Crystallization of Active Pharmaceutical Ingredients in the Presence of Heterosurfaces
Raquel Arribas Bueno
Thesis presented for the award of Doctor of Philosophy (PhD)
Supervisors: Prof. Kieran Hodnett, Dr. Sarah Hudson, and Dr. Peter Davern
Submitted to the Faculty of Science and Engineering, University of Limerick, Ireland, January 2018
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DECLARATION
I declare that the work presented in this thesis herein, is entirely my own work and has not been submitted to this or any other university. Due reference and acknowledgment has been made, when necessary, to the work of others.
______Raquel Arribas Bueno
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TABLE OF CONTENTS ABSTRACT ...... 6
LIST OF PUBLICATIONS ...... 8
ABBREVIATIONS AND NOTATIONS ...... 11
1. CHAPTER 1: INTRODUCTION
1.1.General Introduction ...... 17 1.2.Components of a Drug ...... 18 1.3.Stages of the Drug Manufacturing Process ...... 18 1.4.The Crystal ...... 20 1.5.Crystallization ...... 22 1.6.Polymorphism ...... 40 1.7.Bioavailability ...... 43 1.8.Crystallization of API’s in the presence of excipients ...... 45 1.9.Scope of this Project ...... 48 1.10. References ...... 50
2. CHAPTER 2: MATERIALS AND ANALYTICAL TECHNIQUES
2.1.Materials ...... 55 2.1.1. Acetaminophen ...... 55 2.1.2. Fenofibrate ...... 56 2.1.3. Lactose ...... 58 2.1.4. Mannitol ...... 59 2.1.5. Microcrystalline cellulose ...... 60 2.1.6. Carboxymethyl cellulose ...... 61 2.1.7. Polycaprolactone ...... 62 2.1.8. Mesoporous silica ...... 65 2.1.9. Polycarbonate ...... 64 2.1.10. Polymethyl methacrylate ...... 64 2.1.11. Polytetrafluoroethylene ...... 65 2.1.11. Methanol ...... 65 2.2.Analytical techniques ...... 66 2.2.1. Powder X-Ray diffraction ...... 66 2.2.1.1. The crystal ...... 67
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2.2.1.2 Diffraction peaks and Miller indices ...... 68 2.2.1.3. Diffractometer ...... 72 2.2.1.3.1. Reflection mode ...... 72 2.2.1.3.2. Transmission mode ...... 73 2.2.1.4. Diffraction pattern ...... 73 2.2.1.5. XRD analysis conducted in this work ...... 74 2.2.2 Scanning electron microscopy (SEM) ...... 74 2.2.2.1. SEM analysis conducted in this work ...... 76 2.2.3. In situ SEM-Raman and Raman spectroscopy...... 77 2.2.3.1. Raman spectroscopy ...... 77 2.2.3.1.1. Raman analysis conducted in this work ...... 79 2.2.3.2. In situ SEM-Raman...... 79 2.2.3.2.1. In situ SEM-Raman analysis conducted in this work ...... 80 2.2.4. Ultraviolet-visible spectrophotometer (UV-Vis) ...... 80 2.2.4.1. UV-Vis analysis conducted in this work ...... 81 2.2.5. Solid-state Nuclear Magnetic Resonance Spectroscopy (SSNMR) ...... 82 2.2.5.1. SSNMR analysis conducted in this work...... 83 2.2.6. iControl LabMax ...... 83 2.2.6.1. Focussed beam reflectance measurement (FBRM) ...... 84 2.2.6.2. In situ Fourier Transform Infrared (FTIR) ...... 85 2.3.References ...... 88 3. CHAPTER 3: INFLUENCE OF PROCESS PARAMETERS ON THE HETEROGENEOUS NUCLEATION OF ACTIVE PHARMACEUTICAL INGREDIENTS ONTO EXCIPIENTS ...... 91 3.1.Abstract ...... 92 3.2.Introduction ...... 92 3.3.Experimental ...... 96 3.4.Results ...... 101 3.5.Discussion ...... 117 3.6.Conclusion ...... 121 3.7.References ...... 122 4. CHAPTER 4: HETEROGENEOUS CRYSTALLIZATION OF FENOFIBRATE ONTO PHARMACEUTICAL EXCIPIENTS ...... 124 4.1.Abstract ...... 125
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4.2.Introduction ...... 125 4.3.Experimental ...... 129 4.4.Results and discussion ...... 137 4.5.Conclusion ...... 160 4.6.Supplementary information ...... 160 4.7.References ...... 160 5. CHAPTER 5: CRYSTALLIZATION OF ACTIVE PHARMACEUTICAL INGREDIENTS IN THE PRESENCE OF POLYMER COUPONS ...... 164 5.1.Abstract ...... 165 5.2.Introduction ...... 165 5.3.Materials ...... 168 5.4.Methods ...... 169 5.5.Results ...... 175 5.6.Discussion ...... 191 5.7.Conclusion ...... 196 5.8.References ...... 196 6. CHAPTER 6: COMPARATIVE STUDY OF ACETAMINOPHEN AND FENOFIBRATE CRYSTALLIZATION...... 198 6.1.Abstract ...... 199 6.2.Introduction ...... 199 6.3.Materials ...... 202 6.4.Methods ...... 202 6.5.Equations ...... 208 6.6.Results ...... 212 6.7.Discussion ...... 224 6.8.Conclusion ...... 231 6.9.References ...... 232 7. CONCLUSIONS ...... 235 8. ACKNOWLEDGMENTS ...... 238
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ABSTRACT
This research investigates the mechanism of the heterogeneous nucleation of active pharmaceutical ingredients (APIs) in the presence of different ‘heterosurfaces’, i.e. excipients and polymers films.
In the first part of the work, shown in Chapter 3, acetaminophen (AAP), α/β-lactose (α/β-
Lac), and methanol (MeOH) were selected as the model active pharmaceutical ingredient, excipient, and solvent, respectively. The excipient suspended in a supersaturated solution of
AAP in MeOH was used as a heterogeneous surface (“seed”), and parameters influencing the heterogeneous nucleation of AAP, such as (a) the AAP solution/excipient contact time, (b) the
AAP supersaturation, and (c) the AAP to excipient loading, were varied to demonstrate how the nucleation rate and the degree of crystallization can be manipulated to control the particle size and the balance between nucleation and growth.
With the knowledge taken from the first part of the work, a different crystallization system was studied in Chapter 4. A poorly water-soluble API, fenofibrate (FF), with a much longer induction time was used to determine whether the processing parameters for heterogeneous
API nucleation in the presence of different excipients could be optimised to decrease the induction time, increase the rate of API nucleation and control the API particle growth process such that the API’s dissolution rate could therefore be improved. The excipients were found to strongly enhance FF’s nucleation rate during its crystallization from supersaturated MeOH solutions relative to the rate observed in the absence of the excipients; this was accompanied by a pronounced reduction in the induction time for FF from > 22 hours in the absence of excipients to ca. 15 minutes in their presence at optimum conditions. Furthermore, the choice of excipient and process conditions can be used to reduce particle size and thus improve dissolution rates of these poorly water-soluble APIs.
With the aim of understanding the effect of the surface topography and wettability of
‘heterosurfaces’ on the crystallization of APIs, static polymer coupons with different
6 | P a g e wettability properties and surface roughness were used as ‘heterosurfaces’ in Chapter 5.
Polymers with high wettability facilitated a pronounced reduction in the induction time compared to that observed during homogeneous nucleation, with evidence that the nucleation initially started on the polymer surface. In addition, the reduction in the induction time was slightly higher when the polymer surface was covered with grooves instead of simply being presented as a relatively smooth flat surface. A homogeneously dispersed layer of small (<50
m) API particles on polymer surfaces can be obtained in less than 5 minutes using this approach. Such API-coated polymer surfaces could be potentially used as medical devices with ancillary medical substances.
Finally, Chapter 6 provides an overview of the mechanism of heterogeneous crystallization by combining previous results with additional data to provide a clearer understanding of this type of crystallization process. As such, the crystallization of AAP and
FF in the presence and in the absence of excipients was studied in detail by monitoring the processes at a 500 mL scale using FBRM and FTIR probes. An explanation for the heterogeneous crystallization of APIs on excipient surfaces is offered in terms of intermolecular functional group complementarity, molecular volume and solubility is given, which may be helpful to consider for the selection of APIs and excipients in the design of future heterocrystallization experiments.
Graphical abstract: SEM image of FF crystallized on the surface of a Polycaprolactone particle
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LIST OF PUBLICATIONS:
This thesis is based on the work in the following papers:
I. Influence of Process Parameters on the Heterogeneous Nucleation of Active
Pharmaceutical Ingredients onto Excipients
Raquel Arribas Bueno, Clare M. Crowley, Benjamin K. Hodnett, Sarah Hudson,
and Peter Davern
Organic Process Research & Development 2017 21 (4), 559-570
DOI: 10.1021/acs.oprd.6b00425
II. Heterogeneous Crystallization of Fenofibrate onto Pharmaceutical Excipients
Raquel Arribas Bueno, Clare M. Crowley, Benjamin K. Hodnett, Sarah Hudson,
and Peter Davern
Accepted: Crystal growth and design
Related Conference contributions and oral presentations (not included in thesis):
Arribas Bueno, R., Crowley C., Hudson, S., Davern, P., Hodnett, B. K.,
“Crystallization of APIs onto excipients”. Poster presented at: British
Association of Crystal Growth (BACG 2014), July 2014, University of Leeds,
Leeds, UK
Arribas Bueno, R., Crowley C., Hudson, S., Davern, P., Hodnett, B. K.,
“Crystallisation of Acetaminophen in the Presence of Excipients” Poster
presented at: Faraday discussion, March 2015, University of Leeds, Leeds, UK
Arribas Bueno, R., Crowley C., Hudson, S., Davern, P., Hodnett, B. K.,
“Crystallisation of Acetaminophen in the Presence of Excipients” Oral
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presentation at British Association of Crystal Growth (BACG 2015), July 2015,
University of London, London, UK
Arribas Bueno, R., Crowley C., Hudson, S., Davern, P., Hodnett, B. K.
“Crystallization of Fenofibrate in the presence of Excipients”. Poster presented
at: International School of Crystallization (ISC), May 2016, University of
Granada, Granada, Spain
Arribas Bueno, R.; Murphy B., Verma V., Crowley C., Hudson, S., Davern, P.,
Hodnett, B. K. “Heterogeneous Nucleation of APIs onto Excipient
Matrices” Poster presented at: 68th Chemistry Colloquium in Cork, June 2016,
University Collage Cork, Cork, Ireland
Arribas Bueno, R.; Crowley C., Hudson, S., Davern, P., Hodnett, B. K.
“Crystallization of Fenofibrate in the presence of Excipients”. Poster presented at
Crystal Growth of Organic Materials (CGOM12), July 2016, University of
Leeds, Leeds, UK
Arribas Bueno, R.; Crowley C., Hudson, S., Davern, P., Hodnett, B. K.
“Dissolution of Fenofibrate Prepared by Heterogeneous Nucleation”. Oral
presentation at British Association of Crystal Growth (BACG 2016), July 2016,
University of Leeds, Leeds, UK
Verma V., Arribas Bueno, R.; Crowley C., Hudson, S., Davern, P., Hodnett, B.
K. “Effect of Dispersed Excipients on the Nucleation of Active Pharmaceutical
Ingredients” Oral presentation at British Association of Crystal Growth (BACG
2017), July 2017, University of Manchester, Manchester, UK
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Arribas Bueno, R.; Crowley C., Hudson, S., Davern, P., Hodnett, B. K. “ Tuning
process parameters for the heterogeneous nucleation of active pharmaceutical
ingredients on excipients” Oral presentation at 20th International Symposium of
Industrial Crystallization (ISIC20), September 2017, University College Dublin,
Dublin, Ireland
Verma V., Arribas Bueno, R.; Crowley C., Hudson, S., Davern, P., Hodnett, B.
K. “Impact of Scale-up and Dispersed Excipients on the Nucleation of Active
Pharmaceutical Ingredients” Oral presentation at 20th International Symposium
of Industrial Crystallization (ISIC20), September 2017, University College
Dublin, Dublin, Ireland
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ABBREVIATIONS AND NOTATIONS
Abbreviations: API Active Pharmaceutical Ingredients AAP Acetaminophen BCS Biopharmaceutical Classification System CCDC Cambridge Structural Database CLD Chord Length Distribution CMC Carboxymethyl cellulose CNT Classical Nucleation Theory CP/MAS13 C-NMR Cross-Polarization Magic Angle Spinning Carbon-13 Nuclear Magnetic Resonance
D-Man D-Mannitol DTGS Deuterated Triglycine Sulfate FBRM Focused Beam Reflectance Measurement FDA Food and Drug Administration FF Fenofibrate FTIR Fourier Transform Infrared Spectroscopy GBR Guided Bone Regeneration Gluc Glucose GRAS Generally Regarded as Safe Excipients HBD Hydrogen Bond Donors HBA Hydrogen Bond Acceptors HCl Hydrochloric acid MCC Microcrystalline Cellulose MeOH Methanol MCT Mercury Cadmium Telluride MSMPR Mixed-Suspension Mixed-Product Removal MSZW Metastable Zone Width PC Polycarbonate PCL Polycaprolactone PMMA Poly(methyl methacrylate) PTFE Polytetrafluoroethylene
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PSD Particle Size Distribution PXRD Powder X-Ray diffraction SEM Scanning Electron Microscopy
SiO2 Mesoporous silica SSNMR Solid-State Nuclear Magnetic Resonance Spectroscopy UV/vis Ultraviolet-Visible Spectroscopy XRD X-Ray diffraction -Lactose -Lac -Lactose -Lac -Lac -Lactose -LMH -Lactose Monohydrate Notations: μ Chemical potential of supersaturated state * μ Chemical potential of saturated state c Solution concentration c* Equilibrium concentration or solution solubility
∆퐺푇 Difference between the free energy of the system in its final and initial states ∗ ∆퐺푇 Free energy cost of creating the critical nucleus
∆퐺퐹퐼푁퐴퐿 Final free energy 푛 Number of molecules ∆μ Difference in chemical potential between a molecule in solution and that in the bulk of the crystal phase r Radius of the nucleus 훾 Surface free energy or interfacial energy S Supersaturation k Boltzmann constant R Ideal gas constant
푁퐴 Avogadro’s number T Temperature
푉푚 Molecular volume r* Critical radius J Nucleation rate for homogeneous 푍 Zeldovich factor
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푗 Rate at which molecules attach to the nucleus causing it to grow 휌 Number density of molecules
퐴1, 퐴1,퐻퐸푇 Nucleation rate constants for homogeneous and heterogeneous nucleation, respectively dr/dt Time derivative kc, kD, k1, k', k" Growth constants S – 1
∆μc Critical supersaturation ti Induction time tr Relaxation time tn Time required for the formation of a nucleus tg Time required for the nucleus to grow to a detectable size
푘푔 and g Growth constants
푘푏 and b Nucleation constants A Crystal area per unit mass of solvent
퐽퐻퐸푇 Nucleation rate for heterogeneous nucleation ∗ ∆G 퐻푂푀 Free energy cost of creating the critical nucleus for homogeneous nucleation ∗ ∆G 퐻퐸푇 Free energy cost of creating the critical nucleus for heterogeneous nucleation
휌퐼 Number density of foreign particles times the number of places a critical nucleus can form on each foreign particle
휌퐹푃 Number density of foreign particles 휀 Number of places a critical nucleus can form on each foreign particle. n An integer representing the order of the diffraction peak λ Wavelength
E1-E0 Radiation energy h Plank constant ν Frequency dhkl Inter-plane distance h k l Miller plane a, b, c Lattice parameters 2 Diffraction angle in the XRD ncident angle in the XRD A Absorbance
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I Intensity of light reflected from a sample
I0 Intensity of light reflected from a reference material b Path length c Sample concentration csupernatant Concentration of API remaining in the supernatant mmethanol Methanol mass mexcipient Excipient mass
Tcry Crystallization temperature
Tsat Saturation temperature
휌퐴푃퐼 Density of the API
Mw Molar mass
D50 Median diameter
D90 Particle size distribution which are the intercepts for 90% of the volume
D10 Particle size distribution which are the intercepts for 10% of the volume
D90 – D10 Span
푡∞ Time after the end of the API crystallization
푡0 Time before the API starts to crystallize
∆퐻푏푖푛푑 Binding energy between molecule 1 and molecule 2
∆Hmolecule1+molecule2 Total electronic energy in a system which contains molecule 1 and molecule 2
∆퐻푚표푙푒푐푢푙푒1 Total electronic energy in a system which contains molecule 1
∆퐻푚표푙푒푐푢푙푒2 Total energy in a system which contains molecule 2 d Dispersion coefficient p Polarization HF Hartree–Fock method Contact angle
solid Solid surface energy
solid-liquid Interfacial energy
liquid Liquid surface energy C Mersmann equation constant tg Time required for a single crystal to grow to a certain size tmax Time where a maximum in the chord length was observed t0 Time where an increase in the chord length was observed
푉푛푢푐푙푒푢푠 Volume of a critical nucleus
14 | P a g e tm Time required for the addition of a molecule to a crystal
Vp Volume of a particle at tg
푡푛푢푐푙푒푢푠 Time required for the formation of a critical nucleus
Eaads Activation energy for adsorption
Eades Activation energy for desorption
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CHAPTER 1: Introduction
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1.1. General Introduction
In medicine manufacture, the Active Pharmaceutical Ingredients (APIs) are crystalline for the most part, which need to be ‘formulated’ or packed into a tablet that can be taken orally.
Therefore, the size and the shape of the API crystals are very important as the crystalline material once formed still needs to undergo the rest of the manufacturing processes. It is also very important to make sure that your crystalline material remains stable and does not change its form1-2.
Control of crystal morphology can be achieved by changing operating process conditions such as the use of different solvents, or by tailor-made additives. In the context of pharmaceutical solids, the solvent-induced crystal habit modification approach is limited by the solvent toxicity and cost, crystallisation efficiency, and the purity requirements of the final product. Hence, the use of additives as crystal habit modifiers of APIs is starting to play an important role in the pharmaceutical industry1.
This project proposes a process in which the API is crystallized by cooling crystallization in the presence of a suspended excipient. The aim is the promotion of nucleation and growth of the API on or close to the surface of the excipient particles. By tuning process parameters, the desired API particle size and polymorphic form are sought, with a final dissolution rate of the drug similar to the marketed one. Furthermore, employing generally regarded as safe excipients (GRAS) as additives to achieve the desired size and shape of the API represents a practical alternative for such a highly regulated industrial sector as the pharmaceutical industry. Figure 1 shows a scheme of the process performed.
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Figure 1: Scheme of the process performed
Below, the components of a drug and the stages of the current drug manufacturing process will be introduced 3 and explain how this new approach can potentially reduce the processing steps with a consequent reduction in costs:
1.2. Components of a Drug
A drug is compounded from an API, which is the ingredient in a pharmaceutical drug that is biologically active, and the excipients, which are ingredients other than the API used to improve the physical properties of the drug. Ideally, excipients should be inert and they are used as diluents, binders, compression aids, granulating agents, disintegrants, dissolution agents and coating agents. The percentage of excipient in a drug depends on the API properties, such as the compression, disintegration and dilution properties and the dose required.
1.3. Stages of the Drug Manufacturing Process3
1. Synthesis of the APIs:
Most APIs are produced by a chemical synthesis, typically in organic solvents which can
usually be recycled. Depending on the complexity of the molecule required, synthesis of
APIs might need multi-step complex chemistry utilizing a range of processing
18 | P a g e technologies. The products of the reaction are often washed, to remove impurities, filtered and dried.
2. Crystallization:
This is a very important purification technique. This is carried out to isolate the API and ensure there are minimum levels of impurities and that the crystals have the correct size, shape and physical properties. Seeding is generally used to promote the crystallization.
3. Formulation steps:
The next stages of the manufacturing processes are the formulation stages. These stages ensure that the medicine is prepared in an accurate dose and is in a form which can be conveniently administrated to the patient, e.g. tablets, capsules, creams, pastes, gels or ampoules for injections.
Milling and sieving: Raw materials (API and excipients) are milled and sieved to
make sure that the physical size of the solid particles is correct and uniform
throughout.
Mixing and blending: This involves ensuring that the API is thoroughly mixed with
the excipients to make a stable, palatable and effective final product with the
required API concentration. This mix should be homogeneous. The blending might
be dry or wet. Wet blending uses water where possible.
Granulation: This stage involves mixing/ pressing the powders to make the small
particles stick together. This way tiny particles adhere together to form some multi-
particulate entities called granulates. This stage is used primarily in the
manufacturing of tablets. It is an important stage as it ensures that the mix of
powders does not segregate. This might happen due to the powders having different
particle sizes or densities.
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Drying: This can take place through a variety of ways, such as: in ovens, on beds or
dryers using vacuum, spraying or freezing, or using infrared or microwave
techniques.
Tablet pressing: The tablets are pressed using direct compression of the blended
materials.
Tablet coating: This gives the tablet more physical strength and delays the tablet
breaking up once it is swallowed. There are different types of coatings which will
do different things. For example, aqueous based or solvent-based coatings might be
used and this will typically allow the tablets to disintegrate in the stomach.
Cellulose might be used to prevent it from being digested in the stomach. Sugar
coatings are very typically used but now very thin polymer films are starting to be
used.
The proposed approach focuses on the crystallization step in an attempt to improve manufacturing methods and minimize the number of formulation steps. Then, if amenable to scale-up, this proposed method offers the potential to eliminate the need to (a) mill bulk APIs, and (b) blend milled APIs with excipients.
To understand properly the following project some basic concepts will be introduced.
1.4. The Crystal
Crystals consist of three-dimensional (3-D) repeating patterns of atoms, molecules or ions.
They can be described by defining the item to be repeated, the motif, which can be an atom, a molecule or a collection of molecules or ions and the symmetry operations that are the ways in which the motif is repeated. The lattice describing the scheme of repetition is now a 3-D array of points and the unit cell is the smallest repeating unit within this 3-D structure. The unit cell is fully described by six lattice parameters, comprised of the three lengths of the unit cell (a, b, c) and the three inter-axial angles (). Considerations of the relative magnitude of these
20 | P a g e parameters gives rise to the definition of the seven crystal systems. Having extra lattice points in the face- or body-centred sites produced the 14 unique Bravais lattice types spread over these seven systems. Examination of these seven crystal systems reveals that the unit cells become progressively less symmetrical upon moving from cubic through orthorhombic to triclinic4. Figure 2 shows the seven crystal systems.
Figure 2: Seven crystal systems depending on the type of unit cell and the crystal classes5 It is also possible to refine the seven crystal systems in terms of the symmetry elements which they possess. These elements represent various combinations of rotation, mirror, translation and inversion and together with the Bravais lattice type define the full 3-D arrangement of atoms, molecules or ions within a given structure as expressed by its space group. The symmetry exhibited in a unit cell is also reflected by the physical and chemical properties of the resulting macroscopic crystal. Symmetry is evident in properties such as crystal growth rates and crystal shape and surface chemistry. Visualization of the crystal structure of a drug molecule can be challenging particularly when the molecular mass is large4.
In crystallography, a form is defined as a set of equivalent faces resulting from the crystal symmetry. All the forms present on a crystal represent the morphology of the crystal. However,
21 | P a g e the concept of morphology alone does not fully cover the external form of the crystal, which is contained in the notion of crystal habit. The concept of habit includes the notion of face extension 6. Although crystals can be classified according to seven crystal systems, the relative sizes of the faces of a particular crystal can vary considerably 7. Figure 3 shows an example of different crystal habits.
Figure 3: Crystal habits illustrated for hexagonal crystals7 1.5. Crystallization
Crystallization as defined by Chen et al. as an important separation and purification process employed to produce a wide variety of materials 2. It can also be defined as a natural or artificial process of formation of solid crystals from a solution, melt or more rarely deposited directly from a gas 8.
1. Supersaturation
As is the case for all separation processes, crystallization takes place when a driving force
is applied, with supersaturation being the driving force for crystallization. Supersaturation
is central to crystallization processes as it influences factors such as nucleation, growth and
transformation kinetics. After dissolving the chemical species in a solvent, the solution
must be supersaturated in order to observe nucleation and growth 6. Supersaturation can be
defined in different ways. As Mangin et al. explain, supersaturation is the difference
between the chemical potential of the solute molecules in the supersaturated and saturated
22 | P a g e states respectively (μ – μ*), while Chadwick et al. define the supersaturation as the normalized distance of the actual concentration from equilibrium ((c – c*)/c*), where c = concentration of the crystallizing solute, and c*= solubility of the crystallizing solute 9. It can also be defined as the supersaturation as the measure of the concentration distance from equilibrium (c – c*)10-11, but the most common form is as the ratio of concentrations (c/c*) 6.
Then in this thesis supersaturation is defined as c/c*.
These factors in turn affect the size distribution, grain size, purity and final form of the crystals. Large particles are generated at low supersaturations, whereas small particles are generated at high supersaturations12.
Supersaturation can be achieved by various methods, with solution cooling, addition of a second solvent to reduce the solubility of the solute, chemical reaction (precipitation), solvent evaporation and change in pH being the most common methods used in industry.
2. Nucleation and growth
In the process of crystallization of a compound at least two stages must be distinguished: (i) the formation of a critical nucleus (nucleation) and (ii) its subsequent growth.
Nucleation mechanism
Nucleation is defined as the series of atomic or molecular processes by which the atoms or molecules of a reactant phase rearrange into a cluster of the product phase. Critical nuclei is the minimum size that must be formed by atoms or molecules clustering together before a new-phase inclusion is stable and begins to grow 13. These assemblies have a critical size above which they can grow and below which they are unstable and dissolve. Understanding the fundamentals of nucleation is crucial to the control of crystallization processes, including the control of the nucleation and transformation of polymorphs and solvates, the isolation of metastable solid phases in confined spaces, the orientation of polymorphs using
23 | P a g e solid substrates that template certain crystal structures, and the understanding of transformations during dissolution of metastable solid phases10, 14.
Nucleation mechanisms can be divided into two main categories, as shown in Figure 4, namely primary and secondary. Primary nucleation is the formation of nuclei from solution, whereas secondary nucleation is the formation of nuclei brought about by the presence of like crystals 10.
NUCLEATION
Primary Secondary
(Induced by crystals, seeding)
Homogeneous Heterogeneous (Spontaneous) (Induced by foreign particles) Figure 4: Types of nucleation Primary nucleation can be formed by homogeneous or by heterogeneous mechanisms.
Homogeneous nucleation occurs spontaneously and generally with much more difficulty, in a uniform solution. On the other hand, heterogeneous nucleation occurs on the surface of foreign particles. These particles serve to reduce the energy barrier required for the creation of a new solid-liquid interface 10. This in turn, reduces the activation energy required, making heterogeneous nucleation easier to produce.
Two mechanistic schools of thought can be identified in the area of current crystal nucleation research as summarized in Figure 5. First, there is the school of Classical
Nucleation Theory (CNT), which considers that, in supersaturated solutions, concomitant density and order fluctuations lead to the formation of clusters within which the molecular packing reflects all possible polymorphs of the solute: crystal nuclei have the same structure as mature crystals. On the other hand, a “non-classical” crystal nucleation
24 | P a g e pathway, such as a two-step mechanism, has also been proposed. In this two-step mechanism, crystalline order is preceded by the separation of a dense, disordered liquid phase, and fluctuations in density are disconnected from fluctuations in order: initial clusters are liquid-like, and crystalline order appears over time 15.
Figure 5: Structural models of cluster formation during crystal nucleation 15 Energetics
According to nucleation theory, the work necessary to form a cluster of n molecules is the difference between the free energy of the system in its final and initial states plus a term related to the formation of an interface between the nucleus and the solution 7, 13. This can be expressed by (assuming a spherical nucleus):
2 ∆GT = −푛∆μ + 4πr 훾 Equation 1
Where:
∆μ is the difference in chemical potential between a molecule in solution and that in the bulk of the crystal phase, n is the number of molecules, r is the radius of the nucleus,
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and 훾 is the surface free energy or interfacial energy
Following thermodynamics ∆μ can be expressed as:
∆μ = 푁퐴 kT ln (S) = R T ln(S) Equation 2
where k is the Boltzmann constant, T is the absolute temperature, S is the supersaturation
ratio, 푁퐴is Avogadro’s number and R the molar gas constant.
If each molecule in the crystal occupies a volume Vm (molecular volume), then each
4 휋푟3 nucleus will contain ∗ molecules, with r being the radius of the nucleus of the crystal. 3 푉푚
Equation 3 will then take the following form:
3 4 πr 2 ∆GT = − ∗ 푁퐴 kT ln (S) + 4πr 훾 Equation 3 3 푉푚
4휋푟2훾
∗ ∗ ∆퐺푇 푎푡 푟
3 4 휋r ∆퐺푇 − 푁퐴 kT ln (S) 3 푉푚
∆퐺퐹퐼푁퐴퐿 = 푒푞푢푖푙푖푏푟푖푢푚
Figure 6: (a) Total free energy versus cluster size. (b) Nucleation rate as a function of supersaturation (showing the critical supersaturation)16
Figure 6 shows a plot of ∆GT as a function of r; it can be seen how the function reaches a
maximum, which represents the energetic barrier that needs to be surpassed to achieve
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∗ * nucleation ∆퐺푇. The value of r at this maximum (r ) is defined as the critical radius or
nucleus size7, 13. Both values are defined by:
2γ푉 r∗ = 푚 kTlnS Equation 4
16 π γ3 푉2 ∆퐺∗ = 푚 푇 3k2T2ln2S Equation 5
It has been proved that the value of the critical radius (r*) decreases (as well as that of
* 17 ∆GT ) as the supersaturation increases , meaning that the probability of having nucleation
in a given system will be higher at higher supersaturations.
The rate of nucleation (i.e., the number of nuclei formed per unit time per unit volume) can
be expressed by an Arrhenius-type equation 13:
∆퐺 ∗ − 푇 J = 휌 푍 푗 e kT Equation 6 where the prefactor is the product of the three terms: the number density of molecules, , the rate at which molecules attach to the nucleus causing it to grow, j, and the Zeldovich factor Z, which is the probability that a nucleus at the top of the activation energy barrier will go on to form a crystal. The number density of molecules is essentially the number of possible nucleation sites per unit volume, as for homogeneous nucleation the nucleus can form around any one of the molecules present. An upper bound on the rate at which molecules attach to the nucleus causing it to grow, j, is provided by the diffusion-limited
∆퐺 ∗ − 푇 ∗ flux onto the nucleus. While in the exponential factor, e 푘푇 , ∆퐺푇 is the free energy cost of creating the critical nucleus, the nucleus at the top of the activation energy barrier. By definition the probability of an event occurring is proportional to the exponential of minus the free energy cost of the event over the thermal energy kT. Thus, the exponential factor comes from the very low probability of forming a nucleus at the top of the barrier18.
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Combining Equation 5 and 6 together and grouping the constant, J, can be defined as follows 19:
3 2 ∗ 16 π γ 푉푚 ∆퐺푇 − 3 3 2 − 퐽 = 휌 푍 푗 e 3푘 T ln S = 퐴1e kT Equation 7
A typical plot of J as a function of supersaturation (S) is depicted in Figure 6b. It can be seen in this plot that the nucleation rate is virtually zero until a critical value of supersaturation is achieved, after which the rate increases exponentially. The metastable zone (which will be defined later) is delimited by this critical supersaturation (∆μc).
Equation 4 and 5 show that both ∆G* and r* depend heavily on the surface free energy (훾), so any process that modifies this value will have an effect on the possible viability of the nucleation process. It has been proved that in the presence of a foreign substrate the decrease in the value of γ therefore reduces the values of ∆G* and r∗ at constant supersaturation17, that is, making nucleation more favourable.
A decrease in γ will also decrease the value of the critical supersaturation (∆μc), since the
nucleation rate is also dependent on the surface energy (Equation 6 and Equation 7). This
will make heterogeneous nucleation more viable than homogeneous nucleation at low
supersaturation conditions. The reduction of the surface energy will be the highest when the
best match between the substrate and the crystallizing substance is achieved. This situation
is created, of course, when both the substrate and the crystallizing substance are the same
(seeding crystallization), referred to as secondary nucleation. This mechanism will be more
favourable than both heterogeneous and homogeneous nucleation and thus produced at
lower supersaturations.
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Growth mechanism
Crystal growth is the series of processes by which a growth unit is incorporated into the surface of a crystal, causing an increase in size. Growth occurs once the nuclei are formed and exceed the critical size; from there they become crystals. Hereafter, we recall the basic principles of crystal growth6. Growth kinetics and mechanisms depend on external factors
(medium, temperature, supersaturation, and hydrodynamics) and on internal factors
(structure, bonds, and defects).
Crystals grown from solution typically exhibit regular, planar faces characterized via Miller indices. Although appearing flat to the naked eye, these crystalline surfaces are rarely so at the molecular level. The various features which make up the nanoscopic surface topography of crystal faces are intimately involved in the mechanisms by which they grow.
The mechanism of growth may be defined as the manner in which atoms or molecules attach to the growing crystal surface. The growth rate is a function of supersaturation and the transition between stable growth on atomically smooth surfaces and unstable growth at the roughened growth interface. Two idealized types of lateral mechanisms can be distinguished, namely (i) screw dislocation and (ii) 2-D surface nucleation, as shown in
Figure 7.
i
ii
Figure 7: Schematic diagram of the growth of a crystal on (i) screw dislocation and (ii) 2-D surface nucleation20
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The morphology of a crystal depends on the growth rates of different crystallographic faces. Some faces grow very fast and grow out to extinction and have little or no effect on the crystal form; while slow growing faces persist. The growth of a given face is governed by the crystal structure and defects and by the environmental conditions 15.
The various faster growing surfaces with smaller relative areas are most likely to be prone to surface roughening and, for example, fast growing needle-shaped crystals may tend to incorporate impurities selectively at their face ends if the growth process on these interfaces is not carefully controlled4.
The growth medium influences the growth kinetics of the faces in different ways. First of all, the solvent is adsorbed to a greater or lesser extent by different faces and thus selectively slowing down their growth rates. Solubility also plays a role: the higher the solubility, the higher the growth rate21. The growth medium also influences solvation, desolvation, and complex formation. If not predetermined by the process, variations in temperature also produce different growth rates. Lastly, hydrodynamics, or more precisely the relative velocity of the solution compared to the crystal, is an important parameter.
When the solution is quiescent, the face grows slowly at a rate determined by the molecular diffusion of the solute towards the crystal. The growth rate of the face increases with the flow velocity of solution to the crystal. However, there is still a diffusional limitation; growth rates tend very quickly towards a plateau and thus reaches an upper limit determined by the phenomena at the crystal surface6.
A rate law is an equation of the form22:
Growth rate = (a constant) × (a function of concentration(s)) Equation 8
A rate may be a flux (j) of matter (unit, mol/m2s) or a linear velocity v (unit, m/s). In the time derivative dr/dt; r is defined as the radius of a sphere with the same volume as the
30 | P a g e crystal. An elementary rate law is one which is determined by a single rate-determining step. The elementary rate laws are determined as follows corresponding to the mechanisms
1 to 722-24:
1) Transport by convection in solution: dr/dt = kc (S – 1) = kc
2) Transport by diffusion through solution: dr/dt = kD (S –1) = kD
3) Adsorption at the crystal solution interface: dr/dt = k1 (S – 1) = k1
4) Mitigation within the adsorption layer (two-dimensional diffusion): dr/dt depends on the surface geometry
5) Adsorption at a step (ledge) in the crystal surface: dr/dt = k' × (step density) (S – 1) = k'
×
6) Mitigation along the step (one-dimensional diffusion): dr/dt depends on step geometry
7) Integration at a kink in a step: dr/dt = k" × (kink density) (S – 1) = k" ×
* Where: c = concentration of the crystallizing solute, c = solubility of the crystallizing solute, c – c* = supersaturation, S = c/c*, = saturation ratio, = S – 1= relative supersaturation and kc, kD, k1, k', k" are constants. All the activity coefficients were neglected.
The first two processes are the so-called transport processes, whereas 3 – 7 are referred to as surface processes. Since these different steps normally occur in series, the slowest process will control the overall crystal growth. Therefore, growth can be transport (when step 1 and 2 are the slowest) or surface controlled (when steps 3 – 7 are the slowest)16.
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3. Induction time and Metastable Zone Width (MSZW)
Induction time is defined as the time elapsed from the instance of achievement of supersaturation to the detection of the first crystallization event at a constant supercooling25,
4. Its value will thus depend on the setting of t = 0 and the technique used to detect the formation of crystals. The induction period can be influenced by factors such as supersaturation, agitation, presence of impurities and viscosity. Mullin 7 defined the induction time as:
풕풊 = 풕풓 + 풕풏 + 풕품 Equation 9
The induction time is separated into three periods: tr is the relaxation time, required for the systems to achieve a quasi-steady-state distribution of molecular clusters; tn is the time required for the formation of a nucleus; and tg is the time required for the nucleus to grow to a detectable size.
Supersaturated solutions are metastable when crystallization ultimately occurs after this induction time. This process is inhibited by a kinetic barrier. Every solution has a maximum limit to which it may be supersaturated before it becomes unstable and crystallization spontaneously occurs (induction time is negligible). The region between the saturation curve and this unstable boundary is called the metastable zone (MSZ). The MSZ is ideal for controlled crystal growth10, 11.
The metastable zone width (MSZW) is usually defined for a solution as the supercooling at which the first crystallization event is detected when the solution is cooled at a constant rate. The measurement of the metastable zone width (MSZW) for a solute/solvent system is also required to better understand the appropriate crystallization approach. Control of the metastability of a mixture is crucial, as this will have implications on purity, particle size and crystal form.
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If concentration is plotted against temperature for any given crystallization three regions are observed (Figure 8)25:
A stable or undersaturated region where crystallization cannot occur.
A metastable region where the solution is supersaturated to a degree and where
crystallization will take place after a time.
An unstable region where the solution is further supersaturated and where
spontaneous crystallization without a time delay is expected.
The supersolubility (metastable zone limit) and the solubility curves are also known as the cloud and clear points, respectively. The width of the metastable region (MSZW) is known to be dependent on various process parameters, such as concentration, presence of impurities, cooling rate, and level of agitation. The measured MSZW is also known to be affected by the nucleation detection technique utilised (ultrasound velocity, focused beam reflectance measurement (FBRMs), or turbidity)11.
Figure 8: Diagram showing the different regions of a concentration versus the temperature profile for a typical API.
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Within the metastable zone, nucleation is quite controlled and crystals are able to grow with a steady supply of solute molecules without the formation of other nuclei. Ideally the
MSZW should be large enough to provide a stable region for crystal growth, but not so large that it presents a barrier to growth.
In the unstable region, controlling the crystal growth to macroscopic dimensions is not possible. Thus in this region depending on the degree of supersaturation, very small particles can be produced11. Additionally, the cooling rate also plays an important role, with fast cooling (small induction times), producing a large MSZW that leads to small crystals, and slow cooling producing a narrower MSZW likely to lead to larger and high quality crystals26.
4. Cooling crystallization
In cooling crystallization the supersaturation is generated by a step decrease in temperature.
Depending on how the solution is cooled a different end product may be obtained.
In this work cooling crystallization was used to create supersaturation. The temperature was rapidly decreased from the saturation temperature to a fixed crystallization temperature.
The supersaturation decreased as the solution desupersaturated, creating the highest supersaturation in the beginning.
The important phenomena that occur during crystallization and therefore solution desupersaturation can be described by the following quantities:
the nucleation rate [number of nuclei/(m3s)]; the number of new crystals formed per
unit time and volume of suspension
the growth rate [m/s]; the rate at which the size of the crystals increases
Thus, the balance equation for such a process is defined in Equation 8:
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풅∆풄 풅풆풔풖풑풆풓풔풂풕풖풓풂풕풊풐풏 풓풂풕풆 = = 풌 ∆풄풃 + 풌 푨∆퐜품 풅풕 풃 품 Equation 10
풃 푵풖풄풍풆풂풕풊풐풏 풓풂풕풆 = 풌풃∆풄 Equation 11
퐠 푮풓풐풘풕풉 풓풂풕풆 = 풌품푨∆퐜 Equation 12 where ∆c = c – c* is the difference between actual and saturation concentration. kb and b are nucleation constants, kg and g are the growth constants, and A is the crystal area per unit mass of solvent. This last term will depend on an area shape factor, on the crystal size and on the number of seed crystals27.
A good knowledge of how all these factors influence the crystallization is important to better understand the behaviour of the different systems.
5. Heterogeneous crystallization
As it was defined previously, heterogeneous crystallization occurs on the surface of foreign particles. The presence of these foreign particles reduces the activation energy required to form a new phase. Then, the metastable zone width is generally smaller for heterogeneous crystallization as compared to homogeneous crystallization.
In 1997 Espitalier et al. 19 measured the rate of nucleation of ketoprofen in pure acetone which was correlated using Equation 7. The surface free energy () was evaluated from the slope of the line resulted after representing ln (1/J) against (T-3 (lnS)-2). The graph showed two straight lines with a change in the slope observed for the change from homogeneous
(high supersaturations) to heterogeneous (low supersaturations) nucleation. After our own calculations from the represented graph, the value of A1 for primary nucleation was found to be 9-fold larger than for secondary nucleation. Thus, the surface free energy () for homogeneous nucleation was 2-fold larger than that for heterogeneous nucleation in this particular system. This result gives us an idea of the order of the reduction in the surface free energy by the addition of foreign particles.
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A special kind of heterogeneous crystallization, epitaxial crystallization, which was first observed in 1981 by Boistelle et al.28 can be defined as the heterogeneous nucleation and growth of inorganic/organic crystals of a particular orientation upon a separate/different crystalline surface, the substrate. The metastable substance nucleates on the other crystalline surface of the substrate beyond a threshold supersaturation. Epitaxial polymorphic growth is due to the resemblance of the substrate and crystal form along certain orientations; epitaxial nucleation should not be confused with ordinary heterogeneous nucleation of polymorphs on top of each other29. The substrate acts as a seed crystal, and the deposited film may lock into one or more crystallographic orientations with respect to the substrate crystal. If the overlayer either forms a random orientation with respect to the substrate or does not form an ordered overlayer, this is termed non-epitaxial growth30. The success of epitaxial nucleation is governed by the ability of the surface in question to order prenucleation aggregates or clusters so that nucleation becomes favourable. Understanding the mechanisms controlling epitaxial ordering is fundamental to controlling such final properties of crystalline materials such as solubility, optical properties, and electrical conductivity31. Therefore understanding the mechanisms driving epitaxy, at a molecular level, is critical.
There are some parameters that can affect the epitaxial nucleation on crystal surfaces, such as:
1. Crystal planes (Ledge-directed epitaxy)
For many years scientists have scratched surfaces to promote crystallization32.
Crystals are observed to nucleate along the scratches32-35. Despite the popularity of
this technique, the understanding of why crystals nucleate in scratches is very
limited.
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In 1993, Carter et al. 36 introduced the term ‘ledge-directed epitaxy’ that involves a lattice match between the substrate and the growing phase along the (1) ledge plane, and (2) equivalent dihedral angles of the substrate ledge sites and (3) a pair of aggregate planes whose identity is assigned on the basis of the structure of the mature crystal. Attractive van der Waals forces at the substrate-aggregate interface will rely on the molecular corrugation of the substrate and aggregate planes contacting the substrate during nucleation. These forces will be greater when the topographies of corrugated substrate surfaces and aggregates are in perfect registry, or if the interfacial surfaces of the substrate and aggregate are molecularly flat on the scale of the aggregate size (Figure 9). Furthermore, the ledge site orientation will dictate the orientation of the growing phase. Ledge-directed epitaxy is observed when the crystallographic planes of the substrate and aggregate have high packing densities and low molecular corrugation, and when the interfacial interactions predominantly involve non-directional dispersive forces. Conversely, crystal substrates with large dipolar contributions render nucleation less favourable and topographic orientation effects less likely.
