Thermostability of Vaccines in the Indian Context: A Review of Progress and Perspectives

Vaccination saves millions of lives each year, and the effectiveness of immunization programs relies heavily on the stability of vaccines. Since most vaccines are sensitive to temperature variations, it is essential to maintain a cold chain and store them consistently between 2°C and 8°C throughout the entire process—from manufacturing to administration—to preserve their potency [1]. Thermal sensitivity is a major factor                influencing both the potency and overall quality of vaccines. To ensure their stability, cold chain systems have been implemented. However, even within these systems, vaccines remain vulnerable to temperature excursions, which can compromise their effectiveness. Several factors contribute to failures in maintaining cold chain integrity, including fuel shortages, malfunctioning or outdated refrigeration units, and inadequate adherence to proper cold chain protocols—all of which can lead to temperature fluctuations [2].The thermal sensitivity impact on the distribution of vaccine worldwide and concern to the philanthropic organizations, government Institutions, health authorities and vaccine industry attempting to enhance the distribution of vaccines [3]. Aluminium-based adjuvants have traditionally been used to enhance vaccine thermostability; however, growing concerns regarding their potential neurotoxic effects and possible links to autoimmune disorders have led to the exploration of alternative strategies. To ensure the stability and safety of vaccines, the World Health Organization (WHO) has established specific guidelines for their evaluation [4]. These guidelines offer a structured framework for determining appropriate storage conditions and shelf life, and they help identify various factors that can influence vaccine stability [5]. Various methods are available for estimating the shelf life of vaccines, many of which are outlined by the International Council for Harmonisation (ICH) guidelines [6]. Shelf-life estimation is commonly performed using statistical models, including both linear and non-linear regression analyses, often supported by pourability tests. To enhance the accuracy and robustness of the ICH procedures, additional approaches such as quantile regression, consideration of batch effects, and mixed model tolerance interval methods have been suggested [ 7,8]. n the past, lyophilization has been used to produce dry powder formulations of live attenuated vaccines, significantly improving their stability by reducing moisture content to typically below 3% [ 9]. Despite the use of drying techniques, many vaccines still depend on cold chain storage. Subunit vaccines containing aluminium salt adjuvants are particularly challenging to lyophilize, as the process can compromise immunogenicity and alter particle size. To overcome these limitations, alternative drying methods such as vacuum-foam drying, spray-freeze drying, supercritical fluid drying, and spray drying have recently been explored to enhance both the thermostability and flexibility of vaccine formulations [10–12]. The formulation of vaccines plays a pivotal role in the overall development process, including approval, testing, and large-scale production. It involves transforming the vaccine antigen into a medicinal product suitable for administration. The formulation process encompasses several key stages—from the initial identification of immunogenic components to the creation of a stable, effective vaccine. This includes developing stability-indicating assays (such as potency testing), characterizing the chemical and physical properties of antigens, optimizing the route of administration and choice of adjuvants, and designing formulations that improve the vaccine’s stability, immune response, and shelf life. A primary objective in vaccine formulation is to enhance immunogenicity using adjuvants, which not only amplify the immune response but also guide it effectively toward both humoral and cellular immunity.

1.Types of thermostability of vaccines

Understanding how stable a vaccine is, particularly how quickly its potency decreases at a specific temperature, is important for determining its proper storage conditions. [13]. Among the vaccines commonly administered in the UIP, adsorbed diphtheria and tetanus toxoids exhibit the highest stability, while OPV is highly heat-sensitive. Once reconstituted, vials without preservatives (such as most live virus vaccines) should be discarded within one hour, whereas those containing preservatives may be used for up to three hours or until the end of the aimmunization session [14].

A] Diphtheria and tetanus toxoids

Adsorbed diphtheria and tetanus toxoids, whether in monovalent form or as part of combination vaccines, are among the most stable vaccines in common use. They can tolerate elevated temperatures for extended storage periods; however, freezing can alter both their appearance and potency. Freezing of adsorbed vaccines such as DPT, DT, TT, and HB is strictly contraindicated, as it can lead to reduced immune response or increased local reactions. This effect is not due to the toxoids themselves, but to the aluminium-based adjuvant, which undergoes structural changes upon freezing. The freezing point of adsorbed DTP vaccines lies between –5°C and –10°C, and the time required to freeze depends on the vial’s dose volume and ambient temperature—typically about 110 to 130 minutes at –10°C. Once frozen, aluminium oxide loses its colloidal form and breaks into crystalline particles, which can cause aseptic abscesses at the injection site and render the vaccine ineffective. Thawed frozen vaccines often display granular or flaky particles, and when shaken, these settle within 30 minutes, leaving a sediment beneath a clear liquid column. This sedimentation pattern, known as the “shake test,” confirms prior freezing. Studies have shown that potency loss is minimal when the vaccine is stored for up to 1.5 years at 18°C, for 6–12 months at 24°C, and for 2–6 months at 37°C, as outlined in Table I.[15,16,17,18].

