3.1. High-Speed Electrospinning of β-Galactosidase
The broadly applied single-needle electrospinning is not capable of the mass production of fibers, and therefore, its productivity is far from the needs of commercial pharmaceutical manufacturing. In this research, HSES was used to increase the throughput of the technology. Based on the results of our previous study [21
] on placebo systems, a matrix solution composed of 7.65 w
% PVA, 0.57 w
% PEO, and 15.30 w
% mannitol was selected for the experiments with β-galactosidase to achieve a grindable fibrous product.
The placebo system was supplemented with β-galactosidase powder so that the enzyme would be 20 w
% of the solid product (Table 1
Even though it was possible to obtain enzyme-containing fibers by electrospinning of this solution, the high solid content caused premature drying of the material, which resulted in blocking of the spinneret and it needed to be cleaned regularly during the electrospinning process. To address this problem, the amount of mannitol in the system was reduced so that, together with β-galactosidase, their amount would equal the amount of mannitol in the placebo system (Table 1
). Electrospinning could be performed seamlessly using the optimized solution composition, which suggests that decreasing the amount of mannitol in the matrix leads to a better processability. However, in our earlier study, it was shown that when the sugar alcohol content in the fibers was decreased below a critical concentration, fiber grindability deteriorated [21
]. Due to this, the mannitol amount was not decreased further in the present work.
The feeding rate used in the electrospinning experiment with the optimized composition was 30 mL/h, which is about 30 times higher than what is achievable with single-needle electrospinning for aqueous solutions [21
]. The obtained fibrous mat was easily removable from the aluminum foil used on the collector (Figure S1
). The product was examined by means of SEM. The fibrous nature of the produced β-galactosidase-containing sample can be seen in Figure 3
A. Bead-free fibers were obtained with diameters around 1–5 µm, but submicronic fibers were also observable.
3.3. Characterization of the Fibers
In order to reveal the physical state of the different materials in the fibers, DSC, XRPD, and Raman examinations were carried out. The reference PEO and PVA are semi-crystalline polymers (glass transition temperature of PVA could be detected at 46.1 °C), which was confirmed by the DSC (Figure 4
) measurement. The reference β-galactosidase powder did not show any significant peak (except for water loss). The reference δ-mannitol had a sharp melting peak at 165.9 °C, even though other researchers detected two peaks (the first belonging to the melting of the δ polymorph, followed by the fast recrystallization to the more stable β polymorph, with a second melting peak) [25
]. Similarly, the fibrous material had two endothermic peaks at 148.8 °C and 160.8 °C, which probably belong to mannitol. During the DSC run, a melting point depression was seen (165.9 °C → 148.8 °C) due to the submicronic mannitol crystals with a large specific surface [27
], which was probably followed by recrystallization to a more stable form and the melting of it [25
]. Based on these results, it can be concluded that all fiber components are amorphous except mannitol.
In order to confirm the results obtained by DSC, XRPD measurements were performed. According to the diffractograms, only mannitol was crystalline in the fibers, showing the characteristic peaks of the δ-polymorph. This polymorph of mannitol has been shown to be the least stable at ambient conditions [28
] and it can transform into the α- or β-polymorph [26
], which can be found in the physical mixture of the electrospinning matrix and β-galactosidase (Figure 5
). During drying (e.g., spray drying), the formation of α- and β-mannitol is expected [29
]. However, in the fibers, δ-mannitol can be found, which might be ascribed to the even faster drying with ES (and therefore, no possibility for rearrangement into a stable form) or to the presence of the other substances.
