Novel approach for overcoming the stability challenges of lipid-based excipients. Part 2: Application of polyglycerol esters of fatty acids as hot melt coating excipients

Journal Pre-proofs Research paper Novel Approach for Overcoming the Stability Challenges of Lipid-Based Excipients. Part 2: Application of Polyglycerol Esters of Fatty Acids as hot melt coating excipients Sharareh Salar-Behzadi, Carolina Corzo, Diogo Gomes Lopes, Claudia Meindl, Dirk Lochmann, Sebastian Reyer PII: DOI: Reference:

S0939-6411(20)30020-5 https://doi.org/10.1016/j.ejpb.2020.01.009 EJPB 13214

To appear in:

European Journal of Pharmaceutics and Biopharmaceutics

Received Date: Revised Date: Accepted Date:

28 September 2019 14 January 2020 20 January 2020

Please cite this article as: S. Salar-Behzadi, C. Corzo, D. Gomes Lopes, C. Meindl, D. Lochmann, S. Reyer, Novel Approach for Overcoming the Stability Challenges of Lipid-Based Excipients. Part 2: Application of Polyglycerol Esters of Fatty Acids as hot melt coating excipients, European Journal of Pharmaceutics and Biopharmaceutics (2020), doi: https://doi.org/10.1016/j.ejpb.2020.01.009

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Novel Approach for Overcoming the Stability Challenges of Lipid-Based Excipients. Part 2: Application of Polyglycerol Esters of Fatty Acids as hot melt coating excipients

*Sharareh Salar-Behzadi1,2, Carolina Corzo1,2, Diogo Gomes Lopes1, Claudia Meindl3, Dirk Lochmann4 Sebastian Reyer4 1Research 2Institute

Center for Pharmaceutical Engineering GmbH, Graz, Austria

of Pharmaceutical Sciences, Department of Pharmaceutical Technology, University of Graz,

Graz, Austria 3Center 4IOI

for Medical Research, Medical University of Graz, Graz, Austria

OLEO GmbH, Witten, Germany

*Corresponding author: Sharareh Salar-Behzadi Research Center for Pharmaceutical Engineering GmbH, Inffeldgasse 13, 8010 Graz, Austria Tel: +43 316 87330948 e-mail: [email protected]

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Abstract: The application of hot melt coating (HMC) as an economic and solvent-free technology is restricted in pharmaceutical development, due to the instable solid-state of HMC excipients resulting in drug release instability. We have previously introduced polyglycerol esters of fatty acids (PGFAs) with stable solid-state (Part 1). In this work we showed a novel application of PGFAs as HMC excipients with stable performance. Three PGFA compounds with a HLB range of 5.1 to 6.2 were selected for developing immediate-release formulations. The HMC properties were investigated. The viscosity of molten lipids at 100°C was suitable for atomizing. The DSC data showed the absence of low solidification fractions, thus reduced risk of agglomeration during the coating process. The driving force for crystallization of selected compounds was lower and the heat flow exotherms were broader compared to conventional HMC formulations, indicating a lower energy barrier for nucleation and lower crystallization rate. Lower spray rates and a process temperature close to solidification temperature were desired to provide homogeneous coating. DSC and X-ray diffraction data revealed stable solid state during 6 months storage at 40°C. API release was directly proportional to HLB and indirectly proportional to crystalline network density and was stable during investigated 3 months. Cytotoxicity was assessed by dehydrogenase activity and no in vitro cytotoxic effect was observed.

Keywords: polyglycerol esters of fatty acids, hot melt coating, lipid-based excipients; solid-state, stability, stable release profile

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1- Introduction Solvent-free fluid bed hot melt coating (HMC) is a well-known manufacturing technology for coating of oral pharmaceutical dosage forms [1-6]. This technology is favored for taste-masking but also for development of modified released profiles by application of lipid-based excipients as coating material. Basically, the process consists of maintaining the core material in a fluidized state in a fluid bed container via an adjustable air flow. The lipid-based excipient (LBE) as coating material is sprayed onto the fluidized core material in a molten state and crystallizes on the surface of core to form a coating layer [7]. The outstanding advantage of this technology is given by using the molten coating material and thus, being solvent-free due to the avoidance of organic and aqueous solvents. This results in a faster and more costeffective manufacturing process, since time consuming evaporation steps, costly solvent recovery and deposal, common in film coating, are not required. Moreover, lipid-based compounds as the most frequently applied HMC excipients are naturally occurring materials that are predominantly digestible with “Generally Recognized as Safe” (GRAS) status. In a patient-centric product development strategy, hot melt coating of multiparticulate systems is particularly advantageous because of the excellent slippage property of lipid-based excipients, causing a smooth and pleasant mouth feeling [1,8]. This can be used for improving the adherence of pediatric and geriatric population and patients suffering from dysphagia to therapy and for development of “direct to mouth” taste-masked multi-particulate products filled in stick for fast administration of the dosage form. An excellent example for the latter application is an available product containing N-acetylcysteine as mucolytic agent [9]. Despite these advantages, fluid bed HMC still does not find its well-deserved attraction in pharmaceutical industry, which is mostly due to the complex and instable solid state of LBEs. This complexity is famously the result of the hierarchical structure of lipids in molecular, nano-, and micro-level that dominates the macroscopic properties of these molecules [10-14].

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The most common LBEs are triacylglycerols (TAGs), diacylglycerols (DAGs) and monoacylglycerols (MAGs), acylglycerol derivates, free fatty acids, fatty alcohols, etc. [7,15]. Based on their chemical structure and environmental conditions, these materials can crystalize in several crystalline structures with different thermodynamic stabilities, with the tendency of transformation into the thermodynamically more stable form. This polymorphic transformation and crystalline growth often result in the alteration of macroscopic properties and thus, in the impairment of long-term stability of hot melt coated formulations [14,16-20]. Another issue is the hydrophobic nature of lipid-based excipients, when an immediate release formulation is desired. In previous works we pointed out these issues and extensively studied the effect of polymorphic transformation, alteration in the microstructure and crystalline growth of lipids on the performance of pharmaceutical products produced via hot melt coating technology [8, 14, 19, 21]. In a recent work (Part 1) we introduced a chemical group of potential LBEs; polyglycerol esters of fatty acids (PGFAs), which are the hydroxyethers of glycerol esterified with fatty acids with outstanding solid state stability. We also described in that work that the hydrophilicity-lipophilicity balance (HLB), melting point and the melt viscosity of the compounds belonging to PGFAs can be adjusted by varying the number of polyglycerol moieties, the chain length of the fatty acids and the free hydroxyl positions (full/partial esterification of hydroxyl groups). In the current work we screened the feasibility and processability of selected PGFA molecules; PG3C16/C18 partial, PG4-C18 partial and PG6-C18 partial, with a HLB range of 5.1 to 6.2 as potential HMC excipients. The aim was to develop immediate release and taste-masked multi-particulate systems of Nacetylcysteine (NAC) as API, with stable performance. The data showed the advanced performance of selected PGFAs as HMC compounds. The possibility to select the HLB for tailoring the API release profile privileged the advantage of a monophasic system and thus avoiding the addition of emulsifiers with the consequence of phase separation, lipid crystalline growth and thus instability of the pharmaceutical end 4