B] Pertussis Vaccine

The thermostability of this vaccine is presented in Table I. Freezing affect. the same way as DPT. Although no specific data exist for the acellular pertussis vaccine, its stability is expected to be comparable to other protein-based vaccines—showing relatively good heat stability, low tolerance to freezing, and a shelf life of about 2–3 years when stored at 2°C to 8°C. [19].

World Health Organization (WHO): Vaccine Storage and Handling Guidelines, various vaccine position papers (2015–2018).

C] Hepatitis B Vaccine

The hepatitis B vaccine is formulated as a liquid suspension containing purified hepatitis B surface antigen (HBsAg) adsorbed onto aluminium salts. When stored between 2°C and 8°C, it maintains stability for many years, with an average shelf life of around four years, although no absolute maximum has been established. Research indicates that heating the vaccine to various temperatures for different durations does not significantly affect its safety or effectiveness. In one investigation, products from three manufacturers retained stability for at least one year at ambient temperatures of 20°C to 26°C. Additionally, one brand remained stable and effective even after being exposed to 45°C for one week or 37°C for one month. [20]. 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.

D] Measles Vaccine

In recent years, notable advancements have been achieved in enhancing the heat stability of the measles vaccine, largely due to established WHO guidelines and the incorporation of effective stabilizing agents. [21]. The WHO criteria specify that: (i) a freeze-dried measles vaccine must contain a minimum of 1,000 live virus particles per dose after being incubated at 37°C for seven days, and (ii) any reduction in viral titer during this process should not exceed 1 log??. Freeze-dried measles vaccine in its lyophilized form is highly stable and can withstand freezing and refreezing without damage. However, once reconstituted, its potency declines rapidly at higher temperatures. As shown in Table I, the reconstituted vaccine remains effective for about 24 hours when stored at 4°C and for approximately 16–24 hours at 26°C. [22]. Due to the risk of contamination, the reconstituted vaccine should be used within six hours, or for a single immunization session, regardless of the storage temperature. During this time, it must be kept away from high temperatures and protected from light.

E] BCG Vaccine

BCG was the first vaccine to have a WHO-defined heat stability requirement. However, standardizing its stability profile is challenging due to variations in strains and manufacturing processes. Each vaccine lot should undergo accelerated degradation testing. Research indicates that prolonged storage at higher temperatures can decrease post-vaccination allergic reactions and reduce the size of lesions at the injection site. Most freeze-dried BCG vaccines remain stable when stored between 0°C and 8°C. [23]. The stability of BCG vaccine depends on factors such as the lyophilization process, the type of stabilizer used, and the quality of ampoule sealing. Vaccines sealed under vacuum demonstrate greater stability compared to those sealed with nitrogen or argon. [24]. BCG vaccines sealed under vacuum maintain higher stability than those sealed with nitrogen or argon. Additionally, products stored in rubber-stoppered vials tend to be less stable than those preserved in ampoules. [25]. BCG vaccine should be packaged in amber-coloured glass ampoules to shield it from exposure to ultraviolet and fluorescent light. [26].

F] Oral Polio Virus Vaccine (OPV)

Although OPV is among the least stable vaccines, its durability has been enhanced in recent years through the addition of stabilizers such as magnesium chloride. An Indian study reported that the half-life of various OPV formulations was approximately 4.3 days at 22°C and 1.7 days at 36°C.[27]. OPV from most manufacturers can remain stable for up to two years when stored at –20°C, for more than six months at 2°C to 8°C, and for over 48 hours at 37°C. Vials in current use may be kept at 2°C to 8°C in the central section of a refrigerator. The freezing point ranges from –6.6°C to –8.1°C, and at freezer temperatures around –5°C, OPV may not fully solidify. Studies have shown that repeated thawing and refreezing—up to 180 cycles within the –25°C to 2.5°C range—does not reduce the vaccine’s titer.[28].It should be noted that in the study, the temperature did not exceed 2.1°C. In real-world settings, interruptions in the cold chain can expose vaccines to much higher temperatures. Therefore, these findings apply only when thawed vaccine remains within the temperature range of a properly functioning refrigerator. In practice, repeated thawing of OPV should be avoided. According to WHO guidelines, OPV should not be stored at 0°C to 8°C in health centres for longer than one month, and transport at these temperatures should not exceed one week. [29]. The introduction of Vaccine Vial Monitors (VVMs) has simplified the process of assessing vaccine stability. The heat stability of OPV is influenced by several factors, including the virus type, the stabilizer used, the pH of the suspension, the seal integrity of the vial, and the volume of air space above the vaccine.