Figure 9: Schematic representation of nucleation at a ledge in which molecules adsorbed on the surface and migrate to the ledge, resulting in the formation of a prenucleation aggregate at the ledge More recent studies performed by Page et al.37 studied via computer simulation, the heterogeneous nucleation of a crystal in a wedge-shaped groove. It was concluded that nucleation is faster when the wedge angle is such that a defect-free unstrained piece of the crystal fits perfectly into the wedge.
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2. Nanoconfinement
Recent investigations have shown that crystal nucleation under porous confinement
can alter nucleation kinetics, phase stabilization, polymorphism, and crystal
orientation38-43.
One recent example is the study done by Lopez-Mejias et al.38 who tracked a range
of nanopore geometries used as heteronucleants printed on biocompatible polymers.
It was concluded that the use of geometrically imprinted biocompatible surfaces as
heteronucleants nanopores can alter the nucleation kinetics, due to the angle
matching between the intrinsic angle present in the growing crystals and the angle at
the corners of the nanopore. Besides, some nanopores were able to provide kinetic
control over the formation of the polymorph that fits on the nanopore versus the
thermodynamically more stable form.
3. Functional groups and lattice matching:
Molecular functionality is known to play an important role in heterogeneous
nucleation. Both polymorphism and crystallographic orientation can be controlled
by changing the functional groups present at the surface of the substrate. It is
hypothesized that this is due to subtle changes in the strength and directionality of
the intermolecular interactions, such as hydrogen bonding, between the substrate
and prenucleation aggregate. The effect of lattice matching between the
heterosurface and the overlying crystal face is also important in order to understand
the epitaxial ordering upon these surfaces and in particular surface morphology.
There are three modes of lattice registry possible: commensurism, coincidence, and
incommensurism. Commensurism and coincidence correspond to a total or partial
lattice match, respectively. Incommensurism signifies a lattice mismatch. A partial
(coincident) or total (commensurate) lattice match between the two opposing lattice
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planes is thought to significantly lower the nucleation free energy barrier due to
precisely matched interactions between a substrate and prenucleation aggregates
adsorbed at the surface31.
In 2012, Chadwick et al. explored the effects of the molecular functionality of
crystalline surfaces for heteroepitaxial nucleation31. -Lactose monohydrate and D-
mannitol were used as crystalline substrates onto which acetaminophen was
crystallised. The addition of the excipients produced a fivefold reduction in the
induction time. Nucleation of APIs was preferential on substrates whose surface
functionality and hydrogen bonding preferences matched with those of API, with
lattice matching playing a secondary role. Chadwick et al. proposed that
functionality matching highly influences the epitaxial ordering of the APIs on the
substrate surface, emphasising the importance of utilising as many hydrogen
bonding groups as possible to stabilize the prenucleation aggregate.
Energetics
For heterogeneous nucleation the rate of nucleation can be expressed as18:
∆퐺 ,퐻퐸푇∗ − 푇 퐽퐻퐸푇 = 휌퐼 푍 푗 e kT Equation 13
Where:
number density of sites for heterogeneous nucleation:
ρI = ε × ρFP + ρ Equation 14
Where:
휌퐹푃: the number density of foreign particles.
휀: the number of places a critical nucleus can form on each foreign particle.
j: the rate at which molecules attach to the nucleus causing it to grow.
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Z: Zeldovich factor, which is the probability that a nucleus at the top of the barrier will
go on to form a crystal.
∆퐺 ∗ − 푇,퐻퐸푇 e 푘푇 : free energy cost of creating the critical nucleus for heterogeneous
nucleation.
It has been proved that in the presence of a foreign substrate the decrease in the value of γ
∗ therefore reduces the values of r* and ∆퐺푇 at constant supersaturation (Equations 4 and 5).
∗ Then ∆퐺푇,퐻퐸푇 will not depend only on the supersaturation but also on the nature of the
surface of the foreign particle. As foreign particles are presumably rather variable it is
expected the barrier to vary from nucleation site to nucleation site. In addition, the
prefactor ρI that is the number density of sites for heterogeneous nucleation, as defined in
Equation 14, contains not the number density of molecules ( but the number density of
foreign particles times the number of places a critical nucleus can form on each foreign
particle. Thus, comparing Equation 5 and Equation 13 nucleation will be more favourable
for heterogeneous nucleation due to an increase in the density sites for nucleation and to a
decrease in the free energy cost of creating the critical nucleus.
1.6. Polymorphism
Polymorphism, as defined by Rodriguez-Spong et al., is the ability of a crystalline substance to exist in different molecular arrangements and/or different molecular conformations, each displaying different molecular characteristics14.
As different polymorphs display different physical properties such as density, melting point and dissolution rate, polymorphism is vitally important to the manufacture of chemicals, in particular, pharmaceuticals. Production of an unwanted polymorph will give a product that most likely will not satisfy the intended purpose or processing characteristics of the required
API 44.
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The variation in the physical properties of a solid, such as crystal habit, density, solubility, hardness, colour, optical properties, melting point or chemical reactivity play an essential part in the formulation and the manufacture of the solid and in the application of the formulated product, in particular, pharmaceuticals43, 45. All industries producing a pure or formulated solid, in particular the pharmaceutical industry, understand that polymorphism generates potentially very interesting applications. For example, the hardness of a crystal can favour granulation or conversion into pills. Conversely, the undesired crystallization of excipients in a formulation during freeze-drying can have a negative impact on the quality of the product 6.
The knowledge of solid-state properties in an early stage of drug development helps to avoid manufacturing problems, to fine-tune the performance of drugs and provides space for innovation. By definition, every new crystal form is novel and not possible to predict how many different crystal forms can be prepared, how to prepare them and the properties of unknown crystal forms.
Thus, a change in polymorphic form can modify the bioavailability and the shelf-life of a compound, e.g. the case of Ritonavir. Ritonavir is an antiretroviral drug from the protease inhibitor class used to treat HIV infection and AIDS produced by Abbot Laboratories. It was discovered in 1992 and approved by Food and Drug Administration (FDA) in 1996. Only one polymorph (Form I) was known at that time. The drug was formulated as semi-solid capsules.
In 1998 many capsules failed the dissolution test and further precipitation in the capsules occurred due to the presence of a new polymorph (Form II). The Form II were brought into the laboratory and thereafter it became impossible to get Form I. Form II investigation team visited the Italian plant still producing Form I and soon significant amounts of Form II started to show up in that plant. The new crystal form (Form II) was unusually stable and unusually difficult to crystallise, as it had much lower solubility. Abbott had to discontinue the production of the
41 | P a g e drug until they corrected the polymorph problem. Nowadays, there is a regulatory requirement to identify all solid forms of the compound46.
The stable polymorph represents the lowest possible free energy state for the system and it will always be the polymorph with the lowest solubility in a solution system. Other polymorphs are termed metastable and will attempt to transform to the stable form. Knowledge of the possible transformations in a polymorphic system is essential when designing a process to isolate a desired polymorphic form. Isolation of a metastable polymorph will require operating conditions which prevent the transformation, whereas isolation of the stable form will require operating conditions which ensure the transformation has gone to completion44.
The ability of individual polymorphs to undergo polymorphic transformations and interchange polymorph form is a vast area which is very much dependent on the nature of the compound under study.
1. Solvates and hydrates
In amorphous and crystalline forms, a solid drug may be anhydrous or a solvate/hydrate.
When a solid form contains a solvent, it is known as a solvate. The presence of residual
solvent may affect dramatically the crystalline structure of the solid depending on the type
of intermolecular forces that the solvent may have within a crystalline solid. When the
solvent is water, it is termed a hydrate.
Due to the frequent presence of water in the environment and its use in solvent blending
during crystallization processes, the formation of hydrated drugs is common. Because the
water molecule is small and able to form hydrogen bonds, it is easily incorporated into the
crystalline lattice of drugs occupying spaces and stabilizing the structure47.
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2. Co-crystals and salts
Co-crystals are crystalline materials composed of two or more different molecules,
typically drug and co-crystal formers (“coformers”), in the same crystal lattice.
Pharmaceutical co-crystals have opened up opportunities for engineering solid-state forms
beyond conventional solid-state forms of an active pharmaceutical ingredient (API), such
as salts and polymorphs. Co-crystals can be tailored to enhance drug product bioavailability
and stability and to enhance the processability of APIs during drug product manufacture.
Another advantage of co-crystals is that they generate a diverse array of solid-state forms
for APIs that lack ionizable functional groups, which is a prerequisite for salt formation10.
Salt formation is one of the primary solid-state approaches used to modify the physical
properties of APIs. However, a major limitation within this approach is that the API must
possess a suitable (basic or acidic) ionisable site. In comparison, co-crystals offer a
different pathway, where any API regardless of acidic, basic or ionisable groups, could
potentially be co-crystallized48.
1.7. Bioavailability
Bioavailability is a measurement of the rate and extent to which a drug reaches the site of action. It depends directly on its solubility, which itself will have a time dependency on the solid phase. A drug can thus become completely ineffective if the amount of substance initially intended to enter the blood circulation system is reduced through low solubility and/or low dissolution kinetics. Moreover, if its apparent solubility is higher than intended, the risks of side effects are increased6. The rate limiting factor to absorption of these drugs from the gastrointestinal tract is the dissolution rate from the dosage form. An incomplete dissolution in
49 the gastrointestinal tract can severely restrict bioavailability . The selection of the commercial solid form, shape and size and associated crystallisation process is one of the key milestones in order to improve the bioavailability2. In addition, from a manufacturing point of view,
43 | P a g e equidimensional crystals (such as spheres or cubes) are usually preferred in industry as they have better handling and processing characteristics such as flowability and compactability1.
Amidon et al. classified APIs into four groups based on their solubility and permeability 50.
Drug dissolution and gastrointestinal permeability are two parameters controlling the extent and rate at which drug absorption takes place. Typically, a drug must be dissolved before absorption can take place in the body. It is difficult to estimate absorption data due to the complexity of the processes in the gastrointestinal tract and complex pharmacokinetics. The biopharmaceutical classification system (BCS) is used to predict in vivo drug performance from in vitro measurements of permeability and solubility. Figure 10 shows the different classes of drugs according to this classification. A drug is considered highly soluble when the highest dose strength is soluble in ≤ 250 mL water over a pH range of 1 to 7.5 at 37.5°C, and is considered highly permeable if the absorption of an orally administered dose in humans is
>90% when determined using mass balance51. For oral drug delivery BCS class I is the most desirable, however each API is different and each approach has to be edited to meet the other demands of the drug and its desired product profile. Although the crystalline form of a drug usually provides better stability and purity, the lattice energy, that is the energy required to break apart the lattice of a crystal, can hinder the dissolution of these crystalline forms52.
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Figure 10: Biopharmaceutical classification system (BCS)53. 1.8. Crystallization of API’s in the presence of excipients
Estimation of drug–excipient interactions is a crucial step in preformulation studies of drug development to achieve consistent stability, bioavailability and manufacturability of solid dosage forms54. Many studies in the past years have been focused on the study of drug- excipient interactions55-57 and compatibility58-59 and analytical tools for compatibility assessment of APIs60-66. However, some researchers have investigated the crystallization of
APIs in the presence of excipients9, 31, 39, 49, 67-69. Some of them are shown below:
In 2009, Zimmermann et al. studied a typical antisolvent method in the presence of stabilisers49. The effects on the physicochemical properties after the adsorption of pharmaceutical excipients onto microcrystals of siramesine hydrochloride were tracked.
Microcrystals with optimised physicochemical properties were prepared by precipitation in the presence of excipients, which adsorbed on the particle surface and altered particle size, morphology, and dissolution rate. The poorly water-soluble drug siramesine hydrochloride was
45 | P a g e precipitated by the antisolvent method in the presence of various polymeric and surface active excipients. The results showed that the excipients which most effectively altered the physicochemical properties were the ones which exhibited the greatest affinity for the surface of the drug particles. Powder dissolution studies of six of the resulting particle systems showed a significant increase in percentage dissolved after 15 minutes compared to the starting material. Furthermore, only a very small amount of excipient – less than 1.4% w/w – was necessary to exert a pronounced effect on the physicochemical properties of the particles. Thus, the use of excipients to optimize particle properties did not compromise the drug load of the resulting powder.
In 2011, Chadwick et al.9 described the use of a new method for selecting a three dimensional crystalline substrate to selectively crystallise Form II of AAP from solution under conditions which typically yield Form I. The success in achieving this goal provides insights as to the possible mechanisms responsible for controlling heteroepitaxy on three dimensional crystalline substrates. Suitable candidate substrates were found using Cambridge Structural Database
(CCDC) for all structures whose unit cell parameters were a closematch to those of Form II.
This work allows for the rational design and selection of crystalline surfaces to control nucleation and hence polymorphism. The results suggested that only by selecting a crystalline substrate with similar unit cell parameters and molecular functionality to Form II can polymorphic control over AAP be achieved. They also showed that by choosing a crystalline substrate for its crystallographic and functional similarity to a desired polymorphic form, it was able to selectively crystallise Form II of AAP, a polymorph that has previously proven difficult to crystallise from solution.
Again in 2012, Chadwick et al.31 studied the heterogeneous crystallization of acetaminophen
(AAP) on crystalline substrates with differing lattice parameters and surface functionalities.
The results showed that nucleation was preferred on substrates whose surface functionality
46 | P a g e matched with that of AAP even when other substrates exhibited a better lattice match with specific AAP crystal faces.
In 2013, Quon et al. investigated the effect of spherical agglomerates of heterogeneous crystalline substrates on the nucleation of acetaminophen (AAP) 67. Triclinic lactose and D-
Mannitol were used as crystalline substrates. Crystallization of acetaminophen (AAP) in the presence of spherical agglomerates of triclinic lactose displayed enhanced nucleation kinetics when compared to the nucleation kinetics of AAP in the presence of single crystals of triclinic lactose. Crystallization of AAP in the presence of spherical agglomerates and powder D- mannitol showed little difference in the nucleation induction time. Quon et al. attribute this decrease in the induction time in the triclinic Lactose/AAP system to the availability of new crystal faces in the spherical agglomerates, as PXRD measurements of the spherical agglomerates showed additional peaks that were attributed to the growth of additional crystal faces. Single crystal and spherical agglomerates of D-mannitol did not show any significant difference in the available crystal faces. These results suggest that stronger epitaxial matching can be achieved between AAP and lactose when using spherical agglomerates. Molecular computations indicated the epitaxy is driven by a combination of coincident lattice matching and the formation of extensive hydrogen bonding between the two crystal faces. This work demonstrates how the crystal morphology of a crystalline substrate is important in maximizing the effect on nucleation kinetics and that determining the “best” crystal face for controlling epitaxy is paramount in the design of surfaces for control of heterogeneous nucleation.
Most recent studies39, 68-69, performed from 2015 to 2017, were focused in the continuous crystallization of APIs in the presence of excipients with industrial applications. Firstly, Dwyer et al.39 explored the crystallization of APIs, including fenofibrate, in rigid nanoporous media over a broad range of pore sizes allowing a fundamental understanding of the relationship between pore size, crystallinity and bioavailability. The dissolution testing showed enhanced
47 | P a g e dissolution profiles for the nanocrystalline materials confined to different porous matrices.
Again in 2017 68 a dual-stage mixed-suspension, mixed-product removal (MSMPR) crystallizer was designed in which FF was loaded into the porous matrices of different pore sizes in the first stage, and then fed to a second stage in which the crystals were further grown in the pores.
This resulted in high loadings while still producing nanocrystals confined to the pores without the formation of bulk-sized crystals on the surface of the porous silica. In a parallel study done by Yazdanpanah et al.69 a continuous heterogeneous crystallization process was developed in which the API was crystallized directly on the surface of an excipient within the crystallizer.
The product was subsequently dried and formed into tablets. A mixture of excipient and API with a maximum drug loading of 47% was obtained.
1.9. Scope of this Project
This project proposes a new formulation process in which the API is crystallized in the presence of a suspended excipient. The aim is the promotion of nucleation and growth of the
API on the surface of the excipient particles. By tuning process parameters the desired API particle size can be obtained, with a final dissolution rate of the drug similar to the marketed one. Advantages of this approach include a reduction in the induction time, the promotion of crystallisation at lower supersaturations and a reduction in processing steps e.g. milling and mixing. Furthermore, employing generally regarded as safe excipients as additives to achieve the desired size and shape of the API represents a practical alternative for such a highly regulated industrial sector as the pharmaceutical industry. This project expands the approach used by Chadwick et al.31 whereby the effects of morphology, excipient adsorption and surface composition are examined. In addition, the influence of the API and excipient nature, supersaturation, API/excipient mass ratio and temperature on the API nucleation and growth are investigated.
This entails the following seven key challenges:
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To elucidate the mechanism of heterogeneous crystallization of APIs onto excipients.
The stabilisation of API particles, and control of API crystallinity.
To identify the conditions in which crystallization occurs exclusively on the excipient
surfaces rather than in solution.
To determine the nature of the interactions between the heterogeneously crystallised
API and the excipient.
To manipulate process parameters to arrive at a desired outcome in terms of release of
the API from the excipient.
To increase the dissolution rate of the API to avoid the milling step in the
manufacturing process.
To understand the effect of the different surface properties in heterogeneous
crystallization.
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1.10. References
1. Mirza, S.; Miroshnyk, I.; Heinamaki, J.; Antikainen, O.; Rantanen, J.; Vuorela, P.; Vuorela, H.; Yliruusi, J., Crystal Morphology Engineering of Pharmaceutical Solids: Tabletting Performance Enhancement. Aaps Pharmscitech 2009, 10 (1), 113-119. 2. Chen, J.; Sarma, B.; Evans, J. M. B.; Myerson, A. S., Pharmaceutical Crystallization. Crystal Growth & Design 2011, 11 (4), 887-895. 3. Baxter, T.; Prescott, J., Chapter 28 - Process Development, Optimization, and Scale-up: Powder Handling and Segregation Concerns A2 - Qiu, Yihong. In Developing Solid Oral Dosage Forms, Chen, Y.; Zhang, G. G. Z.; Liu, L.; Porter, W. R., Eds. Academic Press: San Diego, 2009; pp 637-665. 4. Roberts, K.; Docherty, R.; Taylor, S., Materials Science: Solid Form Design and Crystallisation Process Development. In Pharmaceutical Process Development: Current Chemical and Engineering Challenges, Blacker, A. J.; Williams, M. T., Eds. Royal Soc Chemistry: Cambridge, 2011; pp 286-316. 5. Hammond, C.; Hammond, C., Basics of crystallography and diffraction. Oxford: 2001; Vol. 214. 6. Mangin, D.; Puel, F.; Veesler, S., Polymorphism in Processes of Crystallization in Solution: A Practical Review. Organic Process Research & Development 2009, 13 (6), 1241-1253. 7. Mullin, J. W., Crystallization. 4th ed.; Butterworth-Heinemann: Oxford, 2001. 8. Eder, R. J. P.; Schmitt, E. K.; Grill, J.; Radl, S.; Gruber-Woelfler, H.; Khinast, J. G., Seed loading effects on the mean crystal size of acetylsalicylic acid in a continuous-flow crystallization device. Crystal Research and Technology 2011, 46 (3), 227-237. 9. Chadwick, K.; Myerson, A.; Trout, B., Polymorphic control by heterogeneous nucleation - A new method for selecting crystalline substrates. Crystengcomm 2011, 13 (22), 6625-6627. 10. McLeod, J.; Paterson, A. H. J.; Jones, J. R.; Bronlund, J. E., Primary nucleation of alpha-lactose monohydrate: The effect of supersaturation and temperature. International Dairy Journal 2011, 21 (7), 455-461. 11. Mitchell, N. A.; Frawley, P. J., Nucleation kinetics of paracetamol-ethanol solutions from metastable zone widths. Journal of Crystal Growth 2010, 312 (19), 2740-2746. 12. Tung, H. H., Industrial Perspectives of Pharmaceutical Crystallization. Organic Process Research & Development 2013, 17 (3), 445-454. 13. Kashchiev, D., Nucleation : basic theory with applications. Butterworth Heinemann: Oxford; Boston, 2000. 14. Rodriguez-Spong, B.; Price, C. P.; Jayasankar, A.; Matzger, A. J.; Rodriguez-Hornedo, N., General principles of pharmaceutical solid polymorphism: a supramolecular perspective. Advanced Drug Delivery Reviews 2004, 56 (3), 241-274. 15. Davey, R. J.; Schroeder, S. L. M.; ter Horst, J. H., Nucleation of Organic CrystalsA Molecular Perspective. Angewandte Chemie-International Edition 2013, 52 (8), 2166-2179. 16. Cubillas, P.; Anderson, M. W., Synthesis Mechanism: Crystal Growth and Nucleation. In Zeolites and Catalysis, Wiley-VCH Verlag GmbH & Co. KGaA: 2010; pp 1-55. 17. Kashchiev, D.; van Rosmalen, G. M., Review: Nucleation in solutions revisited. Crystal Research and Technology 2003, 38 (7-8), 555-574. 18. Sear, R. P., Nucleation: theory and applications to protein solutions and colloidal suspensions. Journal of Physics: Condensed Matter 2007, 19 (3), 033101. 19. Espitalier, F.; Biscans, B.; Laguérie, C., Particle design Part A: Nucleation kinetics of ketoprofen. Chemical Engineering Journal 1997, 68 (2), 95-102. 20. Hull, D.; Bacon, D. J., Introduction to dislocations. Butterworth-Heinemann: 2001. 21. Wouters, J.; Quéré, L., Pharmaceutical Salts and Co-crystals. Royal Society of Chemistry: 2011. 22. Nielsen, A. E., Rate laws and rate constants in crystal growth. Croatica Chemica Acta 1987, 60 (3), 531-539. 23. Nitsche, R., D. Elwell. H. J. Scheel. Crystal Growth from High-Temperature Solutions. Academic Press 1975; XIV + 630 Seiten. Preis £ 19, 80; $ 52. 25. Kristall und Technik 1976, 11 (3), K28-K29. 24. Lasaga, A. C., Kinetic Theory in the Earth Sciences. Princeton University Press: 2014.
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25. Kubota, N., Effect of sample volume on metastable zone width and induction time. Journal of Crystal Growth 2012, 345 (1), 27-33. 26. Barrett, M.; McNamara, M.; Hao, H. X.; Barrett, P.; Glennon, B., Supersaturation tracking for the development, optimization and control of crystallization processes. Chemical Engineering Research & Design 2010, 88 (8A), 1108-1119. 27. Jones, A. G.; Mullin, J., Programmed cooling crystallization of potassium sulphate solutions. Chemical Engineering Science 1974, 29 (1), 105-118. 28. Boistelle, R.; Rinaudo, C., Phase transition and epitaxies between hydrated orthorhombic and anhydrous monoclinic uric acid crystals. Journal of Crystal Growth 1981, 53 (1), 1-9. 29. Stoica, C.; Verwer, P.; Meekes, H.; Vlieg, E.; van Hoof, P.; Kaspersen, F. M., Epitaxial 2D nucleation of the stable polymorphic form of the steroid 7 alpha MNa on the metastable form: Implications for Ostwald's rule of stages. International Journal of Pharmaceutics 2006, 309 (1-2), 16- 24. 30. Encyclopaedia Britannica, I.; Encyclopaedia Britannica, I., Britannica Concise Encyclopedia. Encyclopaedia Britannica: 2008. 31. Chadwick, K.; Chen, J.; Myerson, A. S.; Trout, B. L., Toward the Rational Design of Crystalline Surfaces for Heteroepitaxy: Role of Molecular Functionality. Crystal Growth & Design 2012, 12 (3), 1159-1166. 32. Dean, J. R., Practical Skills in Chemistry. Prentice Hall: 2002. 33. Sholl, C. A.; Fletcher, N. H., Decoration criteria for surface steps. Acta Metallurgica 1970, 18 (10), 1083-1086. 34. Turnbull, D., Kinetics of Heterogeneous Nucleation. The Journal of Chemical Physics 1950, 18 (2), 198-203. 35. Gutzow, I. S.; Schmelzer, J., The Vitreous State: Thermodynamics, Structure, Rheology, and Crystallization. Springer Berlin Heidelberg: 2013. 36. Carter, P. W.; Ward, M. D., Topographically directed nucleation of organic crystals on molecular single-crystal substrates. Journal of the American Chemical Society 1993, 115 (24), 11521- 11535. 37. Page, A. J.; Sear, R. P., Crystallization Controlled by the Geometry of a Surface. Journal of the American Chemical Society 2009, 131 (48), 17550-17551. 38. Lopez-Mejias, V.; Myerson, A. S.; Trout, B. L., Geometric Design of Heterogeneous Nucleation Sites on Biocompatible Surfaces. Crystal Growth & Design 2013, 13 (8), 3835-3841. 39. Dwyer, L. M.; Michaelis, V. K.; O'Mahony, M.; Griffin, R. G.; Myerson, A. S., Confined crystallization of fenofibrate in nanoporous silica. CrystEngComm 2015, 17 (41), 7922-7929. 40. Diao, Y.; Myerson, A. S.; Hatton, T. A.; Trout, B. L., Surface Design for Controlled Crystallization: The Role of Surface Chemistry and Nanoscale Pores in Heterogeneous Nucleation. Langmuir 2011, 27 (9), 5324-5334. 41. Kim, K.-J.; Mersmann, A., Estimation of metastable zone width in different nucleation processes. Chemical Engineering Science 2001, 56 (7), 2315-2324. 42. Tan, L.; Davis, R. M.; Myerson, A. S.; Trout, B. L., Control of Heterogeneous Nucleation via Rationally Designed Biocompatible Polymer Surfaces with Nanoscale Features. Crystal Growth & Design 2015, 15 (5), 2176-2186. 43. Keel, T. R.; Thompson, C.; Davies, M. C.; Tendler, U. B.; Roberts, C. J., AFM studies of the crystallization and habit modification of an excipient material, adipic acid. International Journal of Pharmaceutics 2004, 280 (1-2), 185-198. 44. Croker, D.; Hodnett, B. K., Mechanistic Features of Polymorphic Transformations: The Role of Surfaces. Crystal Growth & Design 2010, 10 (6), 2806-2816. 45. Beckham, G. T.; Peters, B.; Trout, B. L., Evidence for a size dependent nucleation mechanism in solid state polymorph transformations. Journal of Physical Chemistry B 2008, 112 (25), 7460-7466. 46. Wilkins, L. W., Martin’s Physical Pharmacy and Pharmaceutical Sciences. 5th ed.; 2006. 47. Healy, A. M.; Worku, Z. A.; Kumar, D.; Madi, A. M., Pharmaceutical solvates, hydrates and amorphous forms: A special emphasis on cocrystals. Advanced Drug Delivery Reviews. 48. Yadav, A. V.; Shete, A. S.; Dabke, A. P.; Kulkarni, P. V.; Sakhare, S. S., Co-Crystals: A Novel Approach to Modify Physicochemical Properties of Active Pharmaceutical Ingredients. Indian Journal of Pharmaceutical Sciences 2009, 71 (4), 359-370.
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49. Zimmermann, A.; Millqvist-Fureby, A.; Elema, M. R.; Hansen, T.; Mullertz, A.; Hovgaard, L., Adsorption of pharmaceutical excipients onto microcrystals of siramesine hydrochloride: Effects on physicochemical properties. European Journal of Pharmaceutics and Biopharmaceutics 2009, 71 (1), 109-116. 50. Amidon, G. L.; Lennernäs, H.; Shah, V. P.; Crison, J. R., A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharmaceutical research 1995, 12 (3), 413-420. 51. Baghel, S.; Cathcart, H.; O'Reilly, N. J., Polymeric Amorphous Solid Dispersions: A Review of Amorphization, Crystallization, Stabilization, Solid-State Characterization, and Aqueous Solubilization of Biopharmaceutical Classification System Class II Drugs. Journal of Pharmaceutical Sciences 2016, 105 (9), 2527-2544. 52. Baghel, S.; Cathcart, H.; O'Reilly, N. J., Polymeric Amorphous Solid Dispersions: A Review of Amorphization, Crystallization, Stabilization, Solid-State Characterization, and Aqueous Solubilization of Biopharmaceutical Classification System Class II Drugs. Journal of Pharmaceutical Sciences 105 (9), 2527-2544. 53. Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.; Jarvinen, T.; Savolainen, J., Prodrugs: design and clinical applications. Nat Rev Drug Discov 2008, 7 (3), 255-270. 54. Chadha, R.; Bhandari, S., Drug-excipient compatibility screening-Role of thermoanalytical and spectroscopic techniques. J. Pharm. Biomed. Anal. 2014, 87, 82-97. 55. Wu, Y.; Levons, J.; Narang, A. S.; Raghavan, K.; Rao, V. M., Reactive Impurities in Excipients: Profiling, Identification and Mitigation of Drug–Excipient Incompatibility. AAPS PharmSciTech 2011, 12 (4), 1248-1263. 56. Rowe, R. C.; Sheskey, P. J.; Owen, S. C.; American Pharmacists, A., Handbook of pharmaceutical excipients. APhA/Pharmaceutical Press: London; Chicago, 2009. 57. Desai, S. R.; Shaikh, M. M.; Dharwadkar, S. R., Preformulation compatibility studies of etamsylate and fluconazole drugs with lactose by DSC. Journal of Thermal Analysis and Calorimetry 2003, 71 (2), 651-658. 58. Qiu, Y.; Chen, Y.; Zhang, G. G.; Yu, L.; Mantri, R. V., Developing solid oral dosage forms: pharmaceutical theory and practice. Academic press: 2016. 59. Stability Aspects of Preformulation and Formulation of Solid Pharmaceuticals. Drug Development and Industrial Pharmacy 1984, 10 (8-9), 1373-1412. 60. Pyramides, G.; Robinson, J. W.; William Zito, S., The combined use of DSC and TGA for the thermal analysis of atenolol tablets. Journal of Pharmaceutical and Biomedical Analysis 1995, 13 (2), 103-110. 61. Giron, D., Contribution of thermal methods and related techniques to the rational development of pharmaceuticals—Part 1. Pharmaceutical Science & Technology Today 1998, 1 (5), 191-199. 62. Phipps, M. A.; Winnike, R. A.; Long, S. T.; Viscomi, F., Excipient compatibility as assessed by isothermal microcalorimetry. Journal of Pharmacy and Pharmacology 1998, 50 (S9), 9-9. 63. Newman, A. W.; Byrn, S. R., Solid-state analysis of the active pharmaceutical ingredient in drug products. Drug Discovery Today 2003, 8 (19), 898-905. 64. Mura, P.; Faucci, M. T.; Manderioli, A.; Bramanti, G.; Ceccarelli, L., Compatibility study between ibuproxam and pharmaceutical excipients using differential scanning calorimetry, hot-stage microscopy and scanning electron microscopy1. Journal of Pharmaceutical and Biomedical Analysis 1998, 18 (1–2), 151-163. 65. Aigner, Z.; Heinrich, R.; Sipos, E.; Farkas, G.; Ciurba, A.; Berkesi, O.; Szabó-Révész, P., Compatibility studies of aceclofenac with retard tablet excipients by means of thermal and FT-IR spectroscopic methods. Journal of Thermal Analysis and Calorimetry 2011, 104 (1), 265-271. 66. Compatibility Study Between Naproxen and Tablet Excipients Using Differential Scanning Calorimetry. Drug Development and Industrial Pharmacy 1990, 16 (4), 673-683. 67. Quon, J. L.; Chadwick, K.; Wood, G. P. F.; Sheu, I.; Brettmann, B. K.; Myerson, A. S.; Trout, B. L., Templated Nucleation of Acetaminophen on Spherical Excipient Agglomerates. Langmuir 2013, 29 (10), 3292-3300. 68. Dwyer, L.; Kulkarni, S.; Ruelas, L.; Myerson, A., Two-Stage Crystallizer Design for High Loading of Poorly Water-Soluble Pharmaceuticals in Porous Silica Matrices. Crystals 2017, 7 (5), 131.
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69. Yazdanpanah, N.; Testa, C. J.; Perala, S. R. K.; Jensen, K. D.; Braatz, R. D.; Myerson, A. S.; Trout, B. L., Continuous Heterogeneous Crystallization on Excipient Surfaces. Crystal Growth & Design 2017.
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CHAPTER 2: Materials and analytical techniques
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2. Materials and Analytical Techniques
In this chapter a description of the materials and the main analytical techniques to characterize the samples will be presented.
The materials used include: Acetaminophen (AAP), Fenofibrate (FF), -Lactose (-Lac),
D-Mannitol (D-Man), microcrystalline cellulose (MCC), Carboxymethyl cellulose (CMC),
Polycaprolactone (PCL), Mesoporous silica (SiO2), Polycarbonate (PC) Polymethyl methacrylate (PMMA), Polytetrafluoroethylene (PTFE), and Methanol (MeOH).
The primary characterization techniques used were: Powder X-ray Diffraction (XRD),
Scanning Electron Microscopy (SEM), in situ SEM-Raman and Raman Spectroscopy, ultraviolet-visible spectroscopy (UV/vis), solid-state nuclear magnetic resonance spectroscopy
(SSNMR), and iControl LabMax which includes focussed beam reflectance measurement
(FBRM) and In situ Fourier transform infrared (FTIR) probes. This chapter will give a brief description of these methods.
2.1. Materials
API
2.1.1. Acetaminophen (AAP)
One of the APIs selected was Acetaminophen (AAP), commonly known as Paracetamol.
AAP is classified as a mild analgesic. It is commonly used for the relief of headaches and
other minor aches and pains and is a major ingredient in numerous cold and flu remedies.
In combination with opioid analgesics, AAP can also be used in the management of more
severe pain such as post-surgical pain and providing palliative care in advanced cancer
patients. AAP is an odourless white crystalline powder, with a slightly bitter taste. It
is soluble in organic solvents such as methanol and ethanol but slightly soluble in water and
ether 1.
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AAP is now a generic API produced by a range of pharmaceutical companies. It is a
compound known to have three crystalline polymorphs in addition to existing in an
amorphous form. Commercial AAP consists primarily of the thermodynamically stable
monoclinic (type I) form, even though this polymorphic form has poor compression
properties; the orthorhombic (type II) form is metastable and directly compressible. The
crystal structure of type III has not been determined by X-ray crystallography due to its
high instability 2.
HBD = 2 HBA = 2
Figure 11: Chemical structure of AAP with the number of hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA)
Table 1: Different polymorphs and characteristics of AAP crystal3-4
FI FII FIII4 Amorphous Polymorph FI FII FIII - Space group P21/a Pbca Pca21 - a (Å) 12.930 17.166 11.837 - b (Å) 9.400 11.777 8.560 - c (Å) 7.100 7.212 14.819 - beta 115.90 90.00 No data - Z 4 8.0 4 - Symmetry cell setting Monoclinic Orthorhombic Orthorhombic Amorphous CCDC Ref code HXCAN01 HXCAN08 No data -
In this work, the monoclinic Form I of AAP is used, as it is the commercial form.
2.1.2. Fenofibrate (FF)
The second selected API was Fenofibrate (FF). Fenofibrate is one of the best-known and
most prescribed hyperlipidemia therapeutic agents, which was first commercialized in the
1970s5. Fenofibrate helps reduce cholesterol and triglycerides (fatty acids) in the blood.
High levels of these types of fat in the blood are associated with an increased risk of
56 | P a g e atherosclerosis (clogged arteries)6-7. Fenofibrate is available in several formulations and is sold under several brand names, including Lipantil supra by Abbott Laboratories, the one used in this work8.
Fenofibrate is highly lipophilic with a water solubility of ca. 0.8 μg/mL9. Despite the ultralow solubility, most current commercial products of fenofibrate contain micronized crystalline fenofibrate. Up to now three polymorphic forms of fenofibrate have been reported in the literature, but just two of them are included in the CCDC database3. Form I, with a melting temperature of 80 °C, is the most stable polymorph, which has been used as an API in tablet and capsule formulations. Pure crystalline fenofibrate form I can be obtained from the solvent evaporation method, while the metastable form II, with a melting temperature of 74 °C, can be produced by recrystallization of amorphous fenofibrate from the melt10-11. In 2015 a new polymorph, Form III, with a low melting point of 50 °C, was discovered. This polymorph was obtained via a controlled heterogeneous nucleation method using low quantity (1% w/w) of talc5. Finally, amorphous fenofibrate has a low glass transition temperature (Tg) at approximately −20 °C, indicating high molecular mobility at ambient temperature and this contributes to the unpredictable crystallization behaviour of the amorphous drug from the melt.
HBD = 0 HBA = 3
Figure 12: Chemical structure of FF with the number HBD and HBA
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Table 2: Different polymorphs and characteristics of FF crystals
FI FII FIII5 Amorphous Polymorph FI FII FIII - Space group P1̅ P21/n P1̅ - a (Å) 8.133 13.619 9.4803 - b (Å) 8.239 7.554 9.7605 - c (Å) 14.399 17.880 10.9327 - beta 105.75 92.35 90.352 - Z 2.0 4.0 2.0 - Symmetry cell setting Triclinic Monoclinic Triclinic Amorphous CCDC Ref code TADLIU01 TADLIU02 No data -
Excipients
Six different dispersed excipients were used in this thesis: -Lactose (-Lac), D-Mannitol
(D-Man), microcrystalline cellulose (MCC), carboxymethyl cellulose (CMC), polycaprolactone (PCL) and mesoporous silica (SiO2).
2.1.3. Lactose
Lactose (4-O--D-galactopyranosyl-D-glucopyranose), the milk sugar, is a carbohydrate
comprising one galactose moiety linked to a glucose molecule through a -1,4 linkage. It
exhibits two anomeric forms, -Lactose (-Lac), and -Lactose (-Lac), which differ in
the configuration of the terminal hydroxyl group of the glucose unit. In the solid state,
lactose can be amorphous or crystallized. Five crystalline forms are known: -lactose
monohydrate (-LMH), hygroscopic anhydrous -lac, stable anhydrous -Lac, -Lac and
mixed compounds of the two anomers and -Lactose (-Lac). In this work only
-Lac was used.
-Lactose
When a highly concentrated solution of lactose is crystallised at temperatures above
93.5°C, crystals of β-Lac are exclusively formed. Crystals of pure β-Lac have a
characteristic kite-like form. Particles with crystalline β-Lac are more brittle than α–
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LMH crystals and they do not contain crystal water. -Lac is often referred to as
anhydrous lactose. Industrially, β-Lac is produced by roller drying highly
concentrated lactose solutions. The isomeric purity is approximately 80% β-Lac, the
remaining 20% being anhydrous α-Lac. This type of Lac is used mainly as filler-
binder for tablet production via direct compression processes.
HBD = 8 HBD = 8 HBA = 11 HBA = 11
Figure 13: -Lac and -Lac molecules with the number HBD and HBA
Table 3: Characteristics of the Lac crystals
FII Polymorph FII Space group P21 a (Å) 10.839 b (Å) 13.349 c (Å) 4.954 beta 91.31 Z 2 Symmetry cell setting Monoclinic CCDC Ref code BLACTO 2.1.4. Mannitol
The acyclic sugar alcohol, D-mannitol, is a natural product produced by various plants, algae and fungi, and is one of the classic examples of a compound which crystallises in several polymorphs, often simultaneously. Approximately eight polymorphs of D-mannitol have been claimed over the years; however only three (, and ) are proven to exist, being the most stable 12.
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D-Mannitol
D-Mannitol (Figure 14, Table 4) is a common excipient used in pharmaceutical
tablet formulations. It is mainly used as a diluent in tablet formulations, e.g.
chewable tablets. In lyophilizates, mannitol acts as a filler to produce a
homogeneous, fluffy cake. It is highly soluble in water, well tolerated and exhibits a
low drug interaction potential. Because of its sweet taste and its pleasant texture, the
hexahydric alcohol has become the key excipient in dispersible and orodispersible
formulations. Rapidly disintegrating and dispersing tablets can overcome deficient
swallowing ability of the patients, particularly in pediatrics and geriatrics, and can
play a very important role in improving patient compliance. Various mannitol grades
are available on the market. For direct compression, pre-processed grades such as
spray-dried and granulated mannitol are utilized13. HBD = 6 HBA = 6
Figure 14: D-Mannitol molecule
Table 4: Characteristics of the D-Man crystals with the number HBD and HBA
FII Polymorph FII Space group P212121 a (Å) 8.694 b (Å) 16.902 c (Å) 5.549 beta 90 Z 4 Symmetry cell setting Orthorhombic CCDC Ref code DMANTL07 2.1.5. Microcrystalline cellulose
Microcrystalline cellulose (MCC) (Figure 15) is a purified, partially depolymerized cellulose prepared by treating alpha cellulose (type I), obtained as a pulp from fibrous
60 | P a g e plant material, with mineral acids. Cellulose is the most abundant natural polymer on earth with an annual biomass production of 50 billion tons14. Cellulose consists of linear chains of -1,4-D anhydroglucopyranosyl units. MCC is commonly manufactured by spray drying the neutralized aqueous slurry resulting from the hydrolysis of cellulose. Most commercial grades are formed by varying and controlling the spray drying conditions in order to manipulate the degree of agglomeration (particle size distribution) and moisture content
(loss on drying). MCC is generally considered as the diluent with the best binding properties and it is recognised as one of the preferred binders. In addition to its dry binding properties, and in comparison, to brittle excipients, MCC is self-disintegrating with low lubricant requirement due to its extremely low coefficient of friction and its very low residual die wall pressure. MCC offers other advantages including broad compatibility with
APIs, physiological inertness, ease of handling, and security of supply14.
HBD = 8 HBA = 11
Figure 15: MCC molecule with the number HBD and HBA per monomer
MCC is an amorphous solid, as its powder pattern contains no crystalline diffraction peaks.
2.1.6. Carboxymethyl cellulose
Carboxymethyl cellulose (CMC) (Figure 16) is a cellulose derivative with carboxymethyl groups (-CH2-COOH) bound to some of the hydroxyl groups of the glucopyranose monomers that make up the cellulose backbone. It is an amorphous solid often used as its sodium salt, sodium carboxymethyl cellulose 15. In this work the sodium salt was used. CMC is used in pharmaceuticals as a thickening agent, for example as the
61 | P a g e lubricant in eye drops, and in the oil-drilling industry as an ingredient of drilling mud, where it acts as a viscosity modifier and water retention agent 16.
HBD = 6 HBA = 8
Figure 16: CMC molecule with the number HBD and HBA per monomer
2.1.7. Polycaprolactone
Polycaprolactone (PCL) (Figure 17) is a biodegradable polyester with a low melting point of around 60 °C and a glass transition temperature of about −60 °C. The most common use of polycaprolactone is in the manufacture of speciality polyurethanes.
Polycaprolactones impart good water, oil, solvent and chlorine resistance to the polyurethane produced17.
PCL is degraded by hydrolysis of its ester linkages in physiological conditions (such as in the human body) and has therefore received a great deal of attention for use as an implantable biomaterial. In particular, it is especially interesting for the preparation of long term implantable devices, owing to its degradation which is even slower than that of polylactide. PCL has been approved by the FDA in specific applications used in the human body as a drug delivery device, suture, or adhesion barrier. It is being investigated as a scaffold for tissue repair via tissue engineering, guided bone regeneration (GBR) membrane. It has been used as the hydrophobic block of amphiphilic synthetic block copolymers used to form the vesicle membrane of polymersomes. A variety of drugs have been encapsulated within PCL beads for controlled release and targeted drug delivery18.
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HBD = 0 HBA = 2
Figure 17: Polycaprolactone molecule with the number HBD and HBA per monomer PCL is an amorphous solid as its powder pattern contains no crystalline diffraction peaks.