product. The stable solid state of these molecules resulted in stable product performance. Although the PGFAs in general possess the GRAS status and they are already applied in food (US FDA 21CFR172.854 and EU food additive E475) cosmetic and pharmaceutical industry in the production of the shell of soft gelatin capsules [22], the safety was confirmed by investigation of the viability of Caco-2 cells after exposure to the selected PGFAs.

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2- Materials and methods: 2-1 Materials N-acetylcysteine (NAC), a mycolytic and freely water soluble agent was used as model substance and the crystals were hot melt coated without any preliminary granulations step. PG3-C16/C18 partial (WITEPSOL® PMF 1683) (HLB = 5.1), PG4-C18 partial (WITEPSOL® PMF 184) (HLB value = 5.6) and PG6-C18 partial (WITEPSOL® PMF 186) (HLB value = 6.2) were used as hot melt coating materials. The materials were synthesized and provided by IOI OLEO GmbH. Caco-2 cells from American Type Culture Collection (Rockville, USA) were used for in vitro toxicity studies. The composition of cell culture medium and exposure medium are described below in sample preparation. All other reagents and chemicals were of analytical grade and were used as received.

2-2 Methods 2-2-1 Hot melt coating (HMC) process: The fluid bed hot melt coating was performed using a Ventilus® V-2.5 laboratory system fluid bed equipment combined with an IHD-1 hot melt device (Romaco Innojet GmbH, Germany). A batch of 200 g NAC was used as core material for each HMC trial. Three different batches of NAC were coated using PG3C16/C18 partial, PG4-C18 partial, and PG6-C18 partial as coating material. Percentage of the coating material in the coated NAC particles was 50 (%w/w) for all three batches. The process parameter settings were selected based on preliminary trials, considering physical properties, i.e. melting point, solidification point, and viscosity in molten state of PGFAs. The final selected atomizing air pressure and process temperature were 0.8 bar and 40 °C, respectively. A spray rate of 7.5 g/min was applied for the batch coated with PG3-C16/C18 partial. The two other batches were processed with a spray rate of 5 g/min.

2-2-2 Analytical methods 6

2-2-2-1 Characterization of NAC as core material Particle size measurements: Particle size of NAC was measured before and after HMC using a QICPIC high-speed analysis sensor (Sympatec GmbH, Germany) and a RODOS/L dry disperser. The measurement spectrum range was between 20 to 3000 µm. The feeding rate was 30%, 400 frames per second were taken, the injector diameter was 4 mm and the air pressure was 1 bar. The particle size distribution (PSD) was defined as X90 – X10 (X90/X10). Aspect ratio was calculated as the ratio of the minimal to the maximal Feret diameter, representing the level of the sphericity of particles. A value of one represents a spherical shape.

2-2-2-2 Characterization of PG fatty acid esters as coating material Solid state and microstructure: Differential scanning calorimetry (DSC) Polymorphic behavior and solidification properties of PGFAs were analyzed using a DSC 204 F1 Phoenix (NETZSCH, Germany). Samples of 3-4 mg of different lipids (raw material, as received) were weighed into aluminum pans and scanned at a heating rate of 5 K/min. The temperature was then held for 5 min at 100 °C and cooled down to -20 °C at a cooling rate of 5K/min. A second cycle was performed at same heating rate (T0 sample). All samples were stored at room temperature (RT) and at 40 °C. Thermal behavior and possible polymorphic transformation of stored samples were analyzed after 6 months (6m/RT and 6m/40°C samples). Additionally, samples of pure lipids were prepared and scanned at a heating and cooling rate of 10 K/min, in order to simulate the cooling of lipid in a hot melt coating process as much as possible. Samples were heated up to 100 °C, held for 5 minutes and subsequently cooled down to 20 °C. The driving force (Δµ) for crystallization was calculated from the gathered data, using equation 1 [11]: 7

(equation 1)

Δµ = ΔHmΔT/Tm

Where ΔHm is the melting enthalpy (j/g), ΔT is the difference between melting and crystallization temperature (°C), which is called as super-cooling and Tm (°C) is the melting temperature.

X-ray diffraction analysis The polymorphism and microstructure of selected PGFAs were analysed via simultaneous Small and Wide Angle X-ray Scattering (SWAXS). A point-focussing camera system (S3-MICRO, formerly Hecus X-ray systems Austria, now Bruker AXS, Germany) equipped with thermostated sample spinning and two linear position sensitive detectors to cover the real space resolution ranges of 10–1500 Å (SAXS) and 3.3–4.9 Å (WAXS) was used. The raw material once as received and once after melting and recrystallization (T0 samples) were placed into a glass capillary with a diameter of ~2mm, which was later sealed with wax and placed into the capillary rotation unit. The singular measurements were performed at room temperature with an X-ray exposure time of 1300 sec. After measurements the glass capillaries of T0 samples were stored at RT and 40 °C for 6 months (6m/RT and 6m/40°C samples). The short-spacing was analyzed in wide-angle region to characterize the polymorphic behavior of PGFAs. The lamellar structure, crystallite thickness and growth were characterized by analyzing the long-spacing in small angle region [23]. The average crystallite thickness (D) of PGFAs was estimated based on the first order Bragg SAXS peaks and the Scherrer equation [13]: Kλ

D = FWHMcos(θ)

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(equation 2)

K (Scherrer constant) is a dimensionless number that provides information about the shape of the crystal and in case of the absence of detailed shape information, K = 0.9 is a good approximation. λ is the X-ray wavelength (1.542 ° A). FWHM is the width in radians of the diffraction maximum measured at halfway height between background and peak, typically known as full width at half maximum (FWHM) and θ is the diffraction angle.

