Vaccines are great success in medical science and provide immunity against a variety of infectious diseases. However, vaccines which have prepared from pathogenic microorganisms or their metabolites, always present safety risks. To confirm their safety and efficacy, it is important to choose the right inactivator agent [1]. For decades, inactivated viral vaccines have been applied effectively in immunization programs. As they do not comprise replicating viruses, they are regularly an ideal product class for certain people, such as pregnant women and immunocompromised people [2]. In the vaccine and antiserum industry, inactivated viral preparations are so usable [3].
The current COVID-19 pandemic, caused by the SARS CoV-2 coronavirus, has threatened human lives around the world and continues to spread, affecting thousands of people every day. This disease has been characterized by an acute respiratory illness that leads to severe breathing problems and is very similar to the common flu with severe cough in affected people, which leads to many of them being hospitalized. Additionally, there have been multiple reports of sepsis, blood clots, and multiple organ failure in several individuals infected with the virus. The severity of the disease is much higher in people with underlying diseases such as diabetes, high blood pressure, cancer and respiratory problems [3]. This worldwide pandemic resulting in over 600 million infections and over 6 million deaths [4].
Due to the new nature of the disease, there is currently no drug to definitively treat Covid-19. Assessments have focused on promising drugs including remdesivir, lopinovar/retinovar, favipiravir, hydroxychloroquine, monoclonal antibodies, and vaccines against SARS-CoV-2 infection. Among them, the covid vaccines showed the best effectiveness in control of the epidemy. Antibody-based interventional therapies are very important in the treatment of severe cases. In some of these developments, inactive virus particles are used for immunization accompanied by adjuvants. Large-scale cultures of virus and antigens are critical for the successful production of whole virus vaccines and antisera. This needs complete virus inactivation while causing minimal structural and antigenic destruction to maintain the induction of an antibody response. Vaccines have been developed using different methods including DNA, RNA, viral vectors and subunit proteins against this extremely contagious respiratory disease. The spike protein has been investigated by scientists around the world to develop potential vaccines. Antiviral drugs, antibodies, and vaccines have been advanced by numerous researchers all over the world and entered human clinical trials [3, 5]. Due to prior studies on the prevention and control of seasonal influenza, it has been recommended to produce vaccines based on whole virus particles, comprising attenuated and inactivated virus vaccines. Both inactivated and attenuated virus vaccines have their own disadvantages and complications [6].
SARS-CoV-2 (Wuhan-Hu-1) has been sequenced first on January 5, 2020 and its genome sequence has been submitted to GenBank. SARS-CoV-2, similar to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), is an enveloped, single- and positive-stranded RNA virus. Mature SARS-CoV-2 has four structural proteins, which are envelope (E), membrane (M), nucleocapsid (N) the response of CD4+/CD8 + T-cells [6].
There are several methods for inactivating and spike (S). All these proteins may act as antigens to stimulate neutralizing antibodies and increase viral stocks that are used to produce vaccines and antiserum. Inactivation could be carried out by means of chemical or physical procedures or a combination of the two. A wide range of new and well-established inactivation agents or methods have been defined for successful virus inactivation in vaccine preparation. For example, ascorbic acid, psoralens, gamma and ultraviolet radiation, ethylenimine derivatives, hydrogen peroxide, heat and many others can be mentioned. However, only formaldehyde and β-propiolactone (BPL) have been widely used for decades to inactivate licensed human virus vaccines [7]. SARS-CoV-2 has been reported to be inactivated by temperatures starting at 56°C [8, 9]. BPL-mediated inactivation of SARS-CoV and SARS-CoV-2 has been confirmed [8, 10–12]. Heating is identified to denature proteins which may adversely affect their antigenicity. γ-irradiation has been used in numerous vaccine studies, but has been found to destruct antigens if not appropriately optimized. Although formaldehyde is one of the oldest and most widely available chemical agents for virus inactivation, also destroys the antigenicity of the protein and therefore it is not very desirable. BPL has appeared as a very general chemical agent in different vaccine designs due to its high inactivation potency and moderately little damage to antigens. BPL has been shown to damage the genetic content, but its effect on virion proteins is less well known [3]. Advantages of BPL in comparison to formaldehyde have included significantly shorter inactivation time, lower inactivation temperature which may avoid thermal degradation of main epitopes and less altering of the protein moieties, due to the initial reaction of the compound with nucleic acids [7]. BPL mainly modifies viral DNA or RNA over acylation or alkylation to inactivate the virus [13]. The structure of BPL has been shown in Fig. 1.
