University of Groningen
Blends of trehalose and pullulan to stabilize biopharmaceuticals in alternative dosage forms:
orodispersible films and dissolving microneedles
Tian, Yu
DOI:
10.33612/diss.135502200
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Publication date: 2020
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Citation for published version (APA):
Tian, Y. (2020). Blends of trehalose and pullulan to stabilize biopharmaceuticals in alternative dosage forms: orodispersible films and dissolving microneedles. University of Groningen.
https://doi.org/10.33612/diss.135502200
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CHAPTER 7
Summary, concluding remarks and perspectives
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SUMMARY
Biopharmaceuticals including therapeutic proteins, vaccines, hormones, and blood components have become the fastest growing class of medicines in today’s pharmaceutical markets due to the rapid developments in biomedical sciences and technology [1,2].This is exemplified by the therapeutic proteins approved by the U.S. Food and Drug Administration (FDA) during the period 2011 to 2016, they are indicated for a wide variety of diseases, which can benefit a broad spectrum of patient groups (Figure 7.1) [3].
However, the broader application of biopharmaceuticals is hampered by two major challenges. First, biopharmaceuticals are produced as aqueous solutions or dispersions in which they are unstable during storage as well as transportation outside of the cold-chain. Second, most biopharmaceuticals are administered via the parenteral route which has several drawbacks such as pain at the injection side, risks for needle stick injuries, requirement for trained health care personnel and poor patient compliance due to needle phobia [4].
Figure 7.1 Pie chart showing the distribution of FDA-approved therapeutic proteins (2011–
2016) by therapeutic area (left). Pie chart showing the distribution of secondary therapeutic area for oncology drugs (right) [3].
One of then most applied and successful strategies to stabilize biopharmaceuticals is to dry them in the presence of sugars [5,6]. After drying, the sugar should be amorphous and in the glassy state to obtain a compact coating and minimal molecular mobility of the biopharmaceutical for optimal stabilization. [5–7]. Low molecular weight saccharides are able to form such compact coating. However, the glass transition temperature (Tg) of low molecular weight sugars is relatively low and therefore the glasses of these sugars are physically unstable in particular when exposed to high relative high humidity conditions. On the other hand,
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polysaccharides have a high glass transition temperature, thus having excellent physical stability, but due to their bulky and inflexible nature they fail to form a compact coating around the biopharmaceutical. We hypothesized that with a combination of a low molecular weight sugar and a polysaccharide a compact coating of biopharmaceuticals while having an excellent physical stability can be achieved [8]. In this thesis, combinations of the disaccharide trehalose and the polysaccharide pullulan were explored as stabilizers for biopharmaceuticals. Trehalose was selected because in literature it is generally recognized as the gold standard for stabilization of biopharmaceuticals and pullulan because of its exceptionally high glass transition temperature (261 °C). In addition, pullulan shows excellent mechanical and film forming properties enabling the development of alternative dosage forms.
Having the biopharmaceutical in the dry and stable state, offers the opportunity to develop alternative dosage forms. In this thesis, two different types of dosage forms with improved patient acceptance were developed and discussed, i.e. oromucosal films (buccal or sublingual administration) and microneedles (intradermal administration).
In Chapter 2, a literature review is given on theoromucosal films. The patient-centric design of oromucosal films can result in a lower dosing frequency and thus a high patient compliance. Orodispersible films (ODFs) are used more frequently for the treatment of systemic disorders, while mucoadhesive buccal films (MBFs) are used both for local and systemic diseases. Polymers that are frequently used as basis for ODFs and MBFs are discussed. Different incorporated active pharmaceutical ingredients (APIs) are discussed as well, includingwater-soluble and poorly water-soluble small drug molecules, biopharmaceuticals and herbal plant extracts. Novel printing techniques for oromucosal films are described and the most widely used inkjet printing and 3D printing techniques are summarized. Before incorporating biopharmaceuticals into alternative dosage forms based on trehalose/pullulan blends, the strategy of stabilizing biopharmaceuticals by these combinations of sugars should be evaluated, which was done in a study described in Chapter 3. Blends of trehalose and
pullulan were used to stabilize the model protein β-galactosidase. We found that freeze-dried trehalose/pullulan solutions all showed single Tg values, indicating that the sugars formed homogeneous blends at a molecular level. The high Tg of pullulan (261 °C) was an important property for protein stabilization, since it strongly increased the Tg of the mixtures with trehalose, depending on the trehalose/ pullulan ratio. Tg of trehalose/pullulan blends was well above room temperature even when exposed to high humidity conditions. The storage stability of freeze-dried β-galactosidase without sugar was substantially lower than with trehalose/pullulan blends, even for the pullulan only formulation. Despite these observations,
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pullulan only will not be an optimal stabilizer, as due to its bulky nature, it will be unable to from a tight coating of the protein. As a consequence, the stability of β-galactosidase incorporated in trehalose/pullulan blends gradually decreased with decreased trehalose/pullulan ratios when stored at 30 °C or 60 °C under anhydrous conditions. However, when a β-galactosidase formulation containing trehalose but no pullulan was exposed to 30 °C/56 %RH, trehalose crystallized because the Tg was decreased to below storage temperature. As result, protein stabilization by the sugar was lost. At 56% RH, trehalose/pullulan blends with a weight ratio of 1/2, 1/1, 2/1 and 1/5 outperformed the stabilizing capacities of the trehalose only formulation. Therefore, is was concluded that trehalose/pullulan blends combine the benefits of both sugars, i.e. the high Tg of pullulan and the ability of trehalose to form a compact coating around proteins. These benefits are in particular relevant when the freeze-dried product is exposed to high relative humidity conditions.
