Preparation of H1N1 microneedles by a low-temperature process without a stabilizer

Abstract

During the manufacture of H1N1 microneedles, a stabilizer is usually added to maintain the antigenicity of the vaccine. However, finding a suitable stabilizer is difficult, and the addition of a stabilizer can limit the antigen dose and the addition of an adjuvant because of the limited volume of the microneedles. In this study, the authors evaluated whether H1N1 microneedles could be fabricated without a stabilizer by keeping the production environment at a low temperature.

H1N1 microneedle patches without a stabilizer were prepared in a process that involved maintaining a low temperature of 10 °C. The protective immune response to this method of drug application was investigated by comparing it with traditional intramuscular (IM) immunization and with the use of H1N1 microneedles with a stabilizer.

A process-sensitive antigen, H1N1, was stabilized without the use of a stabilizer in a process that maintained a low temperature of 10 °C. The preparation process consisted of coating and drying processes. In animal experiments, mice were immunized using an array of low-temperature H1N1 microneedles without a stabilizer (LT-MN), and they showed strong antibody responses. Compared to three other application methods of traditional IM immunization, low-temperature H1N1 microneedles with a stabilizer (LT-MN-T), and room-temperature H1N1 microneedles with a stabilizer (RT-MN-T), LT-MN produced comparable results in inducing protective immunity. A plaque reduction neutralization test found that LT-MN and LT-MN-T provided greater immunity compared with IM and RT-MN-T.

A process in which the temperature is maintained at 10 °C can provide successful vaccination with H1N1 microneedles without the addition of a stabilizer. This process can be applied to various temperature-sensitive biologics.

Introduction

The microneedle system is designed to overcome the limitations of the existing transdermal drug delivery system [1], [2], [3]. Microneedles can deliver active ingredients into the skin through the stratum corneum regardless of the molecular weight and polarity of the ingredients [4], [5]. The use of microneedles for vaccinations has been studied and developed because microneedles can deliver the antigen into the skin layer, where the cells that need to receive the antigen are primarily located [6], [7], [8], [9]. Microneedles also provide the advantages of convenience and user compliance by avoiding the pain and fear of needles that patients often experience [10], [11]. Vaccine microneedles have shown efficacy equivalent to intramuscular (IM) injection [12], [13], [14]. Vaccine microneedles have also shown improved storage stability compared to the conventional formulation [15], [16]. However, in all previous studies, vaccine microneedles have required a stabilizer because the antigen was denatured during the process of preparation and storage [17], [18], [19]. An excessive amount of stabilizer was added to stabilize the antigen during the manufacturing process as well as during subsequent long-term storage [16], [20]. When the antigen was formulated for microneedles, great effort was necessary to screen the optimal stabilizer [21], [22]. There are a number of limitations related to the addition of the stabilizer: (1) the limitation of the appropriate co-solvent of the stabilizer and the antigen, (2) the limitation of the solubility of the stabilizer in the solvent, (3) the limitation of the amount of antigen that can be loaded into the formulation with the stabilizer, (4) the decrease in the mechanical strength of the dissolving microneedles, and (5) the phase separation of the formulation during the drying process [15], [23]. In particular, in the case of influenza antigens, the amount of stabilizer was 20 times more than that of the antigen, and the addition of an excessive amount of stabilizer could cause the above-mentioned limitations [12], [24].

The decrease in the stability of the antigen occurs rapidly at the stage where the liquid formulation is made into a microneedle shape and dried [17], [25]. If antigenicity is maintained during the manufacturing process, the addition of a stabilizer is less constrained because the change in antigenicity was mainly caused during the manufacturing process when the vaccine microneedles were stored at room temperature and below room temperature [20], [26]. Hemagglutinin (HA) activity, including H1N1, was stabilized mainly by adding a stabilizer during manufacturing, but HA activity did not change during long-term storage without a stabilizer at room temperature [16]. If HA activity can be maintained during the manufacturing process, the amount of antigen per unit area of microneedle array can be increased by minimization or elimination of the stabilizer.

The low-temperature process of producing vaccine microneedles was introduced for the first time to improve the stability of the antigen in the manufacturing process, resulting in the removal of the stabilizer during the manufacturing process. H1N1 vaccine microneedles were prepared by a low-temperature microneedle manufacturing system. To investigate the improvement in the stability of the antigen as a result of using the low-temperature process, we compared the antigenicity of low-temperature H1N1 microneedles without a stabilizer (LT-MN) to the antigenicity of IM immunization, room-temperature H1N1 microneedles with a stabilizer (RT-MN-T), and low-temperature H1N1 microneedles with a stabilizer (LT-MN-T). We then conducted animal experiments to confirm the improvement in stability by the low-temperature process.