Engenharia Farmacêutica II – Aula 1 e 2
CASE STUDIES
1. CASE STUDY 1 – “Crystallization of Mannitol”
The primary objective of this study is to examine solute crystallization behaviour in one specific system,
water−mannitol, which is directly relevant to pharmaceuticals and biopharmaceuticals (because of lyophilization
processes, …).
METHOD OF CRYSTALLIZATION: Freeze-drying (consider a cooling rate associated with the freeze and then a
heating stage associated with the drying).
A typical solution for freeze-drying contains an active ingredient and several inactive ingredients (excipients),
such as a buffer, a lyoprotector, and a bulking agent. Freezing of a pharmaceutical solution usually starts with
formation of hexagonal ice, while the phase behaviour of the solutes depends on multiple factors, including their
chemical nature, concentration, and temperature/time pathway. In addition to thermodynamic phase relationships,
kinetic aspects of mannitol crystallization are also of critical importance for industrial freeze-drying processes. For
example, temperatures for the onset and completion of crystallization during freezing/warming are of a major
practical significance, because they can provide a direct input into the design of freezing and primary drying steps for
freeze-drying manufacture.
NOTE: In many cases, crystallization of a solute is desirable, as it facilitates the freeze-drying process and also assures
a more consistent long-term stability profile. However, there are also potential problems associated with the
crystallization, including reduced product yield due to vial breakage during manufacture, instability during shelf life if
the crystallization during freeze-drying is incomplete, and formation of a crystalline hydrate (vs an anhydrous
crystalline polymorph); in the latter case, the product could fail the residual water content test, resulting in lot-to-lot
variability and in rejection of (already produced and expensive) drug product lots. Such crystallization-related issues
have been observed for freeze-dried products containing a common pharmaceutical bulking agent, mannitol.
Mannitol, a common pharmaceutical ingredient, exhibits complex polymorphism even in simple binary
mannitol/water mixtures, with four crystalline forms observed: at least three anhydrous polymorphs (α, β, δ) and a
hemihydrate (HH) observed in both frozen and freeze-dried states. In this investigation, time/temperature-resolved
synchrotron X-ray diffraction measurements are performed during freezing and thawing of mannitol/water
mixtures. Mannitol crystallization depends strongly on the cooling rate:
- if the cooling rate is lower than the critical cooling rate, the crystallization is initiated during cooling;
- if the cooling rate is higher than the critical cooling rate, mannitol remains amorphous during freezing and
crystallizes during subsequent heating above −30 °C.
A temperature−composition phase diagram is
constructed, reflecting eutectic and peritectic points and
lower-temperature equilibria involving mannitol
hemihydrate, hexagonal ice, and β-mannitol.
NOTE: Eutectic and peritectic points are specific points on
phase diagrams used to describe the behavior of two or
more components (usually elements or compounds) as
they undergo phase transformations, such as melting or
solidification.
Eutectic point - The eutectic composition is the composition of the mixture at the eutectic point, and the
eutectic temperature is the temperature at which this transformation occurs. At the eutectic point, a liquid
phase transforms directly into two solid phases (or vice versa) at a specific temperature and composition.
Peritectic point - The peritectic composition is the composition of the mixture at the peritectic point, and the
peritectic temperature is the temperature at which this transformation occurs. At the peritectic point, a solid
phase reacts with a liquid phase to form a different solid phase at a specific temperature and composition.
Peritectic points are less common than eutectic points and are often observed in materials where a particular
solid phase transforms into another solid phase via a reaction with the surrounding liquid phase.
The experiment:
The cooling rates were selected based on initial DSC experiments, which indicated mannitol crystallization
during cooling at 0.5 °C/min, whereas the higher cooling rate resulted in amorphous mannitol which crystallized only
during subsequent warming.
