Characterization of the size distribution and aggregation of virus-like nanoparticles used as active ingredients of the HeberNasvac therapeutic vaccine against chronic hepatitis B^*

Virus-like particles (VLPs) are self-assembling multi-protein nanoparticles that mimic the structural organization and conformation of viruses. VLPs are useful in medicine and nanotechnology. Their repetitive molecularly-defined architecture is attractive for engineering multivalency, notably for vaccination. VLPs enable their antigenic and carrier function both by surface display as well as a delivery system for encapsulation, providing versatility as vaccines and in general as a biomedical research tool [1].

Proteins that assemble into VLPs derive from a variety of viruses, including HIV-1 [2–4], rubella virus [5], human papillomavirus [6, 7], Semliki forest virus [8], RNA phages [9], and hepatitis B virus (HBV) [10, 11]. Due to the highly repetitive structure of such VLPs they are very immunogenic for B cells and induce strong and long-lasting IgG responses in the absence of adjuvants [12, 13]. The 20–200 nm size of VLPs facilitates direct drainage to lymph nodes, increasing uptake by antigen-presenting cells and cross-presentation [14, 15]. VLPs also efficiently reach the MHC class I pathway in vivo. Also, the RNA or DNA packaged in VLPs triggers costimulatory signals through Toll-like receptors TLR3/7/8/9 [15, 16]. Considering all the above information, display on VLPs greatly enhances the response to otherwise poorly immunogenic epítopes [14, 17].

Chronic hepatitis B (CHB) infection remains a health problem worldwide with more than 250 million people in this condition. Persistent infection causes serious liver complications like cirrhosis and hepatocellular carcinoma [18]. Taking into account the limitations of current treatments for CHB, the development of a therapeutic vaccine has been considered a promising immuno-therapeutic approach [19]. In this sense, HeberNasvac was designed as a formulation based on the HBV surface and nucleocapsid antigens (HBsAg and HBcAg, respectively) [20]. This formulation is highly immunogenic after mucosal and/or parenteral administration [21, 22]. HeberNasvac have already completed several clinical trials in CHB patients with successful results [23, 24] The active ingredients HBsAg and HBcAg antigens were obtained as VLPs by recombinant methods in Pichia pastoris and E. coli, respectively [20]. In the present work we further studied the physical characteristics of these antigens and their resulting formulation evaluating the size distribution of the preparations using dynamic light scattering (DLS) and transmission electron microscopy (TEM). The pattern of subvisible particles in the vaccine formulation was also studied. In all cases a representative number of batches were studied in a wide range of production dates.

2.1. Samples

2.2. Particle analyzer

2.3. Mathematic and statistic analysis

Mathematic and statistic analysis were used in Delsa Nano C Particle Analyzer software. The principle of the method is based on the fact that the diffusion rate of particles is determined by their size; information about their size is contained in the rate of fluctuation of the scattered light. So, by correlating the fluctuation, we can determine the particle size distribution of the population present. The diffusion coefficient, D, is inversely proportional to the particle size according to the Stokes–Einstein equation

$$\begin{eqnarray}&&D=\frac{{{k}_{\text{B}}}T}{3\pi {{\eta}_{0}}d},\end{eqnarray}$$

where D is the diffusion coefficient, k_B is Boltzmann's constant, T is absolute temperature, η₀ is viscosity and d is the hydrodynamic diameter. The results were compiled and the mean value of size was determined using the peak value as provided by the equipment.

2.4. Transmission electron microscopy

2.5. Subvisible particle measurement

3.1. Particle size distribution by DLS

The source of recombinant HBsAg nanoparticle is the CIGB's industrial facility for Heberbiovac, the vaccine for anti-Hepatitis B prophylaxis during more than 25 years [25]. The HBcAg nanoparticle is also an antigen produced under GMP at the CIGB facilities. The particle size distribution of both VLPs was evaluated by DLS using several batches. Figure 1 shows the DLS results obtained for three individual batches of HBsAg, HBcAg and HeberNasvac; representatives of the batches in table 1.

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A similar size range and relatively similar particle size distribution was observed in all cases; however, a slightly wider particle size distribution was detected in the HBsAg preparations due to the normal polydispersity of samples. Table 1 summarizes the average particle size of all the studied samples. In the case of recombinant HBcAg, HBsAg and HeberNasvac, the results for the three batches are shown in figures 1(a)–(c), respectively. In general, for all the evaluated samples, the particle size distribution was in the range between 20 and 180 nanometers (nm). The average size values were similar between each independent API and the final vaccine formulation (table 1).

