Application Note

An automated, high-throughput approach togenerating cocrystals with solvent-drop grinding (SDG)
Introduction

frac­tion of the experimental space. An automated
SDG method would eliminate the labor-intensive
and time-consuming steps and, when combinedwith a rational high-throughput approach,would enable exploration of a much broader
experimental space. In this appli­cation note, we
describe the development of an automatedhigh-throughput method for cocrystal screening
with Unchained Labs' freeslate system for small
molecule preformulation.

Increasing demand for drug candidates has expanded the range of exploration beyond modifications of ionizable active pharma­ceutical ingredients (APIs). As a result, there is a growing need for
new approaches to polymorphism studies, since
screens that tar­get pharmaceutical salts are limited to this category of materials. Cocrystalization
techniques are an emerging alternative approachfor weakly ionizable or non-ionizable drug candidates. Cocrystal­ization is a particularly attractive
approach because virtually all APIs are potentiallycapable of cocrystal formation. More impor­tantly,cocrystals are known to improve the “druggability”
charac­teristics of an API, such as solubility, bioavailability and stability.1

Experimental conditions
The goal of the study was to create an automatedprocess to replicate the manual method of cocrystal
formation by SDG of glutaric acid/1,2-di(pyridin-4yl)
ethane with MeOH (Figure 1) as grinding solvent.

There are many methods for forming cocrystals,
but solvent-drop grinding (SDG) is the preferredapproach due to a higher prob­ability of generating
cocrystals.1 To increase the chances of formingand selecting optimal cocrystals, a systematicand expansive screening of variables including
different coformers, solvents and solvent
combinations is required. Currently, most SDG
methods are performed manually and involvea number of tedious and time-consuming steps
such as weighing, mortar and pestle grinding
and manual transfer for x-ray diffraction (XRD)
analysis. While some attempts have been madeto automate the method using mechani­cal millingwith steel balls, the compound loading andanalytics are still manual. The time and effort
required for these manual methods make it
difficult — if not impossible — to cover a meaningful

Manual method
Solvent-drop grinding involves adding bothcoformers and a solvent to a mortar and manuallygrinding with a pestle. Due to time constraints
resulting from the manual mortar and pestle
grinding method, no experimental variables otherthan the number of grind­ing cycles were explored.

OO

HOOH

Glutaric AcidN+SDG
MeOHOHONOOH

CocrystalNN

1,2-di(pyridin-4-yl)ethane

Figure 1: Cocrystal formation by SDG of glutaric acid/1,2-di(pyridin-4yl) ethane with MeOH as grinding solvent.

1AN AUTOMATED, HIGH-THROUGHPUT APPROACH TO GENERATING COCRYSTALSRow

MeOHµL

Total materialloadingA

1010 mg

20 mg

B2010 mg

20 mg

C1010 mg

20 mg

D2010 mg

20 mg

E1010 mg

20 mg

F2010 mg

20 mg

G1010 mg

20 mg

H2010 mg

20 mg

# Steelballs

Steelball size

2Small(2.4 mm)
31Medium(3.9 mm)
2Table 1: Screening conditions for development of an automated SDG method. Typicallyscreens would be performed in an 96-well plate format to evaluate 96 conditions inparallel, but this test case only evaluated 16 conditions in a 96-well plate.

Automated method

The experiment designs are stored in the LEA
database and then used by Automation Studio™
to control the system.

The first step in developing an automated method
was to evaluate which combination of variables
would yield full conversion to the desired cocrystal.
The experimental design is shown in Table 1. Typically 96 conditions could be screened at once, but
for this test case only 16 conditions were screened
in a 96-well plate.

Equipment
The SDG method was automated using a freeslate
system (Figures 3–4), which includes all thecapabilities required for this process, including
liquid and solid dispensing, vortexing, weighing
and crystallization using a proprietary crystallizer
assembly (Figure 4).

Variables and experimental conditions• Material loading: [1:1 molar ratio of glutaric acid:
1,2-di(pyridin-4-yl)ethane]

Workflow––10 mg total: 4.2 mg of glutaric acid and 5.8
mg of 1,2-di(pyridin-4-yl)ethane

An overview of the automated workflow is shownin Figure 5.

––20 mg total: 8.4 mg of glutaric acid and 11.6
mg of 1,2-di(pyridin-4-yl)ethane

1 Solid dispense
The 96-well crystallizer assembly is automatically moved to the on-deck balance, and therequested amount of each coformer (glutaricacid and 1,2-di(pyridin-4-yl)ethane) is dispensed (Figure 6).

• Size and number of steel balls for grinding:––Small size (2.4 mm): 2 vs. 3––Medium size (3.9 mm): 1 vs. 2• MeOH (grinding solvent) amount: 10 μL vs. 20 μL

2 Addition of steel balls
Steel balls were added manually to the crystallizer assembly.

• Grinding condition: Vortex with steel balls at
900 rpm for one hour at room temperature
Experiment design

3 Liquid dispense
The plate is sealed and the freeslate adds
MeOH as the grinding solvent to the wells containing the coform­ers and steel balls (Figure 7).

The first step in the automated workflow isto setup the experiment in Library Studio®,which is part of Unchained Labs' Lab Execution
and Analysis (LEA) software suite (Figure 2).
2

AN AUTOMATED, HIGH-THROUGHPUT APPROACH TO GENERATING COCRYSTALSRow

MeOH µL

Total material loading

A1010 mg

20 mg

B2010 mg

20 mg

C1010 mg

20 mg

D2010 mg

20 mg

E1010 mg

20 mg

F2010 mg

20 mg

G1010 mg

20 mg

H2010 mg

20 mg

# Steelballs

Steel ballsize

2

Materials
and parameters

Small(2.4 mm)3

1Medium(3.9 mm)
2

Experimental
steps

Figure 2: The experimental design is entered into Library Studio. The specified recipe includes both chemical (names and amounts) andprocessing (vor­texing) steps in the order they will be executed.