Results and Discussion:
Comparison of the experimental data with the phase diagram reveals that the mannitol crystallization
behaviour does not follow the equilibrium but appears to obey the Ostwald crystallization rule . Novel insights on
equilibrium and kinetics phase relationships in mannitol/water systems could lead to improved formulations and
manufacturing processes for pharmaceuticals and biopharmaceuticals.(…) The phase diagram graphically illustrates
the fact that a coexistence of three crystalline phases (two mannitol forms and ice) in a finite temperature range in a
binary system is prohibited at the equilibrium by the phase rule. The simultaneous observation of two mannitol
forms (both in this study and in multiple literature reports) therefore reflects a nonequilibrium nature of the system
under the conditions of the experiment. Nevertheless, metastable forms can be kinetically stable, in particular in the
case of mannitol, as also observed in this study.
Crystallization during cooling takes place at higher temperatures, above −20 °C, whereas crystallization
during heating commences above −30 °C, coinciding with a sharp decrease in the viscosity of the freeze-
concentrated solution. This difference in the crystallization temperature can have a major practical consequence for
manufacturing pharmaceutical formulations containing mannitol. Thermodynamics relationships in the mannitol−
water system are presented in the form of a T−x phase diagram. The unexpected finding of this study is the
observation of the two-step mannitol crystallization pattern, with the second step taking place close to the eutectic
temperature. The second step could be either due to preferred orientation of crystallites via a change in the crystal
habit, possibly as the result of Ostwald ripening, or to a real increase in the fraction of crystalline phase. While the
former appears to be the more plausible mechanism, the diffraction data do not support it, and therefore, an
increase in the crystallinity cannot be ruled out at this point. The second crystallization step would also be consistent
with a previous report of amorphous mannitol after annealing of the frozen solutions at −10 °C and with the
suggestion that mannitol could form two amorphous phases.
Discussion in Class:
What polymorphs would form if the solution with
20% mannitol concentration was cooled?
2. CASE STUDY 2 – “Crystallization of paracetamol in different polymorphs”
Paracetamol is known to have three polymorphs: stable form I (monoclinic), metastable form II
(orthorhombic), and unstable form III. These can be arranged from the most stable at standard temperature and
pressure as form I >form II > form III.
Below −120°C, the form II polymorph becomes more stable than
form I. Among the polymorphs, the form II notably contains well-defined
slip planes in the crystal structure, which would consequently facilitate
direct compression for tablet manufacturing.
QUESTIONS:
1. Which do you consider to be form I and form II judging by their temperature of fusion (see solubility curve) ?
First it is important to understand which form (I or II) is more soluble. According to the solubility curves, we
can conclude that the Form II (red curve) exhibits a higher solubility curve than the Form I (blue curve). Besides
that, in general, the solubility of a substance in a solvent tends to increase with increasing temperature for many
solid solutes. As you raise the temperature, it provides more energy to the molecules in the solvent and solute,
allowing them to overcome intermolecular forces and mix more readily.
On another hand, the fusion temperature, or melting point, is the temperature at which a substance
transitions from a solid to a liquid phase under a specific set of conditions (typically atmospheric pressure). This
transition involves breaking the intermolecular forces holding the solid together.
The relationship between solubility and fusion temperature is often complex and can vary from one
substance to another. However, some general trends can be observed: Higher Melting Point → Lower Solubility: In
some cases, substances with higher melting points may have lower solubilities at a given temperature because the
solid-state structure is more stable and less likely to dissolve in the solvent. Lower Melting Point, Higher Solubility:
Conversely, substances with lower melting points may exhibit higher solubilities at the same temperature because
the solid-state structure is less stable, making it easier for the molecules to disperse in the solvent.
Thereby, considering that Monoclinic has the highest melting point, it can be considered as the more stable
crystalline structure, which means it would exhibit a lower solubility in comparison to the Orthorhombic, which has a
lower melting point and therefore a higher solubility. As so, the Form I (blue curve, lower solubility) is the
Monoclinic since it has the highest melting point and the Form II (red curve, higher solubility) is Orthorhombic since
it has the lowest melting point.