2.1.8. Mesoporous silica
Silicon dioxide (Figure 18), also known as silica, is an oxide of silicon with the chemical formula SiO2, most commonly found in nature as quartz and in various living organisms. In many parts of the world, SiO2 is the major constituent of sand. SiO2 is one of the most complex and most abundant families of materials, existing as a compound of several minerals and as synthetic product. Notable examples include fused quartz, mesoporous silica, fumed silica, silica gel, and aerogels. It is used in structural materials, microelectronics as component in the food and pharmaceutical industry 19.
Mesoporous silica is a mesoporous form of silica and it has many applications in medicine, biosensors, drug delivery, catalysis, thermal energy storage and imaging20.
Regarding drug delivery, as mesoporous silica has a large surface area of the pores it allows the particles to be filled with a drug 21. Ordered mesoporous silica show potential to boost the in vitro and in vivo dissolution of poorly water-soluble drugs 22.
Figure 18: Silica structure
SiO2 is an amorphous solid, as its powder pattern contains no crystalline diffraction peaks.
The mesoporous silica used in this work has specific surface area = 294 m2/g and a pore size that ranges between 20 to 40 nm.
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Polymers
Three different polymers were used in Chapter 6 of this thesis: Polycarbonate (PC), Polymethyl methacrylate (PMMA) and Polytetrafluoroethylene (PTFE).
2.1.9. Polycarbonate
Polycarbonates (PC) (Figure 19) are a group of thermoplastic polymers
containing carbonate groups in their chemical structures. Polycarbonates used in
engineering are strong, tough materials, and some grades are optically
transparent. Polycarbonate is a durable material. Although it has high impact-resistance, it
has low scratch-resistance. The characteristics of polycarbonate compare to those
of polymethyl methacrylate (PMMA, acrylic), but polycarbonate is stronger and will hold
up longer to extreme temperature 23. Many polycarbonate grades are used in medical
applications and comply with both ISO 10993-1 and USP Class VI standards (occasionally
referred to as PC-ISO)24.
HBD = 0 HBA = 3
Figure 19: Polycarbonate molecule with the number HBD and HBA per monomer 2.1.10. Polymethyl methacrylate
Poly(methyl methacrylate) (PMMA) (Figure 20), also known as acrylic or acrylic glass as
well as by the trade names Plexiglas, Acrylite, Lucite, and Perspex among several others, is
a transparent thermoplastic often used in sheet form as a lightweight or shatter-resistant
alternative to glass. The same material can be utilized as a casting resin, in inks and
coatings, and has many other uses25. PMMA has a good degree of compatibility with
human tissue, and it is used in the manufacture of rigid intraocular lenses which are
implanted in the eye when the original lens has been removed in the treatment of cataracts.
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In particular, acrylic-type contact lenses are useful for cataract surgery in patients that have
recurrent ocular inflammation (uveitis), as acrylic material induce less inflammation26.
HBD = 0 HBA = 2
Figure 20: Polymethyl methacrylate molecule with the number HBD and HBA per monomer
2.1.11. Polytetrafluoroethylene
Polytetrafluoroethylene (PTFE) (Figure 21) is thermoplastic polymer with high-molecular-
weight consisting of carbon and fluorine. PTFE is hydrophobic: neither water nor water-
containing substances wet PTFE. It has one of the lowest coefficients of friction of any
solid. At room temperature PTFE is a white solid27. It is used as a film interface patch for
sports and medical applications, featuring a pressure-sensitive adhesive backing, which is
installed in strategic high friction areas of footwear, insoles, ankle-foot orthosis, and other
medical devices to prevent and relieve friction-induced blisters, calluses and foot
ulceration. Expanded PTFE membranes have been used in trials to
assist trabeculectomy surgery to treat glaucoma28. PTFE can also be used for dental fillings,
to isolate the contacts of the anterior tooth so the filling materials will not stick to the
adjacent tooth29.
HBD = 0 HBA = 4
Figure 21: Polytetrafluoroethylene molecule with the number HBD and HBA per monomer Solvent
2.1.12. Methanol
Methanol (MeOH) (Figure 22, Table 5) is a colourless alcohol, hygroscopic and completely
miscible with water, but with a lower density. It is a versatile solvent, but very toxic and
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extremely flammable. This simple single-carbon alcohol is a volatile solvent and a light
fuel 30, and thus easily removed from API/excipient samples.
Figure 22: MeOH molecule Table 5: Characteristics of methanol 30
Density @ 20 °C (g/cm3) 0.798 Molecular mass (g/mol) 32.04 Boiling point (°C) 64.7 The APIs used in this work are highly soluble in MeOH, whereas the excipients are poorly
soluble in MeOH at the same conditions. That was the main reason for the selection of this
solvent.
2.2. Analytical techniques
2.2.1. Powder X-Ray Diffraction (PXRD) X-rays are produced by bombarding a metal target (Cu or Mo usually) with a beam of electrons emitted from a hot filament (often tungsten). The incident beam will ionize electrons from the
K-shell (1s) of the target atoms and X-rays are emitted as the resultant vacancies are filled by
31 electrons dropping down from the L (2p) or M (3p) levels. This gives rise to K and K lines .
Figure 23: Generator of X-ray 31
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Diffraction occurs when light is scattered by a periodic array with long-range order, producing constructive interference at specific angles. The electrons in an atom coherently scatter X-rays.
We can regard each atom as a coherent point scatterer. The strength with which an atom scatters light is proportional to the number of electrons around the atom 32.
2.1.1.1. The Crystal
The molecules in a crystal are arranged in a periodic array and thus can diffract light. The wavelength of X-rays is similar to the distance between molecules. The scattering of X-rays from molecules produces a diffraction pattern, which contains information about the molecular arrangement within the crystal. Amorphous materials like glass do not have a periodic array with long-range order, so they do not produce a diffraction pattern. The diffraction pattern is a product of the unique crystal structure of a material. The crystal structure describes the molecular arrangement of a material. When the molecules are arranged differently, a different diffraction pattern is produced.
Crystalline materials are characterized by the long range, periodic arrangements of molecules.
The unit cell is the basic repeating unit that defines the crystal structure. The unit cell contains the maximum symmetry that uniquely defines the crystal structure. The unit cell might contain more than one molecule. The crystal system describes the shape of the unit cell. The lattice parameters describe the size of the unit cell. The unit cell repeats in all dimensions to fill space and produce the macroscopic grains or crystals of the material.
Figure 24: Crystal system formed by an assembly of unit cells 32
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Crystal structures focus on symmetry elements to define the molecular arrangement. Symmetry in crystal structures is a product of energy minimization in the molecular arrangement.
Symmetry in the crystal structure often produces symmetry in material properties and behaviour. Symmetry elements are used to define seven different crystal systems, explained in
Chapter 1.
2.1.1.2. Diffraction peaks and Miller indices.
Diffraction peaks are associated with planes of atoms or molecules. Miller indices (hkl) are used to identify different planes. Observed diffraction peaks can be related to planes of molecules to assist in analysing the molecular structure and microstructure of a sample. Parallel planes of molecules intersecting the unit cell define directions and distances in the crystal. The
Miller indices (hkl) define the reciprocal of the axial intercepts. The crystallographic direction,
[hkl], is the vector normal to (hkl). dhkl is the vector extending from the origin to the plane (hkl) and is normal to (hkl).
Figure 25: [1 0 0], [1 1 0] and [1 1 1] faces defined by Miller indices 33
The vector dhkl is used in Bragg’s law, defined in Equation 1, to determine where diffraction peaks will be observed. When analysing XRD data, we look for trends corresponding to directionality in the crystal structure by analysing the Miller indices of diffraction peaks. The position and intensity of peaks in a diffraction pattern are determined by the crystal structure.
nλ = 2 dhkl sin θ Equation 15
68 | P a g e where: n = An integer representing the order of the diffraction peak
λ = Wavelength of the x-ray dhkl = Inter-plane distance
θ = Scattering angle
32 Figure 26: dhkl vector corresponding to the [1 1 0] face of a crystal
The position of the diffraction peaks are determined by the distance between parallel planes of molecules. Bragg’s law calculates the angle where constructive interference from X-rays scattered by parallel planes of molecules will produce a diffraction peak. In most diffractometers, the X-ray wavelength, λ, is fixed. Consequently, a family of planes produces a diffraction peak only at a specific angle 2θ. dhkl is the vector drawn from the origin of the unit cell to intersect the crystallographic plane [h k l] at a 90° angle. Thus, it is the distance between parallel planes of molecules in the family [h k l] and it is a geometric function of the size and shape of the unit cell.
For parallel planes of molecules, with a space dhkl between the planes, constructive interference only occurs when Bragg’s law is satisfied. Additionally, the plane normal [h k l] must be parallel to the diffraction vector, s.
Plane normal, [h k l]: the direction perpendicular to a plane of molecules
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Diffraction vector, s: the vector that bisects the angle between the incident and diffracted
beam32.
Figure 27: Diffraction peaks and their correspondence faces32
At 20.6°, Bragg’s law fulfilled for the [100] planes, producing a diffraction peak.
The [110] planes would diffract at 29.3°; however, they are not properly aligned to
produce a diffraction peak (the perpendicular to those planes does not bisect the incident
and diffracted beams). Only background is observed. In order to measure different
crystallographic directions the sample can be tilted as shown in Figure 28. This is called
asymmetric scan.
The [200] planes are parallel to the [100] planes. Therefore, they also diffract for this
crystal. Since d200 is ½ d100, and according to the Bragg’s law, they appear at 42°.
Figure 28: Asymmetric scan of the [1 1 0] face32
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For every set of planes, there will be a small percentage of crystallites that are properly oriented to diffract (the plane perpendicular bisects the incident and diffracted beams). Basic assumptions of powder diffraction are that for every set of planes there is an equal number of crystallites that will diffract and that there is a statistically relevant number of crystallites, not just one or two 32.
Refining the lattice parameters of the unit cell from X-ray powder diffraction data requires knowing how to assign the Miller indices (h k l) to each diffraction peak, a process known as indexing. Normally for a known material, this is done by comparing the data to that reported in the literature or in a database of diffraction patterns. The Miller indices relate the peak positions or d-spacing to the lattice parameters by an equation specific to the crystal system.
For example, in a structure with an orthorhombic unit cell the relationship is expressed in the following Equation 2.
1 h2+k2+l2 2 = 2 2 2 Equation 16 dh k l a +b +c
Where
dhkl = Inter-plane distance h, k, l = Miller indices a, b, c = Lattice parameters
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2.1.1.3. Diffractometer
Generator
Sample
Figure 29: Parts of a diffractometer32 1. The incident angle,is defined as the angle between the X-ray source and the
sample.
2. The diffraction angle, 2, is defined between the incident beam and the detector.
3. The incident angle is always 1/2 of the detector angle 2. In PANalytical
X’Pert Pro (where ), the sample is fixed and the tube rotates at a rate -°/min and the
detector rotates at a rate of °/min.
4. The diffraction vector (s), the vector that bisects the angle between the incident
and scattered beam, is always normal to the surface of the sample.
Powder diffraction data can be collected using either transmission or reflection geometry, as
shown below. If the particles in the powder sample are randomly oriented, these two
methods will yield similar data34.
2.1.1.3.1. Reflection mode
For reflection PXRD, the tube will direct the X-rays at the sample; the crystal structure
will then deflect the X-rays at the angle towards the detector. The detector, which
rotates will then record the number of counts at a certain angle and then relay this to the
computer. A pattern can then be calculated, showing as peak intensity against angle
(2).
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Figure 30: Reflection mode34
2.1.1.3.2. Transmission
For transmission mode PXRD is the same as reflectance mode PXRD except that the X- rays pass through the sample.
Figure 31: Transmission mode34
2.1.1.4. Diffraction pattern
Peak position: Depends on the unit cell dimensions and the dimension of the
elementary cell.
Peak width: Depends on the stacking faults, residual strain/stress and the
particle grain size.
o No Strain
Figure 32: Peak pattern without stress35
o Uniform Strain (d1-d0)/d0, peak moves, no shape changes
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Figure 33: Peak pattern with uniform stress35
o Non-uniform Strain d1≠constant, Peak broadens
Figure 34: Peak pattern with non-uniform stress35
Peak intensity: Depends on the crystal structure and the content of the
elementary cell.
2.1.1.5. XRD analysis performed in this work
X-ray powder diffractograms were recorded on a Phillips PANanalytical X’Pert MPD PRO diffractometer using nickel-filtered copper CuKradiation source (= 1.5405600 Å) in reflection mode. The CuKdifractometer anode was run under a current of 40 mA and a tension of 40 kV. Dried and sieved samples were placed on a zero background sample holder and scans were performed between 5-40 degrees 2 at a scan rate of 0.005°2/min. The incident beam path had divergence slit with an angle of 1/4° and the diffracted beam path a radius of 240 nm.
2.2.2. Scanning electron microscope
Scanning electron microscope (SEM) is a type of electron microscope, designed for directly studying solid objects, that utilises a beam of focused electrons of relatively low energy as an electron probe that is scanned in a regular manner over the specimen. The action of the
74 | P a g e electron beam stimulates emission of high-energy backscattered electrons and low-energy secondary electrons from the surface of the specimen 36.
It is a very useful technique for crystal characterization, which gives extensive topographic and morphological information. The scanning electron microscope has many advantages over traditional microscopes. The SEM has a large depth of field, which allows more of a specimen to be in focus at one time. The SEM also has much higher resolution, so closely spaced features can be magnified at much higher levels. Because the SEM uses electromagnets rather than lenses, the researcher has much more control in the degree of magnification. All of these advantages, as well as the actual strikingly clear images, make the scanning electron microscope one of the most useful instruments in research today.
The SEM uses electrons instead of light to form an image. A beam of electrons is produced at the top of the microscope by an electron gun. The electron beam follows a vertical path through the microscope, which is held within a vacuum. The beam travels through electromagnetic fields and lenses, which focus the beam down toward the sample. Once the sample is raster scanned by the beam, electrons and X-rays are ejected from the sample37.
Figure 35: Schematic diagram of an SEM37
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Detectors collect these X-rays, backscattered electrons, and secondary electrons and convert them into a signal that is sent to a screen similar to a television screen. This produces the final image.
Figure 36: How the incident beam interact with the sample38
No elaborate specimen-preparation techniques are required for examination in the SEM, and large and bulky specimens may be accommodated. It is desirable that the specimen be rendered electrically conducting; otherwise, a sharp picture will not be obtained. Conductivity is usually achieved by evaporating a film of metal, such as gold, 5–10 nm thick, onto the specimen in a vacuum (such a thickness does not materially affect the resolution of the surface details). If, however, the SEM can be operated at 1–3 kV of energy, then even non conducting specimens may be examined without the need for a metallic coating 39.
2.2.2.1. SEM analysis conducted in this work
The appearance of the particles was examined by SEM (JCM-5700, Carryscope).
Approximately 2-5 mg of dried, unground sample was scattered evenly onto the surface
of an aluminium stub covered with a 12 mm diameter carbon tab. Excess crystals were
removed and the sample was sputter coated with a thin film of conductive gold in an
Emitech K550X sputter coater under vacuum (10-1 mbar) using a current intensity of 20
mA and a deposition rate of 8 nm/min during 80 seconds. After the vacuum argon gas
was introduced. The stub containing the coated sample was then placed in the specimen
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chamber under high vacuum, and the accelerator electron beam was directed onto the
sample and the image was produced. The accelerating voltage used normally was 10
keV. However, lower voltages were sometimes used for imaging.
The habit and the mean size of the API particles crystallized in the presence of the
‘heterosurfaces’ were measured from the micrographs using image analysis (Adobe
Measurement Tool) with at least 20 API particles measured per sample.
2.2.3. In situ SEM-Raman Spectroscopy
Micro-Raman measurements were performed on an InVIA Reflex spectrometer (Renishaw) coupled to an optical microscope (DM2500, Leica) or an SEM (JSM-6510LV, JEOL); the latter being referred to as the SEM-SCA (SEM-Structure & Chemical Analyser). Instrument calibration was performed using the Si (100) peak (520.5 ± 1 cm−1). Spectra were acquired using the 785 nm laser, variable laser power (0.1 – 10 mW), acquisition times (10 – 500 s) and accumulations (1 – 20) over the spectral range of interest. Spectra collection and processing were performed with the WIRE™ 4.1 software (Renishaw).
2.2.3.1. Raman spectroscopy
Raman is the study of fundamental molecular vibrations which provides a finger print of a molecular solid. The distinction between spectra of different polymorphisms is rarely large and is observed as changes in peak shape, intensity, position or absence of bands.
Molecular movements in crystals are only allowed with defined phase relations giving rise to the vibrational modes of the crystal. These collective molecular movements are termed acoustic or optical phonons depending on the generation of a dipole moment during the vibration.
Optical phonons take part in inelastic light-scattering processes due to their induced dipole moment. The interaction of electromagnetic radiation with matter leads to absorption and reflection and light-scattering processes. In Rayleigh scattering, most scattered photons have
77 | P a g e the frequency identical to the incident ones. But a small part of the scattered light may have a higher or lower energy than that of the incident light. This process is known as the Raman effect: the incoming photon excites the scattering matter from its electronic ground state into a virtual state, from which it relaxes under the emission of a Raman scattered photon of lower or higher energy called Stokes or anti-Stokes scattering, depending on the initial and final vibrational levels (Figure 37). These light-scattering processes can be understood within a classical model of the scattering process. Such a classical model can describe the observed
Raman shifts. In a quantum mechanical picture of Raman scattering, the incoming photon either generates an excited phonon or therefore has a lower energy after the scattering process or it annihilates an excited phonon in the solid and thus has a higher energy after the scattering event 40:
Figure 37: Energy-level diagram showing the states involved in Raman spectra40-41. The incident photons will thus interact with the molecule present, and the amount of energy change (either lost or gained) by a photon is characteristic of the nature of each bond
(vibration) present. Not all vibrations will be observable with Raman spectroscopy (depending upon the symmetry of the molecule) but sufficient information is usually present to enable a very precise characterization of the molecule structure. Hence, the amount of energy shift for a
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C-H bond is different to that seen with a C-O bond. By looking at all these various wavelengths of scattered light, one can detect a range of wavelengths associated with the different bonds and vibrations.
2.2.3.1.1. Raman analysis conducted in this work
Samples were analysed using a Renishaw inVia microscope system. The samples were placed and areas of interaction located on the stage of a Leica microscope, with 10, 20 and 50x objective lenses Measurements were made at room temperature using a 514 nm argon laser at
10 % power and the spot laser was less than 5 m. Samples were scanned from 3310-150 cm-1 with an exposure time of 50 seconds. Multiple acquisitions were made to maximise the signal.
2.2.3.2. In situ SEM-Raman
Figure 38: In situ SEM-Raman and Raman spectroscopy
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2.2.3.2.1. In-situ SEM-Raman analysis conducted in this work
A Jeol JSM-6510 LV in conjunction with a Renishaw inVia Raman microscopy system was used to analyse uncoated and coated samples. An accelerating voltage of 1.5 keV was used for the analysis. Approximately 2-5 mg of dried, unground sample was scattered evenly onto the surface of a stub.
The sample analysis was located using the SEM. When the area for Raman analysis was decided the electron beam was turned off. An optimal video image of the sample was then used to focus the laser beam. When the laser beam was focused the Raman spectra was collected.
2.2.4. Ultraviolet-visible spectrophotometer (UV-Vis).
Ultraviolet and visible (UV-Vis) absorption spectroscopy is the measurement of the attenuation of a beam of light after it passes through a sample or after reflection from a sample surface.
Absorption measurements can be at a single wavelength or over an extended spectral range.
The wavelength range of UV radiation starts at blue end of visible light (400 nm) and ends at
200 nm. Ultraviolet absorption spectra arise from transition of electrons within a molecule from a lower level to a higher level. A molecule absorbs ultraviolet radiation of frequency (ν), the electron in that molecule undergo transition from lower to higher energy level. The energy of radiation can be calculated by the following equation:
E1-E0 = h . ν = =h/λ Equation 17
Where,
E1-E0 = radiation energy h = Plank constant = 6.63 × 10-34 m2 kg / s
ν = frequency
λ= wavelength
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The ultraviolet-visible spectroscopy measures the intensity of light passing through a sample
(I), and compares it to the intensity of light before it passes through the sample (I0). The ratio (I/I0) is called the transmittance, and is usually expressed as a percentage (%T).
The absorbance, A, is based on the transmittance42:
A = - log (I/I0) Equation 18
The UV-visible spectrophotometer can also be configured to measure reflectance. In this case, the spectrophotometer measures the intensity of light reflected from a sample (I), and compares it to the intensity of light reflected from a reference material (I0) (such as a white tile). The ratio
I/I0 is called the reflectance, and is usually expressed as a percentage (%R). In this work the
UV-Vis spectrometer is used in transmission mode 42.
The Beer-Lambert law (or Beer's law) is the linear relationship between absorbance and concentration of an absorbing species:
A = ε * b * c Equation 19 where ε is the wavelength-dependent molar absorptivity coefficient with units of M-1 cm-1. b is the path length, and c is sample concentration in M.
2.2.4.1. EUV-Vis analysis conducted in this work
In this work a UV-1800 UV-Vis Spectrophotometer from Shimadzu with double beam was used 43. The absorbance is given as an output and the concentration of the solutions was calculated using the Beer-Lambert law. A calibration curve representing standard samples of known concentration with its corresponding absorbance was initially calculated in order to determine the constants in the Beer-Lambert equation. The concentration of dissolved FF was measured with reference to the UV absorbance for FF at λ = 289 nm.
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2.2.5. Solid-state Nuclear Magnetic Resonance Spectroscopy (SSNMR)
This is a research technique that exploits the magnetic properties of certain atomic nuclei. This type of spectroscopy determines the physical and chemical properties of atoms or the molecules in which they are contained. It relies on the phenomenon of nuclear magnetic resonance and can provide detailed information about the structure, dynamics, reaction state, and chemical environment of molecules. The intramolecular magnetic field around an atom in a molecule changes the resonance frequency, thus giving access to details of the electronic structure of a molecule and its individual functional groups. Most frequently, NMR spectroscopy is used by chemists and biochemists to investigate the properties of organic molecules, although it is applicable to any kind of sample that contains nuclei possessing spin.
Suitable samples range from small compounds analyzed with 1-dimensional proton or carbon-
13 NMR spectroscopy to large proteins or nucleic acids using 3 or 4-dimensional techniques.
The impact of NMR spectroscopy on the sciences has been substantial because of the range of information and the diversity of samples, including solutions and solids44.
Solid-state NMR (SSNMR) spectroscopy is a kind of nuclear magnetic resonance (NMR) spectroscopy, characterized by the presence of anisotropic (directionally dependent) interactions. A spin interacts with a magnetic or an electric field. Spatial proximity and/or a chemical bond between two atoms can give rise to interactions between nuclei. In general, these interactions are orientation dependent. In media with no or little mobility (e.g. crystals, powders, large membrane vesicles, molecular aggregates), anisotropic interactions have a substantial influence on the behaviour of a system of nuclear spins44.
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2.2.5.1. SSNMR analysis conducted in this work
In this work Solid-state Cross-Polarization Magic Angle Spinning Carbon-13 Nuclear
Magnetic Resonance (CP/MAS13 C-NMR) has been used to investigate the structure of two samples. This is a type of SSNMR spectroscopy. Anisotropic interactions between nuclei, which are usually averaged by Brownian motion in liquid samples, cause significant line broadening in solid state NMR, but can be averaged to zero by spinning the sample very rapidly at the magic angle. Cross polarisation is used to enhance the signal from weakly coupled nuclei such as 13C nuclei 45.
2.2.6. iControl LabMax
A 1 L vessel was equipped with Focused Beam Reflectance Measurement (FBRM), a temperature sensor and in situ Fourier Transform Infrared (FTIR), as shown in Figure 39. All the probes were distributed by Mettler Toledo. Measurements from these instruments were recorded by iControl LabMax SoftwareTM Mettler Toledo and exported to Excel for data processing. A 4-bladed PTFE screw propeller stirrer shaft (Diameter = 9.5 mm, Length =
650 mm) was used to mix the solution. This impeller is known for mixing media in an up-to- down axial flow. A stirring of 175 rpm was selected for all the experiments, as it was enough to keep the solution mixed without forming bubbles or vortices.
Figure 39: Sensors used to track API crystallization. Temperature and supersaturation are solution state properties that can be manipulated; the chord count is a solid state property that we use to characterize the crystals46.
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2.2.6.1. Focussed beam reflectance measurement (FBRM)
In FBRM, a focused laser beam spinning at a high speed propagates into the slurry through a sapphire window mounted on the tip of a cylindrical probe. When the laser beam intersects the edge of a particle, some of it is backscattered to the detector installed in the same probe, and induces a rise signal in the circuit until it reaches the opposite edge of the particle. A chord length is thus registered. The product of risetime and tangential velocity of the spinning laser beam is a chord length. The measurement range of chord length depends on the scanning speed of the laser beam and is divided into a fixed number of linear channels in the hardware. Each count of chord length is recorded in a corresponding channel and a chord length distribution
(CLD) is thus generated. Chord length counts grouped by channels are the primary data provided by FBRM. In addition, the control interface provides a variety of weighted or unweighted statistics of the primary data, e.g. total counts of chord lengths in all channels, mean chord length, median, standard deviation of CLD, etc., which are different statistical presentations of the primary data. It is logical to expect a relationship between the total counts of chord length and the number of particles, and a linkage between CLD and PSD. Total counts of chord lengths have been found to change linearly and then nonlinearly with solids concentration as solids concentration varies from low to high. However, the number of particles is different from solids concentration in that solids concentration can be broken down into two factors, i.e., the number of particles and particle size. This break down is necessary in order to describe a nucleating and growing system of particles. Based on the working principles of FBRM, a larger particle is more likely to be detected than a smaller one and therefore generates more counts. It is of utmost importance to decouple the effects of particle size and the number of particles on chord length counts if a correct relationship is to be constructed47-49.
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Figure 40: (a) The focused beam reflectance method (FBRM) probe technique. (b) Measurement of a particle chord length using the FBRM technique 50
Figure 41: A typical chord length distribution
2.2.6.2. In situ Fourier Transform Infrared (FTIR)
Fourier transform infrared spectroscopy (FTIR) is a technique which is used to obtain an infrared spectrum of absorption or emission of a solid, liquid or gas. An FTIR spectrometer simultaneously collects high spectral resolution data over a wide spectral range. This confers a significant advantage over a dispersive spectrometer which measures intensity over a narrow range of wavelengths at a time 51. The components of a FTIR are explained as follows:
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IR sources
FTIR spectrometers are mostly used for measurements in the mid and near IR regions. For the mid-IR region, 2−25 µm (5000–400 cm−1), the most common source is a silicon carbide element heated to about 1200 K. The output is similar to a blackbody. Shorter wavelengths of the near-IR, 1−2.5 µm (10000–4000 cm−1), require a higher temperature source, typically a tungsten-halogen lamp. The long wavelength output of these is limited to about
5 µm (2000 cm−1) by the absorption of the quartz envelope. For the far-IR, especially at wavelengths beyond 50 µm (200 cm−1) a mercury discharge lamp gives higher output than a thermal source 52.
Detectors
Mid-IR spectrometers commonly use pyroelectric detectors that respond to changes in temperature as the intensity of IR radiation falling on them varies. The sensitive elements in these detectors are either deuterated triglycine sulfate (DTGS) or lithium tantalate
(LiTaO3). These detectors operate at ambient temperatures and provide adequate sensitivity for most routine applications. To achieve the best sensitivity the time for a scan is typically a few seconds. Cooled photoelectric detectors are employed for situations requiring higher sensitivity or faster response. Liquid nitrogen cooled mercury cadmium telluride (MCT) detectors are the most widely used in the mid-IR. With these detectors an interferogram can be measured in as little as 10 milliseconds. Uncooled indium gallium arsenide photodiodes or DTGS are the usual choices in near-IR systems. Very sensitive liquid-helium-cooled silicon or germanium bolometers are used in the far-IR where both sources and beamsplitters are inefficient53.
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Beam splitter
Figure 42: Simple interferometer with a beam-splitter and compensator plate 53 An ideal beam-splitter transmits and reflects 50% of the incident radiation. However, as any material has a limited range of optical transmittance, several beam-splitters may be used interchangeably to cover a wide spectral range. For the mid-IR region the beamsplitter is usually made of KBr with a germanium-based coating that makes it semi-reflective. KBr absorbs strongly at wavelengths beyond 25 μm (400 cm−1) so CsI is sometimes used to extend the range to about 50 μm (200 cm−1). ZnSe is an alternative where moisture vapor
−1 can be a problem but is limited to about 20μm (500 cm ). CaF2 is the usual material for the near-IR, being both harder and less sensitive to moisture than KBr but cannot be used beyond about 8 μm (1200 cm−1). In a simple Michelson interferometer one beam passes twice through the beamsplitter but the other passes through only once. To correct for this an additional compensator plate of equal thickness is incorporated. Far-IR beamsplitters are mostly based on polymer films and cover a limited wavelength range.
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REFERENCES
1. Naeem, M.; Mahmood, A.; Khan, S.; Shahiq, Z., Development and evaluation of controlled-release bilayer tablets containing microencapsulated tramadol and acetaminophen. Tropical Journal of Pharmaceutical Research 2010, 9 (4). 2. Price, C. P.; Grzesiak, A. L.; Matzger, A. J., Crystalline polymorph selection and discovery with polymer heteronuclei. J. Am. Chem. Soc. 2005, 127 (15), 5512-5517. 3. Groom, C. R.; Bruno, I. J.; Lightfoot, M. P.; Ward, S. C., The Cambridge Structural Database. Acta Crystallographica Section B 2016, 72 (2), 171-179. 4. Perrin, M.-A.; Neumann, M. A.; Elmaleh, H.; Zaske, L., Crystal structure determination of the elusive paracetamol Form III. Chemical Communications 2009, (22), 3181-3183. 5. Tipduangta, P.; Takieddin, K.; Fábián, L.; Belton, P.; Qi, S., A New Low Melting-Point Polymorph of Fenofibrate Prepared via Talc Induced Heterogeneous Nucleation. Crystal Growth & Design 2015, 15 (10), 5011-5020. 6. Yang, L. P. H.; Keating, G. M., Fenofibric Acid. American Journal of Cardiovascular Drugs 2009, 9 (6), 401-409. 7. Wong, T. Y.; Simó, R.; Mitchell, P., Fenofibrate – A Potential Systemic Treatment for Diabetic Retinopathy? American Journal of Ophthalmology 2012, 154 (1), 6-12. 8. Limited, M. I. H. http://www.medicines.ie/medicine/11695/SPC/Lipantil+Supra+145mg+film-coated+tablets/. 9. Jamzad, S.; Fassihi, R., Role of surfactant and pH on dissolution properties of fenofibrate and glipizide—A technical note. AAPS PharmSciTech 2006, 7 (2), E17-E22. 10. Heinz, A.; Gordon, K. C.; McGoverin, C. M.; Rades, T.; Strachan, C. J., Understanding the solid-state forms of fenofibrate – A spectroscopic and computational study. European Journal of Pharmaceutics and Biopharmaceutics 2009, 71 (1), 100-108. 11. Górniak, A.; Wojakowska, A.; Karolewicz, B.; Pluta, J., Phase diagram and dissolution studies of the fenofibrate–acetylsalicylic acid system. J. Therm. Anal. Calorim. 2011, 104 (3), 1195-1200. 12. Fronczek, F. R.; Kamel, H. N.; Slattery, M., Three polymorphs (alpha, beta and delta) of D-mannitol at 100 K. Acta Crystallographica Section C-Crystal Structure Communications 2003, 59, O567-O570. 13. Wagner, C. M.; Pein, M.; Breitkreutz, J., Roll compaction of granulated mannitol grades and the unprocessed crystalline delta-polymorph. Powder Technol. 2015, 270, 470-475. 14. Thoorens, G.; Krier, F.; Leclercq, B.; Carlin, B.; Evrard, B., Microcrystalline cellulose, a direct compression binder design environment-A review. Int. J. Pharm. 2014, 473 (1-2), 64- 72. 15. Biswal, D.; Singh, R., Characterisation of carboxymethyl cellulose and polyacrylamide graft copolymer. Carbohydr. Polym. 2004, 57 (4), 379-387. 16. Hollabaugh, C. B.; Burt, L. H.; Walsh, A. P., Carboxymethylcellulose. Uses and Applications. Industrial & Engineering Chemistry 1945, 37 (10), 943-947. 17. Labet, M.; Thielemans, W., Synthesis of polycaprolactone: a review. Chem. Soc. Rev. 2009, 38 (12), 3484-3504. 18. Bhavsar, M. D.; Amiji, M. M., Development of Novel Biodegradable Polymeric Nanoparticles-in-Microsphere Formulation for Local Plasmid DNA Delivery in the Gastrointestinal Tract. AAPS PharmSciTech 2008, 9 (1), 288-294. 19. Iler, R. K., The Chemistry of Silica: Solubility, Polymerization, Colloid and Surface Properties and Biochemistry of Silica. Wiley: 1979. 20. Nandiyanto, A. B. D.; Kim, S.-G.; Iskandar, F.; Okuyama, K., Synthesis of spherical mesoporous silica nanoparticles with nanometer-size controllable pores and outer diameters. Microporous Mesoporous Mater. 2009, 120 (3), 447-453.
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21. Trewyn, B. G.; Nieweg, J. A.; Zhao, Y.; Lin, V. S. Y., Biocompatible mesoporous silica nanoparticles with different morphologies for animal cell membrane penetration. Chem. Eng. J. 2008, 137 (1), 23-29. 22. Mellaerts, R.; Mols, R.; Kayaert, P.; Annaert, P.; Van Humbeeck, J.; Van den Mooter, G.; Martens, J. A.; Augustijns, P., Ordered mesoporous silica induces pH-independent supersaturation of the basic low solubility compound itraconazole resulting in enhanced transepithelial transport. Int. J. Pharm. 2008, 357 (1), 169-179. 23. Parvin, M.; Williams, J., The effect of temperature on the fracture of polycarbonate. Journal of Materials Science 1975, 10 (11), 1883-1888. 24. Powell, D. G. Medical Applications of Polycarbonate. https://web.archive.org/web/19990223191619/http://www.devicelink.com/mpb/archive/98/09/0 03.html. 25. Smith, W. F.; Hashemi, J., Foundations of materials science and engineering. McGraw-Hill: 2011. 26. Meyers, R. A., Molecular biology and biotechnology: a comprehensive desk reference. John Wiley & Sons: 1995. 27. Sperati, C. A.; Starkweather Jr, H., Fluorine-containing polymers. II. Polytetrafluoroethylene. Springer: 1961. 28. Wang, X.; Khan, R.; Coleman, A., Device-modified trabeculectomy for glaucoma. Cochrane Database of Systematic Reviews 2015, (12). 29. Dunn, W.; Davis, J.; Casey, J., Polytetrafluoroethylene (PTFE) tape as a matrix in operative dentistry. Operative dentistry 2003, 29 (4), 470-472. 30. Institute, M., Methanol Safe Handling Manual. http://www.methanol.org/getattachment/05f122f1-c9d3-47d8-8c16-5a5d39717cbe/Methanol- Safe-Handling-Manual-Final---English.pdf.aspx, 2013; p 207. 31. Loye, H. z., X-Ray Diffraction how it works what it can and what it cannot tell us. University of South Carolina: 2001; p 34. 32. Speakman, S. A., Basics of X-Ray Powder Diffraction. http://prism.mit.edu/xray. 33. Fu, L.; Kane, C. L.; Mele, E. J., Topological insulators in three dimensions. Phys. Rev. Lett. 2007, 98 (10), 106803. 34. Warren, B. E., X-ray Diffraction. Courier Corporation: 1969. 35. Suryanarayana, C.; Norton, M. G., X-Ray Diffraction: A Practical Approach. Springer US: 2013. 36. Joy, D. C. Scanning electron microscope (SEM) 37. Goldstein, J.; Newbury, D. E.; Echlin, P.; Joy, D. C.; Romig Jr, A. D.; Lyman, C. E.; Fiori, C.; Lifshin, E., Scanning electron microscopy and X-ray microanalysis: a text for biologists, materials scientists, and geologists. Springer Science & Business Media: 2012. 38. Sediako, A. D.; Soong, C.; Howe, J. Y.; Kholghy, M. R.; Thomson, M. J., Real-time observation of soot aggregate oxidation in an Environmental Transmission Electron Microscope. Proceedings of the Combustion Institute 2017, 36 (1), 841-851. 39. Boyde, A.; Wood, C., Preparation of animal tissues for surface‐scanning electron microscopy. Journal of Microscopy 1969, 90 (3), 221-249. 40. Mestl, G., In situ Raman spectroscopy - a valuable tool to understand operating catalysts. Journal of Molecular Catalysis a-Chemical 2000, 158 (1), 45-65. 41. Collette, T. W.; Williams, T. L., The role of Raman spectroscopy in the analytical chemistry of potable water. J. Environ. Monit. 2002, 4 (1), 27-34. 42. Skoog, D. A.; Holler, F. J.; Crouch, S. R., Principles of instrumental analysis. Cengage learning: 2017. 43. shimadzu Shimadzu Scientific Instruments. http://www.ssi.shimadzu.com/products/product.cfm?product=uv1800_3.
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44. Zhang, S.-Y.; Wang, M.-T.; Liu, Q.-H.; Hu, B.-W.; Chen, Q.; Li, H.-X.; Amoureux, J.- P., Determination of coordination modes and estimation of the 31P-31P distances in heterogeneous catalyst by solid state double quantum filtered 31P NMR spectroscopy. PCCP 2011, 13 (13), 5617-5620. 45. Larsson, P. T.; Hult, E.-L.; Wickholm, K.; Pettersson, E.; Iversen, T., CP/MAS 13C- NMR spectroscopy applied to structure and interaction studies on cellulose I. Solid State Nucl. Magn. Reson. 1999, 15 (1), 31-40. 46. Griffin, D. J.; Kawajiri, Y.; Rousseau, R. W.; Grover, M. A., Using MC plots for control of paracetamol crystallization. Chem. Eng. Sci. 2017, 164, 344-360. 47. Yu, Z. Q.; Chow, P. S.; Tan, R. B. H., Interpretation of Focused Beam Reflectance Measurement (FBRM) Data via Simulated Crystallization. Organic Process Research & Development 2008, 12 (4), 646-654. 48. Jonasz, M.; Fournier, G., Light Scattering by Particles in Water: Theoretical and Experimental Foundations. Elsevier Science: 2011. 49. Barrett, P.; Glennon, B., In-line FBRM Monitoring of Particle Size in Dilute Agitated Suspensions. Particle & Particle Systems Characterization 1999, 16 (5), 207-211. 50. Greaves, D.; Boxall, J.; Mulligan, J.; Montesi, A.; Creek, J.; Dendy Sloan, E.; Koh, C. A., Measuring the particle size of a known distribution using the focused beam reflectance measurement technique. Chem. Eng. Sci. 2008, 63 (22), 5410-5419. 51. Griffiths, P. R.; De Haseth, J. A.; Winefordner, J. D., Fourier Transform Infrared Spectrometry. Wiley: 2007. 52. Smith, D. R.; Morgan, R. L.; Loewenstein, E. V., Comparison of the Radiance of Far- Infrared Sources. J. Opt. Soc. Am. 1968, 58 (3), 433-434. 53. Chalmers, J. M.; Griffiths, P. R., Handbook of Vibrational Spectroscopy. Wiley: 2002.
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CHAPTER 3:
EXAMINATION OF PROCESS
PARAMETERS FOR THE HETEROGENEOUS
NUCLEATION OF ACTIVE
PHARMACEUTICAL INGREDIENTS ON
EXCIPIENTS
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3.1. ABSTRACT
It is known that chemical and physical compatibility between a heterosurface and the crystallizing molecule promotes heterogeneous nucleation. In this work acetaminophen (AAP),
-Lactose (-Lac) and methanol (MeOH) are selected as the model API, excipient and solvent, respectively. The excipient – suspended in a supersaturated solution of AAP in MeOH
– was used as a heterogeneous surface (‘seed’), and parameters influencing the heterogeneous nucleation of the AAP such as (a) AAP solution/excipient contact time, (b) AAP supersaturation, and (c) AAP to excipient loading were tuned to demonstrate how the nucleation rate and degree of crystallization can be manipulated to control the particle size and the balance between nucleation and growth. The optimal crystallization experiment was carried out at a modest supersaturation to avoid homogeneous nucleation, ensuring that at that supersaturation under the same conditions but in the absence of -Lac no nucleation occurs up to 2 hours. During the heterogeneous crystallization AAP particles nucleate on the -Lac surface and then grow uniformly producing small AAP particles (<15 m) in a robust manner such that the particle size distribution (PSD) can be maintained constant over a wide variety of contact times, supersaturations, and AAP loadings (%).
3.2. INTRODUCTION
The successful development and commercialization of an Active Pharmaceutical Ingredient
(API) requires adequate processability, stability, and bioavailability, ultimately leading to better patient outcomes. However, APIs with desired biological activities rarely exhibit adequate physical properties to meet all of these requirements. Crystal engineering – the design of molecular solids in the broadest sense – is gaining increased attention within the pharmaceutical industry because it enables preparation of materials with tailored physical properties 1. The selection of the commercial solid form, shape and size from a designed
92 | P a g e crystallization process is one of the key milestones in the development of any new chemical entity, not only from an API manufacturing perspective, but also from a drug product processing performance and stability perspective 2. Control of crystal morphology can be achieved by solvent selection and/or tailor-made additives1 and by changing operating process conditions i.e. temperature decreasing rate, mixing and seeding. In the context of pharmaceutical solids, the solvent-induced crystal habit modification approach is limited by solvent effectiveness, crystallization efficiency and the purity requirements of the final product.
Employing “generally regarded as safe” excipients represents a practical alternative for the achievement of a desired crystallographic form, particle size and shape in the highly regulated pharmaceutical industry 1.
The literature reports indicate that the presence of foreign particles serves to reduce the energy barrier required for the creation of a new solid-liquid interface 3 and that the nucleation of crystalline phases on molecular crystal substrates can be controlled by the following key factors:
1. the chemical compatibility between a heterosurface and the crystallizing molecule, such
as hydrogen bonding, that promotes nucleation 4-7.
2. the surface topography of the substrate which plays an important role in the nucleation
by influencing the nucleation kinetics (nanoconfinement) 8.
3. the compatibility of crystal structures between the heterosurface and the nucleate (ledge-
directed epitaxy) due to symmetry constraints 9.
Previous studies have provided fundamental insights into heterogeneous nucleation using foreign substrates 4-7, 10-11. However, only some of these studied the factors that can influence the rate of heterogeneous nucleation 12 and just one of them with the purpose of improving industrial pharmaceutical processes 14. In addition, many researchers have mainly looked at how compatible surfaces with nanoscale features, such as Langmuir monolayers, self-
93 | P a g e assembled monolayers (SAMs) and synthetic polymers, can increase the nucleation rate and influence the crystal habit and the interfacial orientation of the API 5, 8, 15-19. However, most of them use non-biocompatible materials that are not approved for human ingestion 20-22.
Acetaminophen (AAP) (Figure 43(a)) is a generic API produced by a range of pharmaceutical companies with three known crystalline polymorphs in addition to existing in an amorphous form. Commercial AAP consists primarily of the thermodynamically stable monoclinic form
(Form I), even though this polymorphic form has poor compression properties 23. The monoclinic form of AAP was used throughout this experimental study, and no polymorphic transformation was observed during any of the nucleation experiments 24. Some current AAP crystallization processes are based on cooling crystallization from solution 25-27, however the crystallization time can be extended to 8 hours. In some other processes seeding is used to promote AAP crystallization and to control the final crystal size 28-29.