To evaluate the molecular interactions, FTIR spectroscopy was applied on the samples (Figure 6
). PVA and mannitol molecules contain free hydroxyl groups (which can act as potential proton donors for hydrogen bonding) and β-galactosidase possesses numerous different groups that can act as potential proton donors or receptors. Therefore, hydrogen bonding might occur in the fibers. Characteristic absorption peaks of β-galactosidase are at 3298 cm−1
due to OH stretching and at 2939 cm−1
due to CH stretching. The absorption bands at 1651 cm−1
indicate the CONH vibration, and the 1541 cm−1
peak is the NH bending vibration of the β-galactosidase structure [30
]. These peaks indicate the protein nature of β-galactosidase. PVA has a broad absorption band from OH at 3319 cm−1
, bands from stretching vibrations of CH2
/CH groups at 2941/2910 cm−1
and from C=O at 1736 cm−1
(characteristic of the carbonyl group of polyvinyl acetate), together with deformation bands of CH2
/CH at 1437/1375 cm−1
, and CO stretching vibrations at 1096 cm−1
and 1261 cm−1
]. Characteristic absorption bands of PEO include the band at 2893 cm-1
due to symmetric and antisymmetric CH stretching, and bands at 1468 cm−1
bending) and 846 cm−1
rocking). The band in PEO at 1104 cm−1
indicates asymmetric COC stretching [32
]. Mannitol showed the characteristic peaks of the OH group at 3289 cm−1
and the CH stretching at 2936 cm−1
. Multiple characteristic absorption bands of δ-mannitol can be observed in the 500–1500 cm−1
region, which can also be seen in the spectrum of the electrospun sample, indicating the presence of the crystalline δ polymorph in the fibers [25
]. The characteristic bands of β-galactosidase, PVA, and PEO either disappeared or appeared shifted in the spectrum of the HSES fibers, indicating molecular interaction (presumably hydrogen bonding) between the components.
The local distribution of the components in the ground fibers was analyzed by Raman mapping. For accurate dosing, homogeneity of the enzyme in the formulation is required. According to the Raman chemical map (Figure 7
A), β-galactosidase seems to be uniformly distributed in the ground fibers as very small differences in color are seen.
The Raman mapping results also confirmed that the electrospun fibers mainly contained δ-mannitol. The characteristic peaks of the δ polymorph are shown in Figure 7
B and these are in good agreement with data reported by others in the literature [33
]. The ground electrospun fibers were reanalyzed by DSC, XRPD, and Raman after one year of storage at 4 °C. Even though the δ-polymorph is the least stable among the mannitol polymorphs, no recrystallization was observed in the fibers after this extended storage.
It has been previously shown that sugars and sugar alcohols can interact with water vapor and they have different water sorption capacities based on their physical state [35
]. Amorphous sugars tend to absorb large amounts of water into their bulk structure, whereas crystalline sugars interact with water based on surface adsorption only. Water can act as a plasticizer in electrospun fibers and consequently, the water content of the electrospun materials influences their grindability significantly. It has been shown that a water content below 8% ensures acceptable grindability of sugar-containing fibers [21
]. It was also shown that the physical state of excipients could impact the grindability, with crystalline mannitol eliminating the need for post-drying. The water content of the β-galactosidase-containing fibrous sample measured by the loss on drying (LOD) method was 6.0%. Presumably, this relatively low water content is due to the crystalline nature of mannitol in the fibers.
3.4. Tableting and Long-Term Stability Study of the Tablets
As the marketable final form of a lactase enzyme is preferably a tablet, the purpose of this study was not only to investigate the processability of enzyme-containing electrospun fibers, but also to produce tablets without losing the achieved advantages (i.e., activity preserved after processing). The fibrous powder was mixed with MCC, mannitol, and crospovidone, and the powder mixture was subsequently tableted (Figure S2
). The main compression force was ~8 kN in this experiment. The composition of the produced tablets can be found in Table 2
Enzyme activity was measured after HSES, grinding, and tableting to assess the effect of the processing steps on β-galactosidase. The activity of a stable enzyme formulation was measured parallel to each sample to serve as a reference. The results are depicted in Figure 8
. No significant difference can be seen between the activity of the reference enzyme and the electrospun and processed β-galactosidase, which suggests that the drying conditions with HSES are so gentle temperature wise that no degradation of this protein-type drug is seen.
Ensuring long-term stability of biopharmaceutical products is one of the main challenges in their pharmaceutical use and new formulations thus need to stabilize biopharmaceuticals to maintain their activity during storage. The storage stability of the electrospun and tableted β-galactosidase was compared with a reference enzyme formulation. The tablets of electrospun β-galactosidase were kept at 4 °C and room temperature and their activity were measured after 1 month, 3 months, 6 months, and 1 year. The periodic activity measurements showed that the enzyme remained stable in the tablets at both 4 °C and 25 °C, even after one year of storage (Figure 8
). This result shows that the processable matrix containing PVA, PEO, and mannitol is suitable for stabilizing β-galactosidase in the long term.