2. Which form is easier to crystallize (justify)?
According to the professor, the Form II is easier to crystallize since it’s considered to be the first to
precipitate (has a lower melting point and a higher solubility curve which probably means that the energetic barrier
is smaller, which eases the crystallization process). However, Polymorph II is a metastable form, meaning it can
convert into the more stable Polymorph I over time.
3. How can you produce Form I without contamination from
form II?
A solution would be seeding, which in fact is a way to
control the crystallization process. As so, the seeding should be
above the blue solubility curve of Form I and below the red
solubility curve of Form II.
Note that the metastable zone is the region in the phase
diagram in which the supersaturation is sufficiently low to avoid
spontaneous nucleation. In seeded crystallization processes, the
seed crystals should be added within the metastable zone in order
to avoid spontaneous nucleation. The metastable zone is always
above the solubility line and then limited by a supersaturation limit
where there is a high probability of spontaneous nucleation.
4. How can you produce Form II without contamination from form I?
As so, the seeding should be above the red solubility curve of Form II.
5. Study the influence of particle size of 1 g of seeds, that can precipitate 100 kg of paracetamol ?
Not answered.
3. CASE STUDY 3 – “Crystallization of Ritonavir – the worst case scenario”
Polymorphism of a pharmaceutical agent needs to be well understood and carefully controlled during
product development. Ritonavir (RTV), a protease inhibitor marketed as Norvir, is an antiviral drug active against
human immunodeficiency virus (HIV). Being a strong cytochrome P450 3A (CYP3A) inhibitor, ritonavir interacts with
many other drugs that are primarily metabolized by CYP3A leading to mutually increased exposures.
Ritonavir was discovered at Abbott Laboratories and introduced to the market in 1996. Only one crystal form
of ritonavir was identified during development of the compound and 240 lots of Norvir capsules were produced with
no stability problems. In mid-1998, however, several lots of capsules failed the dissolution requirement and when
capsule contents were examined using microscopy and X-ray powder diffraction, a new polymorph was identified
that had greatly reduced solubility compared to the original crystal form. This new form, referred to as form II, is an
example of conformational polymorphism, which occurs when different conformational isomers of a compound
crystallize as distinct polymorphs. Within weeks this new polymorph began to appear throughout both the bulk drug
and formulation areas. Since the manufacture of Norvir semi-solid capsules formulation involved the preparation of
a hydroalcoholic solution of ritonavir which although not saturated with respect to form I was 400% supersaturated
with respect to form II, the sudden appearance and dominance of this dramatically less soluble crystal form made
this formulation unmanufacturable. Additionally, Norvir oral solution could no longer be stored at 2–8°C without the
risk of crystallization. These factors combined to limit inventory and seriously threatened the supply of this life
saving treatment for AIDS. It was necessary to immediately reformulate Norvir. It is well known in the
pharmaceutical industry, particularly among the solid-state community, for its unexpected development of a more
stable polymorph Form II, in mid-1998, leading to the discontinuity of the marketed formulation . Although finding
the most stable polymorph is important in drug product development as demonstrated by the RTV case,
discovering/characterizing all crystal forms and understanding the solid-state landscape are also important,
especially for subjects like structure-property relationship and polymorph prediction.
Form II of ritonavir was found to be both unusually stable and at the same time unusually difficult to
crystallize.
Although the polymorph (form II) corresponding to the “cis” conformation is a more stable packing
arrangement, nucleation of the polymorph requires the formation of a less stable conformation in solution,
clustering of this less stable conformation and subsequent crystal growth. This nucleation process, even in the
presence of form II seeds, is energetically unfavored except in highly supersaturated solutions. For this reason, form
II may have never been identified except for the coincidence of a solution which was very highly supersaturated (∼
400%) with respect to the polymorph and an unknown nucleation enhancer, possibly a related compound capable of
heterogeneously seeding the solution. No control of crystal form was required for ritonavir’s original formulation as
directed in the International Conference on Harmonization (ICH) Guidelines.