Figure 1 Structure of β-propiolactone (BPL)
In several studies, the half-life of BPL at 37˚C has been shown to vary from 24 to 32 minutes and at 4˚C from 16 to 20 hours. Virus inactivation by BPL is achieved to undetectable levels in 15 minutes at 37˚C and in 8 hours at 4˚C [14]. BPL has recently been used in developing inactivated SARS-CoV-2 vaccine preparation. The results have illustrated that SARS-CoV-2 incubation (1x 106 pfu) in solution with 0.5% BPL for 16 hours at 4°C followed by 2 hours incubation at 37°C led to complete inactivation of infectious SARS-CoV-2 and then hydrolysis of all residual BPL which is necessary for insurance of complete hydrolysis of BPL in order to avoid cytotoxicity to mammalian cells [8].
BPL has been reported to be carcinogen, which should be given more attention in the clinic. Due to BPL structure, it contains two types of active carbon atoms, carbonyl carbon atom and β-carbon atom, which can react with various nucleophiles and may cause toxicity in the human body. Therefore, it is essential to develop a high-sensitivity method for the determination of BPL residue in vaccines. BPL residue must not be detected in purified samples to confirm the purification step and quality control of the product. The study of Shuo Lei, et al about BPL residue determination in inactivated rabies vaccines has demonstrated presence of BPL in three unpurified samples batches, but has not been detected in the purified samples, which has indicated qualification of the test samples [1].
Inactivated vaccines against SARS-CoV-2 have been developed by some vaccine manufacturers, including Sinopharm BIBP vaccine which produced by China National Pharmaceutical Group (Sinopharm) and Beijing Institute of Biological Products (BIBP) (a Vero cell-based, aluminum hydroxide-adjuvanted, β-propiolactone-inactivated vaccine based on HB02 strain) [2] and Covaxin (India's first vaccine which have developed by Bharat Biotech in collaboration with the Indian Council of Medical Research (ICMR) and the National Institute of Virology (NIV)[5].
Numerous analytical methods have been reported for determining of BPL residue in vaccines, mostly high-performance liquid chromatography (HPLC) and gas chromatography (GC). Though, as a result of weak BPL ultraviolet (UV) absorption, it is not recommended to be determined by HPLC with a UV detector. It has been reported that the GC method for BPL determination can give more reliable results [1]. In order to further optimize the determination of the negligible amount of BPL, an analytical method based on GC-MS has been developed [1], although the optimal device conditions and sample preparation details are not mentioned in that study. GC is a standard analytical method which has been applied in development and quality control in industry, including drug residues determination. Coupling MS with GC can be the most effective method for the complex mixtures analysis because of the selectivity and structural information of MS. This combination, increases the sensitivity of identification and elucidation of the compounds structure. In all types of GC-MS, as in other GC ionization detectors, ions are created by electron or chemical ionization but now are classified by molecular weight (or mass-to-charge m/z ratio). Semi-volatile and volatile compounds can be separated by GC, but cannot be identified, but it is possible with MS [15].
Due to the newness of the covid vaccines and the urgency of their entry into the market due to the high mortality of this disease, no complete studies have been done on them, especially on their quality control, and still the regulators and pharmacopoeias have not set the BPL limit for these inactivated vaccines and hence the need for further investigation and studies in this field is felt. Therefore, in order to ensure the safety of these vaccines, it seems necessary to design methods to evaluate the amount of BPL remaining in inactivated covid vaccines after purification. We deal with this in this study, which is a kind of post marketing study.