The aim of the study described in Chapter 4, was to incorporate biopharmaceuticals in
ODFs based on trehalose/pullulan blends. Aqueous solutions of trehalose/pullulan at different
ratios with a model protein (either lysozyme or β-galactosidase) together with small amounts of glycerol and Tween 80 were used to prepare ODFs by either air-drying or freeze-drying. It was found that lysozyme was a relatively stable protein. Lysozyme could be incorporated in ODFs without loss of activity and remained stable for at least 4 weeks at 30 °C/0% RH, irrespective of the production method or trehalose/pullulan ratio. β-galactosidase, however, showed a clear trend towards increased stability when the trehalose/pullulan ratio was increased. Furthermore, β-galactosidase incorporated in ODFs by freeze-drying showed a better process stability, whereas air-drying showed a better storage stability. Probably due to the porous structure and therefore a larger specific surface area, β-galactosidase would degrade faster in freeze-dried ODFs than in non-porous, dense air-dried ODFs. In conclusion, the study described in Chapter 4 demonstrates the potential of the trehalose/pullulan blend as stabilizer
in protein containing ODFs.
The aim of the study described in Chapter 5, was to incorporate H5N1 whole
inactivated influenza virus vaccine (WIV) in ODFs to develop a stable vaccine formulation intended for immunization via the oral cavity. At first, we prepared a plain hypromellose (HPMC) based ODF, onto which a vaccine solution containing sugar was pipetted followed by air- or vacuum-drying. We evaluated whether trehalose only (at different concentrations) or trehalose/pullulan blend can be used to stabilize WIV when incorporated in ODFs. It was observed that sugars (trehalose only or a blend of trehalose and pullulan) can significantly
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increase the stability of WIV during production of the ODFs when compared with WIV without sugars. Surprisingly, during storage of the ODFs at challenging conditions (60 °C/0% RH or 30 °C/56% RH) for 4 weeks only a marginal loss of WIV activity was found in all cases, i.e. also for the formulations without trehalose/pullulan. Control experiments indicated that by pipetting the WIV only solution onto the ODF, the ODF most like partially dissolved locally resulting in WIV being incorporated in a matrix of the film components after drying. The main constituent of the ODF, HPMC, was apparently capable of stabilizing WIV during storage. In conclusion, ODF may be a suitable and potential alternative for delivering stabilized WIV to the oral cavity.
In Chapter 6, WIV was incorporated in dissolving microneedles based on
trehalose/pullulan blends. The dissolving microneedles were prepared by pouring the trehalose/pullulan solution containing WIV into the PDMS mold, followed by centrifugation at 3750 rpm at 4 °C for 3 h and air-dried at 37 °C overnight. The dissolving microneedle arrays were sharp and stiff, showing high penetration efficiency in ex vivo experiments using human skin. In addition, due to the high aqueous solubility of trehalose and pullulan, the microneedle arrays exhibited fast dissolution, i.e. within 15 mins. After storage at anhydrous conditions up to 37 °C for 4 weeks, microneedle arrays remained their mechanical integrity with almost 100 % penetration efficiency. However, their mechanical integrity lost dramatically after exposure to more humid conditions (56% RH for 4 weeks). Furthermore, WIV incorporated in dissolving microneedles based on trehalose/pullulan showed excellent process and storage stability, although there was a slight reduction of hemagglutination titers after production, which was probably due to the drying process at 37 °C overnight. After storage at 4 °C, 25 °C and 37 °C for 4 weeks, WIV incorporated in dissolving microneedles remained its stability, whereas WIV dispersed in phosphate buffered saline fully lost hemagglutinating capacity of WIV. In conclusion, our research demonstrates the suitability of trehalose/pullulan-based dissolving microneedles for influenza vaccine delivery.
