Stability of whole inactivated influenza virus vaccine during coating onto metal microneedles

https://doi.org/10.1016/j.jconrel.2012.12.002Get rights and content

Abstract

Immunization using a microneedle patch coated with vaccine offers the promise of simplified vaccination logistics and increased vaccine immunogenicity. This study examined the stability of influenza vaccine during the microneedle coating process, with a focus on the role of coating formulation excipients. Thick, uniform coatings were obtained using coating formulations containing a viscosity enhancer and surfactant, but these formulations retained little functional vaccine hemagglutinin (HA) activity after coating. Vaccine coating in a trehalose-only formulation retained about 40–50% of vaccine activity, which is a significant improvement. The partial viral activity loss observed in the trehalose-only formulation was hypothesized to come from osmotic pressure-induced vaccine destabilization. We found that inclusion of a viscosity enhancer, carboxymethyl cellulose, overcame this effect and retained full vaccine activity on both washed and plasma-cleaned titanium surfaces. The addition of polymeric surfactant, Lutrol® micro 68, to the trehalose formulation generated phase transformations of the vaccine coating, such as crystallization and phase separation, which was correlated to additional vaccine activity loss, especially when coating on hydrophilic, plasma-cleaned titanium. Again, the addition of a viscosity enhancer suppressed the surfactant-induced phase transformations during drying, which was confirmed by in vivo assessment of antibody response and survival rate after immunization in mice. We conclude that trehalose and a viscosity enhancer are beneficial coating excipients, but the inclusion of surfactant is detrimental to vaccine stability.

Introduction

Microneedle patches have been studied as a novel means to administer vaccines with increased immunogenicity and simplified logistics [1], [2], [3]. The increased immunogenicity is believed to be due to targeting antigen delivery to the skin, which has a rich population of antigen-presenting cells and extensive lymphatic drainage [4], [5], [6], [7], [8]. The simplified logistics arise from the easy administration of the vaccine (potentially by minimally trained healthcare personnel or by patients themselves), avoidance of dangerous hypodermic needles and sharps disposal, and small package size to reduce storage, transportation and disposal volumes.

Most research on microneedle patches has emphasized the use of microneedles measuring hundreds of microns in length that are often made of metal and are coated with a vaccine formulation [9], [10], [11], [12], [13]. Upon insertion into skin, the vaccine formulation dissolves off the microneedles into the skin within minutes, after which the microneedle patch is removed and discarded. Vaccines are typically coated onto microneedles by applying a liquid formulation that rapidly dries onto the microneedle surface, leaving a thin, solid film of vaccine and coating excipients.

It is well known that biopharmaceuticals can be destabilized during a drying process [14]. Sophisticated chemical formulations and drying protocols have been developed to protect these sensitive compounds during drying by lyophilization and other methods. However, these methods often cannot be directly transferred to use during coating of microneedles because (i) added excipients must be compatible with the coating process, (ii) the total amount of excipients applied to the microneedles must be small (e.g., 1 mg per patch) and (iii) microneedle coatings air-dry quickly (within minutes), leaving little time to modulate drying conditions.

The goal of this study was to investigate the factors and mechanisms involved in influenza vaccine destabilization during the coating of microneedles, especially those associated with the coating drying process. From our study on the stability of influenza vaccine coated onto microneedles during long-term storage, we know that thermodynamic processes in the solid coating, such as crystallization and phase separation, play a major role in vaccine activity loss [15]. The tendency of disaccharides to crystallize, as well as the suppression of this process using polymers, has been discussed previously in the context of influenza vaccines [16]. We also found that the surface properties of the coated substrate have an effect on these processes. Finally, we further hypothesize that osmotic stress during the drying process may affect vaccine stability.

These processes are all influenced by the composition of the coating formulation. In our lab, we often coat microneedles using aqueous formulations containing a surfactant (e.g., poloxamer) to facilitate good wetting of the microneedle surface, a viscosity enhancer (e.g., carboxymethyl cellulose) to increase coating thickness and a disaccharide sugar (e.g., trehalose) to stabilize the vaccine during drying [17]. Trehalose has been widely used as a stabilizer of biomolecules under low water conditions [18]. However, trehalose, and any other solute present at high concentration, can also function as an osmolyte to generate osmotic stress on the vaccine during drying of the coating.

This study sought to determine how each of these excipients affects stability of a whole inactivated influenza virus vaccine. We therefore tested a number of different coating formulations with different concentrations of each excipient. Dried coatings were then characterized using a hemagglutination (HA) assay as a measure of antigen preservation in vitro to determine vaccine activity [19], [20], light microscopy and X-ray powder diffraction (XRD) to find evidence of crystallization or phase separation, stopped-flow light scattering (SFLS) to observe osmotic shrinking behavior of the virus, and transmission electron microscopy (TEM) to find structural damage to the virus. In vivo immunogenicity and challenge experiments were also carried out in mice to confirm vaccine activity levels.

Section snippets

Preparation of vaccine

Influenza A virus (A/PR/8/34) vaccine was prepared as described previously [21].

Coating formulation and in vitro vaccine stability tests

To identify initial vaccine destabilizing factors, influenza vaccine stability tests were performed on titanium (Ti) plates before being tested on Ti microneedles. This model has been used previously due to the simplified sample preparation and has been shown to be equivalent to coating on microneedles [15]. The coating formulation used in all experiments was composed of one or more of the following:

Functional activity of whole inactivated/live influenza vaccine coated on Ti plates

Our first experiments assessed the effect of coating formulation on the functional activity of influenza vaccine. Coating formulations contained different viscosity enhancer (CMC) and surfactant (Lutrol) concentrations. Functional hemagglutinin activity of the vaccine was assessed via HA assay after drying one day at ambient conditions. Plasma-cleaned Ti plates were used to simulate Ti microneedle surfaces to enable higher-throughput experimentation.

Drying influenza vaccine (either inactivated

Discussion

In this study, the stability of influenza vaccine-coated microneedles was investigated in vitro and in vivo. The purpose of this work was to identify vaccine destabilizing factors during the microneedle coating/drying process, investigate the role of excipients in the current microneedle coating formulation, and design a strategy to maintain protective efficacy of the vaccine during drying.

Conclusions

In this work, stability of influenza vaccine during coating onto microneedles was investigated. We examined the role of excipients used in current microneedle coating formulations, focusing on the stabilizing effect of sugar, osmotic pressure, phase transformations of the coating, and suitability of the coating formulation to coat microneedles. This work showed that 1) trehalose significantly protects influenza vaccine during drying, and that 2) osmotic pressure increase caused by high sugar

Competing interests

M.R.P. is an inventor of patents that have been licensed to companies developing microneedle-based products, is a paid advisor to companies developing microneedle-based products, and is a founder/shareholder of companies developing microneedle-based products. This potential conflict of interest has been disclosed and is being managed by the Georgia Institute of Technology and Emory University.

Acknowledgments

This project was primarily carried out at the Institute for Bioengineering and Bioscience and the Center for Drug Design, Development and Delivery at the Georgia Institute of Technology. Animal studies and associated analytic work were carried out at Emory University. This work was financially supported by NIH grants EB006369 (M.R.P.), AI0680003 (R.W.C.), AI093772 (S.M.K.), and AI087782 (S.M.K.). The authors acknowledge Donna Bondy for administrative support of this research, Dr. Seong-O Choi

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