Twenty-four years after RTV Form II materialized, we herein report a new polymorph, Form III, discovered via
melt crystallization. We, serendipitously, discovered Form III in an effort to study crystal nucleation of amorphous
RTV. It is the least stable form, monotropically, among the three polymorphs. The three polymorphs have different
conformations and hydrogen bonding motifs.
1. What was the Influence of polymorphism on properties (the issues) in Ritonavir ?
In the case of Ritonavir, the different polymorphs can have distinct solubility profiles, with some polymorphs
being less soluble than others. This can impact the drug's bioavailability, as less soluble forms may not be as readily
absorbed in the body. Additionally, stability concerns arise because metastable polymorphs can convert into more
stable forms over time, leading to changes in the drug's properties during storage.
2. How was this problem controlled?
Form II may never have been identified except for the coincidence of a solution which was very highly
supersaturated (∼ 400%) with respect to the polymorph and an unknown nucleation enhancer, possibly a related
compound capable of heterogeneously seeding the solution. No control of crystal form was required for ritonavir ’s
original formulation as directed in the International Conference on Harmonization (ICH) Guidelines. As any other
crystallization process, here in this case the control is more focused on the crystallization process and its conditions.
4. CASE STUDY 4 – “Control of Carbamazepine Polymorphism During Rapid Cooling Crystallization
(HOMEWORK)
In pharmaceutical research, due to its diverse solid forms including five anhydrous polymorphs, one
dihydrate form and many solvates and cocrystals.
Monoclinic form III is thermodynamically stable below 70 °C, whilst forms I, II and IV are metastable,
though their crystallization kinetics can dominate in solution. It has been previously reported that both
supersaturation ratio and solvent can determine the polymorphic nucleation of CBZ. The result indicated that
higher supersaturation ratios lead to higher crystallization rate, thus favouring the formation of form II, which
is the least stable form of CBZ.
Scope of work: The aim of this work is to investigate the nucleation kinetics and transformation process of
CBZ polymorphs in different solvents in a series of supersaturation ratios. To prepare supersaturated solutions,
the solubility of stable form III in ethanol, 2-propanol, acetone, acetonitrile, nitromethane and toluene at
temperatures from 10 to 50 °C was measured using a gravimetric method. After that, the crystallization
experiments of CBZ were conducted in ethanol, 2-propanol, acetone, acetonitrile, nitromethane and toluene at
25 °C at different supersaturation ratios (S = 1.6 to 4.0), and magnetic stirring is considered during nucleation
and transformation. The nucleation domains of CBZ polymorphs were determined with respect to
supersaturation ratios and solvents.
Experimental apparatus: two water baths (F32,
Julabo), two glass jacketed crystallizers, two
thermometers, two magnetic stirrers (IKA) and two
glass vials, where dissolution and crystallization took
place. The purposes of the left bath and right bath are
to control the temperature for dissolving and
nucleating CBZ in the glass vials, respectively.
CBZ solutions with different supersaturation ratios
(S = C/ C*, C being the initial concentration and C* the
solubility at the crystallization temperature T) were
prepared by adding the required amount of CBZ form
III into the 25 mL glass vial which contained the intended amount of solvents, including ethanol, 2-propanol,
acetonitrile, nitromethane, toluene and acetone. The mixture was heated at 10 °C above the saturation
temperature for 5 h at a stirring speed of 300 rpm until the solids dissolved. The solution was then rapidly cooled
to the crystallization temperature to generate supersaturation by sending the left glass vial into the right
crystallizer. The glass vial was hermetically sealed to avoid any evaporation of the solvent during the experiment.
Crystallization experiments were carried out at initial supersaturation ratios from 1.6 to 4.0 at 25 °C. So basically,
the strategy was seeding with the stable form III (right), then heating up the solution for dissolution of the solids
and after that cool it to generate supersaturation (attention: the
seeds are added always in the cooling step).
