hand, are not very useful for vaccine storage and delivery. Partial freezing can last for 20 hours, but cooled packs are way worse; they can only last for 12 minutes.

The vial location tests confirmed that the corner vials are in danger of freezing. In a vaccine carrier, two frozen water-packs are touching the corner vaccine vials which, alone, caused freezing risk. While the center vials exhibited variable temperature reading, in most cases, they did not drop below 0°C (+32°F).

The temperatures of the water-packs do affect the temperatures of the carriers and everything in them. How the water-packs are prepared in the freezer, and how they are properly conditioned, have a direct impact on the efficiency of the vaccines. To prevent freezing damage, the location of the vials in the carriers also plays a role. The nearer they are to the frozen water-packs, the more likely they may become freeze damaged. Handlers and health personnel must always be careful when dealing with these scenarios, so that vaccines are at their best when administered.


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