CONCLUDING REMARKS AND PERSPECTIVES
The blends of trehalose and pullulan are attractive candidates for the stabilization of biopharmaceuticals. Drying methods for trehalose/pullulan blends used in this thesis were freeze-drying, air-drying and vacuum drying. An often used drying method in pharmaceutical production is spray drying [9]. Spray drying of protein solutions together with trehalose/pullulan blends could yield interesting results. A model to predict the distribution of
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protein and sugar in an evaporating droplet during spray-drying was developed by Grasmeijer et al. [10]. They found that during spray drying a solution containing trehalose bovine serum albumin (BSA), the two components were not homogeneously distributed in the drying droplets; i.e. at the surface the concentration of BSA was relatively high. This phenomenon could be explained by the smaller molecular size and thereby faster diffusion rate of trehalose. In addition, due to its surface active properties, BSA has a tendency to accumulate at the interface [10]. For these two reasons, the surface of the ultimately obtained powder particles will be enriched with BSA. Being located at the surface, these protein molecules cannot be completely coated by sugar molecules. This implies that a large fraction of BSA is not optimally stabilized. The use of trehalose/pullulan blends may enhance protein stabilization by spray-drying due to its much higher molecular weight. Pullulan has a much slower diffusion rate than trehalose by which a more homogeneously distribution of protein and sugar might be achieved. Apart from sugars, amino acids have also been applied to stabilize biopharmaceuticals in during drying and subsequent storage [11,12]. Also combinations of sugars and amino acids are potential stabilizing excipients in biopharmaceutical formulations [13,14]. As amino acids have an even lower molecular weight than trehalose, they may provide an even more intimate coating than trehalose. Blends of amino acids and pullulan probably could provide even better stabilizing effects compared to the blend with trehalose but this needs to be further investigated [15].
Delivery of biopharmaceuticals via the oral cavity by using ODFs is a promising alternative to the conventional injection route which has recently gained considerable interest in the scientific community [16,17]. However, administration of biopharmaceuticals to the oral cavity using ODFs has to our best knowledge never been addressed. Incorporation of biopharmaceuticals in ODFs implies that they have to be brought into the dry state, which requires stabilizing excipients. Application of blends of trehalose and pullulan appeared to be successful in this respect although incorporation of WIV in the HPMC based film forming matrix was also found to stabilize the vaccine. In this thesis, solvent casting was used to produce these ODFs and pipetting techniques were used to apply the biopharmaceuticals. However, alternative techniques may be used to produce ODFs containing biopharmaceuticals. For example, novel printing techniques have recently been explored for the production of ODFs. There are three kinds of printing techniques: inkjet printing [18,19], 3D printing [20,21] and roll-to-roll printing [22,23] (Chapter 2). Advantages of these printing techniques are the high
dosing precision and the possibility of personalized medicine as the dose can be easily adjusted. However, as the conventional fuse deposition modeling 3D printing require high temperatures,
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they are not suitable for production of ODFs with biopharmaceuticals incorporated. In our opinion, it might be feasible to use a 3D-Bioplotter® for this purpose, as described in the ‘expert opinion’ section of Chapter 2 [24]. Therefore, further research related to the development of a
suitable printing technique for biopharmaceuticals on ODFs is required.
Dissolving microneedles are minimally invasive approach to intradermal delivery of drug, allowing for complete and rapid dissolution of encapsulated drug. Various materials including hyaluronan [25,26], carboxymethylcellulose [27] and dextran [28] have been frequently used as matrix materials in dissolving microneedles. To our best knowledge, we were the first to use trehalose/pullulan combinations as matrix in dissolving microneedles. Trehalose/pullulan-based microneedles showed sufficient mechanical strength to penetrate the human skin ex vivo. This combination of a di- and polysaccharide was also able to preserve the activity of incorporated WIV during production and subsequent storage. Although these in vitro and ex vivo are promising, the potential of these formulations should be proven in future in vivo studies. Apart from influenza vaccines, trehalose/pullulan based dissolving microneedles may also be a suitable delivery system for other vaccines (e.g. hepatitis B and polio) and therapeutic proteins. Furthermore, as dissolving microneedles enable to penetrate stratum corneum, they may also be suitable for painless penetration of buccal or sublingual mucosa. Using dissolving microneedles to deliver vaccine to oral mucosa would be an interesting topic which needs further investigations.
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