Results:
It is presented the intrinsic dissolution rates of the different
CBZ polymorphs. We can conclude that the Form II (which is the less
stable polymorph) is the one that presents a higher dissolution rate.
On the contrary, and as it was expected, the Form III was the one
who presented a lower dissolution rate, which translates into its high
stability.
The crystallization of CBZ
polymorphs was conducted in ethanol, 2-
propanol, acetonitrile, nitromethane,
toluene and acetone in a supersaturation
range of 1.6 to 4 at 25 °C. All nucleation
experiments were carried out under the
observation of the naked eye and
microscope. Once nucleation was confirmed
by the naked eye, the microscope was used
to observe crystals and determine their
polymorphs immediately. The nucleation
domains of CBZ polymorphs are summarized
in Fig. 4.
In this Figure, we can identify the
solubility curve of CBZ in the solvent which is
being analysed and we can also observe the
appearance of the different CBZ polymorphs
according to the supersaturation ratios at 25
°C which is the temperature at which the
crystallization process occurs. We can have a
perception (in a comparative term) of the
concentration of each Form (or mixture of
Forms) that form according to that
supersaturation ratio.
QUESTIONS:
Your customer needs a carbamazepine formulation with high bioavailability, but with a particle size in the
range of 50 microns.
1. What solid form do you suggest to be manufactured based on the data that was made available (explain your
suggestion)?
If the costumer needs a CBZ formulation with high bioavailability, the solid form to be suggested must be
easily dissociated thus being available to do its therapeutic effect. Therefore, the solid form chosen must be
the one that exhibits the higher intrinsic dissolution rate, which is the Form II (the less stable form).
2. Select any two solvents of your choosing (in figure 4) and draw a possible solubility curve for Form II?
To facilitate our work, we must choose the two solvents whereas the Form II forms within a wider range of
supersaturation ratios and thereby, can be found in high concentrations more probably (this can be analysed by the
stars diagrams in each graphic). According to this, probably the two options more viable would be the following
solvents: 2-propanol and nitromethane.
The second aspect of this question concerns the drawing of a solubility curve of the Form II. As we well
know, the polymorphic forms in order to form they have to precipitate and thereby as in this case its concentrations
have to be higher than the solubility of the polymorphic form in the solvent. Besides that, there is always a
supersaturation limit as well, and the polymorphic form usually appears spontaneously immediately after that limit.
As so, a suggestion of this possible solubility curve and supersaturation limit will be presented in the 2-
propanol example:
Notice that, above the supersaturation limit of Form II and above the region of appearance of both Form II
and III, if we resort to those concentrations there is a minor probability to find the Form III of a mixture of Form II
and III. In fact, it is ideal to go even higher in concentrations to assure that whenever the polymorphs form and
consequently there is a decrease in concentration the Form III doesn’t appear.
3. Can the crystallization process be designed for the form that is most desirable? How would you do it (solvent,
concentration, temperatures)? (Discuss possible problems)
Yes, it can. The most desirable form according to the previous costumer it was the Form II (less stable and
thereby with higher dissolution rates). Choosing the solvent wisely, controlling the temperature properly and
controlling the concentration in the range that it is pretended, it is possible. Besides that, the seeding process always
helps with that, in this case with seeds of solid form II.
5. CASE STUDY 5 - Can you select two molecules that may co-crystallize with Aminopyrimidine?
(Draw a sketch of the hydrogen bonding)
There are many possibilities for this structure. By doing an analysis to its
structure and according to cocrystals formation theory (by the establishment of
hydrogen bonds), we can conclude that only the NH 2 group can establish those type
of interactions (selected in red). The other Nitrogen (integrated on the aromatic
ring) can’t because the maximum of interactions/bonds it could establish are
already fulfilled.
An example:
