Indian Pediatrics 2003; 40:311-319 

Thermostability of Vaccines

Cold chain is the system of transporting, storing and distributing vaccine in a potent state at recommended temperature till it is administered to an individual. It is the vital link between the child and immunity in immunisation against vaccine preventable diseases (VPD). However potent a vaccine may be, if cold chain is not maintained from the manufacturer to the place of vaccination, the vaccine efficacy greatly suffers(1). Most vaccines lose their potency by heat and sunlight and hence need protection from both(2).

In spite of better development of safe transport and storage facilities, there are still weak points in the cold chain(3). The avoidable errors are reported even from developed countries and include high temperature during storage and transport, exposure of adsorbed vaccine to freezing temperature, refrigerator without thermo-meters and lack of regular recordings of temperature, refrigerator not being used exclusively for vaccine, failure to discard unused vaccine and use of reconstituted vaccines after exposure at ambient tempera-ture(4). Each exposure of the vaccine to an ambient temperature has a cumulative effect on reducing its potency. This is of concern in view of the fairly frequent reports of VPD occurrence in populations thought to have been well immunized(5).

There is no simple and cheap method that can be used in the field to assess whether an exposed vaccine has retained at least the minimum required potency. The available methods like accelerated degradation test, viral titers and biological assays are costly and time consuming taking several months. But the vaccine vial monitor (VVM) now provided with OPV can indicate the level of heat exposure of that particular vial. Know-ledge of vaccine’s stability, especially of the rate of decline in potency at a given temperature can be helpful in determining its storage requirements.

Vaccine Vial Monitors (VVM)

VVMs, which measure exposure to heat, are time and temperature sensitive labels attached to vials of vaccine at the time of manufacture. Through a gradual colour change they warn about the falling potency. They are designed to meet the vaccine’s heat stability curve as per WHO or manufacturer requirements(6). VVMs were first introduced on OPV vials supplied to UNICEF and WHO in 1996.

The information delivered by a VVM is simple. If the inner square is of lighter colour than the outer reference ring, the vaccine can be used. If the inner square is of the same colour or darker than outer ring, the vaccine should not be used (Fig. 1). The VVMs can be seen as a catalyst for much needed changes in strategies of vaccine distribution via the cold chain. It will definitely reduce distribution costs and increase flexibility in handling of vaccines in the field, thus helping to make operations more effective.
 

Fig 1. Vaccine vial monitor

Operationalisation of Cold Chain

The cold chain is not simply a series of warehouses and refrigerated containers, isothermic boxes and portable ice-boxes, it also involves intermediate phases in which transporters, programme administrators, warehouse workers and vaccinators have a part to play. The cold chain involves two complementary aspects: (i) the set chain represented by the refrigerator, (ii) the mobile chain represented by isothermic boxes and iceboxes, and (iii) the third and an important aspect is the personnel in charge of cold chain(5,7).

Equipments

(a) Walk in cold rooms (WIC): They are located at regional levels and are meant to store vaccines up to 3 months and they serve 4-5 districts.

(b) Deep freezers and domestic/ice lined refrigerators (ILR) (300 L): They are supplied to all districts and WIC locations to store vaccines. They are used for long term storage of vaccine below –20º C and also for making ice packs. OPV and measles vaccines can also be stored in deep freezers. The temperature monitoring is done twice a day with an alcohol thermometer for deep freezer and a dial thermometer in case of ILR.

(c) Small deep freezers and ILRs (140 L) are provided to PHCs and clinics.

(d) Cold boxes or isothermic boxes are well insulated, solid and hermetically (air tight) sealed boxes. They are supplied to all peripheral centers and used for trans-portation of large amount of vaccines and to carry them for several days. They are also useful during electricity failure. Cold accumulators are placed between the boxes of vaccine and the sides of a box. A paper or polythene is kept between vaccine and accumulators to prevent freezing of vaccines because of direct contact.

(e) Vaccine carrier or ice boxes are used to carry small quantity of vaccines for distribution or to carry the vaccines to the outreach place of immunization session. It is surrounded by cold accumulators from inside.

(f ) Day carriers are of the size of a small lunch box and are used to carry still smaller quantity of vaccines, but can be used only for a few hours. Here two fully frozen ice packs are used.

(g) Ice packs should be made of plain water. Never add salt to the water. Water should be filled in the icepack up to the level marked. Dry carbon dioxide can also be used instead of water.

(h) Distribution of vaccines: Only small quantity of vaccine is distributed to the periphery as break in cold chain is common at the periphery because of either lack of knowledge or electricity failure. Vacuum flask is never used for an outreach place.

