Application Note

High-throughput 96-well cocrystal screening
workflow for active pharmaceutical ingredients
Increasingly, poorly soluble drug candidates areentering development, which has made identifying
and selecting stable solid forms of an active pharmaceutical ingredient (API) with enhanced in vivo
absorption more difficult. While pharmaceuticalsalts are the most common choice for solid forms,
cocrystals are also known to improve “druggability”
characteristics such as solubility, dissolution, bioavailability and hydration stability. In addition, cocrystals have advantages in terms of their structure
diversity and the potential for intellectual property
protection.

freeslate system configured for small molecule
preformulation and 96-well crystallization assembly with universal substrate to devise the cocrystallization HTR workflow shown in Figure 1. Following cocrystal formation and analysis by x-ray
powder diffraction (XRPD), the solubility and solid/
solution stability of the cocrystals were measured.
The HTR workflow employed solvent mediatedsonic blending to screen for cocrystal candidates
with optimal properties. This application note
describes the development and validation of a 96well high-throughput cocrystallization workflow
using solvent mediated sonic blending by H. Tanand coworkers.

Since there are a large number of potential coformers for a given API, manual screening andselection of cocrystals is labor intensive, time consuming and requires large amounts of materials.
In addition, it is known that pure cocrystal formation is favored when the API and coformers have
similar saturation levels in the screening solvent.
Therefore, a systematic and expansive explorationof a variety of solvents (e.g., aqueous, organic andaqueous/organic mixtures) increases the chanceof forming pure cocrystal during the screen. Combining many solvents and coformers can resultin an experimental design that would be overwhelming to perform manually. A high-throughput
research (HTR) approach, coupled with rational
experimental design, offers significant advantages
and opens up new opportunities for exploring thisexperimental space.

Use of two model cocrystals
to develop a cocrystallization
workflow employing solvent
mediated sonic blending
Since the crystal structure hydration stability at 98%
relative humidity (RH) and solution stability are known
for both 2:1 caffeine-oxalic acid (CO) and theophyllineoxalic acid (TO) cocrystals, they were used asmodels for the development of a sonic blending HTR
cocrystallization workflow on a freeslate system
configured for small molecule preformulation. To
identify the optimal solvent volume to solid mass ratio,
cocrystals of TO and CO were made using varying
volumes (10, 20 and 40 µL) of six screening solvents:
water, ethanol, 50:50 water:ethanol, 1,4-dioxane,
50:50 dioxane:water and ethyl acetate. To do this,
1 mg of the coformer (oxalic acid) was placed into
each well of a 96-well crystallization assembly withglass universal substrate. After addition of oxalic acid,caffeine or theophylline was dispensed into each

HTR has been widely applied to solubility, crystallization, salt and polymorph screening, but
high-throughput cocrystal screening has beenused on a limited basis due to limitations of cocrystallization techniques at a miniaturized scale.
Scientists at Amgen utilized Unchained Labs’
1

HIGH-THROUGHPUT 96-WELL COCRYSTAL SCREENING WORKFLOW

Selectcocrystalformers

Note: The steps in the dotted box
can be performed in 96-well plate
formatSelectsolvents

Dispense
cocrystal formers(1 mg each)

Add API
in appropriate
molar ratio

Add solvents(10 µL per sample)

Blend by
1) Vortexing
2) Sonicating
3) Centrifuging

Evaluatesolubility andstability in water

Evaluate
solid hydration
stability

Identify
cocrystal hitsby XPRDDry at ambient

Furtherevaluatecocrystal
properties

Selectcocrystalcandidates

Figure 1: Diagram of 96-well high-throughput cocrystal screening workflow with solvent mediated sonic blending
and on-plate solid/solution stability characterization1.

well of the assembly using a targeted molar ratio of
2:1 oxalic acid:API. The assembly was sealed and 10,20 or 40 µL of screening solvent was added to eachwell via a piercing needle. After the screening solvent
was added, the assembly was agitated at 3,000 rpm
for 30 minutes using the on-deck vortexer. Thecrystallization assembly was then removed from thefreeslate and sonicated in a water bath (130 W, 40kHz) for 60 minutes.

and excess solvent in each well was removed bywick-drying with comb filter paper. The crystallization assembly was dismantled and the cocrystals
were analyzed by XRPD directly on the universal
substrate. Following XRPD analysis, the hydration
stability of the cocrystals was measured by placing
the glass universal substrate with cocrystals into a
desiccator containing a saturated solution of potassium sulfate to create a 98% relative humidity environment. Cocrystals were incubated at 25 °C andanalyzed at 4, 7 and 14 days by XRPD. Cocrystals
that were stable for 14 days at 98% relative humidity
were subjected to a 24 hour water slurry experiment
to rank order their solubility and stability in water.