Polarized Light Microscopy (PLM) The microstructure of PGFAs was further characterized via 2-D polarized light microscopy (Olympus BX51M, USA) equipped with a Lumenera Infinity 1B digital camera (Teledyne, Lumenera, ON, Canada). The software Infinity Analyze (Teledyne, Lumenera, ON, Canada) was employed to visualize and process images. A drop (about 10 µL) of melted sample (100 °C) was placed on the heated glass slide and covered with a heated cover slip (100 °C). The sample was then cooled down to 4 °C. This set of samples was stored at room temperature and 40 °C for 6 months. Micrographs of freshly prepared samples (T0) and stored samples (6m/RT and 6m/40°C) were taken.

Hot melt coating properties Viscosity of molten lipid Viscosity of molten PGFAs was measured via a Physica – Modular Compact Rheometer, MCR 300 (Anton Paar GmbH, Austria). Measurements were conducted with a cone-plate system CP-50-2 at constant stress. The samples were directly molten on the plate and viscosity was measured at 100 °C. The measurements were prepared in triplicates.

Volume contraction

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The volume contraction of selected PGFAs calculated by measuring their pycnometer density in powder form (room temperature) and the density of molten PGFAs at 90°C. The pycnometer density of powder was measured using a helium-pycnometer AccuPycTM 1340 (Micrometrics, USA) following the sample preparation by gently crushing the solidified PGFAs into powder. Amounts of 4-5 g powder were filled in sample holder and measured. The density of molten PGFAs was measured after melting the samples in an oven (Binder Q5 FP 53, Germany) at 90 °C and measuring the mass of 1 mL molten material. All measurements were prepared in triplicates.

Water uptake and erosion Sample preparation and measurements were undertaken as described in [19]. Briefly, raw PGFAs as received were melted and casted into pre-heated (100°C) silicon molds (40X10X2mm) to form slabs of about 750mg. Each slab was weighted (m0) and placed into a glass vial containing 15 mL 37°C ultra-purified water at 37°C (Milli-Q, Merck Life Science, Germany). The slabs were withdrawn after 24 hours and the excess of water on the surface was carefully removed. The slabs were then accurately weighted (mw) and dried at 40°C until constant weight (md). The water uptake (%WU) and the erosion (%E) were calculated as described in [19].

2-2-2-3 In vitro toxicity studies Sample preparation Selected PGFAs were sieved between 50-200 µm. A dispersion of 0.5 mg/ml of each PGFA was prepared by dispersing 3 mg of material in 6 ml of fasten simulated intestinal fluid (FaSSIF, Biorelevant.com LTD, United Kingdom) or cell culture medium, which was Minimum Essential Medium (MEM). The samples were shaken for 24 h at 37°C and 260 rpm and consequently filtered using a Büchner Flask system and

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vacuum to obtain clear solutions. Samples were tested at the same day. The solubility of substances in these two media was measured gravimetrically.

Cell culture and exposure to PGFAs Caco-2 cells were cultured in MEM, 20% fetal bovine serum, 1% non-essential amino acids (NEAA) and 1% penicillin-streptomycin at 37°C in humid air atmosphere containing 5% CO2 in 75 cm2 cell culture flasks. Cells were then seeded on transwell insert (0.4 µm pore size; 1.13 cm2) on a 12-well plate (Greiner Bioone) at a density of 0.5 x 106 cells per insert with 500 µL and 1500 µL medium in the upper and in the lower compartment. When cell monolayers had reached a transepithelial electrical resistance (TEER) value of >300 Ω*cm2 (18 days) cells were exposed to 500 µl of filtrates of each PGFA in MEM or in FaSSIF (pure samples) for 24 hours. The pure samples were further diluted with FaSSIF or MEM in the volumetric ratios of 1+1, 1+4 and 1+9. Cells were also exposed to 500 µl of each dilution. Caco-2 cells without mucin coating were exposed to PGFA filtrates in MEM. This method will be thereafter called "conventional model". For the experiments in FaSSIF, cells were coated with mucin (40mg/ml) for 30 min and the method is termed as "FaSSIF/mucus model".

In vitro cytotoxicity screening of caco-2 cells after exposure to PGFAs TEER measurements Disruption of tight junctions as indication for a toxic action was studied by measuring TEER values before and after exposing to PGFAs. After 24h of exposure the solution was removed and cells were rinsed three times with PBS. After addition of 500 µl of fresh cell culture medium TEER values were measured using an EVOM STX-2-electrode (World Precision Instruments) connected to a Millicell® ERS Voltohmmeter (World Precision Instruments).

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Dehydrogenase activity (Formazan bioreduction) After exposure of the cells to the samples and measurement of the TEER values the CellTiter 96 Aqueous Nonradioactive Cell Proliferation Assay (Promega) was used according to the manufacturer's instructions. 100 μL of the combined MTS/PMS solution was added to 500 μL fresh medium in each well and plates were incubated for 1 hour at 37°C in the cell incubator. Absorbance was read at 490 nm on a plate reader (SPECTRA MAX plus 384, Molecular Devices).

2-2-2-4 Release of API from hot melt coated microcapsules Drug release test of coated particles containing 600 mg of NAC; corresponding to the daily dose of this API were carried out in a USP apparatus II offline system (ERWEKA DT820LH, Germany), using 900 mL ultra-purified water at 37 °C as dissolution medium with a paddle speed of 100 rpm (n = 3). Additionally, the drug release of particles coated with PG3-C16/C18 partial was analyzed in simulated gastric fluid (SGF) with the pH of 1.2 and in the fasted-state simulated gastric fluid (FaSSGF, biorelevant.com), in order to investigate the effect of phospholipids and bile salt on the release of API. The FaSSGF medium was prepared according to manufacturer instructions. An immediate release profile of at least 85 % release of API within the first 30 minutes of dissolution test was selected as specification. Samples were taken after 5, 15, 30, 45, 60 and 90 minutes. At each sampling point 1.5 mL of the dissolution medium was manually taken and filtered (MCE membrane, 0.22 µm pore size; Merz Brothers GmbH, Austria) into HPLC vials. The removed medium was not substituted but considered in the calculations. The API content assay was performed using the method described in [14]. The NAC concentration in dissolution and content assay samples were measured using a Waters 2996 PDA Detector 195 High-performance liquid chromatography (HPLC) system with an auto-sampler, using the method described in [14]. The zero-order phase of the release profiles was fitted into the equation 3 to calculate the zero-order constant (k): 12

mt/mꚙ.= k (t – t0)

(equation 3)

in which mt (mg) is the amount of drug released at time t (min) and mꚙ (mg) is the amount of drug released at equilibrium. The rate of dissolution of 600 mg pure NAC in each medium was evaluated.

2-2-2-5 Stability studies Samples of lipids in DSC pans, X-ray diffraction capillaries and spread on glass slides for PLM analysis were stored at RT and 40 °C, as described above. The thermal behavior, solid state and microstructure of lipids were analyzed after storage. Hot melt coated particles were filled in plastic bags, sealed and stored under two different conditions; accelerated condition (AC) (40 °C, 75 % r.h.) and long term condition (LT) (25 °C, 60 % r.h.). The release of NAC from coating was measured by performing dissolution tests after 3 months of storage. The release profiles after storage under accelerated conditions were compared with the initial release profile of NAC using the similarity factor (f2) [24]:

f2 =50 x log ([1 + 1/n Σ(Rt – Tt)2)]-0.5x 100)

(equation 4)

n is the number of measured time points, Rt is the dissolution value (%) of the reference batch (T0) at time t and Tt is the dissolution value (%) of the test batch (3m/AC) at time t. Dissolution profiles considered as similar if f2 was between 50 and 100.

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3- Results and discussion 3-1 Physical properties of coated particles The median particle size (X50) of NAC as core material was 524.3 (±0.48) µm with a particle size distribution (X90/X10) of 1.49 and an aspect ratio of 0.659. After HMC process, the median particle size of coated particles was in the range of 712 – 750 µm with the size distribution of X90/X10 = 1.47, which was around the size distribution of uncoated particles and demonstrates the absence of agglomeration during the coating process. The aspect ratio of coated particles was increased (a50 = 0.74), indicating that the coating process leads to more spherical-shaped particles.

3-2 Solid state The solid-state of PGFAs has been described in details in Part 1 of these studies by analyzing some representative molecules, among them PG3-C16/C18 partial. Below, the solid state of this molecule together with the unpublished data on the solid states of PG4-C18 partial and PG6-C18 partial are briefly described.

Polymorphic characterization and nanostructure of selected PGFAs Polymorphism of selected PGFAs was characterized using DSC and WAXS. The obtained DSC data are depicted in Figure 1. These data show a single melting peak in the heating cycle of each selected PGFA (raw material), reflecting the existence of only one polymorphic form. After crystallization, the samples were exposed to the second heating cycle (T0 samples) to confirm the absence of polymorphic transition of PGFAs after heat treatment. The samples were stored at RT and 40 °C for 6 months. The unchanged thermograms revealed no polymorphic alteration and no phase separation in the system for all three PGFAs. 14

The presence of one crystalline structure and the absence of polymorphism were afterwards confirmed by the SWAXS signals. The obtained WAXS signals of selected PGFAs are shown in Figure 2 (right-hand diffractograms), which are related to the short spacings within the cross-sectional packing of the hydrocarbon chains in each molecule, called sub-cell. These data showed that all selected PGFAs have a peak at the reciprocal lattice spacing q of 1.515 Å-1, which corresponds to a short-spacing of d = 4.15 Å by using the equation d = 2π/q (equation 5). The obtained value of 4.15 Å associates with the α form of TAGs [25]. Considering the fact that the sub-cell re-arrangement in the solid state can be defined as polymorphism, the stable WAXS signal and the corresponding short spacing of 4.15 Å after storage of samples under both RT and 40 °C conditions (6m/RT and 6m/40 °C in Figure 2) confirmed the absence of polymorphism and the stable solid state of these compounds. For a detailed description of this stability, which is a common phenomenon in PGFA molecules, please refer to the Part 1 of these investigations.

The long spacing of selected PGFAs, described as the distances between the planes formed by the methyl end-groups in each molecules, was determined using SAXS data. The diffractograms in Figure 2 (left-hand) revealed a main peak at the reciprocal lattice spacing q equal to 0.0986, 0.981, and 0.092 Å-1 for PG3C16/C18 partial, PG4-C18 partial and PG6-C18 partial, respectively. Based on these data and using equation 5, a lamellar thickness of 63.7, 64.0, and 68.1 Å has been calculated for PG3-C16/C18 partial, PG4-C18 partial and PG6-C18 partial, respectively (Table 1). Please note that a lamellar structure incorporates a repetitive sequence of hydrocarbon chains with the polar groups (glyceryl moiety) placed inside, and therefore the lamella thickness is approximately a multiple of the chain length. Considering a length of 1.524Å for each Csp3–Csp3 bond, the approximate hydrocarbon chain length within PGFAs composed by palmitic acid (C16) and stearic acid (C18) is roughly 23Å and 26Å, respectively. As described

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in Part 1 of this study, the obtained lamellar thicknesses can be addressed to a 2L configuration and the increasing polar fraction of the PGFA molecules (increased PG moieties and free OH groups). Generally, a time-dependent alteration in the SAXS pattern and lamella thickness indicate polymorphism (causing a tilt of the hydrocarbon chains) or phase transitions [25]. In this study, the SAXS patterns of 6m/RT and 6m/40 °C samples and thus the calculated lamellar thicknesses remained unchanged (Figure 2 and Table 1), confirming the absence of polymorphic alteration of PGFAs and phase separation.

The crystallite size and the number of stacked lamellas to build the crystallites were quantified using Scherrer equation and are depicted in Table 1. The alteration on the number of lamellas, namely crystallite growth, was therefore monitored during the storage of PGFAs. A very slight increase in crystallite size was observed after 6 months storage, which was more predominant for PG6-C18 partial combined with the increasing of one lamella. The release profile of NAC from microcapsules after storage remained stable for all three PGFAs.

Microstructure The microstructure of lipids corresponds to the morphology of their crystal clusters. Once primary lipid crystals are formed, the crystals aggregate or grow into each other to form the crystal clusters [26]. It is important to note that the size and shape of crystal clusters are affected by sample treatment and processing conditions. Different crystallization temperatures or cooling rates can result different shape and size of crystalline of the same lipid [26, 27]. In order to provide a qualitative comparison between different PGFAs and tripalmitin (representative for TAGs), the sample preparation was constant for all materials. The well-organized crystal clusters of tripalmitin with a spherulitic structure can be observed in Figure 3. The crystal clusters of selected PGFAs are also shown in this figure. PG3-C16/C18 partial crystallines 16

showed the same well-organized maltese-cross spherulitic structure grown radially, which is known for TAGs [28], however, with significantly smaller crystalline size, compared to tripalmitin. The size of crystal clusters decreased further for PG4-C18 partial and PG6-C18 partial, with a combination of disordered spherulitic-shape and grainy-structured clusters for PG4-C18 partial, and small needle-like clusters for PG6-C18 partial. As described in Part 1, the shift of well-organized, spherulitic-shaped crystal clusters of tripalmitin to small disordered and needle-like clusters of PG6-C18 partial can be correlated to available free hydroxyl groups in PGFA partials, with increased number for PG6-C18 partial > PG4-C18 partial > PG3C16/C18 partial . An increase in glyceryl moiety in PGFA partials results in increased number of free –OHs with less dense crystalline networks. These observations were confirmed by the solidification data, showing the sharpest crystallization endotherm due to the highest driving force for crystallization for tripalmitin (PPP) and decreasing the values in the following manner: PPP > PG3-C16/C18 partial > PG4-

C18 partial = PG6-C18 partial. These data are shown in Table 2 and will be further discussed in in the next section. This indicates the faster initial nucleation for PGFA partials. The qualitative comparison between the microstructures after 6 months storage at RT or 40 °C (6m/RT and 6m/40°C) are also shown in Figure 3. The slight calculated increase in the crystallite size after storage presented in Table 1 was obvious in the PLM images of stored PG3-C16/C18 partial. The microstructures of two other PGFAs remained stable. These observations was matched with the DSC and SWAXS data, showing the absence of polymorphism and insignificant crystallite growth.

3-3 Hot melt coating properties of PGFAs The selected PGFAs with HLB values ≥5 were used as HMC excipients to provide an API immediate release profile. PG3-C16/C18 partial has a HLB value of 5.1, the HLB values of PG4-C18 partial and PG6-C18 partial are 5.6 and 6.2, respectively. The hot melt coating properties of these PGFAs, in terms of viscosity in 17

molten state, melting solidification data extracted from DSC measurements and water uptake of meltcasted stabs are listed in Table 2. The same data for tripalmitin and the mixture of tripalmitin with 10%w/w of polysorbate 65 are also added to the table to provide a direct comparison between selected PGFAs with TAGs and an already existing HMC immediate release formulation [8].

Generally, hot melt coating as a solvent-free coating technology requires certain properties of coating excipients. Due to the fact that such excipients are mostly LBEs and sprayed onto the surface of feed material in a molten state to solidify and form a coating layer, properties such as viscosity of the molten material, melt transition range and LBE volume contraction play an important role in the processability and coating quality [5, 7, 15, 29]. As a rule of thumb, a viscosity of molten lipid lower than 300 mPa.s at temperatures of approximately 20 to 40 °C higher than the melting temperature of lipid is required for an immaculate transport of molten lipid towards the pneumatic nozzle and to avoid the clogging of nozzle during atomization [5, 7, 15, 29]. Please note that the type and design of the pneumatic nozzle play a crucial role on the atomization of coating liquid and the required viscosity. More information on the design of pneumatic nozzles can be found elsewhere [30-34]. After reaching the surface of feed material, the molten lipid will be spread on the surface and solidified. The coating quality depends on the crystallization properties and the volume contraction of lipids. An accelerated crystallization process is desired to reduce the tackiness of coating lipid and thus to reduce the risk of agglomeration of feed material during coating process. At the same time an accelerated crystallization process combined with a sharp solidification enthalpy indicates a fast nucleation rate, which can impair the coating quality or even lead to spray congealing of coating material in the process container before reaching the feed material. The tackiness of coating lipid and the risk of agglomeration can increase by using LBEs with high volume contraction. Thus, a balanced crystallization process and volume contraction is desired. It is suggested to apply excipients with as low volume 18

contraction as possible, to avoid tackiness but to provide desire spreading of coating on the core surface and to minimize coating imperfections affecting the API release [5, 7, 15, 29]. As listed in Table 2, the measured viscosities of PGFAs were overall lower than 300 mPa.s, which suggests desired processability of these excipients through the pneumatic nozzles, independent of the nozzle type [5,7,15,29]. The viscosities of molten PGFAs showed a Newtonian flow characteristic and the highest value belonged to PG6-C18 partial. The viscosity of PGFAs increased with the increased length of polyglycerol moiety, being 27.1, 34.2 and 44.1 mPa.s for PG3-C16/C18 partial, PG4-C18 partial, and PG6-C18 partial, respectively. This can be explained with the lower mobility of longer polyglycerol moieties and resulting higher number of hydroxyl groups for hydrogen bonding [35]. This is also the reason for higher viscosity of selected PGFAs compared to the viscosity of pure tripalmitin at 100 °C. The melting onsets of PGFAs were between 54.2 °C for PG3-C16/C18 partial, and approximately 60 °C for PG4-C18 partial and PG6-C18 partial within a desirable range for processing of these materials as hot melt coating excipient [29]. Increasing the melting temperature is directly correlated with the length of hydrocarbon chain of fatty acids in TAG. The same principle is counted for PGFAs. Besides, polyglycerols with certain number of glycerol moieties and the same fatty acid chain possess a higher melting temperature if they are fully esterified. For instance, the melting temperature of PG6-C18 full is higher than the melting temperature of PG6-C18 partial. The increasing number of polyglycerol moieties does not result in increasing the melting temperature of compound. This explains the melting behaviour of selected PGFAs in this study, being in the same range for PG4-C18 partial and PG6-C18 partial and the decreased melting onset for PG3-C16/C18 partial. The solidification peaks for PG3-C16/C18 partial, PG4-C18 partial and PG6-C18 partial were 45.4 °C, 54.33 °C and 52.23 °C, respectively, without additional low solidification fractions. This enables hot melt coating using process temperatures of 40 °C, which is an important criterion for coating of thermosensitive APIs. Moreover, the absence of low solidification fractions reduces the risk of agglomeration during the coating 19

process. The solidification properties for each material were further defined as the sharpness of the exothermic curve of crystallization in terms of full width at half maximum (FWHM) value of each curve using OriginPro 2019b (OriginLab Corporation, USA) and the driving force (Δµ) for the crystallization. The FWHM values were similar for PGFAs and significantly higher for tripalmitin and its mixture with 10%w/w polysorbate 65. The Δµ was 12.4 (±1.4), 14.14 (±0.9), and 22.3 (±3.4) j/g for PG6-C18 partial, PG4-C18 partial and PG3-C16/C18 partial, respectively. The Δµ values for the crystallization of tripalmitin and its mixture were 93.1 (±5.5) and 81.6 (±0.3) j/g respectively. A higher driving force and sharper heat flow exotherm indicate a higher energy barrier for initiating the nucleation. Once initiated, the crystallization happens in a faster rate [11, 36]. This phenomenon can be observed for tripalmitin and its mixture with polysorbate 65 compared to PGFAs. Comparing PGFAs with each other, the driving force for crystallization is higher for PG3-C16/C18 partial. In a hot melt coating process the initial nucleation of PGFAs is faster because of their lower driving force and lower ΔT (difference between melting and crystallization temperature) compared to tripalmitin and its mixture. At the same time due to their lower crystallization rate, PGFAs have more time to spread on the surface of the core material. With optimal setting of process parameters (mainly spray rate, atomization air pressure and process temperature), this behavior of PGFAs can be used for a homogeneous coating without the risk of agglomeration. Please note that the broadness of the thermal events and the nucleation rate are also dependent on selected heating and cooling rate. The selected rate of 10 K/min, in order to simulate the fast cooling of melt in a HMC process as much as possible results in broader thermal events. The volume contraction was about 10 – 12 v/v% for all measured PGFAs and was slightly lower than the volume contraction of tripalmitin and tristearin, however without statistical significance. The water uptake of selected PGFA partials was significantly higher than the water uptake of pure tripalmitin at 37 °C (Table 2) and it was ranged from 10.47 %w/w to 24.17 %w/w. The lowest value of 10.47 %w/w belonged to PG3-C16/C18 partial and the highest value to PG6-C18 partial, which is directly 20

correlated with the increased level of hydrophilicity and HLB value associated with the increased polar fraction (PG moiety) in these molecules. For all selected PGFAs the erosion was below 2 %w/w. Using PGFA partials as hot melt coating materials, these results suggest a drug release mechanism through the coating based on following three steps: (1) water penetration through the coating towards the API core; (2) drug dissolution; and (3) drug diffusion through the liquid located in the PGFAs as coating towards the medium [37]. It can be also concluded that tailoring a drug release profile should be possible by varying the hydrophilicity of the PGFA molecule, resulting in a tailored level of water penetration into lipid coating, taking the solubility of coated API into account.

3-4 In vitro toxicity studies PG3-C16/C18 partial and PG4-C18 partial were very slightly soluble in MEM with a solubility of 0.3 (±0.06) and 0.52 (±0.04) mg/ml, respectively. PG6-C18 partial was slightly soluble with a value of 1.56 (±0.05) mg/ml, which is due to its higher HLB value. The solubility of PGFAs was improved in FaSSIF medium, because of the composition of this medium containing lecithin and sodium taurocholate. A solubility of 0.57 (±0.08), 4.2 (±0.7) and 11.8 (±1.2) mg/ml corresponding to very slightly, slightly and sparingly soluble (USP42) was measured for PG3-C16/C18 partial, PG4-C18 partial and PG6-C18 partial, respectively in FaSSIF. TEER values for Caco-2 cells prior to the exposures were 687 ± 141 Ω * cm2. After exposure of cells to PGFAs in the conventional model no changes in TEER were observed, and in the FASSIF/mucus model the TEER values were 130-200 Ω * cm2 higher than the initial values, however the difference was statistically not significant. The higher TEER values in FaSSIF/mucus model can be due to the mucus overlay and this phenomenon was also described by other working groups. TEER values in the not mucus-producing HT29 cells and in the mucus producing HT-29 MTX parental line were reported to be 110 and 437 Ω * cm2, respectively [38]. Gagnon et al. [39] reported on significantly lower TEER values of washed HT-29 MTX 21

monolayers compared to the values of unwashed monolayers. In our study, the higher values show that the available amount of taurocholate in FaSSIF medium also did not have an adverse effect on intercellular junctions. Viability of Caco-2 cells decreased after exposure of cells to pure solutions of PG3-C16/C18 partial and PG4-C18 partial in the FaSSIF/mucus model (Figure 4a). Viability values measured after exposure to PGFAs in the conventional model were mostly around 100% (Figure 4b). The slightly increased action of PG3C16/C18 partial and PG4-C18 partial in the FaSSIF/mucus model can be the result of the higher solubility of this compounds in the FaSSIF medium. While the solubility of PG6-C18 partial was more improved in FaSSIF compared to other two PGFAs, the viability of Caco-2 cells was not impaired after exposure to this PGFA in the FaSSIF/mocus model. This can be explained by the slightly increased HLB value of PG6-C18 partial and less lipophilicity of this molecule. The higher lipophilicity of PG3-C16/C18 partial and PG4-C18 partial can increase the risk of perturbation in cell membranes [40]. The mucus itself did not present a strong diffusion barrier for hydrophilic and lipophilic molecules in the study of Poutier et al. [38]. It is, however, not clear whether this applies also for substances like PGFAs with much higher molecular weight. The decreased viabilities of Caco 2 cells after exposure to pure solutions of PG4-C18 partial and PG3C16/C18 partial were >70% of the control cells, and therefore non-cytotoxic according to the ISO 109935 guidelines for cytotoxicity testing [41]. Taking the NAC multi-particulate systems coated with 50 %w/w PGFA as an example and by considering the 600 mg daily dose of NAC, the same amount of PGFA; namely 600 mg would be daily taken. This amount of PGFA would be distributed on the surface of small intestine with approximately 6 m in length, 2.5–3.0 cm in diameter and villi and microvilli, which increase the surface area by 30–600 fold, respectively [42]. These dimensions and the low solubility of PGFAs indicate the non-cytotoxic effect of these compounds in an oral administration.

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3-5 Release profile of API from coating Taste-masked multi-particulate systems with an immediate release profile was the defined specification for hot melt coated NAC microcapsules. Taste masking of NAC powder was considered as achieved, if ≤ 10% of drug was released within the first 5 minutes of the dissolution test [43]. A release of 85% API within the first 30 minutes of in vitro dissolution test was considered as immediate release, according to Ph. Eur. 9.0 guideline. Pure (uncoated) NAC is a freely water soluble substance (1g soluble in 1-10 ml water) (Ph. Eur. 9.0), with the pKa of 3.24 for its carboxylic acid. Figure 5 shows the dissolution of pure NAC and the release profiles of NAC from coatings of selected PGFAs in purified water. Due to the freely water solubility of NAC, the pure substance was completely dissolved within the first 5 minutes. All three coatings could be considered as taste masked, since the percentage of released NAC within the first 5 minutes was overall less than 10% (9.94%, 7.5% and 0.5% from PG6-C18 partial, PG4-C18 partial, and PG3-C16/C18 partial, respectively). However, the taste-masking efficiency of PG6-C18 partial coating was in the borderline and PG3-C16/C18 partial provided an exceptional taste masking with nearly no release of API (0.5%) during the first 5 min. Whereas, the release profile of NAC from coatings with PG4-C18 partial and PG6-C18 partial corresponded to the target of minimum 85% of release within 30 min of dissolution test, the release of NAC from PG3-C16/C18 partial was slower than an immediate release profile. These data were in agreement with the Δµ values and microstructure of crystalline clusters of PGFAs (Δµ being higher and the crystalline clusters being more organized for PG3-C16/C18 partial compared to other two PGFAs), their HLB and the results of water uptake measurements. As described above, increasing the polar fraction by increasing the PG moieties results in the increased hydrophilicity and thus increased HLB of PGFAs in the order of PG3-C18 partial < PG4-C18 partial < PG6-C16/C18 partial. The lowest hydrophilicity provided an excellent taste masking and slower release profile of API, compared to two other selected PGFAs. In the contrary PG6-C18 partial with highest hydrophilicity provided a borderline taste-masking and the

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perfect immediate release profile. Microencapsulations of NAC particles with PG4-C18 partial met both criteria for taste masking and immediate release profile, showing 7.5% release at 5 minutes of dissolution

Compared to purified water, the dissolution of pure NAC was slightly reduced in both SGF and FaSSGF (Figure 6), which can be explained by the chemical structure of this compound with the presence of carboxylic group (pKa = 3.24). The release profile of NAC from PG3-C16/C18 partial coating in these three media followed the same pattern, which indicates that the HCl 0.1N (SGF) and FaSSGF had no influence on the solubilization or water-uptake of PG3-C16/C18 partial coating and thus no effect on drug release. We have shown in part 1 of these studies that buffers with different ion concentrations and pH had no impact on the water-uptake of PGFAs. In the current work (in in vitro toxicity studies), we mentioned the very slight solubility of PG3-C16/C18 partial in FaSSIF, and the increased solubility of PG4-C18 and PG6C18 partial with increased HLB value, because of the presence of phospholipids and sodium taurocholate in FaSSIF medium. These two compounds are also available in FaSSGF, however with lower concentration. None of these media contains lipase. The composition and pH of these two media according to manufacturer (https://biorelevant.com) are listed in Table 3. Generally, in human gastrointestinal tract 540% of lipids are digested by pepsin and gastric lipase of acidic gastric juice. Bile secretions containing bile salts and phospholipids are responsible for stabilizing lipid droplets and removing lipolysis products, and pancreatic lipase further supports the enzymatic digestion [44]. As like acylglycerols, the ester bonds of PGFAs are enzymatically hydrolyzed, and polyglycerols and fatty acid moieties are the products of PGFAs enzymatic digestion [45, 46]. Witzleb et al [47] reported on the increased API release from extrudates of cetyl palmitate and glyceryl monostearate in a biorelevant medium containing pancreatic lipase. No influence of FaSSGF composition on the release of NAC from PG3-C16/C18 partial coating can be explained by the absence of enzymes and low concentration of bile salts in this medium.

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The level of coating for all three cases was 50 %w/w to provide an easier comparison between the release profiles. The level of coating strongly depends on the purposed release profile, solubility of API and the size and size distribution of core material. Considering these factors and due to the different behavior of lipids compared to polymers, application of a range of 10 to 50% of coating in a hot melt coating process can result in desired coating properties [3,8,14,15]. Generally, combining taste masking with immediate release profile needs a fine-tuning of the coating level and HLB value of lipids. Using lipid-based excipients with higher HLB values and higher level of coating is a good compromise. In this study, alteration in coating amount of selected PGFAs and further optimization of process parameters can further improve the release profiles. Other PGFAs with lower HLB can be used for development of hot melt coated products with extended release profile. Considering the results of water uptake of the melt-cast stabs of selected PGFAs combined with the achieved release profiles in purified water suggests a drug release mechanism starting with a lag time for penetration of dissolution medium through the coating towards the API core, followed by drug dissolution and diffusion of dissolved drug through the coating layer towards the medium. A very short lag time was observed for PG4-C18 partial and PG6-C18 partial, followed by a zero-order release kinetic within 5 to 20 minutes of the dissolution test, confirming the diffusional mass transfer of dissolved API through the coating layer as long as a saturated solution exists inside the coating and the perfect sink conditions are fulfilled. In case of coating with PG3-C16/C18 partial the lag time was longer and an API release with zeroorder kinetic was observed within 5 to 45 minutes of the dissolution test with a release constant of 0.2*103

(min-1) (Table 4). The release constants for PG4-C18 partial and PG6-C18 partial for a zero-order release

within 5 - 20 min were significantly higher with the values of 7.4*10-3 (min-1) and 10.6*10-3 (min-1), respectively (Table 4). The release constants were with a good agreement with the level of compact packing of PGFA crystalline clusters, being compacter for PG3-C16/C18partial.

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Stability of drug release profile was given during storage at room temperature and accelerated conditions (40 °C) for at least 3 months (Figure 7). The stable release profiles after storage can be observed from these figures and from the calculated similarity factors (f2), comparing the initial release profile of NAC from coating (T0) with the release profile after storage under accelerated condition (3m/AC). As can be seen in Table 5 all values were between 50 and 100, indicating the similarity of release profiles after storage with the initial one.

The correlation between instability in the release profile, causing instable product performance, and the time-dependent structural alterations of lipid-based coating materials has been shown by several working groups [14, 16, 17, 19, 48, 49]. The resulted instable product performance is the main reason that lipidbased formulations and processes particularly hot melt coating still did not find their way in the pharmaceutical industrial manufacturing. In this study, the stable release profiles are perfectly convenient with the stable solid-state of PGFAs. The stable microstructure, the absence of polymorphism and crystalline growth provide a stable coating, which is not affected by environmental stress conditions.

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4- Conclusion While HMC is manifested as the solvent-free coating technology, suitable for green manufacturing and offering a more economic process compared to conventional solvent coating techniques, this process is not still established in the pharmaceutical industry. This is mainly due to the necessity of using lipid-based excipients for a HMC process and the lack of such excipients with stable solid state. The solid state alteration of lipids as coating material causes instable pharmaceutical product performance during the shelf life. In this work, we showed the suitability of selected compounds belonging to the chemical group of PGFAs as hot melt coating excipients with proper physical characteristics and stable solid state. Considering the challenging development of immediate release formulations with lipid based excipients, which in most cases needs biphasic systems or adding emulsifiers to the lipids with the consequence of phase separation and triggering the lipid crystal growth as an additional source of instability, we aimed to screen the suitability of PGFAs for stable immediate release purposes. The data showed the advanced performance of selected PGFAs as HMC compounds. The possibility to select the HLB for tailoring the API release profile allowed the advantage of providing a monophasic system and avoiding emulsifiers. The stable solid state of these molecules resulted in a stable product performance. The GRAS status of PGFAs, their approval as food additives (US FDA 21CFR172.854 and EU food additive E475) and the results of in vitro toxicity studies can be considered for the safety of these compounds for oral administration.