3.2. Morphology and VLP size estimation by TEM

TEM was used to analyze the morphological aspect of the VLPs and measure the individual particle size per sample. In this study we didn't carry out the measurement of the intrinsic agglomerates in each preparation. More than five batches of HBsAg, HBcAg and HeberNasvac were evaluated. Figure 2 comprises representative results for each active ingredient and for the final formulation in three replicates each.

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The HBsAg preparations microphotographs show spherical nanoparticles of approximately 20‒ 30 nm. The presence of dimeric or multiple valence particle agglomerates were observed. The HBcAg nanoparticles are similar to HBsAg in shape and size; however they can be identified by the presence of a high electronic density in the HBcAg central region. The HBcAg preparations also show agglomerated structures. In the HeberNasvac samples we observed that the antigens comprised in the formulation are able to agglomerate although the individual antigens conserve their specific morphology.

3.3. Subvisible particle evaluation

The use of VLPs in vaccination is based on their favorable interaction with immune system cells [1, 14]. The present study characterizes by TEM and DLS techniques the physical aspect and size distribution of the VLPs and protein agglomerates found in the active ingredient preparations as well as in HeberNasvac, and quantifies the agglomerates of VLPs that reach the subvisible range of size using the light obscuration method.

DLS was an important tool for characterizing the size of the nanoparticles and the particle agglomerates of the HeberNasvac drug product and the active ingredients (HBsAg and HBcAg) in solution. The samples analyzed are polydisperse with polydispersity index between 0.15 and 0.25, in all cases less than 0.3.

The average particle size found for HBsAg (55.91 nm) by DLS could be explained by the preferential formation of agglomerates in the preparation. The measurement of the size for independent HBsAg nanoparticles carried out using TEM photographs and Imagen J software rendered a mean size of 23.09 nm, consistent with the previously reported HBsAg size [26, 27]. The association between two or more individual particles was detected by TEM.

The HBcAg individual nanoparticle size calculated by the TEM technique (28.84 nm) was consistent with the reported value for this antigen [28]. The DLS analysis of the active ingredient (40.86 nm), suggested the formation of HBcAg agglomerates. Such agglomerates were also visualized by TEM. The HBsAg particle size is greater than HBcAg determined by DLS.

The measurement by TEM of the mean size of each individual antigen within the HeberNasvac formulation rendered similar results to the antigens measured as active ingredients as expected. However, the final product average particle size (52.49 nm) detected by DLS was similar to that obtained for HBsAg.

In general, the DLS size distribution found in all preparations (20 and 180 nm) can be justified by the formation of protein agglomerates in the active ingredients as well as in the final product. The final size of the agglomerates in all cases may depend on the 200 nm filter pore size at the sterilizing filtration final production step. The agglomeration of both antigens was confirmed by TEM analysis. A similar result has been reported for the HBsAg preparation [29], and was also preliminary reported for the HBcAg and the final formulation [20].

On the other hand, the morphological structure observed by TEM for each VLP correlates with data previously reported by other authors using similar antigen preparations [30, 31], including blood isolated HBV antigens [32, 33]. The presence of a central region of higher electronic density in the HBcAg nanoparticles indicates the presence of encapsulated nucleic acids. This feature of the HBcAg is relevant for vaccine purposes, considering the potent Th1 adjuvant effect that confers the DNA/RNA produced in bacteria [34, 35].

The formation of micrometric aggregates in the final product also constitutes an advantage for this vaccine preparation. It is reported that aggregated structures of sizes ranging between 20‒200 nm are highly immunogenic in animals and humans. The presence of aggregates favors the up-take by antigen presenting cells and the subsequent development of the immune response [14, 15, 36]. The above-mentioned characteristic of the final product is consistent with the promising results obtained for this formulation in the clinical evaluation [23, 24]

Analysis of subvisible particles by the light obscuration method indicates that the final product fulfilled the USP and EP pharmacopeia acceptance criteria for parenteral vaccine preparations. As expected from the size distribution found at the nanometric scale, the higher number of nanoparticles present in the vaccine formulation are between 10 and 25 µm. Although there is a variable amount of particles in both ranges of quantification, the numbers were in the range of acceptability for particles in parenteral formulations. Several batches, representative of a long period of time were analyzed, and the results were reproducible, demonstrating the robustness of the production processes in relation to the studied variables.

In conclusion, the characterization of the particle size distribution in the API and final vaccine formulation is consistent with the reported mean size of recombinant HBsAg and HBcAg particles. The agglomerates of VLPs are detected both by DLS and TEM in the active ingredients as well as in the final product, in both cases with a similar size range and maximum peak of size. The agglomerates of both VLPs can be quantified also by light obscuration and resulted in quantities below the range of acceptability according to major international pharmacopeia.