During sonication, the bath temperature was notallowed to exceed 40 °C. Post sonication, the assembly was centrifuged at 1,650 rpm for 10 minutes andleft to stand overnight at room temperature. The
next day, the crystallization assembly was opened

2

HIGH-THROUGHPUT 96-WELL COCRYSTAL SCREENING WORKFLOW

High-throughput solubility
measurement of caffeine,
theophylline and oxalic acidthe solid forms from the solubility screen to thestarting forms. After removal of the supernatant tomeasure solubility, the remaining solvent in eachwell of the crystallization assembly was aspirated
and discarded. The solid was wick-dried withcomb filter paper and analyzed by XRPD directlyon the glass universal substrate. Table 1 shows
the solubility results and remaining solid forms ofcaffeine, theophylline and oxalic acid found under
the cocrystal screening conditions.

To measure the solubility of caffeine, theophylline
and oxalic acid in the various screening solvents,
50 mg of caffeine, theophylline or oxalic acid and500 µL of the screening solvent were dispensed
into each well of the crystallization assembly using
the freeslate. The crystallization assembly wassealed, removed from the deck, sonicated for 60
minutes, equilibrated at room temperature until
the next day and centrifuged. Next, 100 µL of
supernatant from each well was transferred into
tared glass vials in a separate 96-well plate. Vials
were vacuum-dried and weighed with a resolutionof 0.01 mg. The solubility of caffeine, theophylline
and oxalic acid ranged from 3.9 mg/mL to over
100 mg/mL at room temperature. In addition
to solubility, the Amgen scientists compared
Solubility(mg/mL)/
residue form

WaterCaffeine

HTR cocrystallization resultsusing solvent mediated sonicblending
The objective of this project was to develop anHTR cocrystallization workflow using solvent
mediated sonic blending. Table 2 shows theresults of the cocrystal screen at different

Ethanol

Water:
Ethanol(50:50)

1,4-Dioxane

Dioxane:Water(50:50)EthylAcetate

24.1/CM*

6.0/C

30.9/CM*

18.7/C32.4/CM*

7.2/C

Theophylline

7.0/TM

3.9/T

18.0/TM

4.7/T

18.2/TM

4.6/T

Oxalic acid

93.9

>100>100>100>100>100Table 1: Solubility and residual solid forms of caffeine, theophylline and oxalic acid in the screening solvents.1 C: caffeineanhydrate, CM*: caffeine monohydrate, gradually converted to caffeine anhydrate when exposed to ambient conditions,
T: theophylline anhydrate, TM: theophylline monohydrate.

Reactant

Caffeine (4.0 mg)

Theophylline (4.3 mg)

Water

Ethanol

Water:
Ethanol(50:50)

1,4-Dioxane

Dioxane:Water(50:50)EthylAcetate

10CO

CO

CO

CO

CO

20CO

CO

CO

CO

CO

40

CO

CO

CO

CO

CO

10TO+TM

TO

TO+TM

T+TO

TO

20

TM

T

TMTO

TO

40TM

T

TM

TO

TO

Table 2: Cocrystal screening results of caffeine and theophylline with 1 mg oxalic acid.1 CO: caffeine-oxalic acid cocrystal,
T: theophylline anhydrate, TM: theophylline monohydrate, TO: theophylline-oxalic acid cocrystal.

3

HIGH-THROUGHPUT 96-WELL COCRYSTAL SCREENING WORKFLOW

volumes of screening solvent. While it has beenreported that caffeine anhydrate converts to a
known monohydrate in the presence of water, CO
cocrystals formed in quantitative yield with all thesolvents and solvent volumes tested. However,for TO, depending on the solvent used and thesaturation level of theophylline in the solvent,different cocrystals were formed.

was consistent with previously reported resultsfor CO and TO cocrystals under water slurry
conditions. Based on the solubility data, both
caffeine and theophylline were solid-saturated
under the cocrystal screening conditions andoxalic acid was completely dissolved under all
conditions except for 10 µL water. Therefore, 10 µL
was selected as the solvent volume for the sonicblending workflow because that volume would
maximize the saturation level of oxalic acid in thescreening solvent and enhance the chance offorming pure cocrystals.