Refrigerator

Domestic refrigerator is used for short-term storage of vaccines. The usual temperature of an ordinary chamber of the refrigerator is between 4ºC to 10ºC and that of ice chamber or freezer compartment is between 0ºC to –4ºC. Tips for proper functioning of the refrigerator are provided below(1,2,5,7).

Keep the refrigerator away from sunlight and at sufficient (10 cm) distance from the wall. Place cold accumulators in freezer compartment. They can be of use during electricity failure or in iceboxes. The shelves should be filled with water filled plastic bottles, which help to maintain temperature during electricity breakdown. These bottles are not to be used for drinking purpose. The temperature of the refrigerator is recorded at least twice a day. The length of electricity breakdown is to be noted and during this period vaccines are to be protected by putting them in isothermic boxes. When a layer of ice measuring 5 mm or more forms on the sides of the freezer compartment, the refrigerator should be defrosted. During defrosting, the vaccines should be temporarily placed in an isothermic box.

Tips for proper storage

No foods or drinks or other drugs are to be kept in a refrigerator. It is to be used exclusively for storage of vaccines. To maintain the temperature, it is opened only when necessary. No vaccine should be placed in the door panel or in baffle tray (baffle tray may contain water and this may freeze to ice). Direct contact of vaccine with ice is to be avoided. An empty space must be left between packages for free air circulation. Minimum stock of vaccine is to be kept, say for a month only. Avoid freezing of diluents as the vial may burst when frozen. Correct storage of vaccines in clinical set up is shown in Fig. 2.

Thermostability of Vaccines

Knowledge of a vaccine’s stability, especially the rate of decline in potency at a given temperature, can be helpful in determining the storage requirements(3). Adsorbed diphtheria and tetanus toxoids are the most stable of the vaccines commonly used in UIP and OPV is the most sensitive to heat. Opened vials which have not been fully utilized after reconstitution should be discarded within one hour, if no preservative is present (most live virus vaccine) or within 3 hours, or at the end of the session when the vaccines containing preservative are used(4). Reconstituting the vaccine with warm diluent may be harmful and vaccine loses its potency within hours.

Diphtheria and tetanus toxoids

Adsorbed diphtheria and tetanus toxoids in monovalent form or as components of combined vaccines are the most stable of the commonly used vaccines. They are stable at elevated temperatures, even for long periods of storage, but they may change their appearance and potency when frozen. The freezing of adsorbed vaccines (DPT, DT, TT, HB) constitutes an absolute contraindication for their use as they are associated with a reduced immune response or an increased incidence of local reaction. This is not because of the characteristics of the toxoids them-selves, but because of an aluminium-based adjuvant, which changes its structure. The freezing point for adsorbed DTP vaccine is between –5ºC and –10ºC. The freezing time depends upon the number of doses in the vial and the temperature. It takes about 110 to 130 minutes at –10ºC. When vaccine is frozen, the aluminium oxide loses its colloidal structure and is broken down into crystalline parts, which may cause aseptic abscesses at the injection site and make the vaccine ineffective. Frozen adsorbed vaccines contain granular or flaky particles when thawed. When shaken, they sediment within 30 minutes, leaving a deposit below a column of transparent fluid. This indicates that the vaccine has been frozen (shake test)(7,8). The thermostability is as shown in Table I. Several studies showed an insignificant decrease in potency when the vaccine was stored for 1.5 years at 18º C, for 6-12 months at 24º C and for 2-6 months at 37º C(9,10).

Pertussis Vaccine

Thermostability of this vaccine is as described in the Table I. The effect of freezing the vaccine is same as with DPT. There are no data available for acellular pertussis vaccine and so the stability profile similar to that of other protein vaccines is to be expected i.e., relatively good thermostability, poor resis-tance to freezing and shelf life of 2-3 years at 2ºC to 8ºC(11).

Table I__Stability of Commonly Used Vaccines(3)

Vaccine	Storage temperature (ºC)
	0-8	22-25	35-37	Over 37
Tetanus and diphtheria toxoids as monovalent or component of combined vaccines	Stable for	Stable for Months	Stable for	At 45ºC stable for
	3-7 years		weeks	2 weeks. At 53ºC
				loss of potency
				after few days. At
				60-65ºC loss of
				potency after
				few hours.
Hepatitis B	Stable for 2-4	Stable for months	Stable for weeks	At 45ºC Stable
	years			for days
Measles	Stable for 2	Satisfactory	Satisfactory	At 41ºC: 50%
	years	potency up to 50%	potency up to 50%	loss of potency
		for at least 1 month	for at least 1 week,	after 2-3 days;
			but may lose 20%	at 54ºC: 80%
			for 1-4 days and	loss after 1 day.
			50% for 2-6 days
			exposure
Pertussis	Stable for 18-24	Stability varies:	Stability varies:	At 45ºC:
	months inspite of	some vaccines	some lose 50%	about 10% loss of
	continuous slow	stable for 2 weeks	during storage for	potency per day.
	decrease in		one week	At 50ºC: rapid
	potency			loss in potency.
BCG	Stable for 1 year	Stability varies: 20-	Stability varies:	Unstable: at
		30% loss of	20% loss of	70ºC 50% loss
		viability during 3	viability during 3-	during 30
		month exposure	14 day exposure	minute exposure
OPV	Stable for 6-12	Some vaccines may	Unstable: VVM	Very unstable:
	month	retain titers for 1-2	in use. Loss of	At 41ºC 50%
		week exposure	satisfactory titer in	loss in one day.
			1-3 days.	At 50ºC loss of
				satisfactory titer
				after 1-3 hour
				exposure.
Rabies HDCV	Stable for 3-5	Retained	Stable for 4 weeks	No data available
	years	immunogenicity for
		up to 11 weeks
Live oral	Needs	Prolonged storage	Rapid decrease in	No data available
tyhpoid Ty21a	refrigeration,	resulted in	viable count.
	shelf life	progressively lower	Retains minimum
	depends upon	viable count	potency after 12
	residual moisture		hours exposure
	content

 

Hepatitis B Vaccine

HB vaccine is a liquid suspension consisting of purified hepatitis B surface antigen (HBsAg) adsorbed on aluminium salt. At temperatures of 2ºC to 8ºC, HB vaccine appears to be stable for many years, but the upper limit of storage life has not been defined (average being 4 years). Several studies revealed that vaccine heated up to different temperature for a varied time did not alter reactogenicity and was equally effective. In one study, at room temperature of 20ºC to 26ºC, vaccines from three manufacturers were found stable for at least one year. One brand was found to be stable and effective after exposure at 45ºC for one week and 37ºC for one month(12). It is thus in the upper range of heat stability, together with tetanus and diphtheria toxoids. Although HB vaccine is extremely heat stable, there are not yet enough data to recommend using it entirely outside the cold chain. There is however scope for developing a management instruction that would allow removal of the vaccine from the cold chain in emergencies or in outreach activities of short duration. This vaccine is not to be frozen as with other adsorbed vaccines. The freezing point for HB vaccine is about –0.5ºC.

Measles Vaccine

In recent years significant progress has been made in improving heat stability of measles vaccine because of laid down WHO criteria as given below and use of effective stabilizers(13). The criteria are: (i) freeze dried vaccine should retain at least 1000 live virus particles in each dose at the end of incubation at 37ºC for seven days, and (ii) if during such process titer is decreased, then it shall have done so by not more than 1 log10.

Freeze dried measles vaccine in lyophilized form is extremely stable and is not damaged by freezing and refreezing. But it quickly loses its potency when reconstituted and kept at elevated temperatures. Thermo-stability of measles vaccine is as shown in the Table I. The reconstituted measles vaccine remains potent for 24 hours at 4ºC and for 16 to 24 hours at 26ºC(14). But because of possibility of contamination it is not used for more than 6 hours i.e. for a single session, at whatever temperature it is stored. Even during this period it should be protected from elevated temperature and from light.

BCG Vaccine

BCG was the first vaccine for which a WHO requirement for heat stability was established. But the standardization of the stability and studies on it are complicated because of different strains and manufacturing methods. Accelerated degradation test should be conducted on each lot of vaccine. Studies have shown that longer storage of vaccine at elevated temperature reduces post vaccination allergy and size of vaccination lesions. Most freeze dried BCG vaccines are stable at temperatures of 0ºC to 8ºC(15).

The stability of the BCG vaccine is affected with lyophilisation, stabilizer used and proper sealing of vaccine ampoules. BCG vaccine sealed under vacuum is more stable than one sealed with nitrogen and argon(15). BCG vaccines in rubber stoppered vials have a lower stability than those conserved in ampoules(16). BCG vaccine should be packed in amber glass ampoules to protect it from ultraviolet and fluorescent light(17).

As with other lyophilized vaccines, BCG vaccine also should be discarded after 4-6 hours of reconstitution because of risk of contamination due to lack of preservative and loss of potency.