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Conflict of Interest Statement: The Austrian COMET Program and IOI Oleo GmbH Germany funded this research. The research is free of bias. The authors declares that there is no conflict of interest. Acknowledgements: This work was funded through the Austrian COMET Program by the Austrian Federal Ministry of Transport, Innovation and Technology (BMVIT), the Austrian Federal Ministry of Economy, Family and Youth (BMWFJ), the State of Styria (Styrian Funding Agency SFG) and the Austrian Research Promotion Agency (FFG) as part of the project "LIMASIF" within the "Industrienahe Dissertation" program. The authors thank Michael Stehr and Hendrik Woehlk from IOI Oleo GmbH Hamburg for the overall support.

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Figures legend Figure 1- DSC thermograms of PG3-C16/C18 partial, PG4-C18 partial, and PG6-C18 partial as received (Raw, first heating cycle), T0 (second heating cycle) and after 6 months storage at RT (6m/RT) and 40 °C (6m/40 °C). Exotherms are pointing upwards; dashed lines indicate cooling cycle, solid lines indicate heating cycles Figure 2- SWAXS pattern of selected PGFAs; a=untreated samples, as received, b=T0 sample, c=6m/RT, d=6m/40°C Figure 3- Microstructure of tripalmitin and selected PGFAs after melting and solidification (Time 0), and microstructure of PGFAs after 6 months storage at RT and 40°C (6m/RT and 6m/40°C) Figure 4- Viability of Caco-2 cells according to dehydrogenase activity after 24h exposure to PGFAs using a) FaSSIF/mocus model, medium=FaSSIF, b) conventional model, medium=MEM. Control: Cells exposed to FaSSIF only (a) and MEM only (b) were set as 100%. Abbreviation: P, partial Figure 5- Dissolution of pure NAC and release profile of NAC from microcapsules coated with 50%w/w PG3C16/C18 partial, PG4-C18 partial and PG6-C18 partial after HMC process (T0) Figure 6- Dissolution of pure NAC and release profile of NAC from microcapsules coated with 50%w/w PG3C16/C18 partial in purified water, SGF and FaSSGF Figure 7- Release profile of NAC from microcapsules at T0 and after 3 months storage under long-term condition (3m/LT) and accelerated condition (3m/AC)

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Tables legend Table 1- Lamella thickness, crystallite size and number of stacked lamella of selected PGFAs after melting and solidification (T0) and after 6 months storage at RT and 40 °C (6m/RT, 6m/40°C) Table 2- HMC properties of selected PGFAs, tripalmitin (PPP) and a mixture of PPP with 10%w/w polysorbate 65. Table 3- Composition and pH of FaSSIF and FaSSGF (https://biorelevant.com) Table 4- Zero-order constant (k) and correlation coefficient (R2) of release profiles obtained from fitting the experimental data to zero order phase Table 5- Similarity factor f2 of dissolution profile of stored samples under AC for 3 months (3m/AC) compared with the dissolution profile of the samples after manufacturing (T0)

36

37

38

39

40

41

42

43

44

Table 1- Lamella thickness, crystallite size and number of stacked lamella of selected PGFAs after melting and solidification (T0) and after 6 months storage at RT and 40 °C (6m/RT, 6m/40°C)

Lamella thickness (Å) Crystallite size (nm) Number of stacked lamellae (/)

PG3-C16/C18 partial T0 6m/ 6m/ RT 40°C 63.7 63.7 63.5

PG4-C18 partial T0 6m/ 6m/ RT 40°C 64.0 63.2 63.7

PG6-C18 partial T0 6m/ 6m/ RT 40°C 68.1 68.1 67.5

23.0 4

26.0 4

29.0 4

24.4 4

27.8 4

45

26.1 4

27.4 4

33.2 5

36.3 5

Table 2- HMC properties of selected PGFAs, tripalmitin (PPP) and a mixture of PPP with 10%w/w polysorbate 65.

PG3C16/C18 partial PG4-C18 partial PG6-C18 partial PPP PPP+10 % PS 65

Viscosity at 100°C (mPa.s) 27.1

Tm onset (°C) 54.2 ±0.6

ΔHm

34.2

60.33± 0.06 59.26± 0.12 66.5 ±0.36 64.2 ±0.35

44.1 9.5 9.38

ΔHc ( j/g) 100.88± 3.7

FWHM of solidif. enthalpy 7.04±0.28

ΔT (Tm-Tc) (°C) 13.36 ±1.96

Δµ

(j/g) 98.2±2. 68

Tc peak (°C) 45.4 ±1.01

89.99 ±1.64 77.47 ±5.11 199.6 ±8.93 171.5 ±1.99

54.33 ±0.25 52.23 ±1.36 37.63 ±0.63 36.26 ±0.35

97.08 ±1.47 86.69 ±5.59 131.83± 6.96 123.43± 1.33

6.41±0.31

10.13 ±0.61 10.03 ±1.59 32.9 ±1.34 32.9 ±0.55

14.15 ±0.9 12.4 ±1.4 93.06 ±5.52 81.6 ±0.27

46

7.21±0.44 4.45±0.52 5.34±0.24

(j/g) 22.33 ±3.4

Water uptake (%w/w) 10.47 ±0.76 15.92 ±1.83 24.17 ±0.1 0.5 -

Table 3- Composition and pH of FaSSIF and FaSSGF (https://biorelevant.com)

Taurochlate (mM) Phospholipids (mM) Sodium (mM) Chloride (mM) Phosphate (mM) pH

FaSSIF 3 0.75 148 106 29 5

FaSSGF 0.08 0.02 34 59 1.6

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Table 4- Zero-order constant (k) and correlation coefficient (R2) of release profiles obtained from fitting the experimental data to zero order phase Sample

K x 10-3 (min-1)

R2

PG3-C16/C18 partial

0.2

0.994

PG4-C18 partial (50w/w%)

7.4

0.997

PG6-C18 partial (50w/w%)

10.6

0.992

(50w/w%)

48

Table 5- Similarity factor f2 of dissolution profile of stored samples under AC for 3 months (3m/AC) compared with the dissolution profile of the samples after manufacturing (T0) f2 3m/AC compared to T0 PG3-C16/C18 P

73.42

PG4-C18 P

69.83

PG6-C18 P

65.43

49