The milk sugar, lactose (4-O--D-galactopyranosyl--D-glucopyranose) is a carbohydrate comprising one galactose moiety linked to a glucose molecule through a -1,4 linkage. It exhibits two anomeric forms, -lactose and -lactose, which differ in the configuration of the terminal hydroxyl group of the glucose unit (Figure 43(b)). Crystals of pure β-lactose have a characteristic kite-like form. The -Lactose (-Lac) used in this study, confirmed by
Powder X-ray Diffraction (PXRD), was approximately 80% β-lactose, the remaining 20% being anhydrous α-lactose. This type of lactose is used as a filler-binder for tablet production via direct compression processes 30.
As suggested by Chadwick et al. 6, the many hydroxyl groups on sugar excipients like -
Lac show functional group complementarity to AAP’s hydroxyl and amide functional groups.
This complementarity influences -Lac’s ability to sequester incipient AAP nuclei. AAP and
-Lac were therefore selected as the model API and model excipient respectively for this
94 | P a g e experimental study. Additionally, -Lac presents a rough surface containing many concave recesses which may also promote the AAP crystallization via ledge-directed epitaxy 9 or confinement 8. Finally, methanol (MeOH) was chosen as a suitable crystallization solvent for the heterogeneous nucleation of an API onto an excipient because it is an established crystallization solvent for AAP 31 yet dissolves -Lac only sparingly at the temperatures of interest in this study.
(a)
(b)
Figure 43: Chemical structure of (a) acetaminophen and (b) -Lac. The aim of this study was to tune the following process parameters to demonstrate how the nucleation rate and degree of crystallization can be manipulated and thus controlled: (a) API solution/excipient contact time, (b) API supersaturation, and (c) API to excipient loading.
When translated to the industrial scale, such control may potentially improve solids properties to the extent that the requirement for downstream processing via milling might be avoided.
However, this process will required additional modelling to overcome the limitations due to mass and heat transfer at industrial scale 32.
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3.3. EXPERIMENTAL
3.3.1. Materials
Methanol (MeOH, > 99.9 %), acetaminophen (AAP) (paracetamol, ≥ 98 %) and -
Lactose (-Lac) (≤ 20% α-anomer; ≥ 99% total lactose) were supplied by Sigma-Aldrich and used ‘as received’. The PXRD diffractogram of the ‘as received’ AAP confirmed it to be the monoclinic Form I polymorph (CCDC HXACN01), while the corresponding diffractogram of the ‘as received’ -Lac matched that of the pure -Lactose (CCDC BLACTO) and from the relative peak intensities was determined to be > 80 wt-% -Lactose.
3.3.2. Methods
3.3.2.1. Solubility of AAP and -Lac in MeOH
The solubility of AAP and -Lac was measured in MeOH between 5 and 35 °C; each solubility measurement was performed in triplicate. Excess solids were added to approximately
20 mL of MeOH, placed in a temperature-controlled water bath (± 0.1 °C) at the required saturation temperature (Tsat) and agitated with a magnetic stirrer at 500 rpm for 24 hours.
Agitation was then stopped and the suspensions allowed to settle for more than 1 hour. The concentrations of the dissolved AAP or -Lac were then determined using the dry mass method, i.e., three aliquots of the clear supernatant were transferred to pre-weighed vials fitted with pre-weighted plastic screw lids and PTFE seals using pre-heated (Tsat + 5°C) syringes and
0.2 m Nylon filters and the total mass recorded. The lids were then removed, the solvent evaporated at room temperature in a ventilated laboratory hood, and the vials then transferred to an oven at 40 °C until a constant weight was achieved; the visual appearance of the samples was monitored during drying. The amount of AAP or -Lac present in the supernatant, expressed in terms of concentration as g solute/kg MeOH, was then calculated 33.
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3.3.2.2. Determination of the metastable zone width (MSZW) of AAP in MeOH
All MSZW experiments were conducted in triplicate at three different Tsat (20, 25 and 30
°C) in a HEL PolyBLOCK system at a constant agitation rate of 200 rpm using a PTFE-coated magnetic stirrer. Saturated AAP-MeOH solutions were prepared in accordance with the solubility data: 200 mL of MeOH and the appropriate amount of AAP were placed in the crystallizer of the HEL PolyBLOCK and heated to 10 °C above the saturation temperature
(Tsat) for 1 hour to ensure complete dissolution. Three heating/cooling cycles per batch and at least three batches were examined at a cooling rate of 1 °C/min. Each crystallizer in the HEL had an internal reactor temperature probe and a turbidity probe enabling the crystallization and dissolution to be monitored.
3.3.2.3. Determination of the induction time for AAP in MeOH in the absence of -
Lac
Stock solutions of AAP in MeOH, saturated at 25 °C, were placed in a water bath at Tsat +
5 °C (30 ± 0.1 °C) and agitated at 400 rpm with a PTFE-coated magnetic stirrer for ≥ 12 hours.
20 mL aliquots of the saturated solution were then transferred to 25 mL vials (n=20), sealed with PTFE-lined lids, using pre-heated syringes and filters (PTFE, 0.2 m). The vials were equilibrated at Tsat + 5 °C (30 ± 0.1 °C, 200 rpm, ≥ 12 hours) prior to quench-cooling to the desired crystallization temperatures (Tcry). Agitation was maintained via a PTFE-coated magnetic stirrer at 200 rpm throughout the isothermal treatment. The induction time of each vial (defined as the time when the first crystals of AAP are observed to crystallize in the first vial) was measured using a webcam (Microsoft life cam, wide angle f/2.2, HD Lens 720 p HD,
30 FPS, Autofocus widescreen). The induction time was measured at Tcry of 5, 10, 15 and 20
푐 °C corresponding to supersaturations (S) of 1.56, 1.39, 1.25 and 1.12 respectively (S = , 푐∗ where c = the initial concentration of AAP in MeOH in g AAP/kg MeOH and c* = the
97 | P a g e equilibrium concentration of AAP in MeOH at Tcry in g AAP/kg MeOH). All measurements were performed in triplicate.
3.3.2.4. Crystallization of AAP from MeOH in the presence of -Lac
20 mL aliquots of the saturated AAP-MeOH solutions were prepared at a Tsat of 25 °C as described in Section 2.3. Following equilibration at Tsat + 5 °C the vials were transferred to a water bath at Tcry and held for 15 minutes prior to addition of -Lac. Following the addition of -Lac the suspensions were agitated at 700 rpm, with a PTFE-coated magnetic stirrer, and held isothermally at Tcry. All crystallizations were performed in triplicate. The following parameters deemed capable of influencing the crystallization of AAP in the presence of -
Lac were examined:
(a) the contact time between the AAP-MeOH solution and the -Lac,
(b) the supersaturation of the AAP-MeOH solution, and
(c) the amount of AAP available to crystallize from the AAP-MeOH solution in the
presence of the -Lac, where all supersaturation is consumed via either
heterogeneous or homogeneous nucleation, i.e. the maximum attainable AAP loading
(% w/w) which is defined as follows in Equation 20:
∗ [푐 – 푐 ]× 푚푚푒푡ℎ푎푛표푙 푚푎푥푖푚푢푚 푎푡푡푎푖푛푎푏푙푒 퐴퐴푃 푙표푎푑푖푛푔 (% 푤⁄푤) = ∗ × 100 Equation 20 [푐 – 푐 ]× 푚푚푒푡ℎ푎푛표푙+ 푚푒푥푐𝑖푝𝑖푒푛푡
where:
c = initial concentration of AAP prior to addition to the excipient (g AAP / kg MeOH)
* c = equilibrium concentration of AAP at Tcry obtained from solubility data presented in the Results section (section 3.4.1.) (g AAP / kg MeOH)
mmethanol = mass of MeOH (kg)
98 | P a g e
mexcipient = mass of excipient (g)
3.3.2.5. Treatment of the slurries generated following the crystallization of AAP from
MeOH in the presence of -Lac
Following the required contact time between the AAP-MeOH solution and the suspended particles of -Lac, agitation of the resultant slurry was stopped and the solid fraction was allowed to settle. The supernatant and solid fraction were separated by vacuum filtration
(Büchner funnel + 2.5 m cellulose filter paper), and the solid fraction was dried in an oven at
50 °C and atmospheric pressure to a constant weight (> 24 hours).
3.3.2.6. Characterisation of the supernatant and the isolated solid fractions
a. Quantification of the amount of AAP in the supernatant
The concentration of AAP in the supernatant, expressed as g AAP/kg MeOH, was
determined as described in Section 2.1. From this, the percentage desupersaturation was
calculated using Equation 21:
푐 − 푐 % 푑푒푠푢푝푒푟푠푎푡푢푟푎푡푖표푛 = 100 × [ 푠푢푝푒푟푛푎푡푎푛푡 ] Equation 21 푐− 푐∗
where:
csupernatant = concentration of AAP remaining in the supernatant (g AAP/kg MeOH) c = initial concentration of AAP (g AAP / kg MeOH)
* c = equilibrium concentration of AAP at Tcry (g AAP / kg MeOH)
Additionally, as required, the actual AAP loading (% w/w) was determined according to
Equation 3:
푎푐푡푢푎푙 퐴퐴푃 푙표푎푑푖푛푔 (% 푤⁄푤) = 푚푎푥푖푚푢푚 푎푡푡푎푖푛푎푏푙푒 퐴퐴푃 푙표푎푑푖푛푔 (%푤/푤) × % 푑푒푠푢푝푒푟푠푎푡푢푟푎푡푖표푛 100 Equation 22
99 | P a g e b. Analysis of the solid fractions
i. PXRD
PXRD diffractograms were recorded on a Phillips PANanalytical X'Pert MPD PRO
diffractometer using a Cu radiation source (=1.541 nm) at 40 mA and 40 kV. Scans
were performed in the range 5 – 40° 2 at a scan rate of 0.005° 2/min.
ii. SEM
The habit of the isolated particles was examined by SEM (JCM-5700 and JSM-
6510LV (JEOL)). Samples were gold-coated (SI50B, Edwards) and the surface
appearance of the ‘as received’ -Lac, MeOH-washed -Lac and isolated particles
compared. The mean size of the AAP particles crystallized in the presence of the
suspended -Lac were measured from the micrographs using image analysis (Adobe
Measurement Tool) with at least 20 AAP particles measured per sample.
iii. In situ SEM-Raman Spectroscopy
Micro-Raman measurements were performed on an InVIA Reflex spectrometer
(Renishaw) coupled to an optical microscope (DM2500, Leica) and an SEM (JSM-
6510LV, JEOL); the latter being referred to as the SEM-SCA (SEM-Structure &
Chemical Analyser). Instrument calibration was performed using the Si (100) peak
(520.5 ± 1 cm−1). Spectra were acquired using the 785 nm laser, variable laser power
(0.1 – 10 mW), acquisition times (10 – 500 s) and accumulations (1 – 20) over the
spectral range of interest. Spectra collection and processing were performed with the
WIRE™ 4.1 software (Renishaw).
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3.4.RESULTS
3.4.1. Solubility of AAP and α/β-Lac in MeOH and determination of the MSZW for the
crystallization of AAP from MeOH
Figure 44 shows (i) the experimentally determined and literature solubility values for AAP in MeOH over the range 5 to 35 °C, (ii) the experimentally determined solubility curve for -
Lac in MeOH over the range 5 to 35 °C, (iii) the corresponding MSZ limit determined at a cooling rate of 1 °C/min over the range 20 to 30 °C, and (iv) data points from the induction time measurements for the crystallization of four different AAP-MeOH solutions at Tcry values that spanned the MSZW giving four different supersaturations.
450
400
350
300
250
MSZ limit (Cooling rate = 1 °C/min) 200 S = 1.56; induction time = 7 min 150 S = 1.39; induction time = 33 min S = 1.25; induction time = 128 min 100 S = 1.12; induction time = 292 min
Solubility (g AAP/kg MeOH) AAP/kg (g Solubility AAP Solubility curve (experimental) 50 AAP solubility curve (literature) lactose solubility (experimental) 0 -5 0 5 10 15 20 25 30 35 40 T (°C)
Figure 44: Experimental and literature solubility values 33 of AAP and of -Lac and the MSZ limit of AAP in MeOH from 5 to 35 °C. Selected saturation data points and their corresponding induction times are also shown (number of vials = 20, volume of each vial = 20 mL). The experimentally determined solubility of AAP compared favourably with that previously reported by Granberg and Rasmuson 33. The ratio for the solubility of AAP to -
101 | P a g e
Lac at 15 °C was 629. Thus, the solubility of -Lac can be neglected compared to that of
AAP.
The MSZW for saturated solutions of AAP in MeOH within the range 20 – 30 °C was ca.
19°C. At Tsat =25 °C, this corresponded to a maximum attainable supersaturation of 1.56, above which AAP crystallizes spontaneously.
3.4.2. Determination of the induction times for the nucleation of AAP from MeOH
solutions in the absence of -Lac at different levels of supersaturation
Figure 45 presents the data obtained during the determination of the induction time for the nucleation of AAP from MeOH solutions in the absence of -Lac at different supersaturations.
No crystallization of AAP (via homogeneous nucleation) was observed in any vial up to 2 hours at a supersaturation of 1.25 (corresponding to a Tcry of 15 °C), and the first vial to show signs of crystallization was only observed after 128 minutes. At higher supersaturations, however, homogeneous nucleation occurred much sooner; for example, the first vial to show signs of crystallization at S = 1.39 (Tcry=10 °C) was observed after 33 minutes, and after just 7 minutes at S=1.56 (Tcry=5 °C). The data clearly indicate that higher supersaturations promote shorter induction times.
102 | P a g e
100
90
80
70
60 S = 1.56
50 S = 1.39
40 S = 1.25
% vials crystallized crystallized vials% S = 1.12 30
20
10
0 0 50 100 150 200 250 300 350 400 450 500 Induction time (min)
Figure 45: Induction times for the nucleation of AAP from MeOH solutions in the absence of excipients at Tsat = 25 °C and S = 1.12 (▲), 1.25 (), 1.39 () and 1.56 (); number of vials = 20.
3.4.3. Influence of contact time on the crystallization of AAP from MeOH solutions in the
presence of -Lac
From a consideration of the solubility curve and MSZ limit for AAP in MeOH, and in addition to the induction time measurements for the crystallization of AAP from supersaturated
MeOH solutions, it was decided to use S = 1.25 (corresponding to a Tcry=15 °C) to examine the influence of the contact time with the excipient on the crystallization of AAP. These conditions were selected with the aim of reducing the likelihood of homogeneous AAP nucleation (and subsequent crystal growth) from the supersaturated MeOH solutions in favor of promoting the selective proliferation of AAP crystal particles via heterogeneous nucleation onto the suspended -Lac particles. The only limitation of working at this low supersaturation is that
103 | P a g e only 17.5 % of the AAP present in solution can be recovered. However, high efficiency of the process is possible by recycling the mother liquor to use for the next crystallization.
Figure 46 presents the desupersaturation profile of supersaturated AAP-MeOH solutions
(S=1.25 at Tcry=15 °C) in the presence of -Lac. In these experiments, the quantity of -
Lac added to the supersaturated AAP-MeOH solutions was such that the maximum attainable
AAP loading was 26 % w/w. The desupersaturation profile indicates that nucleation of AAP occurred within the first 30 minutes of contact with -Lac and that close to full desupersaturation was achieved after 3 hours.
100 50
90 45
80 40
70 35 m) 60 30
50 25
40 20 % desupersaturation 30 15
20 10 MeanAAP particlesize (
10 5
0 0 0 0.5 1 1.5 2 2.5 3 Time (hours) Figure 46: Desupersaturation of AAP-MeOH supersaturated solutions in the presence of suspended -Lac particles at different contact times from 0 to 3 hours (S = 1.25, Tcry = 15 °C, maximum attainable AAP loading = 26 % w/w): influence of the contact time (hours) on the % desupersaturation (■) and the mean AAP particle size (◊).
PXRD diffractograms of the solid fractions isolated at each time interval (Figure 47) displayed diffraction peaks at 14°, 15.8° and 18.2° 2 indicating the presence of the stable monoclinic polymorph of AAP, Form I. These peaks correspond to the (0 0 1), (2 0 1̅) and
104 | P a g e
(2 1 1̅) planes of AAP Form I respectively, and the relative increase in their intensities with time shows good general correlation with the observed desupersaturation profile in Figure 46.
3 h
2 h
1 h 30 min
1 h
30 min
0 h
(1 1 0) (1 1 1)
(ii) 'as-received' -Lac
(0 0 1) (2 0 1̅) (2 1 1̅)
(i) recrystallized AAP
10 15 20 Degrees (2
Figure 47: Comparison of the PXRD diffractograms of (i) AAP recrystallized from MeOH, (ii) ‘as received’ -Lac, and the isolated solid fractions obtained at various time intervals following the addition of -Lac to a supersaturated AAP-MeOH solution (S = 1.25, Tcry = 15 °C).
The isolated solid fractions were analyzed by SEM microscopy and in-situ SEM-Raman
(Figure 48, 49, 50 and 52). The habit and particle size of the isolated solid fractions were compared with those obtained for (a) the ‘as received’ α/β-Lac particles, (b) the MeOH-washed
α/β-Lac particles, and (c) the recrystallized AAP (Figure 48). SEM micrographs of the ‘as received’ α/β-Lac revealed crystal agglomerates of 10 – 150 µm in the longest dimension.
Analysis of individual α/β-Lac particles showed that the agglomerates had numerous smaller
105 | P a g e crystals attached to their surface which varied in habit from elongated-columnar to plate-like; the particles also contained numerous voids giving rise to the extensive surface roughness typically seen for roller-dried lactose 35. Washing the ‘as received’ α/β-Lac with MeOH did not appear to alter the habit of the particles; however it did result in an increase in the population of smaller -Lac crystals (Figure 48 (a) and (b)).
SEM micrographs of the solid fractions isolated after different intervals during the crystallization of AAP from MeOH in the presence of -Lac revealed the presence of particles of a different habit on the surface of the -Lac particles (Figure 49). The micrographs further indicated that (i) these particles were predominantly found on the surface of the -Lac particles rather than existing independently even at longer contact times, and (ii) longer contact times between the -Lac and the AAP-MeOH solutions coincided with the observation of some larger particles on the surface of the -Lac. This latter observation was supported by particle size measurements from the corresponding micrographs (Figure 50). The particles were found to be more defined and more readily discernible on the micrographs of those solid fractions produced following contact times of longer than 30 minutes (Figure 50).
(a) (b) (c)
5 m 20 m 10 m
Figure 48: (a) and (b): SEM micrographs of isolated α/β-Lac washed in MeOH, and (c) SEM micrograph of recrystallized AAP.
106 | P a g e
AAP
AAP AAP 10 m (a) 30 min 10 m (b) 1 h 10 m (c) 2 h 30 min
Figure 49: SEM micrographs of α/β-Lac particles after contact times with the AAP-MeOH solution of (a) 30 min, (b) 1 h, and (c) 2h 30 min. S = 1.25, Tcry = 15 °C. Actual AAP loadings: 7.5 % w/w at 30 min, 17.2 % w/w at 1 h, and 23.8% w/w at 2h 30 min. PSD – number of particles measured per SEM micrograph = 20.
20 m 5 m 5 m
Figure 50: SEM micrographs of α/β-Lac particles following contact with a supersaturated
AAP-MeOH solution for 1.5 h. S = 1.25, Tcry = 15 °C; actual AAP loading = 19.6% w/w.
107 | P a g e
14 12 10 8 2.5 h 6 4 2
Numberof particles 0 0 10 20 30 40 50 14 12 10 8 1 h 6 4 2
Numberof particles 0 0 10 20 30 40 50 14 12 10 8 30 min 6 4 2
Numberof particles 0 0 10 20 30 40 50 Particle size (m) Figure 51: Particle size distribution (based on measurements taken from the corresponding SEM micrographs) of the AAP particles observed on the surface of -Lac after contact times of 30 minutes, 1 hour and 2.5 hours between -Lac and the AAP-MeOH solution at S = 1.25 and Tcry = 15 °C.
Analysis via SEM-Raman (Figure 52) confirmed these particles to be crystals of AAP
(monoclinic Form I). Figure 52 (a) shows illustrative SEM micrographs of an AAP–-Lac agglomerate isolated after a contact time of 2.5 hours between the supersaturated AAP-MeOH solution and the -Lac, and indicates the region from where the Raman spectra were captured. The Raman spectra and high resolution SEM micrographs of five discrete spots within this region are presented in Figure 52 (b). The spectra collected from all five spots confirm the presence of AAP as indicated by the peaks centered at 798 and 858 cm-1 Raman shift. The relative intensity of the AAP peaks compared with the -Lac peak at 878 cm-1 indicates that the concentrations of AAP at spots 1, 2 and 5 are higher than at spots 3 and 4.
Analysis of the micrographs of these regions shows numerous AAP particles in the range 1 – 5
μm.
108 | P a g e
(a)
1 2 4 3 5
(b)
-Lac
AAP
Spot 1
Spot 2
Spot 3
Spot 4
Spot 5
780 800 820 840 860 880 Raman shift (cm-1)
Figure 52 (a): SEM micrographs of an isolated -Lac particle obtained following the crystallization of AAP (contact time = 2.5 hours, Tcry = 15 °C, S = 1.25, actual AAP loading = 23.9 % w/w) indicating the position of spots 1-5 on the particle surface; (b) SEM micrographs and the corresponding Raman spectra at spots 1 – 5; scale bar = 2 μm; region from where each spectrum was collected is indicated with a box in the centre of the corresponding micrograph).
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3.4.4. Influence of supersaturation on the crystallization of AAP from MeOH solutions in
the presence of -Lac
Figure 53 shows the % desupersaturation and the mean AAP crystal particle size for the crystallization of AAP from MeOH in the presence of -Lac at six discrete AAP supersaturations in the range 1.12 – 1.64 and with an API/excipient contact time of 2 hours.
This contact time was considered suitable as it gave % desupersatutations of > 80% with good precision during the earlier ‘contact time’ experiments. The different saturation levels were achieved by performing the crystallizations at the appropriate Tcry; for example, Tcry=20 °C gave S = 1.12, Tcry=15 °C gave S=1.25, Tcry= 10 °C gave S = 1.39, and Tcry=5 °C gave S =
1.56. As before, in each case the amount of added-Lac was adjusted accordingly to keep the maximum attainable AAP loading constant at 26% w/w. A direct consequence of this was the need to add ever-increasing amounts of -Lac to those AAP-MeOH solutions with larger supersaturations, making the resultant suspensions increasingly difficult to agitate effectively.
The plot shows a % desupersaturation in excess of 65 % after 2 hours for all supersaturations examined. As the supersaturation increased from 1.12 to 1.47, the % desupersaturation increased from 67 % to a maximum of 89 %. Thereafter it decreased to 68 % as S approached
1.64.
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100 50
90 45
80 40
70 35 m)
60 30
50 25
40 20
% Desupersaturation % 30 15 MeanAAP particlesize (
20 10
10 5
0 0 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 S Figure 53: Desupersaturation of AAP-MeOH solutions and AAP mean particle sizes produced during crystallizations in the presence of suspended -Lac particles at a constant maximum attainable AAP loading of 26 % w/w and a contact time of 2 hours: influence of the supersaturation (S) on (a) the % desupersaturation (■), and (b) the mean AAP particle size (◊).
111 | P a g e
From the experience gained during the earlier ‘contact time’ experiments, it was possible to
recognize AAP particles on the -Lac surfaces via visual inspection of the SEM micrographs
for the relevant isolated solid fractions. On this basis, no pronounced difference was found in
the range of AAP crystal particle sizes produced across the series of supersaturations, as shown
in Figures 54 and 55. Furthermore, AAP particles which had crystallized independently of the
-Lac were only observed at S > 1.39.
(a) S = 1.12 (b) S = 1.47 (c) S = 1.55
AAP AAP AAP
5 m 20 m 10 m
Figure 54: SEM micrographs of α/β-Lac particles following contact with supersaturated AAP -MeOH solutions at (a) S = 1.12, (b) S = 1.47 and (c) S = 1.55 (contact time = 2 hours, maximum attainable AAP loading = 26% w/w).
40 S = 1.55 30
20
10
Numberparticles of 0 0 10 20 30 40 40
30 S = 1.47
20
10
Numberparticles of 0 0 10 20 30 40 40
30 S = 1.12
20
10
Numberparticles of 0 0 10 20 30 40 Particle size (m)
Figure 55: Particle size distribution (based on measurements taken from the corresponding SEM micrographs) of the AAP particles observed on the surface of -Lac after a contact time of 2 hours between -Lac and AAP-MeOH solutions at S = 1.12, 1.47 and 1.55.
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3.4.5. Influence of the maximum attainable AAP loading (% w/w) on the crystallization of
AAP from MeOH solutions in the presence of -Lac
When studying the influence of the maximum attainable AAP loading on the crystallization of AAP from MeOH in the presence of -Lac, a supersaturation of 1.25 and a contact time of 2 hours were chosen because (i) the supersaturation of 1.25 (for Tcry=15°C) sat approximately midway along the MSZW (Figure 44), and (ii) no homogeneous nucleation of
AAP had been observed up to 2 hours at S=1.25 during the earlier induction time measurements. Therefore, a series of experiments was carried out (S=1.25, Tcry = 15 °C, contact time = 2 hours) whereby the % desupersaturation was monitored in circumstances where the maximum attainable AAP loading was varied by adjusting the amount of -Lac added to the supersaturated AAP-MeOH solution. As such, increasing the amount of -Lac added to supersaturated MeOH solutions containing fixed amounts of AAP (at S=1.25) meant decreasing the maximum attainable AAP loading of the resultant solid fractions, and vice versa.
The amount of -Lac added was varied such that the maximum attainable AAP loading of the solid fractions at full desupersaturation ranged from ca. 8 to 84 % w/w. In this context, the amount of -Lac added to the 20 mL aliquots of supersaturated AAP-MeOH solutions varied from 0.2 g to 12 g (giving maximum attainable AAP loadings of between ca. 84 and 8% w/w respectively), although agitation of the resultant suspensions became increasingly difficult as more -Lac needed to be added. Figure 56 plots the % desupersaturation and the mean AAP crystal size obtained after a contact time of 2 hours as a function of the maximum attainable
AAP loading. For all maximum attainable AAP loadings examined, the presence of suspended
-Lac particles increased the % desupersaturation after 2 hours of contact time compared to that observed for homogeneous nucleation in the absence of the excipient, with maximum attainable AAP loadings between ca. 23 and 68 % w/w affording % desupersaturations in the
113 | P a g e range ca. 87 – 95%. Particle sizes of ca. 5 – 15 m were obtained in a range of AAP loadings from ca. 23 to 68 % w/w. However, larger AAP particle sizes were observed above 68 % w/w.
-Lac (wt %) 100 90 80 70 60 50 40 30 20 10 0 -10 100 100
80 80 m)
60 60
Zone 1 Zone 2 Zone 3 40 40 % desupersaturation %
20 20 mean AAP crystal ( size crystal AAP mean
0 0 0 10 20 30 40 50 60 70 80 90 100 110 maximum attainable AAP loading (% w/w) Figure 56: Desupersaturation of AAP-MeOH solutions in the presence of suspended -Lac particles at S = 1.25, Tcry = 15 °C and a contact time = 2 hours: influence of the maximum attainable AAP loading (% w/w) on the % desupersaturation (■), and the mean AAP particle size (◊). Again, SEM micrographs of the resultant solid fractions were visually examined to establish the presence and size of any AAP crystals formed, and also their relative proximity to the -Lac particles. As illustrated in Figures 57 and 58, this examination indicated that (i) the mean AAP crystal size increased gradually as the maximum attainable AAP loading increased, and (ii) AAP crystals were more likely to exist independently for maximum attainable AAP loadings greater than 51 % w/w.
114 | P a g e
23 % w/w 30 % w/w AAP
AAP
20 m 20 m 51 % w/w 78 % w/w
AAP AAP
50 m 20 m Figure 57: SEM micrographs of the solid fractions obtained following a contact time of 2 hours between suspended α/β-Lac particles and supersaturated AAP-MeOH solutions (S = 1.25, Tcry = 15 °C) designed to produce maximum attainable AAP loadings of 23, 30, 51 and 78 % w/w.
115 | P a g e
10 78 % 8 6 4 2 0 Number of particlesNumberof 0 5 10 15 20 25 30 35 40 45 50 55 60 8 51 % 6 4 2 0 Number of particlesNumberof 0 5 10 15 20 25 30 35 40 45 50 55 60
6 30 %
4
2
0
Number of particlesNumberof 0 5 10 15 20 25 30 35 40 45 50 55 60
23 % 2
0
Number of particlesNumberof 0 5 10 15 20 25 30 35 40 45 50 55 60 Particle size (m)
Figure 58: Particle size distribution (based on measurements taken from the corresponding SEM micrographs) of the AAP particles observed on the surface of -Lac after a contact time of 2 hours between suspended α/β-Lac particles and supersaturated AAP-MeOH solutions (S = 1.25, Tcry = 15 °C) designed to produce maximum attainable AAP loadings of 23, 30, 51 and 78 % w/w.
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3.5. DISCUSSION
In practical terms this study sought to examine the influence of varying the following three parameters on the crystallization of a model API, namely AAP, from supersaturated MeOH solutions in the presence of a model excipient which was predominately insoluble, namely -
Lac:
(i) the API/excipient contact time,
(ii) the API supersaturation, and
(iii) the maximum attainable API loading (% w/w).
(i) the influence of API/excipient contact time
The API/excipient contact time had a pronounced influence on the crystallization of AAP from a supersaturated MeOH solution in the presence of -Lac in terms of the extent of desupersaturation produced (Figure 46). As such, ca. 20% desupersaturation occurred within the first 30 minutes (at S=1.25, Tcry=15 °C) when -Lac was present. Thereafter, the % desupersaturation rose to ca. 80% after 2 hours. In contrast, during the induction time measurements performed under the same conditions of S and Tcry but in the absence of -
Lac, crystallization of AAP was not observed for up to 2 hours (Figure 45). Clearly, the presence of the suspended excipient, with its available heterosurfaces, facilitates the more rapid onset of AAP nucleation by reducing the associated free energy barrier. Almost 100% desupersaturation was achieved by 3 hours of API/excipient contact, though the rate of desupersaturation was seen to decrease considerably over time as ever-less supersaturation remained to ‘drive’ the crystallization to completion (i.e. to equilibrium saturation). PXRD diffractograms of the isolated solid fractions (Figure 47) confirmed the presence of AAP Form
I, while the prominence of the (2 0 1̅) peak suggests some element of preferred orientation.
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From an assessment of the corresponding SEM micrographs for these isolated solids fractions, the very small AAP crystal particles (ca. 2 – 3 m) initially observed on the surface of the excipient were seen to give way to more well-defined AAP particles of quite a consistent size
(ca. 10 m) at contact times of 1 hour and longer. Additionally, very few ‘independent’ AAP crystal particles were observed on the micrographs, even at longer contact times. These observations suggest a desupersaturation / crystallization process dominated by the heterogeneous nucleation of AAP onto the excipient surfaces in the early stages. Over time, the process evolved to one where growth of the formed nuclei became more prominent and mostly occurred on the surfaces of the AAP particles already attached to the excipient. That these AAP particles did not continue to grow appreciably as the contact time lengthened suggests that an ample stock of AAP nuclei was available to consume the remaining supersaturation in a uniform manner.
The effect that the excipient has on the desupersaturation and on the maximum attainable
API loading (% w/w) will be discussed at 2 hours. At this time point the degree of desupersaturation is > 80 %. The fact that at this time the desupersaturation is near completeness, but not fully consumed will help to see if changing these variables will increase or decrease the efficiency of the process to recover the maximum API available to crystallize.
Besides, it is understood that at 2 hours all the nucleation and some growth have occurred
(Figures 46 and 51), thus the API particle size has already been stabilized and at the correct point to be studied.
(ii) the influence of the API supersaturation
Desupersaturations levels of ca. 66 – 89% were observed across the range of AAP supersaturations examined (1.12 – 1.64) during the crystallization after 2 hours of AAP from supersaturated MeOH solutions in the presence of suspended -Lac particles. The
118 | P a g e desupersaturation profile (Figure 53) initially rises to a maximum of ca. 89% corresponding at to supersaturations in the range 1.4 – 1.5. Beyond this, the observed fall-off in desupersaturations to 67.6% at S=1.64 is likely due to the comparatively larger quantities of
-Lac particles (> 0.2 g/mL of MeOH) necessarily added to the supersaturated MeOH solutions at these higher supersaturations in order to maintain the constant maximum attainable
AAP loading of 26 % w/w. The resultant poorer mixing of these thicker suspensions likely caused some sedimentation of the -Lac particles, thus reducing their wettability and hindering the desired heterogeneous nucleation/growth of AAP despite the stronger ‘driving force’ provided by the higher supersaturations. Indeed, this driving force appears to have had a limited influence on the range of AAP particle sizes obtained. While acknowledging that this may be due in part to the observed poorer mixing at higher values of S, the range remained quite consistent regardless of the supersaturation used, with small particles (< 15 m) predominating at all supersaturation levels examined (Figure 55). This suggests that the available surface area of -Lac (as defined by the constant maximum attainable AAP loading of 26% w/w at each supersaturation) was sufficient to facilitate an initial surge of heterogeneous nucleation of AAP which thereafter transitioned to crystal growth in a broadly uniform manner during the remainder of the 2 hours. Similar to the earlier ‘contact time’ experiments, very few ‘independent’ AAP crystal particles were seen on the SEM micrographs of the isolated solid fractions for the series of supersaturations examined. This again supports the view that heterogeneous nucleation predominates.
(iii) the influence of varying the maximum attainable API loading (% w/w)
As shown in Figure 56, it is possible to define three general zones in relation to the variation of % desupersaturation as a function of the prevailing maximum attainable AAP loadings (% w/w) for the crystallization of AAP from supersaturated MeOH solutions (S =
119 | P a g e
1.25, Tcry = 15 °C) and constant API/excipient contact time (2 hours). Zone 1 sees a rise in the extent of desupersaturation from ca. 60 to 90% as the maximum attainable AAP loadings increases from 8 % w/w to 15% w/w and onwards to 23% w/w. In this context, the lower % desupersaturations seen at lower maximum attainable AAP loadings are likely the consequence of poor mixing within the thick suspensions caused by the larger amounts of -Lac necessarily added to generate these lower maximum attainable AAP loadings. As before, the resultant sedimentation of -Lac particles has likely reduced their ability to better contact the supersaturated AAP-MeOH solution, thus adversely impacting the desupersaturation process.
An examination of the SEM micrographs (Figure 57) of the isolated solid fractions revealed that the Zone 1 crystallization conditions generated small AAP crystals (typically < 5 μm) that resided predominantly on the excipient surface. As such, despite the issues of poor mixing and sedimentation, it would appear that sufficient nucleation sites existed on those available excipient surfaces to consume much of AAP’s supersaturation via a mechanism where heterogeneous nucleation took precedence over crystal growth.
Zone 2, which extends from a maximum attainable AAP loading of ca. 23% to ca. 68% w/w, is characterised by satisfactory mixing of the API-excipient suspensions – an attribute which likely facilitated the observed average desupersaturation of almost 91% after 2 hours.
Though AAP crystal particle sizes typically remained small within this zone (6 – 12 μm), a gradual shift towards slightly larger AAP particle sizes as the maximum loading increased was observed within Zone 2 (Figure 56). This suggests the emergence of crystal growth as a viable contender to heterogeneous nucleation as the dominant contributor to desupersaturation for
AAP at higher maximum attainable AAP loadings.
Within Zone 3, where maximum attainable AAP loadings are greater than 68% w/w, the above trend towards larger AAP crystals becomes more pronounced (Figure 56) and the related
120 | P a g e particle size distributions broaden (Figure 57). To some extent, this may be due to the relative paucity of excipient surfaces at these high maximum attainable AAP loadings, leading to a reduced number of available nucleation sites, thus encouraging more crystal growth. Zone 3 also sees a decline in the extent of desupersaturation with increasing maximum attainable AAP loadings, with desupersaturations dropping to the low/mid-70% range as loadings increase past ca. 80% w/w. Extending the API/excipient contact times beyond 2 hours might reasonably be assumed to raise these levels of desupersaturation up to those obtained in Zone 2. On this point, the coincidence of enhanced crystal growth at desupersaturations that are lower than those in
Zone 2 suggests that the rate of crystallization via crystal growth in Zone 3 may be greater than the corresponding rate in Zone 2 where heterogeneous nucleation likely plays a more prominent role.
Taken together, and while readily acknowledging the occasional challenges encountered with physical agitation of some crystallization slurries, the above study illustrates that a degree of control may be exercised over the particle size of AAP crystals produced via heterogeneous nucleation onto the surface of suspended particles of -Lac. In particular, the crystallization process showed good robustness over quite a broad intermediate range of maximum attainable
AAP loadings in terms of the desupersaturations obtained and AAP crystal particle sizes produced.
3.6. CONCLUSIONS
Heterogeneous nucleation of AAP onto -Lac produces small AAP particles (< 15 m) in a robust manner such that the PSD can be maintained constant over a wide variety of contact times, supersaturations, and AAP loadings. By varying the API supersaturation, the maximum attainable API loading and the API/excipient contact time for supersaturated solutions and suspended excipients particles, it has been shown that there are optimal ranges for
121 | P a g e supersaturation and maximum attainable API loading capable of producing consistently small
API particles at high levels of desupersaturation. This highlights the importance of tuning process parameters for heterogeneous nucleation in the presence of solid excipient carrier particles.
3.7. REFERENCES
1. Mirza, S.; Miroshnyk, I.; Heinamaki, J.; Antikainen, O.; Rantanen, J.; Vuorela, P.; Vuorela, H.; Yliruusi, J., Crystal Morphology Engineering of Pharmaceutical Solids: Tabletting Performance Enhancement. Aaps Pharmscitech 2009, 10 (1), 113-119. 2. Chen, J.; Sarma, B.; Evans, J. M. B.; Myerson, A. S., Pharmaceutical Crystallization. Crystal Growth & Design 2011, 11 (4), 887-895. 3. McLeod, J.; Paterson, A. H. J.; Jones, J. R.; Bronlund, J. E., Primary nucleation of alpha-lactose monohydrate: The effect of supersaturation and temperature. International Dairy Journal 2011, 21 (7), 455-461. 4. Quon, J. L.; Chadwick, K.; Wood, G. P. F.; Sheu, I.; Brettmann, B. K.; Myerson, A. S.; Trout, B. L., Templated Nucleation of Acetaminophen on Spherical Excipient Agglomerates. Langmuir 2013, 29 (10), 3292-3300. 5. Chadwick, K.; Myerson, A.; Trout, B., Polymorphic control by heterogeneous nucleation - A new method for selecting crystalline substrates. Crystengcomm 2011, 13 (22), 6625-6627. 6. Chadwick, K.; Chen, J.; Myerson, A. S.; Trout, B. L., Toward the Rational Design of Crystalline Surfaces for Heteroepitaxy: Role of Molecular Functionality. Crystal Growth & Design 2012, 12 (3), 1159-1166. 7. Zimmermann, A.; Millqvist-Fureby, A.; Elema, M. R.; Hansen, T.; Mullertz, A.; Hovgaard, L., Adsorption of pharmaceutical excipients onto microcrystals of siramesine hydrochloride: Effects on physicochemical properties. European Journal of Pharmaceutics and Biopharmaceutics 2009, 71 (1), 109-116. 8. Lopez-Mejias, V.; Myerson, A. S.; Trout, B. L., Geometric Design of Heterogeneous Nucleation Sites on Biocompatible Surfaces. Crystal Growth & Design 2013, 13 (8), 3835-3841. 9. Carter, P. W.; Ward, M. D., TOPOGRAPHICALLY DIRECTED NUCLEATION OF ORGANIC-CRYSTALS ON MOLECULAR SINGLE-CRYSTAL SUBSTRATES. J. Am. Chem. Soc. 1993, 115 (24), 11521-11535. 10. Chadha, R.; Bhandari, S., Drug-excipient compatibility screening-Role of thermoanalytical and spectroscopic techniques. J. Pharm. Biomed. Anal. 2014, 87, 82-97. 11. Liu, X. Y., Heterogeneous nucleation or homogeneous nucleation? The Journal of Chemical Physics 2000, 112 (22), 9949-9955. 12. Söhnel, O., Some Factors Influencing the Rate of Heterogeneous Nucleation of Strontium Sulphate. Kristall und Technik 1981, 16 (6), 651-654. 13. Callahan, C. J.; Ni, X.-W., An investigation into the effect of mixing on the secondary nucleation of sodium chlorate in a stirred tank and an oscillatory baffled crystallizer. CrystEngComm 2014, 16 (4), 690-697. 14. Myerson, A. S.; Trout, B. L.; JENSEN, K. D.; PERALA, S. R. K.; TESTA, C. J., Methods and systems for continuous heterogeneous crystallization. Google Patents: 2016. 15. Frostman, L. M.; Ward, M. D., Nucleation of Molecular Crystals beneath Guanidinium Alkanesulfonate Monolayers. Langmuir 1997, 13 (2), 330-337. 16. Hiremath, R.; Basile, J. A.; Varney, S. W.; Swift, J. A., Controlling Molecular Crystal Polymorphism with Self-Assembled Monolayer Templates. Journal of the American Chemical Society 2005, 127 (51), 18321-18327.
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17. Yang, X.; Sarma, B.; Myerson, A. S., Polymorph Control of Micro/Nano-Sized Mefenamic Acid Crystals on Patterned Self-Assembled Monolayer Islands. Crystal Growth & Design 2012, 12 (11), 5521-5528. 18. Lopez-Mejias, V.; Knight, J. L.; Brooks, C. L.; Matzger, A. J., On the Mechanism of Crystalline Polymorph Selection by Polymer Heteronuclei. Langmuir 2011, 27 (12), 7575-7579. 19. Tan, L.; Davis, R. M.; Myerson, A. S.; Trout, B. L., Control of Heterogeneous Nucleation via Rationally Designed Biocompatible Polymer Surfaces with Nanoscale Features. Crystal Growth & Design 2015, 15 (5), 2176-2186. 20. Diao, Y.; Myerson, A. S.; Hatton, T. A.; Trout, B. L., Surface Design for Controlled Crystallization: The Role of Surface Chemistry and Nanoscale Pores in Heterogeneous Nucleation. Langmuir 2011, 27 (9), 5324-5334. 21. Chayen, N. E.; Saridakis, E.; Sear, R. P., Experiment and theory for heterogeneous nucleation of protein crystals in a porous medium. Proceedings of the National Academy of Sciences of the United States of America 2006, 103 (3), 597-601. 22. MCPHERSON, A.; SHLICHTA, P., Heterogeneous and Epitaxial Nucleation of Protein Crystals on Mineral Surfaces. Science 1988, 239 (4838), 385-387. 23. Nichols, G.; Frampton, C. S., Physicochemical Characterization of the Orthorhombic Polymorph of Paracetamol Crystallized from Solution. J. Pharm. Sci. 87 (6), 684-693. 24. Discrimination of Acetaminophen Polymorphs Using Raman Chemical Imaging. ChemImage: PA, 2010; p 2. 25. Preparation technology for paracetamol. Google Patents: 2015. 26. Preparation process of paracetamol. Google Patents: 2015. 27. 对乙酰氨基酚新晶型、其制备方法及复方氨酚烷胺制剂 Acetaminophen new polymorphs, their preparation and formulation of Compound Paracetamol and Amantadine Hydrochloride. Google Patents: 2016. 28. Myerson, A. S.; Wong, S. Y., Devices and methods for crystallization. Google Patents: 2014. 29. Müller, M.; Meier, U.; Wieckhusen, D.; Beck, R.; Pfeffer-Hennig, S.; Schneeberger, R., Process Development Strategy to Ascertain Reproducible API Polymorph Manufacture. Crystal Growth & Design 2006, 6 (4), 946-954. 30. Lefebvre, J.; Willart, J. F.; Caron, V.; Lefort, R.; Affouard, F.; Danede, F., Structure determination of the 1/1 alpha/beta mixed lactose by X-ray powder diffraction. Acta Crystallographica Section B-Structural Science 2005, 61, 455-463. 31. Gordon, A. J.; Ford, R. A., The chemist s companion : a handbook of practical data, techniques, and references. Wiley: New York, 1972. 32. Pohar, A.; Likozar, B., Dissolution, Nucleation, Crystal Growth, Crystal Aggregation, and Particle Breakage of Amlodipine Salts: Modeling Crystallization Kinetics and Thermodynamic Equilibrium, Scale-up, and Optimization. Industrial & Engineering Chemistry Research 2014, 53 (26), 10762-10774. 33. Granberg, R. A.; Rasmuson, A. C., Solubility of paracetamol in pure solvents. J. Chem. Eng. Data 1999, 44 (6), 1391-1395. 34. Sarkari, M.; Brown, J.; Chen, X.; Swinnea, S.; Williams Iii, R. O.; Johnston, K. P., Enhanced drug dissolution using evaporative precipitation into aqueous solution. Int. J. Pharm. 2002, 243 (1–2), 17-31. 35. pharma, D., Lactose: Some basic properties and characteristics. [email protected], p 12.