While pure TO cocrystals formed under anhydrous
conditions (e.g., dioxane and ethyl acetate), a
mixture of TO cocrystals and TM were formed
when the screening solvent contained water. This

Reactant: AMG 517 Form A

Water

Water:
Ethanol(50:50)Ethanol

1,4-Dioxane

Dioxane:Water(50:50)EthylAcetate

Sorbic Acid
Citric Acid
Fumaric Acid
Maleic Acid
L-Malic Acid
L-Tartaric Acid
Succinic Acid
AMG 517(Form A)

Glutaric Acid
Adipic Acid
Glycine
L-AsparticAcid
L-GlutamicAcid
Benzoic Acid
Benzamide
Nicotinamide

Table 3: Cocrystal screening results of AMG517 with 15 coformers.1 AMG 517 or AMG 517 and conformer were blended with 10 µLof solvent. Formation of the cocrystal was detected by XRPD and hydration stability of the cocrystals formed was monitored by
XRPD as the samples were subjected to 98% RH for two weeks. A blank cell indicates no cocrystal formed after blending. The
symbol indicates formation of the cocrystal which was stable at 98% RH. The symbol indicates formation of the cocrystal
which was not stable at 98% RH.

4

HIGH-THROUGHPUT 96-WELL COCRYSTAL SCREENING WORKFLOWValidating an integratedhigh-throughput cocrystal
screening workflow
Amgen compound AMG517 was used to validate
the HTR cocrystal workflow with solvent mediated sonic blending. Ten of the fifteen coformers
selected were known to form cocrystals by either
solvent-drop grinding or solution crystallization.
Cocrystallization with the other five coformers had
not been attempted before. The results using thesame set of six solvents are shown in Table 3. For
the 15 coformers screened, all 10 previously identified cocrystals were found and two new cocrystals
were discovered. The two coformers that formed
new cocrystals were nicotinamide and fumaricacid. Once XRPD analysis was completed, thecocrystals on the glass universal substrate weretreated to 98% relative humidity to evaluate their
hydration stability. Three cocrystals were unstable
and were not analyzed further. The remaining cocrystals were stable for two weeks and were studied further for their solution stability and solubility.

analysis, they can subsequently be subjected toadditional experiments. In this case, the hydration
stability of the cocrystals was evaluated by placing
the glass universal substrate with cocrystals into a
desiccator containing a saturated solution of potassium sulfate to create a 98% relative humidity
environment. Cocrystals were analyzed after 4, 7
and 14 days by XRPD and those that demonstrated a hydration stability of greater than two weeks
were studied further. Amgen scientists validated
this workflow using a poorly soluble compound,
AMG517 and 15 coformers. Ten previously known
cocrystals were identified and two new cocrystals
were discovered during the validation. Nine of thetwelve cocrystals were stable for two weeks under
98% relative humidity and were studied further for
their solution stability and solubility. By using an
automated HTR approach, multiple APIs, coformers and solvents were studied under the same
cocrystallization conditions simultaneously, which
saved time and labor, minimized API usage andenabled the exploration of an expanded designspace.

Conclusion

References

Using the freeslate system, crystallization assembly with glass universal substrate and TO and CO
as model cocrystals, Amgen scientists devised an
HTR cocrystallization workflow based on solvent
mediated sonic blending. For XRPD analysis,
manual manipulation of delicate cocrystals wasavoided by analyzing the crystals directly on theglass universal substrate of crystallization assembly. Since the cocrystals are not affected by this

1 Reprinted from International Journal of Pharmaceutics, Vol 441, Issue 1-2, Van Luu, JananJona, Mary K. Stanton, Matthew L. Peterson,
Henry G. Morrison, Karthik Nagapudi, Helming
Tan, High-throughput 96-well solvent mediated sonic blending synthesis and on-plate solid/solution stability characterization of pharmaceutical cocrystals, p. 356–367, (2012) withpermission from Elsevier

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