G] Mumps and Rubella Vaccine

The heat stability of the mumps vaccine is comparable to that of measles. In Indian MMR formulations, the mumps component remains stable at 37°C for up to 21 days. Freeze-dried monovalent rubella vaccines and the rubella component in combination vaccines exhibit a low rate of degradation, with rubella being more stable than the other components in the combined formulation.

H] Hepatitis A Vaccine

This is an inactivated vaccine formulated with aluminium as an adjuvant. Studies have shown no reduction in immunogenicity when stored at 37°C for up to three weeks. [30].

I] Haemophilus influenzae type B (Hib) Vaccine

The lyophilized PRP-T Hib vaccine remains stable for up to 36 months when stored at refrigerator temperatures and for at least 24 months at 25°C. Once reconstituted, it should be used within six hours. The liquid form of Hib vaccine maintains stability for 24 months under refrigerated conditions.

J] Typhoid Vaccine

The Vi polysaccharide vaccine is exceptionally stable and can remain effective without a cold chain, even in tropical climates, which is a key advantage. Its immunogenicity is preserved for up to six months at 37°C and for three years at 22°C. Nonetheless, refrigeration is recommended to reduce the risk of degradation. [31]. The live oral typhoid vaccine Ty21a should be stored at 2°C to 8°C. In testing, three vaccine lots retained their potency after being kept at 37°C for 12 hours. Further evaluation showed that storage for seven days at 20°C to 25°C still met potency standards. [32].

K] Varicella Vaccine

This vaccine is highly light-sensitive and can be easily inactivated, so it must be protected from direct light both before and after reconstitution. It maintains better stability at –1°C, remains stable for up to two years when stored between 2°C and 8°C, and should be used within 30 minutes after reconstitution. [33,34].

L] Inactivated Poliovirus Vaccine (IPV)

Heat stability varies among inactivated poliovirus types, with type 1 being the most sensitive. When thiomersal is used as a preservative, overall stability declines. For type 1, the D-antigen content falls sharply after 20 days at 24°C and becomes undetectable after 20 days at 32°C. In contrast, types 2 and 3 show greater resilience. Trivalent IPV can remain stable for 1 to 4 years when stored at 2°C to 4°C. [35].

MATERIAL AND METHODS

1.Calcium Phosphate as an Adjuvant in Vaccine Thermostability

C] Thermostability Enhancement

Calcium phosphate as an adjuvant enhances both the physical and chemical stability of vaccines at higher temperatures more effectively than conventional aluminum-based adjuvants like alum. It safeguards the antigen by binding it to its surface, which helps preserve the vaccine’s effectiveness even under heat exposure. Studies have demonstrated that vaccines formulated with calcium phosphate maintain their immune-stimulating properties when stored at temperatures exceeding the typical cold chain range of 2-8°C, making them especially suitable for use in tropical regions such as India.

D]  Biocompatibility and Safety

Calcium phosphate is both biocompatible and biodegradable, with a non-toxic nature. Compared to alum, it tends to produce fewer local reactions and side effects, which enhances the overall acceptance of vaccines formulated with it.

E] Immune Response Enhancement

It functions as a reservoir that gradually releases the antigen over time. This controlled release helps in generating robust humoral and cellular immune responses. Additionally, it activates both Th1 and Th2 pathways, which are essential for sustained and effective immunity.

F] Applications in Thermostable Vaccines

Calcium phosphate has been successfully utilized in vaccines containing diphtheria, tetanus toxoids, and other protein subunit antigens. It shows great potential for vaccines requiring storage beyond stringent cold chain requirements. Studies from vaccine producers in India emphasize CaP as a favored adjuvant for thermostable vaccine formulations because of its affordability and enhanced stability.

G] Manufacturing and cost barriers

Developing vaccines that can tolerate higher temperatures often involves specialized stabilization approaches, including freeze-drying (lyophilization), spray-drying, or embedding the antigen in sugar-based protective matrices. While effective, these methods add layers of technical complexity and raise production expenses. For large-scale public immunization programs in India, such cost increases can be prohibitive. Consequently, manufacturers frequently opt for conventional formulations designed for cold-chain storage, as these allow high-volume production at a lower unit cost. [42].

H] Regulatory and programmatic constraints

Even when a thermostable formulation is developed, extensive stability testing and regulatory approval are required before it can be deployed. India’s vaccine procurement and distribution systems are designed around an existing cold-chain infrastructure, making the integration of new thermostable products slower unless supported by national policy and funding. [43].

I] Environmental logistic challenges in India

 Many regions in India experience extended periods of extreme heat, with temperatures often surpassing 40 °C. In rural and hard-to-reach locations, inconsistent electricity supply further limits reliable refrigeration. These conditions heighten the chances of vaccines being exposed to damaging heat during transportation or field immunization activities. While heat-stable formulations would help mitigate these risks, limited commercial incentives and insufficient focused research mean that most vaccines still rely on conventional cold-chain storage. [44].