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CHAPTER 4:
Heterogeneous Crystallization of Fenofibrate
onto Pharmaceutical Excipients
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4.1. ABSTRACT
The crystallization of fenofibrate (FF) from methanol (MeOH) was carried out in the presence of the following dispersed excipients: α/β-Lactose (α/β-Lac), D-Mannitol (D-Man), microcrystalline cellulose (MCC), carboxymethyl cellulose (CMC), silica (SiO2) and polycaprolactone (PCL). More control was achieved over the nucleation and crystal growth of the FF particles in the presence of excipients relative to its conventional crystallization using
FF seed. Each of the excipients was found to strongly reduce the FF induction time during its crystallization from supersaturated MeOH solutions relative to the rate observed in the absence of the excipients; there was a pronounced reduction in the induction time for FF from > 22 hours in the absence of excipients to ca. 15 minutes in their presence at optimum conditions.
Additionally, the FF particle size can be optimized by adjusting the FF loading (% w/w) and the crystallization temperature. The small FF particles generated via crystallization in the presence of excipients displayed an enhanced initial dissolution rate relative to that observed for FF particles generated in the presence and absence of seed. SiO2-FF composites obtained by this approach presents the best dissolution result of all the tested samples almost matching with the commercial nanomilled FF (Lipantil® Supra). Thus, the process parameters of heterogeneous crystallisation in the presence of pharmaceutical excipients can reduce induction times and control API particle size.
4.2. INTRODUCTION
It is estimated that at least 40% of active pharmaceutical ingredients (APIs) are poorly water soluble, leading to problems such as poor and highly variable bioavailability 1. Common solid formulation approaches to overcome these difficulties and improve the dissolution rates of these drugs include increasing specific surface area of API crystals 2, co-crystal formation 3, polymorphism, salt-formation, reduction in crystallinity, and the addition of excipients 2.
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Among these techniques, increasing surface area by reducing the crystal particle size and/or modifying the crystal habit are very common approaches 4-5.
There are two general approaches to particle size reduction, namely the ‘bottom-up’ or the
‘top-down’ approach. ‘Top-down’ processes involve disintegration methods, such as milling and homogenization 2 and are the most commonly used in the industrial context. However, these techniques are energetically costly 6. In the literature, there are several ‘bottom-up’ approaches to produce small crystals including: controlled crystallization and precipitation after solvent removal via evaporation or freeze-drying 7-8, the ‘hydrosol’ method 9, supercritical fluid methods 10-11, cryogenic spray processes 12, and anti-solvent precipitation . However,
‘bottom up’ approaches can produce an amorphous material or an undesired polymorphic form which can create problems for long term stability 4. In addition, they can be difficult to scale up. Thus, the ‘bottom-up’ approach has not yet been established as a successful commercial technology 13. That said, the last two decades have seen a shift from empirical formulation efforts to an engineering approach based on a better understanding of particle formation in these processes which may enable future commercial development 14.
In pharmaceutical formulation, several processing steps can be involved, such as feeding, blending, milling, wet or dry granulation, drying, tableting and coating. In an attempt to improve manufacturing methods and minimize the number of processing steps required a
‘bottom up’ approach is developed here. API particles of a poorly water-soluble drug are produced in the low micron range (< 15 μm) via the cooling heterogeneous crystallization of the API in the presence of dispersed excipient particles. If amenable to scale-up, this proposed method offers the potential to eliminate the need to (a) mill bulk APIs, and (b) blend milled
APIs with excipients.
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Figure 59: Chemical structure of FF
Table 6: Different polymorphs and characteristics of FF crystals 15-16
FI FII FIII16 Amorphous
Polymorph FI FII FIII -
Space group P1̅ P21/n P1̅ -
a (Å) 8.133 13.619 9.4803 -
b (Å) 8.239 7.554 9.7605 -
c (Å) 14.399 17.880 10.9327 -
beta 105.75 92.35 90.352 -
Z 2.0 4.0 2.0 -
Symmetry cell setting Triclinic Monoclinic Triclinic Amorphous
CCDC Ref code TADLIU01 TADLIU02 No data -
Fenofibrate (FF), an oral medication used to treat high cholesterol levels, Figure 59, was selected as the model API for this present study. It is known to have three crystalline low melting point polymorphs in addition to existing in an amorphous form (Table 6)16. Form I is the most stable polymorph, and is used as an API in tablet and capsule formulations. It has high intestinal permeability but poor solubility in water. It therefore exhibits irregular absorption in the body and is classified as a Class II drug according to the Biopharmaceutics Classification
System (BCS). Specifically, it is poorly soluble in water (<0.5 μg/mL at 25.0 °C), very soluble in methylene chloride and slightly soluble in other organic solvents 17. Other studies have previously examined different ways of improving the dissolution kinetics of FF. For example,
Tierney et al. investigated the growth of FF particles after antisolvent precipitation and freeze- drying, being able to precipitate and stabilize submicron-sized FF particles using additives 8.
Bouledjouidja et al. studied the impregnation of FF on mesoporous silica using supercritical
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18 CO2, achieving high impregnation yields with a low degree of crystallinity . Both, Tierney et al. and Bouledjouidja et al. were successful in improving FF’s dissolution rates. Dwyer et al. explored the crystallization of FF confined in nanoporous silica using different porous sizes, obtaining nanocrystalline FF with different dissolution properties depending on the silica pore size 19. In this last work, solvent evaporation was used to crystallize the FF in the silica matrices focusing on the control of the particle size and crystallinity of FF. Here an alternative approach to that of Dwyer et al.19 is presented, focusing on the crystallization of FF from solution in the presence of dispersed excipients. As such, this study expands the previous research by giving some insight into the impact of heterogeneous nucleation on the reduction of the induction time and on the control of the FF particle size.
Other studies have provided fundamental insights into heterogeneous crystallization using foreign substrates 20-25. Chadwick et al. showed that the nucleation of crystalline phases of acetaminophen (AAP) onto molecular crystal ‘hetereosurface’ substrates can be influenced by
(a) compatibility between the respective lattice parameters, and (b) the chemical and physical compatibility between the ‘hetereosurface’ and the crystallizing molecule 22. Furthermore,
Quon et al. studied the nucleation of AAP in the presence of spherical agglomerates of excipients to find that the specific availability of new crystal faces on the spherical agglomerates enhanced the nucleation of AAP in some cases 24. Alternatively, Ebrahimi et al. present a new formulation technique in which AAP is incorporated into porous lactose by spray drying. The powders prepared using this formulation technique were analysed by X-Ray diffraction (XRD) and Differential Scanning Calorimetry (DSC) to determine their crystallinity. The results showed the presence of nanocrystalline AAP inside the lactose pores with a delivery performance during dissolution studies that was comparable with that of existing formulations 26.
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However, the above studies did not examine the effect of varying key processing
parameters to minimize the particle size of an API with a view to improving the API’s
dissolution rate. In our previous study with AAP 27, processing parameters such as API
solution/excipient contact time, API to excipient loading, and supersaturation level were varied
to demonstrate how the induction time, crystallization rate and the extent of crystallization of
API can be manipulated in the presence of an excipient to give consistently small API particles.
In this present study, a different crystallization system was studied using a poorly water-soluble
API with a much longer induction time to determine whether the processing parameters for
heterogeneous API crystallization in the presence of excipient could be optimized to decrease
the induction time, increase the rate of API nucleation and control the API particle growth
process such that the API’s dissolution rate could be improved.
4.3. EXPERIMENTAL
4.3.1. MATERIALS
Table 7 summarized various attributes of the ‘as received’ materials used in this study.
Table 7: Attributes of the various ‘as-received’ materials used in this study CCDC Purity Material Abbreviation Supplier Crystallinity Reference Code Polymorph (%) Match Methanol MeOH > 99.9 Sigma-Aldrich - - - Hydrochloric acid HCl 37 Sigma-Aldrich - - - Fenofibrate FF ≥ 98 Sigma-Aldrich Crystalline TADLIU01 Form I -Lactose (≤ 30% α-anomer) -Lac ≥ 99 Sigma-Aldrich Crystalline BLACTO β Microcrystalline cellulose MCC - VWR/MERCK Amorphous - -
-D-Mannitol D-Man ≥ 98 Sigma-Aldrich Crystalline DMANTL07 Carboxymethyl cellulose, MERCK CMC - Amorphous - - Sodium salt Millipore Polycaprolactone PCL > 99.9 Sigma-Aldrich Amorphous - - Silica 2 Aeroperl (specific surface area = 294 m /g; SiO2 - Amorphous - - 300 Pharma pore size = 20 to 40 nm) Commercial Fenofibrate Tablet Commercial BGP Products 35 Nanocrystalline TADLIU01 Form I (Lipantil® Supra) FF Ltd.
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4.3.2. METHODS
4.3.2.1 Determination of the solubility of FF and the six excipients in MeOH
The solubility of FF in MeOH was measured between 10.0 and 30.0 °C, and that of each of the six excipients was measured between 5.0 and 35.0 °C. Each solubility measurement was performed in triplicate according to the following procedure. Excess solids were added to approximately 20 mL of MeOH, placed in a temperature-controlled water bath (± 0.1 °C) at the required saturation temperature (Tsat) and agitated with a magnetic stirrer at 500 rpm for 24 hours. Agitation was then stopped and the suspensions allowed to settle for more than 1 hour.
The concentration of the dissolved FF or excipient was then determined using the dry mass method 27. The amount of solute present in the supernatant, expressed in terms of concentration as g solute/kg MeOH, was then calculated according to the method of Granberg et al.28.
4.3.2.2 Determination of the induction time and the metastable zone width (MSZW) for
the crystallization FF from MeOH
Initially, stock solutions of FF in MeOH (86 g/kg MeOH), saturated at 27.0 °C, were placed in a water bath at Tsat + 3.0 °C (30.0 ± 0.1 °C) and agitated at 400 rpm with a PTFE- coated magnetic stirrer for ≥ 12 hours. 20 mL aliquots of saturated solution were then transferred to 25 mL vials, sealed with PTFE-lined lids, using pre-heated syringes and filters
(PTFE, 0.2 m). Thereafter, these filtered aliquots were used in the following induction time and MSZW experiments.
4.3.2.2.1 Induction time
Seven vials were equilibrated at 30.0 ± 0.1 °C, 200 rpm for ≥ 12 hours prior to quench-
cooling to the crystallization temperature (Tcry). Agitation was maintained via a PTFE-
coated magnetic stirrer at 200 rpm throughout the isothermal treatment. The induction time
(defined as the time when the first crystals of FF are observed to crystallize in the first vial)
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was measured using a webcam (Microsoft life cam, wide angle f/2.2, HD Lens 720 p HD,
30 FPS, Autofocus widescreen) at Tcry = 21.9 °C corresponding to a FF supersaturation
푐 level (S) of 1.5 (S = , where c = supersaturated concentration of FF in MeOH in g FF/kg 푐∗
MeOH, and c* = equilibrium concentration of FF in MeOH at Tcry in g FF/kg MeOH).
4.3.2.2.2 Metastable zone width
The metastable zone width (MSZW) was specifically determined for a FF-MeOH
solution saturated at 27.0 ˚C because most of the crystallization experiments in this study
were performed at this saturation level. As such, seven vials were equilibrated at 30.0 ± 0.1
°C and 200 rpm for at least 12 hours prior to cooling at a rate of 0.4 °C/min. Agitation was
maintained via a PTFE-coated magnetic stirrer at 200 rpm throughout the treatment. The
time and temperature at which the contents of each vial crystallized were recorded using a
webcam as in Section 2.2.1 and the average temperature for the onset of crystallization was
used to determine the MSZW.
4.3.2.3 Nucleation experiments in the presence and in the absence of ‘hetereosurfaces’
Vials containing 20 mL aliquots of the saturated FF-MeOH solutions were initially prepared at Tsat as described in Section 2.2. Following equilibration at Tsat + 5.0 °C, the vials were transferred to a water bath at Tcry. Thereafter, these cooled filtered aliquots were treated as outlined below in order to crystallize FF at different processing conditions in the absence and presence of excipients, and in the presence of FF seed.
4.3.2.3.1 In the absence of excipients
FF was crystallized from metastable MeOH solutions at S = 1.5 (21.9 ± 0.1 °C, 200
rpm). The concentration of FF in solution was measured, as explained in Section 2.1, prior
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to the visual onset of nucleation and thereafter at three time points following the
observation of nucleation.
4.3.2.3.2 In the presence FF seed
FF ‘as received’ (Form I; D50 = 360 m, span = 847 m) was used as seed, and was
added to the supersaturated solution at an amount equivalent to 1 % w/w with respect to the
amount of FF available to crystallize at S = 1.5, as defined in Equation 23. Following the
addition of seed, the solution was agitated at 200 rpm with a PTFE-coated magnetic stirrer
and held isothermally at Tcry.
풎풔풆풆풅 푭푭 풔풆풆풅 (풘⁄풘 %) = ∗ × ퟏퟎퟎ Equation 23 [[풄 – 풄 ]× 풎풎풆풕풉풂풏풐풍 ]+ 풎풔풆풆풅 where:
mseed = mass of seed (g) c = initial concentration of FF prior to addition to the excipient (g FF / kg MeOH) * c = equilibrium concentration of FF at Tcry obtained from solubility data (g FF / kg MeOH)
mmethanol = mass of MeOH (kg)
4.3.2.3.3 In the presence of excipients
The required quantity of excipient (either CMC, D-Man, -Lac, MCC, PCL or SiO2)
was added to the supersaturated FF solution. The resultant suspensions were agitated at 700
rpm with a PTFE-coated magnetic stirrer and held isothermally at Tcry. The following
factors, deemed likely to influence the crystallization of FF in the presence of the
excipients, were examined:
(a) the FF-excipient contact time,
(b) the FF supersaturation level,
(c) Tcry, and
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(d) the amount of FF available to crystallize in the presence of an excipient where all supersaturation is consumed via either heterogeneous or homogeneous nucleation, i.e. the FF loading (% w/w), as defined in Equation 24:
[풄 – 풄∗]× 풎 ( ) 풎풆풕풉풂풏풐풍 Equation 24 푭푭 풍풐풂풅풊풏품 풘/풘 % = ∗ × ퟏퟎퟎ [[풄 – 풄 ]× 풎풎풆풕풉풂풏풐풍 + 풎풆풙풄풊풑풊풆풏풕] where:
mexcipient = mass of excipient (g)
Table 8 summarizes the various parameter combinations examined during the
crystallization of FF from metastable MeOH solutions in the presence of FF seed or each of
the six excipients.
Table 8: The various combinations of Tsat, Tcry, S, and FF loading examined for the crystallization of FF from supersaturated MeOH solutions in the presence of different added ‘hetereosurfaces’
Added Supersaturation FF loading T (°C) T (°C) ‘hetereosurface’ sat cry (S) (% w/w) FF seed* 27.0 21.9 1.5 not applicable CMC 16.0 10.0 1.5 7 D-Man 27.0 21.9 1.5 12 16.0 10.0 1.5 7 -Lac 27.0 21.9 1.5 35 27.0 21.9 1.5 35 27.0 21.9 1.5 10 MCC 16.0 10.0 1.5 7 22.0 17.5 1.1 7 15.0 10.0 1.4 29 PCL 27.0 21.9 1.5 7 SiO2 27.0 21.9 1.5 14 *: added at 1 % w/w with respect to the amount of FF available to crystallize at S = 1.5
4.3.2.4 Characterization of the supernatant and the isolated FF-excipient composite solids
Following the required contact time, agitation was stopped and the suspended solids were allowed to settle. The supernatant and solid fraction were separated by vacuum filtration (using a Büchner funnel and 2.5 m cellulose filter paper), and the solid fraction was dried in an oven at 50 °C and atmospheric pressure to a constant weight (> 24 hours).
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c. Quantification of the percentage of FF desupersaturated from solution
The concentration of FF remaining in the supernatant, expressed as g FF/kg MeOH, was determined as described in Section 2.1. From this, the percentage FF desupersaturation was calculated using Equation 25:
풄−풄 푫풆풔풖풑풆풓풔풂풕풖풓풂풕풊풐풏 (%) = ퟏퟎퟎ × [ 풔풖풑풆풓풏풂풕풂풏풕 ] Equation 25 풄− 풄∗ where: csupernatant = concentration of FF remaining in the supernatant (g FF/kg MeOH) c = supersaturated concentration of FF (g FF/kg MeOH)
* c = equilibrium concentration of FF at Tcry (g FF/kg MeOH) d. Analysis of the isolated FF-excipient composite solids iv. Powder X-ray diffraction
Powder X-ray diffractograms (PXRD) were recorded on a Phillips PANanalytical
X'Pert MPD PRO diffractometer using a Cu radiation source (=1.541 퐴̇) at 40 mA and 40 kV. Scans were performed between 5 - 40° 2 at a scan rate of 2.13° 2/min. v. SEM
The habit of the isolated FF-excipient composite solids was examined by SEM (JCM-
5700 and JSM-6510LV (JEOL)). Samples were gold-coated (SI50B, Edwards) and the surface appearance of MeOH-washed excipients, recrystallized FF, FF crystallized in the presence of FF seed, and isolated FF-excipient composite solids were compared. The FF mean particle size was also measured from the micrographs using image analysis (Adobe
Measurement Tool) with 15 to 57 particles being assessed per composite depending on the number of FF particles observed per image. All SEM images shown are representative of the majority of particle sizes and habits observed per sample.
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vi. In situ SEM-Raman Spectroscopy
Micro-Raman measurements were performed on an InVIA Reflex spectrometer
(Renishaw) coupled to an optical microscope (DM2500, Leica) and an SEM (JSM-
6510LV, JEOL) (the latter being referred to as the SEM-SCA (SEM-Structure & Chemical
Analyser)). Instrument calibration was performed using the Si (100) peak (520.5 ±
1 cm−1). Spectra were acquired using the 785 nm laser, variable laser power (0.1 – 10 mW), acquisition times (10 – 500 s) and accumulations (1 – 20) over the spectral range of interest. Spectra collection and processing were performed with the WIRE™ 4.1 software
(Renishaw). vii. Solid state NMR
Carbon-13 solid-state nuclear magnetic resonance (SSNMR) spectra were acquired on a Bruker Avance III HD NMR spectrometer operating at B0 = 9.4 T, with corresponding
1 13 1 13 H and C resonance frequencies of ν0( H) = 400.1 MHz and ν0( C) = 100.6 MHz.
Fenofibrate samples were packed in 4 mm o.d. zirconia rotors with Kel-F caps under ambient atmosphere, and experimental 13C NMR spectra were acquired at natural abundance using a 4 mm triple channel (H/X/Y) Bruker MAS probe operating in double resonance mode. The magic angle was optimised using a rotor packed with KBr and spun at 5 kHz. NMR spectra were referenced to TMS at δiso = 0 ppm by setting the high frequency 13C resonance in adamantane to 38.48 ppm29. The 13C Cross Polarization
Magic Angle Spinning (CP/MAS) NMR spectra were acquired in a single spectral window using the cross-polarization pulse sequence, with a magic angle spinning (MAS) rotor frequency of 10 kHz, a 1H 90° pulse width of 2.5 μs, and 50 kHz 1H decoupling during acquisition. Proton decoupling was carried out with the SPINAL6430 decoupling sequence
1 at 100 %. For each sample the H T1 relaxation time(s) were checked using the saturation
135 | P a g e recovery pulse sequence to ensure that the recycle delay allowed for adequate relaxation between the collection of subsequent transients. 13C CP/MAS spectra were collected using optimised contact times (4 ms and 5 ms for FF and SiO2-FF composite samples, respectively) and relaxation delays (2 s and 5 s for for FF and SiO2-FF composite samples, respectively - at least 1.4 x T1 values). 15,000 scans were collected for the SiO2-FF composite sample and 124 scans for pure ‘as received’ FF (Form I). viii. Dissolution rate studies
The dissolution rates of the various isolated FF-excipient composite solids in 0.1 M HCl with 0.4% w/v Tween-80 were determined at sink conditions of approximately one fifth of the equilibrium FF solubility concentration; these were compared with the corresponding dissolution rates for a ground tablet of a commercial formulation of FF, namely Lipantil®
Supra, 145 mg tablet, BGP Products Ltd. The tablet was ground via mortar and pestle. The dissolution rate of a physical mixture of FF (D50 = 7 m, span = 26 m, produced by grinding FF via mortar and pestle) and MCC was also determined as a control sample.
A solution of 450 mL of 0.1 M HCl with 0.4% w/v Tween-80 was placed in a water bath at 42.0 °C for 24 hours and used as the dissolution medium. These conditions were used to allow for comparison of the determined dissolution rates with those previously reported 8. Following equilibration, the appropriate amount of sample containing 12.5 mg of FF was added to the dissolution medium (450 mL). 1 mL aliquots were withdrawn every 5 minutes during the first 30 minutes and then every 15 minutes thereafter. A 1 mL aliquot was also taken after 24 hours (i.e. complete dissolution) to determine the actual concentration of FF in the original sample. The concentration of dissolved FF was measured with reference to the UV absorbance for FF at λ = 289 nm (Cary 300 Bio).
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4.3.2.5 The use of optical microscopy to monitor the crystallization of FF in the presence
of macroscopic crystals of D-Man
FF was crystallized in the presence of D-Man under an optical polarizing microscope to monitor the crystallization process in real time. The crystallization setup comprised an inverted optical polarizing microscope (Olympus IX53) integrated with an optical digital camera
(Olympus SC100), a PC with image capture and image analysis software (Stream View 1.9.3.) to capture crystal images during the crystallization process. The crystallization vessel was a 0.5 mL cuvette cell (54 mm × 10 mm × 1 mm) submerged in a shallow tank of water which was temperature-controlled by a Haake F3 recirculation bath.
Macroscopic crystals (length = 0.5 – 5 mm) of D-Man were obtained by allowing an unstirred aqueous saturated solution of D-Man (Tsat = 30.0 °C) to cool slowly to room temperature and then stand at room temperature for three days. D-Man crystals were then filtered by vacuum filtration (Büchner funnel and 2.5 m cellulose filter paper), and dried in an oven at 50 °C and atmospheric pressure to a constant weight (> 24 hours).
A solution of FF in MeOH saturated at 27.0 °C was placed in the cuvette cell along with approximately ten D-Man macroscopic crystals which would result in a FF loading close to 80
% w/w. The cell was then submerged in the shallow tank at a Tcry of 21.9 °C. Images and videos were taken during the subsequent nucleation and growth of the FF crystals.
4.4.RESULTS AND DISCUSSION
4.4.1. Solubility of FF and the six excipients in MeOH and determination of the MSZW
The experimentally determined solubility of FF, Figure 60, compares favorably with that previously reported by Watterson and Rasmuson 31. The solubility of FF in MeOH at 25 °C was at least 53 times greater than that of the most soluble excipient (D-Man), Figure 60. As
137 | P a g e such, the solubilities of all six excipients in MeOH (Table 4) were deemed negligible compared with that of FF at the crystallization conditions used in this study.
The MSZW, measured at a cooling rate of 0.4 °C/min, for FF-MeOH solutions saturated at
27.0 °C was 12.5 ± 5.1 °C, Figure 60. This corresponds to a maximum attainable supersaturation of 2.22, after which FF crystallizes spontaneously. Promotion of heterogeneous nucleation and suppression of homogeneous nucleation requires that the supersaturated FF-
MeOH solutions be held within this MSZ, in other words, at a supersaturation of less than 2.22.
FF Lactose MCC D-Man CMC Silica PCL MSZ 180
160
140
120
100 S = 1.5; 21.9° C
80
S = 1.1; 17.5° C 60 Solubility (g/kg MeOH) S = 1.5; 10° C
40
20
0 5 10 15 20 25 30 T (°C)
Figure 60: (i) Experimental solubility values in MeOH for FF between 10.0 and 30.0 °C (♦) and for the excipients between 5.0 and 30.0 °C (■, ∆, ●,▲,+,-), (ii) the estimated MSZ limit (dashed red line) based on an extrapolation through the experimentally determined MSZW data point (×) for FF in MeOH for a saturation temperature (Tsat) of 27.0 °C and at cooling rate of 0.4 °C/min. The points ▲, ♦, and ■ represent the different processing conditions (in terms of S and Tcry) used during the experiments to crystallize FF from MeOH
138 | P a g e
Table 9: Solubility of the six excipients in MeOH at 25.0 °C
Excipient Solubility (g/kg MeOH) D-Man 1.42 ± 0.02 -Lac 0.68 ± 0.003 MCC 0.06 ± 0.007 CMC 0.16 ± 0.018 PCL 0.0007 ± 0.00
SiO2 0.0001 ± 0.00 4.4.2. Induction time and extent of desupersaturation of FF from MeOH solution in the
absence of excipients
During the induction time experiments (20 mL scale, S=1.5), FF was not observed to crystallize in less than 22 hours; indeed, just two out of seven vials nucleated in 40 hours. The
% desupersaturation in the first vial to nucleate was quantified following visual observation of the first nucleation event. At 15 minutes post-nucleation, 79 % desupersaturation was observed, and full desupersaturation was achieved 45 minutes later, i.e. 1 hour post-nucleation. The rate of desupersaturation was seen to decrease considerably over time as ever-less supersaturation remained to ‘drive’ the crystallization to completion (i.e. to equilibrium saturation).
4.4.3. Crystallization of FF in the presence of excipients
(a) (b)
(c) (d)
(e) (f)
Figure 61: Chemical structure of the excipients: (a) -Lac, (b) MCC, (c) D-Man, (d) CMC, (e) PCL and (f) SiO2
139 | P a g e
100
90
80
70
60
50
40 Desupersaturation (%) 30 PCL; 7 %; 21.9 °C 20
10
No FF seed; 21.9 °C 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 Time (hours)
Figure 62: Desupersaturation of FF-MeOH solutions in the absence of seed from induction time experiments (●), and in the presence of the following ‘hetereosurfaces’ during 2 hours of contact: (■) suspended FF seed (1% w/w); (♦) -Lac, (■) MCC and ( ) D-Man at S = 1.5, Tcry = 21.9 °C and 12 % w/w loading; (●) CMC at S =
1.5, 7% w/w loading, Tcry = 10.0 °C, (-) SiO2 at S = 1.5, Tcry = 21.9 °C and 14 % w/w loading, and (×) PCL at S = 1.5, Tcry = 21.9 °C and 7 % w/w loading. RTlnS, the thermodynamic driving force, was in a range of 954 to 994 J.mol-1 for all the experiments.
The addition of -Lac, D-Man, MCC, CMC or SiO2 to a supersaturated solution of
FF in MeOH reduced the induction time of FF in the 20 mL scale from greater than 22
hours (observed in the absence of additives) to less than 15 minutes (Figure 62). This
contrasts sharply with our previous observations for AAP crystallization in similar
conditions (20 ml scale, S = 1.25) on Lac. In that case the reduction in induction
times in the presence of excipients was more modest. For AAP in MeOH the induction
time of the first vial to crystallize (out of 20 vials) in the absence of excipients was 2 hours
and less than 30 minutes in the presence of -Lac.
Each molecule of FF has 3 hydrogen bond acceptors32 and no hydrogen bond donors
(Figure 59), whereas -Lac, D-Man, MCC, CMC and SiO2 each have many hydrogen
140 | P a g e bond donors and acceptors (Figure 61). By contrast the AAP molecules feature both hydrogen bond donors and acceptors. For FF the hydroxyl group hydrogens of the excipients offer an additional type of interaction capable of hydrogen bonding with the oxygens of FF’s ketone, ester and ether functional groups, arguably enhancing the prospect for FF’s nucleation onto the excipients’ surfaces from supersaturated solutions. This functional group complementarity, in particular the addition of hydrogen bonding capability, could reduce the free energy barrier to nucleation and as a consequence lower the induction time or increase the number and lifetimes of pre-critical size clusters. In addition, -Lac presents a rough surface with a high density of grooves, while SiO2
(specific surface area = 294 m2/g) is highly porous 33; both of these attributes may further promote the heterogeneous nucleation of FF. -Lac, D-Man, MCC, CMC and SiO2 thus have the capacity to promote the nucleation of FF through functional group matching and/or surface roughness. Conversely, PCL (which only possesses hydrogen bond acceptors) has no such functional group compatibility with FF and is therefore not expected to promote nucleation via this mechanism. It may still promote heterogeneous nucleation of FF via its rough surface (as seen below in the SEM image in Figure 70a.
The above observations suggest that -Lac, D-Man, MCC, CMC or SiO2 possess ample hydrogen bond donor ability to overcome the MeOH solvent molecules’ ability to solvate FF molecules (also via hydrogen bond donation). In so doing, over time these excipients are therefore able to sequester FF molecules to their surfaces as single molecules or in clusters large enough to ultimately facilitate nucleation. Literature studies have concluded there is a longer lifetime of hydrogen bonding interactions between a molecule in solution and a solid surface than between two molecules in solution 33-35.
Thus, we propose that the interaction of a cluster with a polar or hydrogen bonding group on a macromolecular solid surface should increase the average lifetime of the interaction
141 | P a g e compared to dispersive forces, allowing more stable packing of hydrogen bonded molecules and thus increasing the cluster lifetime 34-35. In water for example, the average lifetime of the interaction between liquid water molecules and solid ice molecules is some value between water molecules in an ice crystal (10-4 s) and that of water liquid molecules
(10-11 s) 36. This can be extrapolated to our system which consists of FF molecules in solution, an excipient surface, and MeOH molecules. Unlike water crystallization, FF molecules, since they do not have hydrogen bond donors, can only interact with each other via Van der Waals interactions. These interactions are weaker than hydrogen bonds, resulting in lifetimes in the order of 0.8 to 3 ps37-38. In addition, FF molecules can also interact with MeOH molecules by hydrogen bonding, resulting in lifetimes in the order of
36, 38 1 to 70 ps . Finally, the lifetime of interactions between CO2 hydrates and Silica surfaces calculated by Bai et al. 39 using molecular dynamics simulations were taken as an estimation of a hydrogen bond lifetime between an excipient surface and a FF molecule.
The lifetime of those interactions ranged between 1 to 160 ns.Thus, FF molecules in solution can strongly interact with OH groups on the excipient surface for a longer period of time than with molecules of MeOH or FF in solution. As the formation of a nucleus generally appears to take in the order of 10-6 to 10-7 s 40-42, the interaction between FF molecules in solution would not be long enough to facilitate the formation of a FF nucleus.
However, the interactions between an excipient solid surface and the FF molecules in solution could endure long enough for the formation of an FF nucleus. As a result, this apparent arrangement of molecules on the excipient surfaces may be the factor responsible for the decrease in the induction time compared to that observed in the absence of excipients. This hypothesis is depicted in Scheme 1.
142 | P a g e
Scheme 1: Schematic representation of the possible interactions during heterogeneous crystallization of FF from MeOH in the presence of Lac
The addition of FF seed produced the most dramatic reduction in induction time, with
almost complete FF desupersaturation (98.5%) being achieved after 15 minutes (Figure
62). As such, when compared with all of the other heterosurfaces examined, FF seed likely
provides the most suitable template for FF nucleation in terms of lattice matching. In the
cases of -Lac, D-Man, MCC and CMC, nucleation of FF was also observed 15 minutes
after their addition. However the extent of FF desupersaturation was lower at this time
compared with the seed: -Lac (70 %), D-Man (63 %), MCC (49 %) and CMC (28 %).
Following nucleation, the initial rate of FF desupersaturation in the presence of -Lac,
D-Man and MCC was broadly comparable with that observed in the presence of FF seed.
Thereafter, even though the corresponding rates of desupersaturation decreased somewhat,
ca. 90% FF desupersaturation was still achieved after 1 hour of total contact time with
each of these three excipients. By contrast, the crystallisation of FF in the presence of
CMC was accompanied by a comparatively lower initial rate of FF desupersaturation, and
a total contact time of 2 hours was required to achieve ca. 90% desupersaturation; this may
be a consequence of the gel that CMC forms when suspended in MeOH, which may have
143 | P a g e hindered the diffusion of FF through the solution. The desupersaturation profile for FF in the presence of SiO2 differs from those of the above four excipients in so far as it has a slightly slower nucleation rate and a lower rate of desupersaturation, and requires a slightly longer contact time to achieve ca. 90% FF desupersaturation. In terms of rationalizing these observations, the results obtained following SEM and 13C SSNMR analysis of the related composite solids (reported later in this chapter) support the view that the crystallization of FF occurred predominantly within the pores of the SiO2, which pore sizes range 20 to 40 nm. As such, the integration of FF into the SiO2 will depend on the absorption properties of the SiO2. In this regard, the mass transfer of FF will depend on the
SiO2 mass resistance, which in turn will depend on film mass transfer, porous diffusion
43 and finally fixation . Thus, the desupersaturation profile for FF in the presence of SiO2 may be explained by poor porous diffusion. Finally, the crystallization of FF in the presence of PCL was accompanied by the poorest desaturation profile of all the excipients examined over the 2 hour contact time. This is likely due to functional group mismatch in so far as PCL and FF only possess hydrogen bond acceptors; as such, PCL possesses no hydrogen bond donors to complement FF’s hydrogen bond acceptors, and viceversa.
Despite this, ca. 20 – 30% FF desupersaturation was observed after 2 hours of contact time. This may be due to the roughened surface topography that PCL exhibits, as evidenced from the results obtained following SEM analysis of the related composite solids (reported later in this paper, Figure 12a).
144 | P a g e
100
90
80
70
60
50
40 % Desupersaturation 30 MCC (7% loading, 10 °C)
20
MCC (35% loading; 21.9 °C) 10
0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Time (hours) Figure 63: Desupersaturation of FF-MeOH solutions at S = 1.5, V=20 mL in the presence of MCC during 2 hours of contact: (♦) Tcry = 10.0 °C and 7 % w/w loading, and (■) Tcry = 21.9 °C and 35 % w/w loading. RTlnS, the thermodynamic driving force, was in a range of 954 to 994 J.mol-1.
Additionally, in the specific case of MCC, no pronounced change in the rate of desupersaturation was observed when performing the crystallization at a combination of high FF loading and Tcry (35% and 21.9 °C, respectively) versus at a combination of low
FF loading and low Tcry (7% and 10 °C, respectively) (Figure 63). This may be rationalized on the basis that even though different FF loadings were used in each experiment, the processing conditions were such that both experiments were performed at the same FF supersaturation (S = 1.5). As such, the difference in crystallization temperature for the two experiments meant that the thermodymanic driving force for nucleation (defined as RTlnS) decreased only marginally from 994 J/mol (for Tcry = 21.9 °C) to 954 J/mol (for Tcry = 10
°C). Therefore, if heterogeneous nucleation behaves as expected from previous reported results 27, a degree of control may be exercised over the nucleation and growth rates during
FF crystallization and thus over the final FF particle size, without any change on the desupersaturation rates.
145 | P a g e
4.4.4. Control over FF particle growth
4.4.4.1. Examination of FF particle sizes for a range of excipients
The habit and particle size of the FF crystallized in the presence of the various
‘hetereosurfaces’ were compared with those obtained for the ‘as received’ FF seed and the
MeOH-washed excipient particles (Figure 64-70). Copious amounts of FF crystals were
formed shortly after seed addition at S=1.5 (Figure 62 and Figure 64b). The cause of the
formation of smaller and better formed particles than the original seed particles was a
consequence of secondary nucleation that was promoted by the seed at this supersaturation44.
SEM micrographs of the isolated composite solids after the FF crystallization in the presence
of -Lac, MCC, D-Man, CMC and PCL show the formation of FF particles clearly
attached to the surface of the excipient particles (Figure 65b, Figure 66b, Figure 67b, Figure
68b and Figure 70b), with few particles of FF found independently of the excipient.
However, no distinct particles of anything other than SiO2 could be detected in the presence
of this excipient, even at the highest magnification (Figure 69b); this suggests that the FF
may have crystallized inside the pores, which have a size range of 20 to 40 nm45. The
presence of FF Form I in all the composite solids at all FF loadings and Tcrys examined, was
confirmed by PXRD, except in the presence of SiO2, where no FF-specific peaks were
detected at normal running conditions.
(a) (b)
FF seed FF
Figure 64: SEM micrographs of: (a) the ‘as received’ FF seed, and (b) the FF crystals produced following crystallization in the presence of FF seed (1% w/w) , S = 1.5, Tcry = 21.9 °C, t = 15 min.
146 | P a g e
(a) (b)
Lac -
Figure 65: SEM micrographs of (a) MeOH-washed -Lac, and (b) -Lac after the crystallization of FF at S=1.5 in the presence of -Lac at 35% w/w loading, 21.9 °C, t = 2h 30. × = FF particles (a) (b)
MCC
Figure 66: SEM micrographs of (a) MeOH-washed MCC, and (b) MCC after the crystallization of FF at S=1.5 in its presence at 35% w/w loading, 21.9°C, t = 30 min. × = FF particles (a) (b)
Man - D
Figure 67: SEM micrographs of (a) MeOH-washed D-Man, and (b) D-Man after the crystallization of FF at S=1.5 in its presence at 12% w/w loading, 21.9 °C, t = 2h. × = FF particles (a) (b)
CMC
Figure 68: SEM micrographs of (a) MeOH-washed CMC, and (b) CMC after the crystallization of FF at S = 1.5 in its presence at 7% w/w loading, 21.9 °C, t = 2h 30. × = FF particles
147 | P a g e
(a) (b)
2 SiO
Figure 69: SEM micrographs of (a) MeOH-washed SiO2, (b) after the crystallization of FF at S=1.5 in its presence at 14% w/w loading, 21.9 °C, t = 1h 30 (a) (b)
PCL
Figure 70: SEM micrographs of: (a) MeOH-washed PCL, (b) after the crystallization of FF at S=1.5 in its presence at 7 % w/w loading, 21.9 °C, t = 1h 30. × = FF particles
Further characterization of the FF-SiO2 composite solids was conducted via PXRD at a longer exposure time and over a shorter range (0.093° 2/min; 13.5 – 17.5° 2), in order to improve the signal to noise ratio. The XRD pattern exhibited a peak that corresponded to FF
Form I. Solid state NMR analysis indicates that the chemical environment of the carbon atoms in the FF-SiO2 composite solid corresponds to that of FF Form I, the same as that found in pure crystalline FF (Figure 72). The absence of peak broadening supports the view that the amount of FF present as an amorphous form is below the limit of detection.
19 Similarly, in the study performed by Dwyer et al. the FF loaded onto SiO2 with the same pore size, was found to be crystalline as determined by XRD.
148 | P a g e
(1 1 -1) (1 -1 1)
(iv) Silica
(iii) Sample: 1 h 30 (FF-Silica: 14 % FF)
(ii) Physical mixture (FF-Silica: 14 % FF)
(i) FF from CCDC (TADLIU) 13.5 14.5 15.5 16.5 2 θ Figure 71: Comparison of the PXRD diffractograms of (i) FF from CCDC database (TADLIU) (ii) Physical mixture of FF and SiO2, 14 % FF loading (iii) the isolated composite solids following the addition of SiO2 to a supersaturated FF-MeOH solution after 1 h 30, 14 % FF loading (iv)‘as received’ SiO2.
(b)
(a)
Figure 72: CP/MAS 13C Solid State NMR spectra of (a) FF as received (Form I) (b) the isolated composite solids following the addition of SiO2 to a supersaturated FF-MeOH solution (FF loading (%) = 14 %) Further analysis of the SEM micrographs via particle size measurements (Figure 73) revealed the rapid growth of the FF particles over time in the presence of all the excipients,
149 | P a g e except SiO2. The growth of the FF particles occurred during the first 30 minutes of excipient contact time, coinciding with the most pronounced rates of desupersaturation (Figure 62).
Thereafter, the FF mean particle size either remained constant or decreased slightly, likely due to the shearing effect during mixing 44. The final mean particle size among the different excipients varied between 40 to 87 m. Plausible explanations for this variability in the FF particle size are the differences in the excipients surface area and in the FF loading.
MCC Lactose-Lac CMC D-Man PCL 160
140
120 m)
100 35 % loading 80 35 % loading 60
Particle size size Particle( 12% loading 7 % loading 40 7 % loading
20
0 0 0.5 1 1.5 2 2.5 Time (h)
Figure 73: Mean FF particle size at different time points from 15 minutes to 2 hours after the crystallization of FF from MeOH solutions at 21.9 °C and S = 1.5, and in the presence of the following excipients (and % FF loadings): ♦ MCC (35% w/w), ■ -Lac (35% w/w), ▲CMC (7% w/w), × D-Man (12% w/w) and ● PCL (7% w/w) The fast growth of FF on the surface of recrystallized D-Man was apparent from an in situ crystallization experiment, Video S1, Figure 74. After 5 minutes, FF nucleated on a particle of D-Man and thereafter continued growing. A clear attachment between the FF crystal and the excipient was observed. The FF loading in this particular experiment was close to 80 % w/w, due to the small quantity of D-Man particles added. As such, there was sufficient FF to nucleate and grow unattached to the D-Man particles possibly as a consequence of secondary nucleation.
150 | P a g e
(a) (b)
FF FF 1 mm 1 mm
(c) (d)
FF FF 1 mm 1 mm
Figure 74: Optical microscope images taken during the crystallization of FF from a MeOH solution in the presence of recrystallized D-Man at S=1.5, Tcry = 21.9 °C, FF loading (%) ~ 80 w/w%, Initial time = 5 minutes (first image); ∆time = 5 sec.
MCC and -Lac were selected as the model excipients for further studies as both induced a strong reduction in the induction time of FF during its crystallization from MeOH solutions.
4.4.4.2. The combined influence of FF loading and crystallization temperature on
the particle size of FF
The combined effect of FF loading and crystallization temperature had a significant influence on the FF particle size obtained during its crystallization from MeOH solutions in the presence of MCC or -Lac (Figure 75). Firstly, the combination of a low FF loading and a low crystallization temperature, i.e. 7 % FF loading and 10.0 °C, led to the formation of small particles of FF with a D50 of ca. 3 m on the surface of both MCC and -Lac
(Figure 75a, 17e). As determined by SEM-Raman, these particles occasionally presented as immature crystals that appeared to coat the excipient particles (Figure 76). This coating was
151 | P a g e only observed at this combination of processing conditions (i.e. low FF loading and low
Tcry). Performing the crystallization at a higher FF loadings of 29 % yet while maintaining a crystallization temperature of 10.0 °C (Figure 75c) generated larger, more well-formed FF particles, with a D50 of ca. 50 m. FF did not coat the MCC or the -Lac in the composite solids prepared under these conditions. When the crystallization temperature was increased to 21.9 °C while maintaining a low FF loading of 10 % (Figure 75b), well-formed FF particles (with a D50 of ca. 35 m and a span of 54 m) were again observed after the crystallization in the presence of MCC. Again, no ‘FF coating’ was observed here. Finally, when the crystallization was performed at a combination of high FF loading (35 %) and at a high crystallization temperature (21.9 °C), the FF particles became significantly bigger, with a D50 of ca. 75 m and a span of 107 m (Figure 75d). In the presence of -Lac (Figure
17f), large FF particles with a D50 and a span of 81 m and 72 m respectively were also observed at high loading (35 %) and high temperature (21.9 °C).
Figure 76 and Figure 77 compare the SEM-Raman analysis of the FF-MCC composite solid isolated using the high FF loading / high crystallization temperature combination with that from a FF-MCC composite solid generated using the combination of low FF loading and low crystallization temperature. The presence of the 1647 cm-1 peak, in the carbonyl stretching region, characteristic of FF Form I, indicates that the particles formed on the MCC particles surface are FF Form I. In addition, the nature of the coating formed around the
MCC particles observed in the SEM images of the isolated composite solids after the crystallization of FF in the presence of MCC at 7% FF loading and 10.0 °C was also determined to be Form I. No carbonyl stretching vibrations shift at 1656 cm-1, which are characteristic of amorphous FF 46, were observed.
The above observations may be explained in terms of the interplay between the nucleation and growth of FF crystals on the surface of the excipients, and on the assumption
152 | P a g e that the lower Tcry of 10 °C is still sufficient to overcome the activation energy barrier for nucleation of FF crystals. This assumption, combined with the comparable nature of the thermodynamic driving forces for FF’s nucleation at both crystallization temperatures, means that nucleation will likely occur as readily at the lower Tcry as it will at the higher Tcry.
The small FF particles and immature FF crystal ‘coatings’ observed only at low loading / low Tcry may therefore be a consequence of the slower growth rate of FF nuclei on the surface of the excipients due to (i) the slower diffusion of FF molecules to the developing
47 nuclei at the lower Tcry, since diffusion is proportional to temperature , and (ii) the relatively lower concentration, and thus reduced availability, of FF molecules at this low loading to feed this slower growth rate.
In comparison with seeded crystallization which showed the formation of large FF particles (D50 = 80 m) (Figure 64), heterogeneous crystallization can form smaller FF particles. By selecting specific process conditions, i.e. 7 % loading and 10.0 °C, a 25-fold reduction in the FF particle size in the best-case scenario has been achieved.
It can be concluded that the combined effect of FF loading and crystallization temperature can have a significant impact on the particle size of FF crystals generated during its crystallization from MeOH solutions in the presence of excipients, with the smallest FF particles forming at a combination of low loading and low crystallization temperature.
153 | P a g e
1.
(a) (b)
Low loadings
(c) (d)
High loadings
Low temperatures High temperatures
2. Low loadings and low temperatures High loadings and high temperatures (e) (f)
Figure 75: SEM micrographs after the crystallization of FF at S = 1.5. 1. in the presence of MCC at 2 h (a) % FF loading = 7%, Tcry =10.0 °C (b % FF loading = 10 %, Tcry =21.9 °C (c) % FF loading = 29 %, Tcry =10.0 °C, and (d) % FF loading = 35 %, Tcry =21.9 °C. 2. in the presence of -Lac at 2 h (e) % FF loading = 7%, Tcry =10.0 °C (f) % FF loading = 35 %, Tcry =21.9 °C (× = FF particle).
154 | P a g e
(a)
Spot 1 Spot 2 Spot 3
Spot 4 Spot 5 Spot 6
(b)
20000 Spot 6
200000 Spot 5
200000 Spot 4
200000 Spot 3
200000 Spot 2
250000 Spot 1
1200000 FF 'as received'
0 1525 1550 1575 1600 1625 1650 1675 1700 Raman shift (cm-1)
Figure 76: (a) SEM micrographs and (b) Raman spectra at different spots (× = spot) of an isolated composite solid particle obtained following the crystallization of FF from MeoH in the presence of MCC at S = 1.5, % FF loading = 7%, Tcry =10.0 °C after 30 min.
155 | P a g e
(a)
Spot 1 Spot 2
1
2
Spot 3 Spot 4
2
1
(b) 40000 Spot 4
15000 Spot 3 (2)
15000 Spot 3 (1)
15000 Spot 2 (2)
15000 Spot 2 (1)
15000 Spot 1
15000 MCC 'as received'
1250000 FF 'as received'
0 1525 1550 1575 1600 1625 1650 1675 1700 Raman shift (cm-1)
Figure 77: (a) SEM micrographs and (b) Raman spectra at different spots (× = spot) of an isolated composite solid particle obtained following the crystallization of FF from MeOH in the presence of MCC at S = 1.5, % FF loading = 35%, Tcry = 21.9 °C after 30 min
156 | P a g e
4.4.5. Dissolution rate studies
During dissolution studies of selected samples, the quantity of FF dissolved after 24 hours did not differ from the value based on the expected loading (Figure 78). The initial dissolution rate of FF after its crystallization from MeOH in the presence of MCC or D-Man at 21.9 °C and at loadings of 35 and 14 %, respectively, where FF particle sizes formed were bigger (D50
= 75 m; span = 107 m and 35 m; span = 105 m, respectively) was slow (0.24 and 0.38 mg/L.min, respectively) compared with the ground tablet of commercial FF (11.75 mg/L.min)
(Table 10). Similar initial dissolution rates were observed after the crystallization of FF by seeding and in the absence of excipients (0.23 and 0.32 mg/L.min, respectively), as the FF particle size was also big (D50 = 80 m; span = 92m and 73 m; span = 50m, respectively).
However, FF crystallized in the presence of MCC or -Lac at 7% of FF loading and at 10.0
°C, corresponding with the smallest FF particle sizes formed (D50 = 3 m; span = 23 m and 3
m; span = 5 m, respectively), present faster initial dissolution rates (0.87 and 0.92 mg/L.min, respectively). The dissolution testing of the physical mixture of milled FF (D50 = 7 m; span =
26 m) and MCC has a similar initial dissolution rate (0.72 mg/mL.min) to that of the FF after the crystallization in the presence of MCC and -Lac at 7% FF loadings and 10.0 °C, as the
FF particle size was similar. This confirms that the enhancement in the initial dissolution rate was mainly due to the reduction in the FF particle size and that the excipients did not give any additional improvement. The initial dissolution rate was found to be inversely proportional to particle size (Figure 79). The formation of smaller FF particles, with larger surface areas, explains this enhancement in the initial dissolution rate 2. However, for all composites of FF with MCC, -Lac or D-Man, the initial dissolution rate is slower than for the commercial FF.
Interestingly, a significant improvement in the dissolution rate was observed for the SiO2-FF composites, having an initial dissolution rate of 6.92 mg/mL.min with 79 % of the FF dissolved
157 | P a g e
in 10 min. This SiO2-FF composites presents the best dissolution result of all the tested samples
almost matching with the commercial nanomilled FF, which initial dissolution rate was 11.75
mg/L.min having 90.8 % of the FF dissolved in 10 min. The major advantage of mesoporous
SiO2 used as excipient for the heterogeneous crystallization of FF lies in its pore size, pore
morphology, and in the surface functional groups, which result in optimized interactions
48 between the FF and the mesoporous SiO2 surface . Furthermore, FF can be loaded into these
pores in a nanocrystalline state, increasing the drug dissolution properties dramatically.
Table 10: Summary of the composites used for the dissolution rate studies including the conditions of their crystallization and the initial dissolution rates. FF loading T t Initial dissolution rate Composite S D50 (wt-%) (°C) (min) (mg. L-1.min-1) from 0-5 min ● Ground tablet of - 34 - - - 11.75 commercial FF
■ FF - SiO2 1.2 14 21.9 1.5 - 6.92 ♦ FF - -Lac 1.5 7 10.0 15 2 0.92 ■ FF - MCC 1.5 7 10.0 30 3 0.87 × Physical mixture 1.5 35 - - 7 0.72 (milled FF-MCC) ● FF - D-Man 1.5 14 21.9 15 35 0.38 + FF recrystallized in the absence of 1.5 100 21.9 1440 73 0.32 excipients ▲ FF - MCC 1.5 35 21.9 30 75 0.24 – FF Seed 1.5 100 21.9 30 80 0.23
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100
90
80
70
60
50
% Dissolved 40
30
20
10
0 0 20 40 60 Time (min) Figure 78: The dissolution profiles of the FF present on the composites shown in Table 5. Dissolution medium: 450 mL of 0.1 M HCl-0.4% w/v Tween 80 at 42.0 °C. Sink conditions: 12.5 mg FF.
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
Initial dissolution rate(mg/L.min) 0.2
0.1
0 0 10 20 30 40 50 60 70 80 90 D (m) 50
Figure 79: Initial dissolution rate of FF, from 0 to 5 minutes at different D50. Dissolution medium: 450 mL of 0.1 M HCl-0.4% w/v Tween 80 at 42.0 °C. Sink conditions: 12.5 mg FF.
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4.5.CONCLUSIONS
It can be concluded that the incorporation of FF seed or suitable excipient surfaces with functional group complementarity with FF into a supersaturated solution forces the FF to nucleate at a more rapid rate than in their absence. This faster rate could be due to a longer lifetime of the interactions between FF molecules in solution and a solid excipient surface than among FF molecules in solution. The fastest rate of desupersaturation was found in the presence of FF seed. However, crystallization via seeding leads to the formation of large particles of FF as growth is the dominant mechanism. By contrast, when crystallized in the presence of the excipients, the combination of FF loading (%) and crystallization temperature can be adjusted to make nucleation the dominant mechanism and thus promote the generation of small FF particles. Due to their reduced particle size, these small FF particles displayed an enhanced initial dissolution rate relative to that observed for FF particles generated in the presence and absence of seed. SiO2-FF composites obtained by this approach presents the best dissolution result of all the tested samples almost matching with the commercial nanomilled
FF.
4.6.SUPPLEMENTARY INFORMATION
My Movie.mp4
Video S1: Crystallization of FF in the presence of recrystallized D-Man at S=1.5, Tcry = 21.9 °C, FF loading (%) ~ 80%, t = 5 – 5.17 min
4.7. REFERENCES
1. Merisko-Liversidge, E.; Liversidge, G. G.; Cooper, E. R., Nanosizing: a formulation approach for poorly-water-soluble compounds. European Journal of Pharmaceutical Sciences 2003, 18 (2), 113-120. 2. Khadka, P.; Ro, J.; Kim, H.; Kim, I.; Kim, J. T.; Kim, H.; Cho, J. M.; Yun, G.; Lee, J., Pharmaceutical particle technologies: An approach to improve drug solubility, dissolution and bioavailability. Asian Journal of Pharmaceutical Sciences 2014, 9 (6), 304-316.
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3. Janjikhel, R. K.; Adeyeye, C. M., Dissolution of Ibuprofen Enantiomers from Coprecipitates and Suspensions Containing Chiral Excipients. Pharmaceutical Development and Technology 1999, 4 (1), 9-17. 4. Savjani, K. T.; Gajjar, A. K.; Savjani, J. K., Drug Solubility: Importance and Enhancement Techniques. ISRN Pharmaceutics 2012. 5. Leleux, J.; Williams, R. O., Recent advancements in mechanical reduction methods: particulate systems. Drug Development and Industrial Pharmacy 2014, 40 (3), 289-300. 6. Parikh, D. M., Handbook of Pharmaceutical Granulation Technology. Taylor & Francis: 1997. 7. de Waard, H.; Hinrichs, W. L. J.; Frijlink, H. W., A novel bottom–up process to produce drug nanocrystals: Controlled crystallization during freeze-drying. Journal of Controlled Release 2008, 128 (2), 179-183. 8. Tierney, T. B.; Guo, Y.; Beloshapkin, S.; Rasmuson, Å. C.; Hudson, S. P., Investigation of the Particle Growth of Fenofibrate following Antisolvent Precipitation and Freeze–Drying. Crystal Growth & Design 2015, 15 (11), 5213-5222. 9. Junghanns, J. U. A. H.; Müller, R. H., Nanocrystal technology, drug delivery and clinical applications. International Journal of Nanomedicine 2008, 3 (3), 295-309. 10. Bleich, J.; Müller, B. W., Production of drug loaded microparticles by the use of supercritical gases with the Aerosol Solvent Extraction System (ASES) process. Journal of Microencapsulation 1996, 13 (2), 131-139. 11. Padrela, L.; Rodrigues, M. A.; Velaga, S. P.; Fernandes, A. C.; Matos, H. A.; de Azevedo, E. G., Screening for pharmaceutical cocrystals using the supercritical fluid enhanced atomization process. The Journal of Supercritical Fluids 2010, 53 (1–3), 156-164. 12. Rogers, T. L.; Johnston, K. P.; Williams, R. O., Solution-Based Particle Formation of Pharmaceutical Powders by Supercritical or Compressed Fluid Co2 and Cryogenic Spray-Freezing Technologies. Drug Development and Industrial Pharmacy 2001, 27 (10), 1003-1015. 13. Sinha, B.; Müller, R. H.; Möschwitzer, J. P., Bottom-up approaches for preparing drug nanocrystals: Formulations and factors affecting particle size. International Journal of Pharmaceutics 2013, 453 (1), 126-141. 14. Vehring, R., Pharmaceutical Particle Engineering via Spray Drying. Pharm. Res. 2008, 25 (5), 999-1022. 15. Groom, C. R.; Bruno, I. J.; Lightfoot, M. P.; Ward, S. C., The Cambridge Structural Database. Acta Crystallographica Section B 2016, 72 (2), 171-179. 16. Tipduangta, P.; Takieddin, K.; Fábián, L.; Belton, P.; Qi, S., A New Low Melting-Point Polymorph of Fenofibrate Prepared via Talc Induced Heterogeneous Nucleation. Crystal Growth & Design 2015, 15 (10), 5011-5020. 17. Granero, G. E.; Ramachandran, C.; Amidon, G. L., Dissolution and Solubility Behavior of Fenofibrate in Sodium Lauryl Sulfate Solutions. Drug Development and Industrial Pharmacy 2005, 31 (9), 917-922. 18. Bouledjouidja, A.; Masmoudi, Y.; Van Speybroeck, M.; Schueller, L.; Badens, E., Impregnation of Fenofibrate on mesoporous silica using supercritical carbon dioxide. International Journal of Pharmaceutics 2016, 499 (1–2), 1-9. 19. Dwyer, L. M.; Michaelis, V. K.; O'Mahony, M.; Griffin, R. G.; Myerson, A. S., Confined crystallization of fenofibrate in nanoporous silica. CrystEngComm 2015, 17 (41), 7922-7929. 20. Chadha, R.; Bhandari, S., Drug-excipient compatibility screening-Role of thermoanalytical and spectroscopic techniques. J. Pharm. Biomed. Anal. 2014, 87, 82-97. 21. Chadwick, K.; Myerson, A.; Trout, B., Polymorphic control by heterogeneous nucleation - A new method for selecting crystalline substrates. Crystengcomm 2011, 13 (22), 6625-6627. 22. Chadwick, K.; Chen, J.; Myerson, A. S.; Trout, B. L., Toward the Rational Design of Crystalline Surfaces for Heteroepitaxy: Role of Molecular Functionality. Crystal Growth & Design 2012, 12 (3), 1159-1166. 23. Liu, X. Y., Heterogeneous nucleation or homogeneous nucleation? The Journal of Chemical Physics 2000, 112 (22), 9949-9955. 24. Quon, J. L.; Chadwick, K.; Wood, G. P. F.; Sheu, I.; Brettmann, B. K.; Myerson, A. S.; Trout, B. L., Templated Nucleation of Acetaminophen on Spherical Excipient Agglomerates. Langmuir 2013, 29 (10), 3292-3300.
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25. Zimmermann, A.; Millqvist-Fureby, A.; Elema, M. R.; Hansen, T.; Mullertz, A.; Hovgaard, L., Adsorption of pharmaceutical excipients onto microcrystals of siramesine hydrochloride: Effects on physicochemical properties. European Journal of Pharmaceutics and Biopharmaceutics 2009, 71 (1), 109-116. 26. Ebrahimi, A.; Saffari, M.; Dehghani, F.; Langrish, T., Incorporation of acetaminophen as an active pharmaceutical ingredient into porous lactose. Int. J. Pharm. 2016, 499 (1–2), 217-227. 27. Arribas Bueno, R.; Crowley, C. M.; Hodnett, B. K.; Hudson, S.; Davern, P., Influence of Process Parameters on the Heterogeneous Nucleation of Active Pharmaceutical Ingredients onto Excipients. Organic Process Research & Development 2017. 28. Granberg, R. A.; Rasmuson, A. C., Solubility of paracetamol in pure solvents. Journal of Chemical and Engineering Data 1999, 44 (6), 1391-1395. 29. Morcombe, C. R.; Zilm, K. W., Chemical shift referencing in MAS solid state NMR. Journal of Magnetic Resonance 2003, 162 (2), 479-486. 30. Fung, B. M.; Khitrin, A. K.; Ermolaev, K., An Improved Broadband Decoupling Sequence for Liquid Crystals and Solids. Journal of Magnetic Resonance 2000, 142 (1), 97-101. 31. Watterson, S.; Hudson, S.; Svard, M.; Rasmuson, A. C., Thermodynamics of fenofibrate and solubility in pure organic solvents. Fluid Phase Equilibria 2014, 367, 143-150. 32. Southan, C.; Sharman, J. L.; Benson, H. E.; Faccenda, E.; Pawson, A. J.; Alexander, Stephen P. H.; Buneman, O. P.; Davenport, A. P.; McGrath, J. C.; Peters, J. A.; Spedding, M.; Catterall, W. A.; Fabbro, D.; Davies, J. A., The IUPHAR/BPS Guide to PHARMACOLOGY in 2016: towards curated quantitative interactions between 1300 protein targets and 6000 ligands. Nucleic Acids Research 2016, 44 (D1), D1054-D1068. 33. Danielli, J. F.; Riddiford, A. C.; Rosenberg, M. D., Recent Progress in Surface Science. Elsevier Science: 2013. 34. Sun, Q.; Harvey, J. A.; Greco, K. V.; Auerbach, S. M., Molecular Simulations of Hydrogen Bond Cluster Size and Reorientation Dynamics in Liquid and Glassy Azole Systems. The Journal of Physical Chemistry B 2016, 120 (39), 10411-10419. 35. Janick, J., Horticultural Reviews. Wiley: 2010. 36. Astley, T.; Birch, G. G.; Drew, M. G. B.; Rodger, P. M., Lifetime of a Hydrogen Bond in Aqueous Solutions of Carbohydrates. The Journal of Physical Chemistry A 1999, 103 (26), 5080- 5090. 37. Smirnov, B. M., Cluster Ions and Van Der Waals Molecules. Taylor & Francis: 1992. 38. Zhang, X.; Zhang, Q.; Zhao, D.-X., Hydrogen Bond Lifetime Definitions and the Relaxation Mechanism in Water Solutions. Acta Physico-Chimica Sinica 2011, 27 (11), 2547-2552. 39. Bai, D.; Chen, G.; Zhang, X.; Sum, A. K.; Wang, W., How Properties of Solid Surfaces Modulate the Nucleation of Gas Hydrate. Scientific Reports 2015, 5, 12747. 40. Mullin, J. W., Crystallization. Butterworth-Heinemann: 2001. 41. Tiller, W. A., The Science of Crystallization: Microscopic Interfacial Phenomena. Cambridge University Press: 1991. 42. Symposium on Industrial, C.; Institution of Chemical, E. In 14th International Symposium on Industrial Crystallization, Rugby, 1999; Institution of Chemical Engineers: Rugby. 43. Fulazzaky, M. A.; Khamidun, M. H.; Omar, R., Understanding of mass transfer resistance for the adsorption of solute onto porous material from the modified mass transfer factor models. Chem. Eng. J. 2013, 228, 1023-1029. 44. de Souza, B.; Cogoni, G.; Tyrrell, R.; Frawley, P. J., Evidence of Crystal Nuclei Breeding in Laboratory Scale Seeded Batch Isothermal Crystallization Experiments. Crystal Growth & Design 2016, 16 (6), 3443-3453. 45. AEROPERL® 300 Pharma Improving the dissolution of poorly soluble APIs. Industries, E., Ed. p 12. 46. Heinz, A.; Gordon, K. C.; McGoverin, C. M.; Rades, T.; Strachan, C. J., Understanding the solid-state forms of fenofibrate – A spectroscopic and computational study. European Journal of Pharmaceutics and Biopharmaceutics 2009, 71 (1), 100-108. 47. Genck, W. J., Temperature effects on growth and nucleation rates in mixed suspension crystallization. 1969.
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48. Maleki, A.; Kettiger, H.; Schoubben, A.; Rosenholm, J. M.; Ambrogi, V.; Hamidi, M., Mesoporous silica materials: From physico-chemical properties to enhanced dissolution of poorly water-soluble drugs. Journal of Controlled Release 2017, 262 (Supplement C), 329-347.
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CHAPTER 5:
Crystallization of Active Pharmaceutical
Ingredients in the Presence of Polymer Coupons
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5.1.ABSTRACT
In this chapter, the effect of the wettability and the surface topography on the promotion of API nucleation from solution was studied by using static polymer coupons (1 cm x 2 cm) with different wettability properties as ‘heterosurfaces’. The crystallization of two model APIs, i.e. fenofibrate (FF) and acetaminophen (AAP) from methanol (MeOH), was carried out in the presence of polycarbonate (PC), polytetrafluoroethylene (PTFE) and poly(methyl methacrylate) (PMMA). A significant reduction in the induction time compared with that for homogeneous nucleation was observed in the presence of PMMA coupons. However, PC and
PTFE coupons did not significantly reduce the induction time. These differences can be correlated with the difference in wettability of the polymers. There was evidence that the nucleation initially started on the PMMA polymer surface. The reduction in the induction time was slightly more when the surface was covered by grooves than for a flat surface. A homogeneously dispersed layer of small (<50 m) API particles on the PMMA coupons can be obtained in less than 5 minutes by using this approach. These polymer coupons containing attached API could be potentially used as medical devices with ancillary medical substances.
5.2.INTRODUCTION
The previous chapters focused on the crystallization of APIs in the presence of dispersed excipients, most of them with functional group complementarity with the API. The effect that
% API loadings, crystallization temperatures and supersaturation have on the nucleation rate and the final API particle size was studied. However, the surface properties, with the exception of the molecular functionality, of the ‘heterosurfaces’, i.e. surface topography and wettability, on the crystallization process was not taken into account.
For many years scientists have scratched surfaces to promote crystallization. Crystals are observed to nucleate along the grooves1-4, or in nanoconfinements 5-10. According to Page et al.
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11 who studied via computer simulation the heterogeneous nucleation of a crystal in a wedge- shaped groove, nucleation is faster when the wedge angle is such that a defect-free unstrained piece of the crystal fits perfectly into the wedge. Carter et al. 12 introduced the term ‘ledge- directed epitaxy’ that involves a lattice match between the substrate and growing phase along the ledge direction, and equivalent dihedral angles of the substrate ledge sites and a pair of aggregate planes whose identity is assigned on the basis of the structure of the mature crystal.
Attractive van der Waals forces at the substrate-aggregate interface will rely on the molecular corrugation of the substrate and aggregate planes contacting the substrate during nucleation.
The wettability of the surfaces was also found to be an important factor that influences heterogeneous nucleation. The rate of nucleation (JHET), as explained in Chapter 1, can be defined as follows13:
∆퐺∗ − 퐻퐸푇 J퐻퐸푇 = 휌퐼 푍 푗 e kT Equation 26 where:
the number density of sites for heterogeneous nucleation, j = the rate at which molecules attach to the nucleus causing it to grow, Z = Zeldovich factor, which is the probability that a nucleus at the top of the activation energy barrier will go on to form a crystal, ∗ ∆퐺퐻퐸푇= free energy cost of creating the critical nucleus for heterogeneous nucleation, k = Boltzmann’s constant, T = Temperature (K)
The free energy will depend not only on the supersaturation but also on the nature of the
‘heterosurface’. Assuming that heterogeneous nucleation is occurring on one single surface, such as a polymer film with a homogeneous surface, if the surface is a smooth infinite plane with a uniform surface, then this barrier would be 2, 14-16:
∗ ∗ ∆G 퐻퐸푇 = ∆G 퐻푂푀 × 푓(θ) Equation 27
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∗ where ∆G 퐻푂푀 is the free energy cost of creating the critical nucleus for homogeneous nucleation and is the angle that the interface between the nucleus and the bulk phase makes with the surface; this is called the contact angle and can be defined17 as follows:
γ −γ cos θ = solid solid−liquid Equation 28 γliquid where:
solid= solid surface energy
solid-liquid= interfacial energy
liquid= liquid surface energy
solid-liquid liquid solid Figure 80: Illustration of contact angles formed by liquid drops on a smooth homogeneous solid 17
The contact angle is determined by the interactions between the surface and the molecules in the nucleus (Figure 80). Attractions between the surface and the molecules that are stronger than those between the molecules in the nucleus will lead to a small angle as the nucleus spreads into a thin droplet to maximize its contact area with the surface. However, if the surface tends to repel the molecules then the nucleus is pushed away from the surface, resulting in a contact angle > 90°. The wettability of a surface increases as the contact angle decreases 17.
A prominent example of nucleation on surfaces, observed on a daily basis, is bubble formation in carbonated drinks at the side walls of a glass or at the surface of a straw. There are many studies in this field that are useful for understanding nucleation onto surfaces in contact with solutions 18-21. Bubbles form in scratches, that act as ‘nucleation sites’, on the surface of a container. Bubbles then grow, and detach, leaving behind a portion of its gas. The bigger the
167 | P a g e scratch or bump, the longer the bubble will be able to ‘hold on’. The rate of bubble production decreases as the level of supersaturation becomes lower. Arguably, the only reason why bubble production continues at this point is because of the existence, now, of gas filled cavities. The existence of metastable gas cavities in the walls of a container makes the nucleation energy barrier for each gas cavity very much lower than for the classical case, given that less interfacial free energy is needed for the cavity to grow to the critical size21. Another factor that was taken into account in these studies for the understanding of gas nucleation from water is the cohesive strength of water which can be significantly reduced at certain solid interfaces22.
In this work different polymer surfaces with different wettability properties were used. The surface of some of the polymers was modified by making different types of grooves on them.
The diversity of grooves along the polymer surfaces will create different contact angles with the solution, increasing in such a way the chances for heterogeneous nucleation.
Polymers are vital parts of many medical implants and prostheses as well as other medical equipment. Examples of medical devices include surgical instruments, catheters, coronary stents, pacemakers, prosthetic limbs, artificial hips/knees, surgical gloves, and bandages23.
Biocompatible polymer surfaces, i.e. PC, PTFE and PMMA were used as ‘heterosurfaces’ for this work with the aim of creating a homogeneously distributed API covering on their surfaces which could be potentially used for medical devices with ancillary medical substances, i.e. selected APIs.
5.3.MATERIALS
Methanol (MeOH, > 99.9 %), chloroform (> 99.9 %) and acetaminophen (AAP)
(paracetamol, ≥ 98 %) were supplied by Sigma-Aldrich, whereas fenofibrate (FF, ≥ 98 %) was supplied by KEMPROTED Limited. The PXRD diffractogram of the ‘as received’ AAP confirmed it to be the monoclinic Form I polymorph (CCDC HXACN01), while the
168 | P a g e corresponding diffractogram of the ‘as received’ FF matched that of the monoclinic Form I polymorph (CCDC TADLIU01). Polytetrafluoroethylene (PTFE) (thickness = 1mm), poly(methyl methacrylate) (PMMA) (thickness = 1mm) and polycarbonate (PC) (thickness =
1.5 mm) films were supplied by Gilbert Curry Industrial Plastics Co. The chemical structure of all the APIs and the polymer films is shown in Figure 81.
PMMA PTFE PC
AAP FF
Figure 81: Chemical structure of PMMA, PTFE, PC, AAP and FF
5.4. METHODS
5.4.1. Polymer coupons fabrication and surface topography modification
Coupons of the polymers were made using a Trotec speedy 400 laser cutter. The dimensions of the coupons were for PTFE and PC: 2 cm × 1 cm × 0.1 cm, and for PMMA: 2 cm × 1 cm × 0.15 cm. Grooves were made on the surface of the PTFE and PC coupons using a blade of 1 cm length and 0.3 mm thickness. For PMMA a distribution of grooves on the surface of these coupons was obtained as follows: PMMA coupons were left in MeOH at 20 °C for 24 hours and then dried at room temperature in a ventilated laboratory hood. Then approximately
5 drops of chloroform were added to their surfaces using a pipette dropper. Finally, the
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PMMA coupons were dried at room temperature in a ventilated laboratory hood until all the chloroform had evaporated.
5.4.2. Contact angle analysis
The contact angle between MeOH and the polymer coupons, i.e. PMMA, PC and PTFE was determined using a KSV Instruments Ltd., CAM 200 compact contact angle meter system, equipped with a C200-30 camera. The data obtained were analyzed using the curve fitting software in the KSV CAM Optical Contact Angle and Pendant Drop Surface Tension Software
Version 4.04. All measures were completed at room temperature with MeOH as the heavy phase (2.2 L) and air as the light phase. Measurements were repeated at least three times for each type of polymer coupon.
5.4.3. Batch crystallization experiments:
Initially, stock solutions of the corresponding API in MeOH were placed in a water bath at
Tsat + 5 °C (Tsat was in the range 23 – 30 °C) and agitated at 400 rpm with a PTFE-coated magnetic stirrer for ≥ 12 hours. Aliquots of 20 mL containing the saturated solution were then transferred to 25 mL vials using pre-heated syringes and filters (PTFE, 0.2 m), and the vials were then sealed with PTFE-lined lids. Thereafter, these filtered aliquots were used in the following nucleation experiments.
5.4.3.1. In the presence of polymer coupons
Vials containing 20 mL aliquots of the saturated API-MeOH solutions were initially
prepared at Tsat. Each pre-weighed polymer coupon was immersed in the corresponding vial
using a Nylon thread before sealing the vial with PTFE-lined lids. Following equilibration at
Tsat + 5 °C the vials were transferred to a water bath at Tcry and agitated at 500 rpm with a
PTFE-coated magnetic stirrer, allowing the API to crystallize in the presence of the polymer
coupons. At a specified contact time, polymer coupons were removed from the vials and
170 | P a g e characterised as explained in section 1.4. A schematic representation of the process is presented in Figure 82.
Figure 82: Schematic representation of the crystallisation process in the presence of a polymer coupon
5.4.3.1.1. Induction time experiments
Five vials, each containing a polymer coupon, held at identical same conditions,
were used for a set of induction time experiments. The induction time (tind) for each vial’s
contents (defined as the time when the first crystals of API are observed to crystallize
either in solution or on the coupon) was measured using a webcam (Microsoft life cam,
wide angle f/2.2, HD Lens 720 p HD, 30 FPS, Autofocus widescreen). The induction
time for supersaturated solutions in the presence of smooth or grooved PC, PTFE and
PMMA coupons was measured. Solutions of FF at S = 1.5 and Tcry = 21.9 °C, or AAP at
S = 1.25 and Tcry = 15 °C were used for these experiments.
5.4.3.1.2. Crystallization of APIs in the presence of polymer coupons
Some experiments were performed in the absence of seed, whereas in others a small
amount of API seed was added to the supersaturated solutions. Both experiments were
performed in the presence of polymer coupons. For seeded crystallization, an amount of
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seed equivalent to 1 % w/w (Equation 29) with respect to the amount of API available to
crystallize at a determined supersaturation was added to each vial once Tcry was reached.
Following the addition of seed, the solution was agitated at 500 rpm with a PTFE-coated
magnetic stirrer and held isothermally at Tcry. FF ‘as received’ (Form I, D50 = 360 m,
span = 847 m) and AAP ‘as received’ (Form I, D50 = 28 m, span = 43 m) were used
as seed for the crystallization of AAP and FF, respectively.
풎풔풆풆풅 푨푷푰 풔풆풆풅 (풘⁄풘 %) = ∗ × ퟏퟎퟎ Equation 29 [풄 – 풄 ]× 풎풎풆풕풉풂풏풐풍 + 풎풔풆풆풅
where:
mseed = mass of seed (g) c = initial concentration of API prior crystallization (g API / kg MeOH) * c = equilibrium concentration of API at Tcry obtained from solubility data (g API / kg MeOH)
mmethanol = mass of MeOH (kg)
Vials containing the supersaturated solution with or without seed and a polymer
coupon were left at Tcry for a defined contact time. Each time a vial was removed from
the water bath, the concentration of API in the supernatant was measured via the dry
mass method, described in section1.4.a, and the quantity of API on the polymer surface
was measured gravimetrically.
5.4.3.2. Crystallization of APIs in the presence of dispersed PMMA particles
PMMA was ground using a pestle and mortar until particles (D = 4.9 ± 2.6 mm) were formed. Those PMMA particles (0.4 g) were added to a supersaturated FF solution (S = 1.5, equivalent to a maximum FF loading = 71 % w/w). The resultant suspensions were agitated at 500 rpm with a PTFE-coated magnetic stirrer and held isothermally at Tcry. Following the required contact time, agitation was stopped and the suspended solids were allowed to settle.
The concentration of the FF remaining in solution was determined using the dry mass method (section 1.4). The supernatant and solid fraction were separated by vacuum filtration
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(using a Büchner funnel and 2.5 m cellulose filter paper), and the solid fraction was dried at room temperature in a ventilated laboratory hood to a constant weight (> 24 hours).
Table 11 summarizes all the experiments performed.
Table 11: The various combinations of Tsat, Tcry, S, and type of crystallization examined for the crystallization of AAP or FF from metastable MeOH solutions in the absence and presence of different polymer coupons
API Polymer Tsat Tcry Supersaturation Type of coupon (°C) (°C) (S) crystallization Grooved 30 21.9 2.0 Unseeded PMMA Grooved 30 21.9 2.0 Seeded PMMA None 30 21.9 1.5 Unseeded Grooved PTFE 27 21.9 1.5 Unseeded Grooved PC 27 21.9 1.5 Unseeded Grooved 27 21.9 1.5 Unseeded PMMA Ungrooved 27 21.9 1.5 Unseeded FF PMMA Grooved 27 21.9 1.5 Seeded PMMA Ungrooved 27 21.9 1.5 Seeded PMMA PMMA 27 21.9 1.5 Unseeded particles Grooved 23 21.9 1.2 Unseeded PMMA Grooved 23 21.9 1.2 Seeded PMMA None 25 15 1.25 Unseeded Grooved 25 15 1.25 Unseeded PMMA Ungrooved 25 15 1.25 Unseeded AAP PMMA Grooved 25 15 1.25 Seeded PMMA Ungrooved 25 15 1.25 Seeded PMMA
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5.4.4. Characterization of the supernatant and API-polymer composites
e. Quantification of the quantity of API on the surface of the polymer coupon
Following a specified contact time, polymer coupons were removed from the vials and
washed with MeOH to prevent crystallization of the API by solvent evaporation. The
solvent remaining on the coupon was left to evaporate at room temperature in a ventilated
laboratory hood until a constant mass was achieved. The mass was measured. The quantity
of API crystallized on the polymer was quantified as follows:
푨푷푰 풐풏 풕풉풆 풄풐풖풑풐풏 (품) = 푨푷푰 풄풐풖풑풐풏 풄풐풎풑풐풔풊풕풆 풘풆풊품풉풕 (품) − 풄풐풖풑풐풏 풘풆풊품풉풕 (품) Equation 30
f. Quantification of the percentage of API desupersaturated from solution
The concentration of API remaining in the supernatant was determined using the dry
mass method (as explained in Chapters 3 and 4). From this concentration, the percentage
API desupersaturation was calculated using Equation 31. With this percentage API
desupersaturation and the quantity of API on the polymer, both the percentage of API
crystallized on the coupon and in solution are calculated using Equation 32 and Equation
33, respectively:
풄−풄 % − 푫풆풔풖풑풆풓풔풂풕풖풓풂풕풊풐풏 = ퟏퟎퟎ × [ 풔풖풑풆풓풏풂풕풂풏풕 ] 풄− 풄∗ Equation 31
푨푷푰 풐풏 풕풉풆 풇풊풍풎 (품) % 풐풏 풕풉풆 풄풐풖풑풐풏 = × ퟏퟎퟎ Equation 32 (풄−풄풔풖풑풆풓풏풂풕풂풏풕)× 풎풎풆풕풉풂풏풐풍
% 풊풏 풔풐풍풖풕풊풐풏 = ퟏퟎퟎ − % 풐풏 풕풉풆 풇풊풍풎 Equation 33
where: csupernatant = concentration of API remaining in the supernatant (g API/kg MeOH) c = initial concentration of API (g API/kg MeOH) * c = equilibrium concentration of API at Tcry (g API/kg MeOH)
g. Analysis of the polymer coupons after crystallization experiments ix. SEM
The polymer coupons after the crystallization of the API were examined by SEM
(JCM-5700 and JSM-6510LV (JEOL)). Samples were gold-coated (SI50B, Edwards) and
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compared. All SEM images shown are representative of the majority of particle sizes and
habits observed on each coupon.
5.5. RESULTS
5.5.1. Contact angle analysis
The highest contact angle with MeOH was found for PTFE followed by PC and PMMA
(Table 12), thus PMMA is the polymer with the highest wettability and PTFE exhibits the lowest wettability.
Table 12: Contact angle between MeOH and PMMA, PC, and PTFE. ) Polymer Contact angle 20.3 ± 1.1 PMMA 34.6 ± 0.8 PC 38.3 ± 1.6 PTFE 5.5.2. Induction time experiments
The induction time for homogeneous nucleation of AAP, at S = 1.25 and Tcry = 15 °C, and
FF solutions, at S = 1.5 and Tcry = 21.9 °C, was > 128 min (as explained in Chapter 3) and
>1320 minutes (as explained in Chapter 4), respectively. The induction times of FF and AAP solutions, at the conditions mentioned before, in the presence of ungrooved and grooved
PMMA coupons were compared (Table 13). Figure 83 shows an example of two vials, one with an ungrooved PMMA coupon and the other with a grooved PMMA coupon, both contained a FF solution after the crystallization of FF for the same time period. The majority of the FF particles that crystallized were attached to the grooved PMMA coupon surface. The amount of FF particles found on the surface of the ungrooved PMMA coupon was lower.
In both FF and AAP systems, a reduction in the average induction time was observed when ungrooved or grooved PMMA coupons were present compared with that for homogeneous nucleation (Table 13). There was an increase in the stochastic nature of the crystallization in the presence of the ungrooved coupons compared with the grooved coupons, shown by a larger
175 | P a g e standard deviation (Table 13). In addition, ungrooved PMMA coupons were less effective in promoting the crystallization of AAP compared with that of the grooved coupons. The average induction time was twice as long for the crystallization of AAP in the presence of ungrooved
PMMA coupons compared with the value in the presence of grooved PMMA coupons. In contrast, the difference in the promotion of the crystallization in the presence of grooved compared with that of ungrooved coupons was less significant during FF crystallization. The average induction time was slightly longer for the crystallization of FF in the presence of ungrooved PMMA coupons compared with that in the presence of grooved PMMA coupons.
The greater reduction in the average induction time for AAP crystallization than for FF crystallization by a change in the surface topography of the PMMA coupons could be due to the difference in chemical functionality of both APIs. Each molecule of FF has 3 hydrogen bond acceptors and no hydrogen bond donors (Figure 81), whereas AAP has 2 hydrogen bond donors and 2 hydrogen bond acceptors25. By comparison PMMA has 2 hydrogen bond acceptors per monomer unit and no hydrogen bond donors. Thus, there is no functional complementarity between FF and PMMA, whereas AAP could hydrogen bond to PMMA.
Since FF cannot hydrogen bond the PMMA, then the grooves would merely facilitate the physical attachment of the FF molecules on the PMMA surface coupon24. However, the presence of grooves on the PMMA surface during AAP nucleation increased the number of active nucleation points that can molecularly interact via functional group complementarity with AAP, thus decreasing the induction time.
The average induction time of FF solutions in the presence of grooved PMMA, PC or
PTFE coupons at the same conditions was observed to be >100, >400, and >525 minutes, respectively; this means a reduction in the induction time of > 795 minutes compared with that of homogeneous nucleation (>1320 minutes). However, there was a stochastic element to the crystallization of FF in the presence of all the polymer coupons because for each set of
176 | P a g e experiments vials crystallized at different times. This stochasticity was more significant when
PTFE or PC coupons were present (Figure 84). In general FF particles were found both on the surface of the polymer coupons and also in suspension for each type of coupon.
Figure 85 correlates the contact angle between MeOH and polymer coupons without grooves with the average induction time for the crystallization of FF from MeOH solution in the presence of the corresponding polymer coupon. The contact angle was found to be linearly proportional to the average induction time.
Table 13: Comparison of the average induction time of FF (S=1.5 and Tcry = 21.9 °C) and AAP (S= 1.25 and Tcry = 15 °C) solutions in the absence and in the presence of grooved and ungrooved PMMA coupons. Average induction time in the Average induction time in the Induction time in the absence presence of PMMA with grooves presence of PMMA without of a polymer coupon (min) (min) grooves (min)
FF 155 ± 54 176 ± 66 > 1320
AAP 37 ± 13 75 ± 28 > 120
(a) (b)
Figure 83: Vials containing a FF solution and (a) an ungrooved, (b) a grooved PMMA coupon after the crystallization of FF for 120 min at S = 1.5 and Tcry = 21.9 °C.
177 | P a g e
grooved PMMA coupon ungrooved PMMA coupon grooved PC coupon grooved PTFE coupon Absence of polymer coupons 100
90 Grooved 80 PMMA Ungrooved 70 PMMA 60 Grooved
50 PC Grooved 40 PTFE 30 % vials crystallized None 20 10 0 0 200 400 600 800 1000 1200 1400 1600 Time (min)
Figure 84: Induction times for the nucleation of FF from MeOH solutions in the absence (●) and in the presence of grooved PMMA coupon (●), ungrooved PMMA coupon (▲), grooved PC coupon (●) and grooved PTFE coupon (●) at S = 1.5 and Tcry = 21.9 °C
45 40 35 ) ° 30 PTFE 25 PC 20 15 Contactangle ( 10 PMMA 5 0 0 200 400 600 800 1000 Average induction time (min)
Figure 85: Correlation between the contact angle of MeOH with the corresponding polymer coupon (PMMA, PC and PTFE) and the average induction time of FF from MeOH solutions at S = 1.5 and Tcry = 21.9 °C in the presence of the corresponding polymer coupon.
5.5.3. Coupon assisted crystallization in the absence of seed
There was an increase over time in the amount of FF found on the grooved PMMA coupons after nucleation of FF from MeOH solution at 21.9 °C, at S = 1.5 or S = 2.0 (Figure
86). The induction times in the presence of grooved PMMA coupons at S = 1.5 and S = 2.0
178 | P a g e were 97 and 30 minutes, respectively. However, there was some FF on the PMMA surface determined by mass before FF particles were detected by visual inspection. SEM analysis indicated the presence of FF particles on the PMMA coupons after 15 minutes (Figure 87).
This amount was larger at S = 2.0 than at S = 1.5 at each time point. The PMMA surface could retain 1.8 mg/cm2 of FF before FF started to crystallize in solution (Figure 86). Once FF started to crystallize in solution, the mass of FF on the coupon continued to increase up to 2 hours.
Figure 87 shows SEM images of the polymer coupons after the crystallization of FF and
AAP at times before the visually observed induction time in the presence of the corresponding polymer coupons. Small, well-formed FF particles were found on the surface of all the polymer coupons (Figure 87a, 7b, 7c). FF particles formed after 15 minutes at S = 1.5 (0.18 mg/cm2) were found to be associated to the PMMA grooves (Figure 87a). Poorly formed AAP particles in the early stage of crystallization were found on the PMMA coupon, which contained just 0.4 mg/cm2 of AAP after 30 minutes (Figure 87d). These AAP particles were not necessarily found on the grooves of the PMMA coupon. From these results it can be said that nucleation of both
FF and AAP occurred on the polymer surfaces. However, it took some time from when the nucleation occurred on the polymer surface until it was detected by the camera (induction time
+ limit of detection of induction time).
179 | P a g e
6 S = 1.5 ) 2 5 S = 2
4 Induction time Induction time 3 at S = 2.0 at S=1.5 of couponof area(mg/cm
2 Limit of visual detection of nucleation 2 cm
1 Massper
0 0 20 40 60 80 100 120 Time (min) Figure 86: Mass of FF per cm2 of coupon area found on grooved PMMA coupons following its crystallization from FF-MeOH metastable solutions at different time points at two different supersaturations (▲ S=2.0; ♦ S=1.5) and Tcry = 21.9 °C. Also shown are the respective induction times which relate to the first visual detection of nucleation.
(a) (b) FF
FF
(c) (d)
FF
AAP
Figure 87: SEM micrographs of grooved (a) PMMA, (b) PTFE and (c) PC after the crystallization of FF at S=1.5 and 21.9°C after 15 min, 220 min and 400 min, respectively and (d) grooved PMMA after the crystallization of AAP at S=1.25 and Tcry =15°C after 30 min.
To put into perspective the significance of API mass, the mass of API per coupon surface area required to create a 50 m homogeneous layer of API particles was calculated using
Equation 34:
180 | P a g e
Mass of API = 푇ℎ푖푐푘푛푒푠푠퐴푃퐼 × ρAPI Equation 34 Scoupon where:
푇ℎ푖푐푘푛푒푠푠퐴푃퐼= 50 m
2 Scoupon =coupon surface area= 4.9 cm
ρAPI = density of the API, which is:
3 26 ρFF =1.18 g/cm
3 26 ρ퐴퐴푃 =1.26 g/cm
For example, the mass of FF and AAP per coupon surface to create a 50 m homogeneous layer is 5.9 mg/cm2 and 6.3 mg/cm2, respectively. This mass will be taken as an approximation to the maximum quantity of API that the polymer coupon can hold before particles start to fall off. As the grooves’ size found on the PMMA surface is approximately 40 m, API particles larger than this size are more likely to get detached from the PMMA coupon.
Additionally, from a measured mass of API per cm2 of coupon surface the equivalent layer thickness can be calculated using Equation 35:
Mass of API 푇ℎ푖푐푘푛푒푠푠퐴푃퐼 = Equation 35 Scoupon×ρAPI
5.5.4. Effect of the surface topography on the attachment of FF particles during seeded
crystallization
As polymer coupons did not reduce the induction time to zero (Figure 84), API seed was added as a way to promote the API crystallization in solution and eliminate the stochastic nature of the induction time.
The crystallization of FF by seeding (1% w/w) in the presence of ungrooved PMMA was compared with that in the presence of grooved PMMA coupons. The aim of this section is to study the effect that the surface topography of the polymer coupon has on the attachment of
181 | P a g e particles. Grooved PMMA coupons were able to attach more FF than ungrooved PMMA coupons (Figure 88 and Figure 90). FF particles detached from each type of coupon during the crystallization process after 5 minutes, as they grew too big.
Mass of FF per cmcm22 (ungrooved surface) % of FF crystals found on the coupon (grooved surface) Mass of FF per cm2cm2 (grooved surface) % of FF crystals found on the coupon (smooth surface) 30 ) 2 16
25 14
12 20 10
15 8
of coupon area (mg/cm area coupon of 6 2 10 4
5 2 % of available FF on PMMA coupon PMMA on FF ofavailable%
Mass per cm per Mass 0 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 35 Time (min) Time (min)
Figure 88: Comparison of (a) the mass of FF per cm2 of coupon area, and (b) the percentage of available FF found on ungrooved and grooved PMMA coupons after nucleation of FF-MeOH metastable solutions by seeding with FF (1 % w/w) at S = 2.0 and Tcry =21.9 °C, number of experiments=1.
(a) (b)
Figure 89: SEM micrographs of (a) grooved and (b) ungrooved PMMA coupons after being in contact with MeOH for 2 hours.
182 | P a g e
(a) (b)
Figure 90: SEM micrographs of (a) grooved and (b) ungrooved PMMA after seeded crystallization of FF (1 % w/w) at S=1.5 and Tcry = 21.9 °C after 5 min.
5.5.5. Seeded crystallization of FF in the presence of grooved PMMA coupons
Nucleation experiments in the presence of grooved PMMA coupons with 1 % w/w FF seed were carried out at three different supersaturations: S = 1.2, S = 1.5 and S = 2.0 and all at Tcry =
21.9 °C. The seeding of solutions at S = 1.5 and S = 2.0 triggered rapid nucleation of FF, primarily in the solution, as opposed to on the coupon, which was followed by full desupersaturation after 30 minutes.
At low supersaturations i.e. S = 1.2 (Figure 91a), the percentage of desupersaturation was
<10 % after 30 minutes. The amount of FF found on the PMMA coupons linearly increased over time, reaching a maximum coverage of 1.9 mg/cm2. In addition, from the total quantity of
FF that crystallized, 56 % appeared attached to the PMMA coupon after 15 minutes. The coverage of the PMMA coupon at 1.9 mg/cm2 is equivalent to a notional layer thickness of 16
m. From 15 minutes onwards at S = 1.2 FF appeared as agglomerates on the PMMA surface
(Figure 92).
At a S = 1.5 (Figure 91b) the amount of FF found on the PMMA coupons increased over time reaching a maximum of 29 mg/cm2 after 30 minutes. There was also an increase in the % of FF crystallized on the coupon and thus a decrease in the % of FF that remained in solution.
This increase in the FF coverage over time means that more FF crystallized on the PMMA coupon or that it is crystallizing in solution and attaching to the PMMA coupon. After 30
183 | P a g e minutes, 29 mg/cm2 of FF was found on the PMMA coupon, formally equivalent to a layer of
246 m. From 15 minutes onwards attached FF was found mainly as large particles (>100 m)
(Figure 92).
At S = 2.0 (Figure 91c) after 10 minutes, 91 % of desupersaturation was observed, which corresponds to 16 mg/cm2 of FF. Between 3 and 10 minutes, during solution desupersaturation, large amounts of FF appeared on the PMMA coupons, ranging 9 to 25 mg/cm2, formally equivalent to a coverage with a thickness of 76 m and 212 m, respectively. A maximum in the % FF crystallized onto the PMMA coupons was observed in this range. Lots of big FF particles were seen from 5 minutes onwards, although PMMA coupons were less well-covered as the time increased (Figure 92). The amount of FF on the PMMA coupons at 15 and 30 minutes remained at ~ 8 mg/cm2. At higher seeded supersaturation levels, the percentage of FF which attached to the PMMA coupons decreased and particles of FF were more likely to be found in solution, Figure 12.
(a) S = 1.2 % of FF crystals found on the coupon % desupersaturation Mass of FF per cm22 cm % in solution 100 40
) 100 2 90 35 90 80 80 30 (mg/cm 70 70 25 60 60
50 20 50
40 40 % crystallized% 15 area coupon of 2 30 30
% Desupersaturation % 10 20 20 5 10 10 Mass percm Mass 0 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Time (min) Time (min)
184 | P a g e
(b) S = 1.5
2 % of FF crystals found on the coupon % desupersaturation Mass of FF cmcm2 % in solution 100 30 100 ) 2 90 90 25 80 80
70 70 20 60 60
50 15 50
40 40 of coupon area (mg/cm area coupon of % crystallized% 10 2 30 30 % Desupersaturation % 20 20 5 10 10
0 0 cm per Mass 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Time (min) Time (min)
(c) S = 2.0 % desupersaturation Mass of FF percm coupon2 area % of FF crystals found on the coupon % in solution 100 30 ) 2 100 90 90 25 80 80 70 20 70 60 60
50 15 50
40 (mg/cm area coupon of 40 2 10 30 crystallized% 30 % Desupersaturation % 20 20 5 10 10 Mass per cm per Mass 0 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 Time (min) Time (min) Figure 91: Left: ♦ % Desupersaturation of FF-MeOH metastable solutions after seeding with FF (1 % w/w) in the presence of a grooved PMMA coupon, and ■ mass of FF per cm2 coupon area found on grooved PMMA coupons. Right: % of FF crystals found on the coupon and in solution. Experiments done at (a) S = 1.2, (b) S = 1.5 or (c) S = 2.0 and Tcry =21.9 °C, at different time points from 0 to 30 min, number of experiments=1.
185 | P a g e
5 min 15 min 30 min
S = 1.2
S = 1.5
S = 2.0
Figure 92: SEM micrographs of grooved PMMA after the crystallization of FF for 5 min, 15 min and 30 min at S = 1.2, S=1.5 and S = 2.0 and Tcry = 21.9 °C.
With the aim of forming a homogeneously dispersed coverage of small FF particles on the
PMMA coupon surface, crystallization in the presence of grooved PMMA coupons was
examined at S = 2.0 and 1% of FF seed for shorter times, 1-4 minutes, i.e. before big particles
are formed. There was a general increase in the mass of FF attached to the PMMA coupons
over time (Figure 93). The coverage increased from 2 mg/cm2 (after 1 minute) to 11 mg/cm2
(after 4 minutes), equivalent to a notional layer of thickness of 17 and 93 m, respectively.
However, there was a stochastic element in the way the particles attached to the PMMA
coupon, which became more pronounced from 1 minute onwards. A homogeneous coverage of
FF on the PMMA coupons was observed after 1 minute and 2 minutes (Figure 94). At 1 minute
these particles were small (30 m) with no big deviations in the particle size. At 2 minutes
186 | P a g e higher amounts of FF were found. However, at 3 minutes FF particles became bigger (>200
m), with a wide particle size distribution.
18
16 ) 2 14
12
10
8 of couponof area(mg/cm 2 6
4 Massper cm 2
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Time (min)
Figure 93: Average mass of FF per cm2 of coupon area found on grooved PMMA coupons over time after nucleation of FF-MeOH metastable solutions by seeding with FF (1 % w/w) at S = 2.0 and Tcry =21.9 °C, number of experiments = 3
187 | P a g e
1 min 2 min 3 min
Experiment 1
Experiment 2
Experiment 3
Figure 94: SEM micrographs of 3 different grooved PMMA coupons for after crystallization of FF in the presence of 1 % w/w FF seed at S=2.0 and Tcry = 21.9 °C for 1 min, 2 min or 3 min. Seeded crystallization of AAP in the presence of grooved PMMA was also carried out.
AAP-MeOH metastable solutions (S = 1.25; Tcry = 15 °C) in the presence of grooved PMMA
coupons were seeded with AAP (1 % w/w). AAP crystallization took longer to complete
compared with that of the FF system (Figure 95), with 81% desupersaturation observed after 30
minutes versus 100 % desupersaturation for FF at S = 1.5 and S = 2.0 after the same time.
There was a linear increase in the amount of AAP on the PMMA coupon from 2 to 10 minutes,
corresponding to maximum on the % of AAP crystallized on its surface. The coverage
increased from 1 mg/cm2 (after 2 minutes) to 5.5 mg/cm2 (after 10 minutes), equivalent to a
notional layer of 7.9 and 43.7 m, respectively. From 10 minutes to 15 minutes the mass of
AAP found on the coupon decreased to 1.1 mg/cm2. This behaviour can be associated with the
188 | P a g e formation of AAP agglomerates that became too large and thus detached, Figure 96. At 5 minutes a homogeneously dispersed coverage of AAP particles between 20 and 100 m was found on the PMMA surface (Figure 96). However, from 10 minutes these particles became bigger and formed agglomerates.
% of FF crystals found on the coupon % desupersaturation Mass of AAP per coupon surface
) % in solution
100 2 100 90 6 90 80 5 80 70 70 4 60 60
50 50 3 40 40 of coupon area (mg/cm area coupon of 2 % crystallized% 30 2 30
% Desupersaturation % 20 20 1 10 10
0 0 0 cm per Mass 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Time (min) Time (min)
Figure 95: Left: ♦ % Desupersaturation of AAP-MeOH metastable solutions after seeding (1 % w/w) in the presence of grooved PMMA coupon, and ■ mass 27 of AAP per cm2 of coupon area found on grooved PMMA coupons. Right: % of AAP crystallized on the coupon, and % of AAP crystallized in solution (S = 1.25 and Tcry =15 °C).
5 min 10 min 30 min
Figure 96: SEM micrographs of grooved PMMA for after crystallization of AAP at S = 1.25 and Tcry = 15 °C for 5 min, 10 min and 30 min (left to right).
5.5.6. Unseeded API Crystallization in the presence of dispersed PMMA particles
During the previous experiments, the PMMA ‘heterosurface’ remained stationary while the supersaturated API solution was agitated around it. Therefore, in order to examine the extent to which the dispersion of the ‘heterosurface’ might influence the API’s crystallization, particles of PMMA with a total surface area similar to that of a PMMA coupon were suspended with
189 | P a g e agitation in the supersaturated API solution. As such, the calculated total available surface area for these suspended PMMA particles (assuming spherical particles) was 5.5 cm2, whereas that for the PMMA coupons was 4.9 cm2. The % desupersaturation during the crystallization of FF from FF-MeOH metastable solutions (S = 1.5; Tcry = 21.9 °C) in the presence of these PMMA particles was measured as shown in Figure 97. 20 % desupersaturation was observed after 30 minutes and 100 % was reached after 60 minutes in the presence of the PMMA particles. The crystallization of FF in the presence of PMMA particles stirred at 500 rpm was faster than that in the presence of static PMMA coupons, which took more than 100 minutes for the solution to start desupersaturating in the absence of FF seed (Figure 84 and Figure 86). SEM micrographs of PMMA particles after the crystallization of FF showed FF particles (20 to 70 m) strongly attached to the PMMA surface (Figure 98).
PMMA particles grooved PMMA coupon
100
90
80
70
60
50
40
% desupersaturation 30
20
10
0 0 10 20 30 40 50 60 70
time (min) Figure 97: Desupersaturation of FF-MeOH metastable solutions in the absence of FF seed in the presence of: ♦ PMMA dispersed particles at different time points, and ■ a static grooved PMMA coupon at S = 1.5, Tcry = 21.9 °C, 500 rpm and 71 w/w % for 15 min, 30 min or 60 min.
190 | P a g e
FF
Figure 98: SEM micrographs of PMMA particles after the crystallization of FF at S = 1.5, Tcry = 21.9 °C, 500 rpm and 71 w/w % FF loading after 60 min.
5.6. DISCUSSION
This work can be divided into two main approaches: (a) grooved coupon-assisted API crystallization in the absence of seed, and (b) seeded crystallization of APIs in the presence of grooved polymer coupons, as shown in Figure 99. The first approach established whether API crystallization began on the polymer coupon or in the solution. The second approach estimated
(i) the extent of attachment of API particles to the polymer coupons after their crystallization in the bulk solution, and (ii) how these API particles grew over time.
Figure 99: Schematic representation of the two crystallization approaches performed in this chapter: (a) coupon-assisted crystallization in the absence of API seed, and (b) seeded crystallization of an API in the presence of grooved polymer coupons
191 | P a g e
5.6.1. Does nucleation start on the polymer coupons or in solution?
Evidence from the SEMs of PMMA coupons after coupon-assisted crystallization of FF in
the absence of any seed at S = 1.5 and S = 2.0 indicates the presence of FF particles on the
PMMA surface, before any FF particles were detected by visual inspection in suspension
(Figure 86). FF particles were found after 15 minutes at Tcry for both supersaturations and the
amount of FF attached increased over time (Figure 86). SEM evidence also indicated that
some FF particles were present on the PTFE and PC coupon surfaces before nucleation in
solution was visually detected (Figure 87b and 87c, respectively). Similar behaviour was
observed for the crystallization of AAP at S = 1.25 where poorly-formed AAP particles in the
early stages of nucleation were found on the PMMA surface before crystallization was
visually detected in solution (Figure 87d). From these observations it can be said that
nucleation of FF and AAP occurred on the polymer surfaces. However, it took more time
after this nucleation had occurred on the polymer surface for further nucleation events to
occur in the bulk solution. This can be explained as follows: the API molecules likely became
‘trapped’ where they crystallized on the polymer surface, due to physical interactions between
the particles and coupon surface24. Thereafter, the API particles remained attached to the
polymer coupon and grew by diffusion of the API molecules in solution to the polymer
surface (due to a concentration gradient) where they crystallized. While this crystallization
continued on the polymer surface, the supersaturation and the driving force in the bulk
solution decreased. No nucleation occurred in the bulk solution until the particles on the
polymer surface grew larger, detached and were suspended where they then acted as seed for
the promotion of the crystallization from the solution 28. Subsequent API particles that
crystallized in solution then attached to the polymer surface as they grew forming
agglomerates over time. Thereafter, these API agglomerates readily detached from the
polymer surface (Figure 99a).
192 | P a g e
Additionally, during seeded crystallization it was observed how API particles
crystallized in the bulk solution, shown by an increase in the % desupersaturation, and then
attached to the polymer coupons (Figure 91 and Figure 96). Over time these API particles
located on the polymer surface grew and formed agglomerates. Thereafter, these API
agglomerates readily detached from the polymer surface (Figure 99b).
It can be concluded that API crystallized (i) directly onto the coupons during unseeded
crystallization and (ii) in solution during seeded crystallization followed by the attachment
of the FF particles to the polymer coupons.
5.6.2. Effect of the polymer coupon’s wettability on the induction time
An explanation for why PMMA lowered the induction time to a greater extent than PC and
PTFE may be due to the wettability of the polymer surfaces 29. The contact angle of PMMA
was significantly lower than that of PC and PTFE. Thus, PMMA’s surface presents better
wettability than the surfaces of either PC or PTFE (Table 12). Additionally, PTFE possesses a
low surface energy and a low friction coefficient, having in most cases very low adhesiveness
for different materials 30. The results support this explanation insofar as they show a direct
correlation between the induction time for the crystallization of FF in the presence of polymer
coupons and the increasing contact angle between MeOH and the polymer coupon (Figure
85). Due to the significant reduction in the induction time in the presence of PMMA coupons,
this polymer was selected for further studies.
5.6.3. Effect of polymer coupon’s surface topography on the crystallization of APIs
To study the effect that grooves have on the reduction of the induction time, the induction
time of FF (S = 1.5) and AAP solutions (S = 1.25) was measured in the presence of
ungrooved and grooved PMMA coupons (Table 13). In both cases the reduction in the
induction time as well as the stochastic nature of the FF crystallization was slightly higher
193 | P a g e
when the PMMA surface was covered with grooves (Figure 84). This result confirms that
grooves helped to promote nucleation. This illustrates the influence of the confinement in the
promotion of the nucleation, which facilitates nuclei formation by providing interaction sites,
as well as angular matching between the surface and the crystallizing molecules in solution5,
12, 31. A significant reduction in the induction time was also seen in the presence of the
ungrooved PMMA coupons compared with that in the absence of any polymer coupon; this
may be due to the adhesive properties of the PMMA and to some imperfections on the
PMMA polymer surface (Figure 89) 32. Additionally, the quantity of FF particles, formed in
bulk solution after seeded crystallization (Figure 99b), that were able to remain attached to
the PMMA surface was lower when the surface did not contain grooves, with FF particles
detaching over time (Figure 88). Thus, it can be said that the attachment of FF particles to a
grooved PMMA surface (after their crystallization in solution) was stronger than that on an
ungrooved surface.
5.6.4. Effect of agitation and polymer coupon’s total available surface area on the
induction time
The crystallization of FF from metastable MeOH solutions (S = 1.5) in the presence of
stirred PMMA particles was performed to study the importance of agitation and the total
available polymer surface area for the promotion of the nucleation. The total available surface
area for the PMMA particles, when assumed to be spherical particles, was 5.5 cm2; this was
slightly higher than that for the polymer coupons, which was 4.9 cm2. In addition, the
roughened surface topography of the PMMA particles, as evidenced from the results obtained
following SEM analysis of the related composite solids (Figure 98), can also be an attribute
for the promotion of the nucleation. The solution started to desupersaturate after 15 minutes
after which there was an increase in the % of desupersaturation, which reached 100 % after
60 minutes (Figure 97). SEM images showed well-formed FF particles which were well-
194 | P a g e
attached to the PMMA particles (Figure 98). Unlike the crystallization in the presence of
PMMA coupons, the PMMA particles are moving in solution creating a good mixing between
the ‘heterosurface’ and the solution, thus improving mass transfer. Once the crystallization
occurred on the PMMA surface, particles were likely to become detached due to a shearing
effect, thus promoting nucleation in solution. It can therefore be can be concluded that the
agitation is an important parameter that helps in the promotion of nucleation, as well as the
previously mentioned surface topography.
5.6.5. How can the quantity of API crystallized on the polymer coupon be controlled?
The optimisation/tuning of supersaturation, contact time and polymer chemistry can
potentially create a homogeneous layer of API particles on the polymer coupon surface, after
their seeded crystallization in solution (Figure 99b).
The PMMA coupons were covered faster with API particles at higher supersaturations.
However, it was not possible to control the size or the extent of these particles by changing
the supersaturation. As such, big particles were seen at all the measured supersaturations, at
contact times of 15 minutes for FF crystallization and 30 minutes for AAP crystallization.
The only way to reduce the API particle size and produce a uniform API layer on the PMMA
surface was to reduce the contact time further. Furthermore, at longer times PMMA coupons
were more likely to become overloaded with API particles which readily detached which only
served to increase the stochastic nature of the crystallization process. Contact times of 2
minutes for FF (Figure 14) and 5 minutes for AAP (Figure 16) were found to be optimal for
achieving a homogeneous coverage of small API particles on the surface of the PMMA
coupons.
195 | P a g e
5.7. CONCLUSIONS
Contact times of less than 2 minute for FF and 5 min for AAP between a polymer surface and a seeded API metastable solution are required to create a homogeneously dispersed coverage of small API particles on the grooved polymer surface by cooling heterogeneous crystallisation. The crystallization of APIs from unseeded metastable solutions initially starts on the polymer surface and continues in solution when the API particles detach from the coupons at high loadings. High wettability of the polymer coupon by the solvent promotes a reduction in the induction time. The agitation of polymers in their dispersed particulate form is also an important artefact for promoting a reduction in the induction time. Grooves on polymer surfaces help to promote both the nucleation, to a small extent, and the attachment of APIs on the surface (after seeded crystallization in the bulk solution).
5.8. REFERENCES:
1. Sholl, C. A.; Fletcher, N. H., Decoration criteria for surface steps. Acta Metallurgica 1970, 18 (10), 1083-1086. 2. Turnbull, D., Kinetics of Heterogeneous Nucleation. The Journal of Chemical Physics 1950, 18 (2), 198-203. 3. Gutzow, I. S.; Schmelzer, J., The Vitreous State: Thermodynamics, Structure, Rheology, and Crystallization. Springer Berlin Heidelberg: 2013. 4. Dean, J. R., Practical Skills in Chemistry. Prentice Hall: 2002. 5. Lopez-Mejias, V.; Myerson, A. S.; Trout, B. L., Geometric Design of Heterogeneous Nucleation Sites on Biocompatible Surfaces. Crystal Growth & Design 2013, 13 (8), 3835-3841. 6. Dwyer, L. M.; Michaelis, V. K.; O'Mahony, M.; Griffin, R. G.; Myerson, A. S., Confined crystallization of fenofibrate in nanoporous silica. CrystEngComm 2015, 17 (41), 7922-7929. 7. Diao, Y.; Myerson, A. S.; Hatton, T. A.; Trout, B. L., Surface Design for Controlled Crystallization: The Role of Surface Chemistry and Nanoscale Pores in Heterogeneous Nucleation. Langmuir 2011, 27 (9), 5324-5334. 8. Kim, K.-J.; Mersmann, A., Estimation of metastable zone width in different nucleation processes. Chemical Engineering Science 2001, 56 (7), 2315-2324. 9. Tan, L.; Davis, R. M.; Myerson, A. S.; Trout, B. L., Control of Heterogeneous Nucleation via Rationally Designed Biocompatible Polymer Surfaces with Nanoscale Features. Crystal Growth & Design 2015, 15 (5), 2176-2186. 10. Keel, T. R.; Thompson, C.; Davies, M. C.; Tendler, U. B.; Roberts, C. J., AFM studies of the crystallization and habit modification of an excipient material, adipic acid. International Journal of Pharmaceutics 2004, 280 (1-2), 185-198. 11. Page, A. J.; Sear, R. P., Crystallization Controlled by the Geometry of a Surface. Journal of the American Chemical Society 2009, 131 (48), 17550-17551. 12. Carter, P. W.; Ward, M. D., Topographically directed nucleation of organic crystals on molecular single-crystal substrates. Journal of the American Chemical Society 1993, 115 (24), 11521- 11535.
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13. Sear, R. P., Nucleation: theory and applications to protein solutions and colloidal suspensions. Journal of Physics: Condensed Matter 2007, 19 (3), 033101. 14. Gorti, S.; Forsythe, E. L.; Pusey, M. L., Growth Modes and Energetics of (101) Face Lysozyme Crystal Growth. Crystal Growth & Design 2005, 5 (2), 473-482. 15. Oxtoby, D. W., Nucleation of First-Order Phase Transitions. Accounts of Chemical Research 1998, 31 (2), 91-97. 16. Manuel Garcıá -Ruiz, J., Nucleation of protein crystals. Journal of Structural Biology 2003, 142 (1), 22-31. 17. Yuan, Y.; Lee, T. R., Contact Angle and Wetting Properties. In Surface Science Techniques, Bracco, G.; Holst, B., Eds. Springer Berlin Heidelberg: Berlin, Heidelberg, 2013; pp 3-34. 18. Discontinuous Bubble Nucleation Due to a Metastable Condensation Transition in Polymer– CO2 Mixtures. The Journal of Physical Chemistry Letters 2013, 1639. 19. Covering Surface Nanobubbles with a NaCl Nanoblanket. Langmuir 2013, 130827132419002. 20. Chappell, M. A.; Payne, S. J., The effect of cavity geometry on the nucleation of bubbles from cavities. The Journal of the Acoustical Society of America 2007, 121 (2), 853-862. 21. Jones, S.; Evans, G.; Galvin, K., Bubble nucleation from gas cavities—a review. Advances in colloid and interface science 1999, 80 (1), 27-50. 22. Ryan, W. L.; Hemmingsen, E. A., Bubble Formation in Water at Smooth Hydrophobic Surfaces. Journal of Colloid and Interface Science 1993, 157 (2), 312-317. 23. Sastri, V. R., Plastics in Medical Devices: Properties, Requirements, and Applications. Elsevier Science: 2013. 24. Tiwari, A.; Garipcan, B.; Uzun, L., Advanced Surfaces for Stem Cell Research. Wiley: 2016. 25. Southan, C.; Sharman, J. L.; Benson, H. E.; Faccenda, E.; Pawson, A. J.; Alexander, Stephen P. H.; Buneman, O. P.; Davenport, A. P.; McGrath, J. C.; Peters, J. A.; Spedding, M.; Catterall, W. A.; Fabbro, D.; Davies, J. A., The IUPHAR/BPS Guide to Pharmacology in 2016: towards curated quantitative interactions between 1300 protein targets and 6000 ligands. Nucleic Acids Res. 2016, 44 (D1), D1054-D1068. 26. https://www.scbt.com/ (accessed 31/10/2017). 27. Allentoft, M. E.; Sikora, M.; Sjogren, K.-G.; Rasmussen, S.; Rasmussen, M.; Stenderup, J.; Damgaard, P. B.; Schroeder, H.; Ahlstrom, T.; Vinner, L.; Malaspinas, A.-S.; Margaryan, A.; Higham, T.; Chivall, D.; Lynnerup, N.; Harvig, L.; Baron, J.; Casa, P. D.; Dabrowski, P.; Duffy, P. R.; Ebel, A. V.; Epimakhov, A.; Frei, K.; Furmanek, M.; Gralak, T.; Gromov, A.; Gronkiewicz, S.; Grupe, G.; Hajdu, T.; Jarysz, R.; Khartanovich, V.; Khokhlov, A.; Kiss, V.; Kolar, J.; Kriiska, A.; Lasak, I.; Longhi, C.; McGlynn, G.; Merkevicius, A.; Merkyte, I.; Metspalu, M.; Mkrtchyan, R.; Moiseyev, V.; Paja, L.; Palfi, G.; Pokutta, D.; Pospieszny, L.; Price, T. D.; Saag, L.; Sablin, M.; Shishlina, N.; Smrcka, V.; Soenov, V. I.; Szeverenyi, V.; Toth, G.; Trifanova, S. V.; Varul, L.; Vicze, M.; Yepiskoposyan, L.; Zhitenev, V.; Orlando, L.; Sicheritz-Ponten, T.; Brunak, S.; Nielsen, R.; Kristiansen, K.; Willerslev, E., Population genomics of Bronze Age Eurasia. Nature 2015, 522 (7555), 167-172. 28. Fulazzaky, M. A.; Khamidun, M. H.; Omar, R., Understanding of mass transfer resistance for the adsorption of solute onto porous material from the modified mass transfer factor models. Chem. Eng. J. 2013, 228, 1023-1029. 29. Chevalier, N. R., Do Surface Wetting Properties Affect Calcium Carbonate Heterogeneous Nucleation and Adhesion? The Journal of Physical Chemistry C 2014, 118 (31), 17600-17607. 30. Horváth, B.; Kawakita, J.; Chikyow, T., Adhesion of silver/polypyrrole nanocomposite coating to a fluoropolymer substrate. Applied Surface Science 2016, 384, 492-496. 31. Lopez-Mejias, V.; Knight, J. L.; Brooks, C. L.; Matzger, A. J., On the Mechanism of Crystalline Polymorph Selection by Polymer Heteronuclei. Langmuir 2011, 27 (12), 7575-7579. 32. A.Lazauskas, A. G., J.Puišo, I. Prosyčevas, S. Ponelytė Morphology and Surface Adhesive Properties of PMMA Films on Pet, Glass and PMMA Substrates. In BALTTRIB' 2011, Lithuania, 2011.
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CHAPTER 6:
Comparative study of Acetaminophen and
Fenofibrate Crystallization
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6.1. ABSTRACT
This chapter provides an overview of the mechanism of heterogeneous crystallization bringing together previous results and additional data to elucidate a better understanding of crystallization process. The roles of chemical functionality, molecular volume and solubility during nucleation of two different active pharmaceutical ingredients (APIs) from methanol
(MeOH) in the absence and in the presence of excipients were studied. Acetaminophen (AAP), a small molecule which has hydrogen bond donors and acceptors and high molar solubility in
MeOH, and fenofibrate (FF), a bigger molecule which only has hydrogen bond acceptors and low molar solubility in MeOH, were used as APIs. Crystallization was tracked on 500 mL scale using FBRM and FTIR probes. Microcrystalline cellulose (MCC) and -Lactose (-Lac), both with a high number of hydrogen bond donors, were used as excipients. MCC and -Lac were found to strongly enhance FF’s nucleation rate to 5.5 nuclei/(m3.s) for each excipient; the rate observed in the absence of excipients was 0.11 nuclei/(m3.s). However, MCC and -Lac do not have as strong an effect during AAP nucleation, where the nucleation rate increased from 0.37 nuclei/(m3.s), in the absence of excipients, to 1.1 and 0.55 nuclei/(m3.s) in the presence of -Lac and MCC, respectively. An explanation in terms of intermolecular functional group complementarity, molecular volume and solubility is given, which may be helpful for the selection of APIs and excipients in the design of future heterocrystallization processes.
6.2. INTRODUCTION
The final dosage form for most drug products consists of a homogeneous mixture of the
API and excipient(s). The downstream process of the drug product manufacturing starts at the crystallization step of the API to purify and isolate it. The isolated solvent-wet crystals are then dried. The intermediate steps include milling, mixing and blending with required excipients, granulation, drying, sieving, and tablet pressing 1-5. These steps consume energy and many
199 | P a g e risks and challenges regarding the quality and uniformity of the product are associated with them 6-7. For every tablet the precise final dosage, content uniformity, composition, mechanical properties, and critical quality attributes are important 2, 8-10.
This work involves the direct crystallization of an API onto a crystalline excipient surface. Nucleation, the first step of crystallization, is an activated process leading to the assembly of molecules held together by intermolecular forces such as van der Waals and hydrogen bonds11. The addition of API crystal seed or ‘heterosurfaces’ such as excipients to supersaturated solutions potentially impacts the rate of nucleation, thus nucleation becomes energetically favourable, due to a decrease in the surface energy. In 1997 Espitalier et al. 12 measured the rate of nucleation of ketoprofen in pure acetone finding a decrease in the surface energy due to a change from homogeneous (at high supersaturations) to heterogeneous (low supersaturations) nucleation. The interfacial energy for homogeneous nucleation was found to be 2-fold larger than that for heterogeneous nucleation in this particular system giving an idea of the order of the reduction in the interfacial energy by the presence of foreign particles.
Most of the previously reported heterogeneous crystallisation studies of organic molecules have been conducted on a small-scale which is well-controlled 13-18. One recent investigation by Yazdanpanah et al.19 studied the dynamic conditions of continuous heterogeneous crystallization in large-scale using three different process designs. The excipient selection and process design parameters were found to have a significant impact on drug loading, avoidance of bulk nucleation and crystallization, control of API crystal shape and size, and process control.
This chapter focuses on batch crystallization of APIs onto excipients, on a larger scale than in previous chapters. It has been shown that compounds with higher solubility have shorter induction times than those with lower solubility as the solute-solvent interfacial energy is generally lower for compounds with higher solubility 20-21. In addition, the interfacial energy
200 | P a g e can be estimated from induction time experiments where the elapsed time between the creation of supersaturation and the formation of a new phase is determined 22-23. Many studies have been done on the effect that different kinetics parameters have on the nucleation rate for homogeneous nucleation20, 24-26. However, there are no studies on the effect that
‘heterosurfaces’ have on the nucleation kinetics of different APIs. In fact, the mechanism by which heterogeneous nucleation occurs is not obvious and may be a consequence of epitaxial interactions13-14, 27-29, nonspecific adsorption30-31, surface topography16, 32-33 and/or intermolecular bonding13, 34.
FF and AAP were selected as model APIs while MCC and -Lac were selected as excipients to act as heterosurfaces. AAP is 10 times more soluble in MeOH than FF at the same temperature. The induction time at 500 mL scale in the absence of excipients of FF in MeOH at
S = 1.5 was 22 hours (shown in Chapter 4), significantly longer than the 1.5 hours for AAP in
MeOH at S=1.25 (shown in Chapter 3). In addition, FF and AAP have different molecular functionalities. AAP has hydrogen bond donors and acceptors, whereas FF only has hydrogen bond acceptors. The selected excipients have a high number of hydrogen bond donors which can interact with both the AAP and the FF molecules. Nucleation kinetics, particle size, solution concentration and degree of API to excipient were examined. Finally, quantum chemical calculations were used to estimate the binding energy between APIs and excipients and explain the observed reduction in the induction time in the presence of excipients.
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6.3. MATERIALS
Table 7 summarized various attributes of the ‘as received’ materials used in this study.
Table 14: Attributes of the various ‘as-received’ materials used in this study
CCDC Purity Material Abbreviation Supplier Crystallinity Reference Polymorph (%) Code Match Methanol MeOH > 99.9 Sigma-Aldrich - - - Acetaminophen AAP > 99.9 Sigma-Aldrich Crystalline HXACAN01 Form I KEMPROTEC Fenofibrate FF ≥ 98 Crystalline TADLIU01 Form I Limited -Lactose -Lac ≥ 99 Sigma-Aldrich Crystalline BLACTO β (≤ 30% α-anomer) Microcrystalline MCC - VWR/MERCK Amorphous - - cellulose
6.4. METHODS
Batch cooling crystallizations were implemented using a LabMaxTM workstation from
Mettler Toledo. The system was equipped with Focused Beam Reflectance Measurement
(FBRM), a temperature sensor and in situ Fourier Transform Infrared (FTIR). Measurements from these instruments were recorded by iControl LabMax SoftwareTM Mettler Toledo and exported to Excel for data processing.
A 4-bladed PTFE screw propeller stirrer shaft (diameter = 60 mm, length = 400 mm) was used to mix the solution. This impeller is known for mixing media in an up-to-down axial flow.
A stirring rate of 175 rpm was selected for all the experiments, as it was enough to keep the solution mixed without forming bubbles or vortices.
Crystal growth and desupersaturation were monitored using the FBRM probe and the in situ IR probe, respectively. The FBRM probe examined the change in counts and chord length distribution (CLD), which includes the mean chord length and the span of a volume-based size distribution that is defined as d90-d10 and gives an indication of how far the 10 percent and 90 percent size distribution points are apart.
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1. Crystallization of AAP
Excess AAP was added to 500 mL of MeOH, placed in a temperature-controlled water bath (± 0.1 °C) at Tsat = 25 °C (AAP solubility at 25 °C = 320 g/kg MeOH) and agitated with a PTFE-coated magnetic stirrer at 500 rpm for 24 hours. Agitation was then stopped and the suspension allowed to settle for more than 1 hour at 25 °C. The solution and solid fraction were separated by vacuum filtration (Büchner funnel + 2.5m cellulose filter paper) and the solution was added to the Labmax vessel.
In situ crystallization of AAP in the absence and in the presence of excipients was tracked. The % desupersaturation was calculated from the change in the peak area at 1515 cm-1 in the FTIR spectrum, as this peak does not overlap any of the MeOH spectrum, during desupersaturation of an AAP-MeOH solution (Tcry =15 °C, S = 1.25) at 15 °C, as shown in Equation 36.
∆ peak area (t) % desupersaturation = (1 − ) × 100 Equation 36 ∆ peak areamaximum
Where:
∆ peak area (t) = peak area (t) − peak area (t0)
∆ peak areamaximum = peak area (t∞) − peak area (t0) t∞ = Time at which available supersaturation has been consumed
t0 = Time at which the API starts to crystallize.
The sequence used to bring the solution to the crystallization temperature using Labmax software was as follows:
1. The solution was heated to 30 °C with a heating rate of 2 K/min.
2. The solution was held at 30 °C for 30 minutes to ensure complete dissolution of the
AAP.
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3. The solution was then cooled to 20 °C with a cooling rate of 1 K/min.
4. The solution was cooled to 15 °C with a cooling rate of 0.5 K/min.
5. The solution was held at 15 °C for 3 hours.
In the presence of excipients:
When the experiment was performed in the presence of excipients, a maximum
AAP loading of 25 % based on the AAP available to crystallize was used. To obtain
this loading 75 g of excipient were added to the 500 mL of AAP solution immediately
after the solution reaches 15 °C. The effect of two different excipients, namely -Lac
and MCC, on the crystallization of AAP was studied.
2. Crystallization of FF
Excess FF was added to 500 mL of MeOH, placed in a temperature-controlled water
bath (± 0.1 °C) at Tsat = 16 °C (FF solubility = 43.95 g/kg MeOH) and agitated with a
PTFE-coated magnetic stirrer at 500 rpm for 24 hours. Agitation was then stopped and the
suspension allowed to settle for more than 1 hour at 16 °C. The solution and solid fraction
were separated by vacuum filtration (Büchner funnel + 2.5m cellulose filter paper) and
the solution was added to the Labmax vessel.
In situ crystallization in the absence and in the presence of excipients was tracked. As
indicated in Equation 36 the % desupersaturation was calculated from the change in the
peak areas of the 1600 cm-1 peak in the FTIR spectrum, as this peak does not overlap any of
the MeOH spectrum, during the desupersaturation of a FF-MeOH solution at S = 1.5 (Tsat =
16 °C and Tcry = 10 °C) and S = 2.0 (Tsat = 22 °C and Tcry = 10 °C).
The sequence used to bring the solution to the crystallization temperature using Labmax
software was as follows:
1. The solution was heated to 25 °C with a heating rate of 2 K/min.
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2. The solution was held at 25 °C for 30 minutes to ensure complete dissolution.
3. The solution was cooled to 20 °C at a cooling rate of 1 K/min.
4. The solution was cooled to 10 °C at a cooling rate of 0.5 K/min.
5. The solution was held at 10 °C for 24 hours.
In the presence of excipients:
When the experiment was performed in the presence of excipients, a maximum FF
loading of 35 % based on the FF available to crystallize was used. To obtain this
loading 11 g of excipient were added to the 500 mL of FF solution immediately after
the solution reaches 10 °C. The effect of two different excipients, namely -Lac and
MCC, on the crystallization of FF was studied.
Analysis of the solid fractions
SEM
The habit of the isolated particles was examined by SEM (JCM-5700 and JSM-6510LV
(JEOL)). Samples were gold-coated (SI50B, Edwards) and micrographs of the solid fractions produced after the crystallization of the corresponding API in the presence and in the absence of excipients were taken. The mean sizes of the AAP and FF particles crystallized in the absence of excipients were measured from the micrographs using image analysis (Adobe
Measurement Tool).
Computational chemistry:
Quantum chemical calculations were performed to optimize the energy of each system using computational chemistry. The Gaussian 0935 package was used to optimize the geometric parameters of the molecules. The methodology of the quantum chemical calculations was based on Hartree–Fock density functional HF 36. From a given input the program calculates molecular orbitals using the Linear Combination of Atomic Orbitals (LCAO) approximation
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(Figure 100). The Hartree–Fock method works as illustrated in the algorithmic flowchart in
Figure 101.
Figure 100: Computational Chemistry Map 37
Figure 101: Algorithmic flowchart illustrating the Hartree–Fock method38
Structures of the molecules that make up each system were constructed in BIOVIA
Material Studio Software and exported in matrix format to be used as input for the quantum chemical calculations. Calculations were applied to investigate (1:1) API–excipient pair interactions. For this purpose, each molecule of either the API or the excipient and of the geometries of the (1:1) API to excipient were calculated and optimized at the HF/6-31G(d,p) level, which gives the total energy of the system at its equilibrium position together with the optimized geometry. Gaussview 0539 was used to visualize the results. The interaction of molecules in the eight following different systems (in terms of the associated binding energy) was studied:
Homogeneous nucleation of AAP: Binding energy between two molecules of AAP.
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Heterogeneous nucleation of AAP in the presence of MCC: Binding energy between a
molecule of AAP and a molecule of glucose (Gluc) which represents the monomeric
unit of cellulose.
Heterogeneous nucleation of AAP in the presence of -Lac: Binding energy
between a molecule of AAP and a molecule of -Lac.
Solvation energy between AAP and MeOH: Binding energy between a molecule of
AAP and a molecule of MeOH.
Homogeneous nucleation of FF: Binding energy between two molecules of FF.
Heterogeneous nucleation of FF in the presence of MCC: Binding energy between a
molecule of FF and a molecule of Gluc.
Heterogeneous nucleation of FF in the presence of -Lac: Binding energy between a
molecule of FF and a molecule of -Lac.
Solvation energy between FF and MeOH: Binding energy between a molecule of FF
and a molecule of MeOH.
The binding energy for each system was calculated via Equation 37:
∆Hbind = ∆Hmolecule1+molecule2 − [∆Hmolecule1 + ∆Hmolecule2 ] Equation 37
Being:
∆Hbind = Binding energy between molecule 1 and molecule 2
∆Hmolecule1+molecule2 = Total electronic energy in a system which contains molecule 1 and molecule 2
∆Hmolecule1 = Total electronic energy in a system which contains molecule 1
∆Hmolecule2 = Total energy in a system which contains molecule 2
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With the aim of calculating the binding energy in each system the following 3D geometries were given as inputs for the quantum chemical calculations: (a) an AAP molecule,
(b) A Gluc molecule, (c) A MeOH molecule, (d) an -Lac molecule, (e) two AAP molecules,
(f) an AAP molecule and a Gluc molecule (g) an AAP molecule and a MeOH molecule, (h) an
AAP molecule and an -Lac molecule, (i) two FF molecules, (j) a FF molecule and a Gluc molecule, (k) a FF molecule and a MeOH molecule, and (l) a FF molecule and an -Lac molecule.
6.5. EQUATIONS The equations used in this chapter are introduced below:
a. Nucleation rate:
1 퐽 = Equation 38 푡𝑖푛푑푉
Classical nucleation theory (CNT)40:
3 2 ∗ 16 휋 훾 푉푚 ∆퐺푇,퐻푂푀 − 3 3 2 − 퐽 = 휌 푍 푗 푒 3푘 푇 푙푛 푆 = 퐴1 푒 푘푇 Equation 39
3 2 ∗ 16 휋 훾퐻퐸푇 푉푚 ∆퐺푇,퐻퐸푇 − 3 3 2 − 퐽퐻퐸푇 = 휌퐼 푍 푗 푒 3푘 푇 푙푛 푆 = 퐴1,퐻퐸푇 푒 푘푇 Equation 40
16 휋 훾3 푉2 푁 ∆퐺 ∗ = 푚 퐴 푇 3푘2푇2푙푛2푆 Equation 41
where:
J and JHET = nucleation rate for homogeneous and heterogeneous nucleation, respectively (number of nuclei/(m3.s))
tind = induction time (s) V = crystallizer volume (m3) 2 훾 푎푛푑 훾퐻퐸푇 = interfacial energy surface (J/m ) k = Boltzmann constant = 1.38×10-23 J/K T = Temperature (K) 41 3 Vm = Molecular volume (m ): -28 3 Vm, AAP = 2.02×10 m
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-28 3 Vm, FF =5.10×10 m S = Supersaturation = c/c* (-) c = initial concentration of API (g API / kg MeOH):
cAAP = 332.1 g/kg at 25 °C
cFF = 43.9 g/kg at 16 °C * c = equilibrium concentration of API at Tcry (g API / kg MeOH): * c AAP = 265.4 g/kg at 16 °C * c FF = 29.3 g/kg at 10 °C 3 A1 and A1, HET = constants (number of nuclei/(m .s))
andthe number density of sites for homogeneous and heterogeneous nucleation, respectively (number of sites/m3)
ρI = ε × ρFP + ρ Equation 42
휌퐹푃: the number density of foreign particles.
휀: the number of places a critical nucleus can form on each foreign particle. Z = Zeldovich factor, which is the probability that a nucleus at the top of the activation energy barrier will go on to form a crystal (-) j = the rate at which molecules attach to the nucleus causing it to grow (molecules/s) ∗ ∗ ∆퐺푇,퐻푂푀 , ∆퐺푇,퐻퐸푇 = Maximum Gibbs free energy for homogeneous and heterogeneous nucleation, respectively, at the top of the energetic barrier (J/mol) 23 -1 NA = Avogadro’s constant = 6.022×10 mol
The nucleation rate equation can also be expressed as a linear equation:
1 16 π γ3 푉2 1 ln ( ) = ln(1/퐴 ) + 푚 퐽 1 3푘3 T3ln2S Equation 43 b. Mersmann equation for calculating the interfacial energy 22, 42:
1 ρ γ = C k T ln ( API ) 2/3 c∗ Equation 44 Vm where: C = constant (-) 3 ρAPI = density of the API (g/m ), which is: 6 3 43 ρFF =1.18×10 g/m 6 3 43 ρ퐴퐴푃 =1.26×10 g/m
209 | P a g e c. Molecules available to crystallize
Molecules available to crystallize per unit volume for each system can be calculated as follows:
(c−c∗)× 휌 Moles available to crystallize/m3 = 푀푒푂퐻 Equation 45 Mw where:
Mw= molar mass (g/mol): 44 Mw,AAP = 151.2 g/mol 44 Mw,FF = 360.8 g/mol 3 3 휌푀푒푂퐻 = MeOH density (kg/m ) = 792 kg/m d. Growth time
For the crystallization of the APIs in the absence of excipients, the growth time was quantified from the chord length distribution taken from the FBRM data, as follows:
tg = tmax − t0 Equation 46 where: tg = Time required for a single crystal to grow to a certain size (s) tmax = Time where a maximum in the chord length was observed (s) t0 = Time where an increase in the chord length was first observed (s)
For the heterogeneous crystallization of the APIs, the growth of the particles cannot be detected in the presence of excipients using the FBRM probe, and so the FTIR probe was used instead. Thus, it was assumed that the API particles started to grow once the solution started to desupersaturate and that the maximum chord length was achieved when the solution completely desupersaturated. e. Growth rate
particle size diameter at t growth rate (m/s) = g Equation 47 tg
210 | P a g e f. Critical radius 40
2γ푉 r∗ = 푚 kTlnS Equation 48 where: r* = critical radius (m) g. Volume of a nucleus
The volume of a nucleus considering it is spherical will be: 4 푉 = π푟∗3 푛푢푐푙푒푢푠 3 Equation 49 h. Time required for the addition of a molecule to a crystal
tm will be defined as the time required for the addition of a single molecule to a crystal at a constant growth rate:
푡𝑔푀푊 푡푚 = Equation 50 ρAPI 푉푝푁퐴 where: tm = Time required for the addition of a molecule to a crystal (s) 3 Vp= Volume of a particle at tg (m ): 4 휋(푝푎푟푡푖푐푙푒 푟푎푑푖푢푠 푎푡 푡 )3 Vp= 3 푔 i. Number of molecules in a nucleus
푉 푛푢푚푏푒푟 표푓 푚표푙푒푐푢푙푒푠 푖푛 푎 푛푢푐푙푒푢푠 = 푛푢푐푙푒푢푠 Equation 51 푉푚 j. Time required to form a nucleus
푡푛푢푐푙푒푢푠 = 푛푢푚푏푒푟 표푓 푚표푙푒푐푢푙푒푠 푖푛 푎 푛푢푐푙푒푢푠 × 푡푚 Equation 52 k. Gibbs free energy for heterogeneous crystallization45-48
∗ ∗ ∆G 퐻퐸푇 = ∆G 퐻푂푀 × 푓(θ) Equation 53 Where: ∗ ∆G 퐻퐸푇 = Gibbs free energy for heterogeneous nucleation ∗ ∆G 퐻푂푀 = Gibbs free energy for homogeneous nucleation = the angle that the interface between the nucleus and the bulk phase f
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6.6. RESULTS
6.6.1. Intermolecular functional group complementarity:
Table 15shows the binding energy in terms of kJ/mol between API-API molecules, API-MeOH molecules and API-excipient molecules from quantum chemical calculations. In addition, the optimized geometries of molecules for each system are shown in in Figure 102 Figure 102. The binding energy between two molecules of AAP was stronger than between two molecules of FF. In terms of type of interactions, molecules of AAP can interact by intermolecular hydrogen bonding, with this interaction slightly weaker than between molecules of AAP and molecules of MeOH (Table 15). In contrast, the interactions among FF molecules which only have hydrogen bond acceptors can only be Van der Waals interactions, with this interaction weaker than between molecules of FF and molecules of MeOH (Table 15), since
MeOH can H-bond to FF. Finally, the interaction between both APIs and the selected excipients is favourable as the computations indicate a significant increase in binding energy released between the API and the excipient molecules. The binding energy of the interaction between API-excipient molecules was stronger than between API-MeOH molecules for both systems (Table 15).
Table 15: Optimized binding energy in kJ/mol between: (a) two AAP molecules, (b) two FF molecules, (c) an AAP molecule and a MeOH molecule, (d) a FF molecule and a MeOH molecule, (e) an AAP molecule and a Gluc molecule, (f) an AAP molecule and an -Lac molecule (g) a FF molecule and a Gluc molecule, and (h) a FF molecule and an -Lac molecule System ∆Hbind (kJ/mol) AAP-AAP -20 FF-FF -10 AAP-MeOH -29 FF-MeOH -25 AAP-Gluc -44 AAP--Lac -47 FF-Gluc -34 FF--Lac -31
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(a) (b) AAP FF
AAP
FF
(c) (d) AAP FF
MeOH MeOH
(e) (f) AAP
FF Gluc
Gluc
(g) Lac (h) Lac
FF
AAP
Figure 102: Optimized geometry of: (a) two AAP molecules, (b) two FF molecules, (c) an AAP molecule and a MeOH molecule, (d) a FF molecule and a MeOH molecule, (e) an AAP and a Gluc molecule, (f) a FF and a Gluc molecule, (g) an AAP and an -Lac molecule, and (h) a FF and an -Lac molecule
6.6.2. Crystallization of AAP and FF from MeOH in the absence of excipients
6.6.2.1. Nucleation
Analysis of the change of the 1515 cm-1 peak in the FTIR spectrum, for AAP
crystallization in the absence of excipients (Figure 103), revealed that the solution started
to desupersaturate once it reached the crystallization temperature. However, as shown by
the increase in the number of counts in Figure 105, it took 1.5 hours to detect AAP
213 | P a g e
particles in solution (induction time). In contrast, for FF crystallization in the absence of
excipients (Figure 103), the solution started to desupersaturate accompanied by an
immediate increase in the number of counts (Figure 107) after 5 hours at Tcry.
The nucleation rate in both systems was calculated using Equation 3812. The results are
shown in Table 16.
100
90
80
70
60
50
40 % Desupersaturation 30
20 % desupersaturation (AAP) 10 % deuspersaturation (FF)
0 0 1 tind, AAP 2 3 4 5 tind, FF 6 7 8 9 10 Time (hrs) Figure 103: % desupersaturation as measured by FTIR probe (1500 cm-1) of (a) an AAP-MeOH solution in the absence of excipient (green trace) (Tcry = 15 °C, S = 1.25 and V = 500 mL), (b) a FF- MeOH solution in the absence of excipient (purple trace) (Tcry = 10 °C, S = 1.5 and V = 500 mL), Red dashed lines = slope of the respective desupersaturation curves.
Table 16: Nucleation rate for the crystallization of AAP (S=1.25, Tcry = 15 °C and V = 500 mL) and FF (Tcry = 10 °C, S = 1.5 and V = 500 mL) from MeOH in the absence of excipients
AAP FF Induction time (s) 5400 18000 Crystallizer volume (m3) 5×10-4 5×10-4 J (nuclei/(m3.s)) 0.37 0.11
The nucleation rate for AAP crystallization was found to be more than 3-fold faster than that for FF crystallization.
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6.6.2.1.1. Interfacial energy
The interfacial energy (훾) can be determined based on two main approaches: those based on the application of the CNT (experimental approach) 40, and those based on simplified thermodynamic models (theoretical approach) 22.
On the one hand, applying the CNT, 훾 for both systems was evaluated. According to Equation 43, the slope of the line obtained after plotting ln (1/J) versus 1/(T3ln2S)
3 2 16 π γ 푉푚 will be equal to 3푘3 . Previous results in Chapter 3 showed the induction time for the crystallization of AAP from MeOH at different saturations (S = 1.56, 1.39 and
1.25), so 훾퐴퐴푃 was calculated with this data. Additionally, with the induction time data taken from the FTIR desupersaturation profiles during the nucleation of FF in the absence of excipients at supersaturations of 1.5 and 2.0, 훾퐹퐹 was also calculated. Both data sets and their corresponding linear equations are shown in Figure 104.
On the other hand, the Mersmann equation (Equation 44), derived on the basis of the Gibbs adsorption isotherm, correlates the crystal-solution interfacial energy with the natural logarithm of the equilibrium solubility. There is some vagueness in the constant C that cannot be clarified because the scattering in the experimental data is too high. A value of 0.414 is recommended for the constant C 49.
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3
2 y = 2E+07x - 3.9144 1
0 0.E+00 1.E-07 2.E-07 3.E-07 4.E-07 5.E-07 6.E-07 7.E-07 8.E-07 9.E-07 -1
ln (1/J) -2
-3
-4 y = 4E+06x - 5.3509 R² = 0.9179 -5
-6 1/(T3ln2S)
Figure 104: Estimation of γAAP and γFF in MeOH for homogeneous nucleation of AAP () and FF () respectively based on the application of the CNT.
Table 17 shows the large difference between the values of the experimentally determined interfacial energy using Equation 43 and the theoretically calculated values obtained from Equation 44, which are in a similar range to reported studies 50. This large difference is a consequence of the vagueness in the constant C of Equation 44. A similar difference between the experimental and the theoretical values for the crystallization of AAP from MeOH was obtained by Omar et. al.50, with the experimental and the theoretical values similar to the ones obtained here. In following calculations, the experimental interfacial energy calculated using Equation 8 will be used as these values are comparable with those previously observed for other organic compounds12, 50-52.
The ∆GT* (Equation 41), calculated using the experimental values of was found to be 1.6-fold larger for the FF system than for the AAP system (Table 17). Then, despite
(i) working at a higher supersaturation in the FF system, and (ii) γAAP ≈ γFF, the energy barrier to overcome in order to initiate crystallization was still larger for the FF
216 | P a g e
system than for the AAP system, thus explaining the former’s lower nucleation rate (J
in Equation 39). As such, J is higher for the AAP system due to the larger exponential
factor for AAP compared to that for the FF system; this is due to the larger volume of a
FF molecule (Vm) compared to that of an AAP molecule as shown in Table 17.
Table 17: Difference between the AAP (S = 1.25, Tcry = 15 °C and V = 500 mL) and the FF (Tcry = 10 °C, S = 1.5 and V = 500 mL) system
γ ∆GT* γ 2 Temperature 3 2 6 2 (mJ/m ) (kJ/mol) S Vm(m ) Vm (m ) (mJ/m ) (°C) (Equation (Calculated with γ (Equation 43) 44) from Equation 43) AAP 15 1.25 2.0×10-28 4.0×10-56 2.5 8.5 8.0 FF 10 1.5 5.1×10-28 2.6×10-55 2.3 9.9 12.6
6.6.2.1.2. Molar solubility of the APIs in MeOH
Additionally, the AAP molar solubility in MeOH was compared to that of FF and
the moles available to crystallize per unit volume in both systems using Equation 45
was calculated. All these terms are shown in Table 18. AAP is 10 times more soluble in
MeOH than FF at 25 °C. This indicates that before nucleation occurred the molecules
available to crystallize, in the AAP system at S = 1.25 were 6 times more densely
concentrated in solution than for the FF system at S = 1.5, as shown in Table 18. This
higher density of solute molecules in solution for the AAP system increases the
likelihood of forming the cluster of molecules responsible for homogeneous nucleation
than for the FF system53.
Table 18: Differences in the mole density for the crystallization of AAP at S=1.25 compared to that of FF at S = 1.5 Moles of API available to Mw Molar solubility at Tsat System crystallize per unit (g/mol) (mol/kg) volume (moles/m3)
AAP 151.16 2.19 350 FF 360.83 0.12 32
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6.6.2.2. Growth
Analysis of the growth rate of both AAP and FF particles during homogeneous crystallization from MeOH was undertaken using the FBRM data (LabMax) and the PSD analysis from the SEM images. From the FBRM data the growth time (tg), defined as the time required for a single crystal to grow to a certain size was quantified for AAP and FF using Equation 46 and shown in Figure 105 and Figure 91, respectively. In addition, the final particle diameter to which AAP and FF particles grew was measured from PSD measurements of the corresponding SEM micrographs after the particles had grown to their maximum size. An approximate growth rate of AAP and FF was calculated (Equation
47) and shown in Table 19.
1200 100
Counts (In the absence of excipients) 90
1000 80 % desupersaturation (In the absence of excipients)
70 800 Mean24 per. chord Mov. length Avg. (Mean particle size) 60 m) 600 50 m) ( 40 10 d - Number Number countsof
400 90 30 d % desupersaturation
20 Mean Chord length ( 200
10 tg, AAP
0 0 tIND, AAP 0 0.5 1 1.5 2 2.5 3 Time (hours)
Figure 105: Desupersaturation and particle size distribution profiles of an AAP-MeOH solution showing (a) the change in counts (blue trace), (b) % desupersaturation as measured by FTIR probe (purple trace), and (c) mean chord length (green trace). (Tcry = 15 °C, S = 1.25 and V = 500 mL), red dashed line = slope of the desupersaturation curve.
218 | P a g e
Figure 106: SEM images of AAP crystallized from an AAP-MeOH solution at S = 1.25, Tcry = 15 °C and time = 3 hour.
300
counts (In the absence of excipient) 100
250 % desupersaturation (In the absence of excipient) 80 200 Mean chord length m)
60 m) 150 ( 10 d - 90 Number Number countsof 40 d 100 % Desupersaturation Meanchord lenght (
50 20
tg, FF 0 0 0 2 4 tIND, FF 6 8 10 12 Time (hrs) Figure 107: Desupersaturation and particle size distribution profiles of an FF-MeOH solution showing (a) the change in counts (blue trace), (b) % desupersaturation as measured by FTIR probe (purple trace), and (c) mean chord length (green trace). (Tcry = 10 °C, S = 1.5 and V = 500 mL), red dashed line = slope of the desupersaturation curve.
Figure 108: A representative SEM image of FF crystallized from a FF-MeOH solution at S = 1.5, Tcry = 10 °C and time = 12 hours
219 | P a g e
Table 19: Approximate growth time, particle size diameter and growth rate of (a) AAP particles crystallized at 15°C, S= 1.25, and (b) FF crystallized at 10°C, S = 1.5, from MeOH
AAP FF Particle diameter at tg ( m) 78±62 299 ± 163 From SEM analysis tg (s) 2520 2280 Growth rate (m/s) ̴ 3.0×10-8 ̴ 1.3×10-7
Using Equations 48 to 52 the approximate values of critical radius (r*), volume of a
nucleus (Vnucleus), number of molecules in a nucleus, time required for the addition of a
molecule to a crystal (tm), and time required for the formation of a nucleus (tnucleus) were
calculated based on previous calculations of γAAP and γFF (from the experimental approach,
Table 4) (Table 20).
Table 20: Approximate critical radius (r*), volume of a nucleus (Vnucleus), number of molecules in a nucleus, time required for the addition of a molecule to a crystal (tm), and time required for the formation of a nucleus (tnucleus) based on previous calculations of γAAP and γFF of (a) AAP particles crystallized at 15°C, S= 1.25 and (b) FF crystallized at 10°C, S = 1.5, from MeOH
AAP FF r* (nm) 1.1 1.4 3 Vnucleus (nm ) 5.1 12.4 tm (ps) 2 0.08 Number of molecules 25 27 in a nucleus tnucleus(ps) 50 2
The growth rate of FF was found to be significantly faster than that of AAP.
6.6.3. Crystallization of AAP and FF in the presence of excipients
6.6.3.1. Nucleation
Table 21 summarizes the nucleation rates of FF and AAP in the presence of the
excipients calculated with the data taken from Figure 109 and 11. The nucleation rate of
FF in the presence of -lac or MCC increased by 50-fold compared to that in the
absence of excipients at the same supersaturations. However, an increase of just 1.5 and 3-
220 | P a g e fold in the nucleation rate of AAP was observed in the presence of MCC and-Lac, respectively, compared to that in the absence of excipients at the same supersaturations.
100
90
80
70
60
50
40
% Desupersaturation % desupersaturation (AAP_α/β-Lac) 30
20 % desupersaturation (FF_α/β-Lac)
10 tg, AAP tg, FF 0 0 tIND, FF 0.5tIND, AAP 1 1.5 2 2.5 3 Time (hrs)
Figure 109: % desupersaturation as measured by FTIR probe of (a) an AAP-MeOH solution in the presence of -Lac (green trace) (Tcry = 15 °C, S = 1.25, 25 % loading and V = 500 mL), (b) a FF- MeOH solution in the presence of -Lac (purple trace) (Tcry = 10 °C, S = 1.5; 25 % loading and V = 500 mL), red dashed lines = slopes of the respective desupersaturation curves.
221 | P a g e
100
90 % desupersaturation (AAP_MCC)
80 % desupersaturation (FF_MCC)
70
60
50
40
% Desupersaturation 30
20
10 tg, FF tg, AAP
0 0 tIND, FF 0.5 tIND, AAP1 1.5 2 2.5 3 Time (hrs)
Figure 110: % desupersaturation as measured by FTIR probe of (a) an AAP-MeOH solution in the presence of MCC (green trace) (Tcry = 15 °C, S = 1.25, 25 % loading and V = 500 mL), (b) a FF-MeOH solution in the presence of MCC (purple trace) (Tcry = 10 °C, S = 1.5; 25 % loading and V = 500 mL), red dashed lines = slopes of the respective desupersaturation curves.
Table 21: Summary of crystallisation parameters for the crystallization of AAP and FF from MeOH in the presence of -Lac and MCC.
AAP+-Lac AAP+MCC FF+-Lac FF+MCC Induction time (s) 1800 3600 360 360 Crystallizer volume (m3) 5×10-4 5×10-4 5×10-4 5×10-4 3 JHET (nuclei/(m .s)) 1.1 0.55 5.5 5.5 JHET/JHOM 3 1.5 50 50
6.6.3.2. Growth
Using Equation 47 the growth rates of FF and AAP in the presence of either MCC or
-Lac were calculated. The growth rate was found to be slower during the crystallization
of both APIs in the presence of excipients compared to that in the absence of excipients
(Table 22). This slower growth rate may be a consequence of a poorer diffusion of the API
molecules in the suspension 54.
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(a) (b)
AAP AAP
(c) (d)
FF
FF
Figure 111: SEM images of AAP crystallized from a AAP-MeOH solution at 25 % AAP loading, at S = 1.25, Tcry = 15 °C, in the presence of (a) MCC at a contact time of 2.5 hours and (b) -Lac at a contact time of 2 hour, and of FF crystallized at 35 % FF loading, S = 1.5, Tcry = 10 °C from a FF- MeOH solution in the presence of (c) MCC at contact time of 45 minutes and (d) -Lac at contact time of 45 minutes.
Table 22: Approximate growth time (tg), particle size diameter, growth rate and time required for the addition of a molecule to a crystal (tm) for the crystallization of (a) AAP at 15°C, S= 1.25, 25 % AAP loading, and (b) FF at 10°C, S = 1.5, 35 % FF loading from MeOH both in the presence of MCC and -Lac
AAP + -Lac AAP + MCC FF + -Lac FF +MCC tg (s) 3600 5000 2160 1044 Particle diameter (m) 25.5 ± 11.1 25.3 ± 12.1 60.3 ± 26.5 75.4 ± 91.6 Growth rate (m/s) 7.1×10-9 5.1×10-9 2.8×10-8 7.2×10-8 푮풓풐풘풕풉 풓풂풕풆 풉풐풎풐품풆풏풆풐풖풔 4.2 5.9 4.6 1.8 푮풓풐풘풕풉 풓풂풕풆 풉풆풕풆풓풐품풆풏풆풐풖풔 tm (ps) 83 115 9.5 2.3
Table 22 also showed the tm for AAP and FF in the presence for -Lac and MCC. As no values for γHET,AAP or γHET,FF were calculated, no estimated values of tnucleus could be
∗ ∗ obtained. However, in general terms, 훾퐻퐸푇 < 훾 because ∆G 퐻퐸푇 < ∆G 퐻푂푀 (Equation
53); thus the maximum value of tnucleus can be estimated. Using Equations 48 to 52 it can be said that the number of molecules in a critical nucleus during heterogeneous nucleation
223 | P a g e
will be lower than for the corresponding number during homogeneous nucleation (i.e. 25
and 27 for AAP and FF respectively, as shown in Table 20); therefore:
푛푢푚푏푒푟 표푓 푚표푙푒푐푢푙푒푠 푖푛 푎 퐴퐴푃 푐푟푖푡푖푐푎푙 푛푢푐푙푒푢푠 푑푢푟푖푛푔 ℎ푒푡푒푟표푔푒푛푒표푢푠 푛푢푐푙푒푎푡푖표푛 < 25
푛푢푚푏푒푟 표푓 푚표푙푒푐푢푙푒푠 푖푛 푎 퐹퐹 푐푟푖푡푖푐푎푙 푛푢푐푙푒푢푠 푑푢푟푖푛푔 ℎ푒푡푒푟표푔푒푛푒표푢푠 푛푢푐푙푒푎푡푖표푛 < 27
The maximum tnucleus for the crystallization of AAP and FF in the presence of -Lac and
MCC according to Equation 52, are shown in Table 23.
Table 23: Maximum tnucleus for the crystallization of critical nucleus (a) AAP at 15°C, S= 1.25, 35 % AAP loading and (b) FF at 10°C, S = 1.5, 25 % AAP loading from MeOH both in the presence of MCC and -Lac System Maximum tnucleus (ps) AAP-MCC 2875 AAP--Lac 2075 FF-MCC 62 FF--Lac 256
6.7. DISCUSSION
6.7.1. Comparative study of the crystallization of AAP and FF in the presence of excipients
The following sections will look at the different parameters influencing heterogeneous crystallization with the aim of finding an explanation for the different crystallization behaviour of these two APIs.
6.7.1.1. Intermolecular functional group complementarity
The presence of intermolecular functional group complementarity between AAP–AAP
compared to its absence among FF molecules is one of the reasons why the nucleation rate
for the AAP system was faster compared to that for the FF system in the absence of
excipients (Table 15). The addition of excipients with functional group complementarity
with the API accelerated the nucleation rate. This acceleration was more pronounced when
the intermolecular functional group complementarity between API–API molecules was
weaker, as seen here in the case of FF.
224 | P a g e
Since API and excipient molecules can interact with each other (via hydrogen bonds, Van der Waals interactions, etc.) the significant increase in the nucleation rate by adding
‘hetereosurfaces’ can be explained in terms of the lifetimes of these interactions. Literature studies have concluded that there is a longer hydrogen bond lifetime between a molecule in solution and a solid surface than that between two molecules in solution 55-57. Thus, it is proposed here that the interaction of a cluster with a polar or hydrogen bonding group on a macromolecular solid surface should increase the average lifetime of the interaction, allowing more time for the API molecules to cluster on the solid surface, thus increasing the probability of reaching a critical size 56-57. Adsorption is always exothermic and for this reason the activation energy for adsorption (Eaads) is always less than the activation energy for desorption
(Eades) (Figure 112). Hence, the lifetime of the adsorbed state is extended.
Figure 112: Schematic representation of the activation energies for adsorption and desorption (Eaads and Eades) In order to have homogeneous nucleation the intermolecular interactions between two molecules should be long enough to facilitate the addition of the required number of molecules to a pre-critical nucleus until it reaches the critical size, i.e:
푙푖푓푒푡푖푚푒 표푓푡ℎ푒 푖푛푡푒푟푎푐푡푖표푛 > 푡푛푢푐푙푒푢푠
In the case of AAP nucleation, AAP molecules can hydrogen bond to each other. For
the FF system the interaction among molecules is primarily a consequence of Van der
225 | P a g e
Waals interactions. Thus, with the values of tnucleus shown in Table 20, it can be concluded
that:
푙푖푓푒푡푖푚푒 표푓퐴퐴푃 푖푛푡푒푟푚표푙푒푐푢푙푎푟 푖푛푡푒푟푎푐푡푖표푛 > 50 푝푠 푙푖푓푒푡푖푚푒 표푓퐹퐹 푖푛푡푒푟푚표푙푒푐푢푙푎푟 푖푛푡푒푟푎푐푡푖표푛 > 2 푝푠
From reported studies, the normal lifetimes for Van der Waals interactions and the
hydrogen bonds between two molecules in solution and also for hydrogen bonds between a
solid surface and molecules in solution are shown in Table 24.
Table 24: Reported lifetimes for different types of interactions.
Type of interaction Lifetime of the interaction (ps) 58-59 Van der Waals 1 to 3 (between molecules in solution) 59-60 H-bond 1 to 70 (between molecules in solution) 61 H-bond 103 to 1.6×105 (between a solid surface and molecules in solution)
According to these reported lifetime values (Table 24) and the calculated values of
tnucleus (shown in Table 20), it can be concluded that there was enough time for the
formation of an API nucleus in the AAP and the FF system. However, this comparison
should be amended with the formal values of tnucleus. Then, in the case of homogeneous
nucleation once the molecules required to form a surface interact with each other for a
sufficient period of time, more molecules will be allowed to join the cluster surface, at a
defined growth rate, causing the formation of a critical nucleus. The induction time will
then depend on the likelihood of molecules interacting with each other which would
depend on the molar solubility of the API.
Due to a slower growth rate for heterogeneous crystallization, the time required for a
molecule to be added to a crystal was larger. Then, the lifetime of the interaction between
an excipient solid surface and the API molecules in solution needs to last longer than for
homogeneous nucleation to allow the formation of an API nucleus on the excipient
226 | P a g e surface. Thus, with the maximum values of tnucleus shown in Table 23, it can be concluded that:
푖푛푡푒푟푚표푙푒푐푢푙푎푟 푙푖푓푒푡푖푚푒 표푓퐴퐴푃 − 푀퐶퐶 푖푛푡푒푟푎푐푡푖표푛 > 2875 푝푠 푖푛푡푒푟푚표푙푒푐푢푙푎푟 푙푖푓푒푡푖푚푒 표푓퐴퐴푃 − 훼/훽– 퐿푎푐 푖푛푡푒푟푎푐푡푖표푛 > 2075 푝푠 푖푛푡푒푟푚표푙푒푐푢푙푎푟 푙푖푓푒푡푖푚푒 표푓퐹퐹 − 푀퐶퐶 푖푛푡푒푟푎푐푡푖표푛 > 62 푝푠 푖푛푡푒푟푚표푙푒푐푢푙푎푟 푙푖푓푒푡푖푚푒 표푓퐹퐹 − 훼/훽– 퐿푎푐 푖푛푡푒푟푎푐푡푖표푛 > 256 푝푠 Knowing that the lifetime of hydrogen bond interactions between solid surfaces and molecules in solution is in the order of 103 to 1.6×105 ps, as shown in Table 24, it can be concluded that there was sufficient time for the formation of a nucleus for all the systems during heterogeneous nucleation. The induction time will then depend on the likelihood of
API molecules interacting with each other or with an excipient particle. As the number density of sites for heterogeneous nucleation (I) is higher than that for homogeneous nucleation () (Equation 42), it will more likely for the API to nucleate in the presence of an excipient surface compared to in its absence.
6.7.1.2. Molar solubility, density of nucleation points and interfacial energy
AAP’s higher molar solubility, compared to that of FF, increases AAP’s relative likelihood to form molecular clusters. Thereafter, in general terms the addition of excipient to any API solution will increase the number of functional groups that can molecularly interact with the API. Thus, in the presence of an excipient, in addition to interacting with other molecules of API in solution, API molecules can also interact with the functional groups located on the excipient’s surface. Taking into account that the density of FF molecules before excipient addition was very low (hence the observed long induction time for homogeneous nucleation), the increase in the number of excipient-based functional groups post-addition significantly enhanced a FF molecule’s likelihood of
227 | P a g e finding a complementary functional group with which to interact, thus lowering the induction time.
Additionally, Equation 40 shows the nucleation rate for heterogeneous nucleation.
Compared to the nucleation rate for homogeneous nucleation (Equation 39), this rate includes terms for ‘the number density of sites for heterogeneous nucleation’ (I) and the interfacial energy for heterogeneous nucleation (γ퐻퐸푇). More excipient was added to the
AAP system (75 g), than to the FF system (11 g) meaning thatI was higher in the AAP- excipient system. This, however, does not explain the more pronounced increase in the nucleation rate during the heterogeneous crystallization of FF compared to that of AAP.
Rather, this observation may be due to a greater reduction in FF’s interfacial energy when going from homogeneous to heterogeneous nucleation (i.e. γHET,FF << γFF) when compared with the corresponding reduction in the case of AAP (i.e. γHET,AAP < γAAP).
6.7.1.3. Wettability and surface topography of the excipients
Chapter 5 demonstrates the importance of the wettability and surface topography of
‘heterosurfaces’ in promoting nucleation. Reported studies have measured the contact angle of MCC and -Lac with water 62-63, which was found to be 67.6 ° and 61.7 °, respectively. However, there is a significant difference in the contact angle of MCC depending on its porosity which can vary depending on the supplier. As such, the difference in wettability together with the difference in surface topography between MCC and Lac may explain the longer induction time of AAP in the presence of MCC compared with that in the presence of -Lac.
228 | P a g e
6.7.2. Competition between nucleation and growth
(a)
(b) (c)
Scheme 1: The interplay between nucleation and growth during the crystallisation process: (a) the general scheme, (b) for AAP, and (c) for FF.
After the analysis of the nucleation and growth rates of AAP and FF from MeOH in the absence and in the presence of excipients the different regions of nucleation and growth are depicted in Scheme 1. In Scheme 1(a), a desirable zone (in terms of the enhanced bioavailability it engenders) is represented in green whereby small API particles are formed at slow growth rates and fast nucleation rates. Additionally, two intermediate zones in which
229 | P a g e either the nucleation or the growth rate is slow are represented in yellow. Finally, an undesired zone, in which big particles formed at fast growth and slow nucleation rates, is represented in red. In the absence of excipients AAP was found to have a moderately fast nucleation and growth rate. After the excipient addition, the AAP nucleation rate was slightly enhanced, whereas the growth rate of the nucleated AAP particles decreased. By contrast, FF in the absence of excipients had a significantly slower nucleation rate, but once nucleation had occurred the FF particles grew rapidly. After the excipient addition the FF nucleation rate was strongly enhanced, whereas the growth rate of the FF particles decreased.
It was observed that the slope of the % desupersaturation line (desupersaturation rate) did not significantly change in the absence compared to the presence of excipients, in both the FF and AAP system at the same conditions (Figure 103, Figure 109 and Figure 110). As the desupersaturation is consumed by nucleation and growth, similar desupersaturation rates but faster nucleation rates implies slower growth rates, thus supporting the results. Consequently, most of the supersaturation was driven by the nucleation event during the heterogeneous crystallization of both APIs in the presence of excipients.
There are some slight variations in the AAP and FF desupersaturation rates depending on the excipient used that are a consequence of the differences in the nucleation and growth rates.
Slower desupersaturation rates were observed for the crystallization of AAP in the presence of
MCC (100 % of desupersaturation achieved after 1.4 hour) compared to that in the presence of
-Lac (100 % of desupersaturation was achieved after 1 hour) due to slower nucleation, as the growth rate was similar in both cases. Additionally, for the crystallization of FF in the presence of -Lac, the desupersaturation rate (100 % of desupersaturation achieved after 15 minutes) was slower compared to that in the absence and in the presence of MCC (100 % of desupersaturation was achieved after 6 minutes) due to a slower growth rate, as the nucleation rate was the same in both cases.
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Going back to Chapters 3 and 4, it was found that by modifying the API loading and the crystallization temperature, the API crystals grew differently, in such a way that for the same contact time API particles were smaller at the combined conditions of low API loadings and low crystallization temperatures. However, no significant change in the desupersaturation rate was observed during API crystallization by decreasing the API loading or crystallization temperatures. Thus, for the same contact time a greater number of API particles grew to smaller sizes at slower growth rates, with the nucleation rate increasing as the growth rate decreased64.
It can be said that the addition of excipients expedites the nucleation rate but slows the growth rate. Additionally, being at the limit where there are enough nucleation points and good mixing can be ensured, by modifying both the API loading and the crystallization temperature, nucleation can be made the predominant mechanism during heterogeneous nucleation of APIs in the presence of excipients, without significant changes in the API desupersaturation rates.
6.8. CONCLUSIONS A heterogeneous crystallization concept in which two different APIs were crystallized in the presence of two selected excipients within a batch-crystallizer at 500 mL scale was investigated. The selected excipients, which can molecularly interact with both APIs, were found to reduce the induction time of the API-MeOH solutions. Heterogeneous nucleation could be very advantageous for a system in which the API has low solubility, large molecular volume, weak API-API molecules interactions and good functional group complementarity with the excipient surfaces, with the aim of increasing its nucleation rate. Additionally, nucleation can be made the dominant contributor to API desupersaturation over the growth during heterogeneous crystallization. This contribution can be modified by changing the API loading and the crystallization temperature.
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44. Southan, C.; Sharman, J. L.; Benson, H. E.; Faccenda, E.; Pawson, A. J.; Alexander, Stephen P. H.; Buneman, O. P.; Davenport, A. P.; McGrath, J. C.; Peters, J. A.; Spedding, M.; Catterall, W. A.; Fabbro, D.; Davies, J. A., The IUPHAR/BPS Guide to Pharmacology in 2016: towards curated quantitative interactions between 1300 protein targets and 6000 ligands. Nucleic Acids Res. 2016, 44 (D1), D1054-D1068. 45. Turnbull, D., Kinetics of Heterogeneous Nucleation. The Journal of Chemical Physics 1950, 18 (2), 198-203. 46. Gorti, S.; Forsythe, E. L.; Pusey, M. L., Growth Modes and Energetics of (101) Face Lysozyme Crystal Growth. Crystal Growth & Design 2005, 5 (2), 473-482. 47. Oxtoby, D. W., Nucleation of First-Order Phase Transitions. Accounts of Chemical Research 1998, 31 (2), 91-97. 48. Manuel Garcıá -Ruiz, J., Nucleation of protein crystals. Journal of Structural Biology 2003, 142 (1), 22-31. 49. Mersmann, A., Crystallization Technology Handbook. CRC Press: 2001. 50. Omar, W.; Mohnicke, M.; Ulrich, J., Determination of the solid liquid interfacial energy and thereby the critical nucleus size of paracetamol in different solvents. Crystal Research and Technology 2006, 41 (4), 337-343. 51. Freitag, K. Preparation of nanodispersions by antisolvent precipitation. lmu, 2010. 52. Fell, J. T.; Efentakis, E., The wetting of powders of acetylsalicylic acid, salicylic acid, phenacetin and paracetamol. Journal of Pharmacy and Pharmacology 1978, 30 (1), 538-541. 53. Tan, L.; Davis, R. M.; Myerson, A. S.; Trout, B. L., Control of Heterogeneous Nucleation via Rationally Designed Biocompatible Polymer Surfaces with Nanoscale Features. Crystal Growth & Design 2015, 15 (5), 2176-2186. 54. Kȩdzierski, M.; Wajnryb, E., Short-time self-diffusion coefficient of a particle in a colloidal suspension bounded by a microchannel: Virial expansions and simulation. The Journal of Chemical Physics 2011, 135 (16), 164104. 55. Danielli, J. F.; Riddiford, A. C.; Rosenberg, M. D., Recent Progress in Surface Science. Elsevier Science: 2013. 56. Sun, Q.; Harvey, J. A.; Greco, K. V.; Auerbach, S. M., Molecular Simulations of Hydrogen Bond Cluster Size and Reorientation Dynamics in Liquid and Glassy Azole Systems. J. Phys. Chem. B 2016, 120 (39), 10411-10419. 57. Janick, J., Horticultural Reviews. Wiley: 2010. 58. Smirnov, B. M., Cluster Ions and Van Der Waals Molecules. Taylor & Francis: 1992. 59. Zhang, X.; Zhang, Q.; Zhao, D.-X., Hydrogen Bond Lifetime Definitions and the Relaxation Mechanism in Water Solutions. Acta Phys.-Chim. Sin. 2011, 27 (11), 2547-2552. 60. Astley, T.; Birch, G. G.; Drew, M. G. B.; Rodger, P. M., Lifetime of a Hydrogen Bond in Aqueous Solutions of Carbohydrates. J. Phys. Chem. A 1999, 103 (26), 5080-5090. 61. Bai, D.; Chen, G.; Zhang, X.; Sum, A. K.; Wang, W., How Properties of Solid Surfaces Modulate the Nucleation of Gas Hydrate. Scientific Reports 2015, 5, 12747. 62. Rulison, C., Wettability studies for porous solids including powders and fibrous materials. Application Note 1996, (302). 63. Kiesvaara, J.; Yliruusi, J., The use of the Washburn method in determining the contact angles of lactose powder. International Journal of Pharmaceutics 1993, 92 (1), 81-88. 64. Genck, W. J., Temperature effects on growth and nucleation rates in mixed suspension crystallization. 1969.
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Conclusions
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It can be concluded that seed or any excipient with functional group compatibility with an
API accelerates its crystallization, lowering the induction time significantly. This decrease in the induction time could be due to a longer lifetime of the interactions between API molecules in solution and a solid excipient surface than among API molecules in solution.
In addition, it has been shown that using this heterogeneous nucleation of APIs onto excipients there are optimal ranges of maximum attainable API loading and crystallization temperatures capable of producing consistently small API particles at high levels of desupersaturation. By tuning process parameters for the heterogeneous nucleation of APIs in the presence of solid excipient carrier particles the API particle size can be controlled, thus allowing the dissolution properties of a poorly-water soluble drug, namely FF, to be modified.
The dissolution rate of FF crystallized in the presence of a selected excipient at conditions where small FF particles were produced was enhanced compared to that of FF crystallized in the presence of a FF seed and in the absence of any seed due to its reduced particle size.
Finally, the surface properties of ‘heterosurfaces’, i.e. their wettability and surface topography, and the mixing between the ‘heterosurfaces’ and the API solution were found have an effect on the promotion of the nucleation of APIs. High wettability of a polymer film is required for a reduction in the induction time. In addition, grooves on polymer surfaces help the promotion of both the nucleation and the attachment of APIs on the surface. Contact times of less than 5 minutes between a polymer surface and a seeded API metastable solution are required to create a homogeneously dispersed coverage of small API particles on the polymer surface. These polymer coupons containing attached API could be potentially used as medical devices with ancillary medical substances.
As an overall conclusion, it can be said that heterogeneous nucleation could be very advantageous for a system in which the API has low solubility, large molecular volume, weak
API-API molecules interactions and good functional group complementarity with the excipient
236 | P a g e surfaces, with the aim of increasing its nucleation rate. Additionally, nucleation can be made the dominant contributor to API desupersaturation over the growth during heterogeneous crystallization. This contribution can be modified by changing the API loading and the crystallization temperature.
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Dream big, work hard,
stay focused, and surround yourself with good people
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Acknowledgments
The work of this thesis has emanated from research conducted with the financial support of the
Synthesis and Solid State Pharmaceutical Centre, funded by Science Foundation Ireland (SFI) under Grant Numbers 12/RC/2275, as well as support from Bernal institute and Department of
Chemical Sciences at the University of Limerick.
I would like to express my deep gratitude to all the people who have helped, supported and inspired me during these four years. You helped making this adventure called PhD unforgettable. Especially to:
Professor Kieran Hodnett, Dr Sarah Hudson and Dr. Peter Davern, my research supervisors, for their patient guidance, enthusiastic encouragement and useful critiques of this research work. I would also like to thank Dr. Clare Crowley, for her advice, assistance and for reviewing early in the project, as well as for helping me with the use of some equipment. My grateful thanks are also extended to the technicians of the Bernal institute and Department of Chemical
Sciences for their help in running some equipment.
I would also like to thank the SSPC group members. You have made my PhD a fun experience.
Apart from the everyday scientific and non-scientific discussions, there are many other good reminds, including soccer on Fridays, weekend plans, conferences, trips, etc. that I will always remember with a smile in my face. You now have a friend to visit in Valladolid (my city) for
‘tapas y cervezas’, so keep practising your Spanish, although you are already quite fluent.
I wish to thank those outside of research: my parents, my sister, my niece and my friends back in Spain, for their support and encouragement throughout my study and for all of your visits and stays during the last 4 years. Time and distance mean so little when someone means so much. You have been helping me in the distance, encouraging me in every obstacle and
239 | P a g e celebrating every success. And as we always say: true friendship does not mean being inseparable; it means being separated and nothing changes.
Finally, to all the new people that I have met in Ireland during these years. You are the most important things that I am getting from this experience.
Muchas gracias a todos!
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