Oral Polio Virus Vaccine (OPV)

Though OPV vaccine is the least stable vaccine, its stability has recently been improved with the use of stabilizers like magnesium chloride. Half-lives of different OPV vaccines when tested in an Indian study were found to be 4.3 days at 22ºC and 1.7 days at 36ºC(18). OPV supplied by most manufacturers are stable for an extended period of up to 2 years at –20ºC, for over 6 months at 2ºC to 8ºC and for over 48 hours at 37ºC. OPV vaccine vial in current use can be stored at 2º to 8º C in the central compartment of the refrigerator. The freezing point varies from –6.6ºC to –8.1°C. Most of the times at the temperature –5ºC of the freezer compartment, OPV may not remain solid. Repeated thawing and freezing up to 180 cycles within range of –25ºC to 2.5ºC temperature did not show any decline in the titer(19). However, here the maximum temperature did not exceed 2.1ºC. Under field conditions, a break in the cold chain can result in vaccines reaching much higher tempera-tures. Consequently these results are valid only for situations where the temperature of thawed vaccine remains in the range found in a refrigerator which is working properly. Repeated thawing of OPV should be avoided for all practical purposes(l). The WHO recommendation is that OPV should not be kept at refrigerator temperatures between 0º C to 8ºC at health centres for more than one month, nor transported at these temperatures for more than one week(20). Use ofVVM has made the problem of recognizing the stability of the vaccine easier.

Thermostability of OPV also depends upon nature of virus type, nature of stabilizer, pH value of virus suspension, tightness of stopper and amount of air space above the vaccine.

Mumps and Rubella Vaccine

Thermostability of mumps is similar to that of measles. The mumps component in Indian MMR shows good stability at 37°C for up to 21 days. The freeze dried monovalent rubella vaccine and the rubella component of combined vaccine show low degradation rate. Rubella is more stable than other components of combined vaccine.

Hepatitis A Vaccine

This is inactivated vaccine and aluminium is used as an adjuvant. No loss of immunogenicity was observed at 37ºC for up to 3 weeks(21).

Haemophilus influenzae typeB (Hib) Vaccine

PRP-T (Lyophilised) Hib vaccine is stable at refrigerator temperatures for 36 months and at 25ºC for at least 24 months. After reconstitution it should be discarded within six hours. Liquid Hib is stable at refrigerator temperature for 24 months.

Typhoid Vaccine

Vi polysaccharide vaccine is highly stable and it does not require a cold chain even in tropical conditions. This is the distinct advantage of this vaccine. Immunogenicity of the vaccine is maintained after storage at 37°C for 6 months and at 22°C for 3 years(22). However it is to be stored in a refrigerator to minimize degradation.

Live oral typhoid vaccine Ty21a is to be stored at 2ºC to 8ºC. Three lots of vaccine stored at 37ºC for 12 hours maintained their potency. Evaluation of vaccine after storage for seven days at 20ºC to 25ºC met the potency requirements(23).

Varicella Vaccine

This vaccine is sensitive to light and is readily inactivated. So it is to be kept away from direct light before and after reconstitution. Stability is better at –1ºC. It is stable for 2 years at 2ºC to 8ºC. It is to be administered within 30 minutes of reconstitution(24,25).

Inactivated Poliovirus Vaccine (IPV)

There are differences in heat stability between various inactivated poliovirus types, with type 1 being most vulnerable. The stability of the vaccine decreases with thiomersal used as preservative. The D-antigen content for type 1 drops significantly after 20 days at 24ºC and is undetectable after exposure to 32ºC for the same period. Type 2 and type 3 are relatively more stable(26). The trivalent IPV is stable at 2ºC to 4ºC for 1-4 years.

Future Trends

Vaccine distribution without a cold chain would considerably simplify the delivery system and make it easier to integrate with drug distribution in developing countries. Sugar-glass drying technology allows vaccines to be made which can be stored and transported routinely at tropical room temperatures(27). Extremes of temperature can be monitored by VVMs.

Long term stabilising ability of trehalose -a disaccharide is made use of in drying and stabilizing technology in vaccine manu-facturing. Dried measles vaccine stabilized with trehalose is found to be stable for two months at room temperature and DTaP for 12 weeks at 60ºC. Only OPV has failed to dry successfully. Though the results are encouraging, the high cost of regulation and lack of a sure market have prevented any sugar dried vaccine product from being licensed.

New multivalent vaccines stabilized with this technology would be regulated for shelf-life storage at temperate or tropical room temperatures. Once all vaccines have been stabilized there will no longer be a need for refrigeration equipment and the associated maintenance. As a consequence the global savings annually will amount to approximately $200 million.

Contributors: KRL conceptualized the manuscript and reviewed the literature. KRL and MKL drafted the paper. MKL will act as the guarantor for the paper.

Funding: None.

Competiting interest: None stated.

Key Messages

• To ensure the optimal potency of vaccines, its storage and maintaining cold chain needs careful attention. • OPV is sensitive to heat and other vaccines are sensitive to freezing. • The stability of vaccines can be ranked: diphtheria, TT, HB showing highest thermo- stability; measles, yellow fever and BCG occupying the middle position and OPV being the most fragile.

 

 References

 

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