Figure 3: The freeslate system is a fully configurable, automatedsystem for preparing, processing and testing chemically complex
samples and mixtures. The freeslate system can be used to automate SDG cocrystal generation using the system’s liquid and solid
dispensing, vortexing and weighing capabilities.

Figure 4: The crystallizer assembly is composed of a 96-wellplate, a glass or quartz plate (universal substrate™) and disposable liner inserts. The steel balls and the compounds will bedeposited in the liner insert. O-rings at the bottom of the insert
will ensure that the material in each well remains isolated. The
cocrystals will be formed on the universal substrate, which canbe removed for analytical testing without further manipulationof the cocrystals.

3

AN AUTOMATED, HIGH-THROUGHPUT APPROACH TO GENERATING COCRYSTALS

1Soliddispense

2
Addition ofsteel balls

34

Liquiddispense

Grinding

5Preparation
for analytics

6

XRD
analysis

Figure 5: Automated method workflow. Steps 1, 3 and 4 are automated by the freeslate for small molecule
preformulation.

AB

HopperFigure 6: (A) The deck of the freeslate with the crystallizer assembly in the on-deck balance. (B) The solid dispensing arm with hopper
containing powder to be dispensed.

Results

4 Grinding
The freeslate vortexes the crystallizer assem­blyat 900 rpm for one hour at room temperature.

Manual method
The XRD results from the manual method areshown in Figure 9. As the desired cocrystal forms,
the peaks related to the individual coformers
gradually disappear over four grinding cycles.
After four grinding cycles, the individual coformer
peaks disappear, indicat­ing complete formation of
the cocrystal.

5 Preparation for analytics
The steel balls are manually removed using a
96-pin magnetic retriever and the crystallizer
assembly is opened as shown in Figure 8.
6 XRD analysis
After the experiment is complete, the universal
substrate con­taining the cocrystals is readyfor analysis by the XRD. The first step is to run
the mapping protocol. This protocol maps theloca­tion of the cocrystals in each well and saves
this information in the LEA database. Once theplate is mapped, the XRD protocol is run. The
XRD will scan each well in the specified locationand store the results in the LEA database.

Automated methodLEA provides complete data management bycollecting and con­solidating data from a widevariety of sources into a single Library ID. For thisautomated SDG method, the Library Studio design,experimental data from the freeslate and analytics
from the Bruker XRD are linked together, allowing
easy data viewing and the ability to track back to4

AN AUTOMATED, HIGH-THROUGHPUT APPROACH TO GENERATING COCRYSTALS

A

Figure 8: (A) Steel ball removal. (B) Opening the crystallizer
assembly.

Figure 7: The freeslate automatically dispenses the required
amount of solvent to the plate.

Lin (Counts)

34000
32000
30000
28000
26000
24000
22000
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0B

Cocrystal peaks

Starting material

4 grinding cycles
3 grinding cycles
2 grinding cycles
1 grinding cycle
Bis-pyridylethane
Glutaric acid

510

15

20

25

30

35

40

2-Theta - ScaleOO

HOOH

Glutaric AcidN+SDG
MeOH

OHON

O
OH

CocrystalNN

1,2-di(pyridin-4-yl)ethane
Figure 9: XRD data from the manual SDG method demonstrates that after four grinding cycles, cocrystal formation
was complete.

5AN AUTOMATED, HIGH-THROUGHPUT APPROACH TO GENERATING COCRYSTALS

Comparison of the optimized conditions (10 mg
total loading, 20 μL of MeOH, with two mediumsteel balls) with the original materials indicatesexcellent conversion to the desired cocrystal as
shown in Figure 13.

the initial experimental design and conditions, as
shown in Figure 10. Data analysis is performed withSpectra Studio™, which allows easy overlay andcomparison of the XRD data.
Using this approach, the data was analyzed todetermine the best conditions for creating cocrystals using the SDG method. The effects ofmaterial loading and volume of grinding solventsare compared in Figure 11. In all cases, the original
coformer peaks are less intense, indicating cocrystal formation. The lower intensity of the original
coformer peaks indicates more complete cocrystalformation. Comparing the four conditions showsthat less total material loading (10 mg) with more
grinding solvent (20 μL) pro­vides more complete
cocrystal formation.

Conclusion
Based on the results of the test cases shown here,
SDG cocrystal screening can be automated using
Unchained Labs' freeslate system configured forsmall molecule preformulation. This automatedmethod clearly identified the impact of different variables on cocrystal formation as well as
the best condition for cocrystal conversion. After
analyzing the XRD data, the following conclusions
were clear:• More steel balls and larger size steel balls resulted in more com­plete cocrystal formation

Comparing the data from the four conditions thatused 20 μL MeOH and a total material loading of
10 mg, we can evaluate the effect of the numberand size of the steel balls on cocrystal forma­tion.
The overlay of these four traces with the original coformers is shown in Figure 12. In this case,trace H6 has a more complete cocrystal formation and the least amount of individual startingma­terial peaks, indicating that more steel balls
and larger size steel balls lead to more effective
cocrystal formation.

• The best cocrystal conversion was seen with
20 μL of MeOH, 10 mg total loading, and two
medium size steel balls (Figure 12, condition H6)• Combined with the other conditions, vortexing at
900 rpm for one hour provides sufficient grinding
to generate cocrystals

Figure 10: LEA software suite links initial experimental design to results, allowing for easy dataviewing and sample tracking.

6

AN AUTOMATED, HIGH-THROUGHPUT APPROACH TO GENERATING COCRYSTALS

450
Original confomer peaks

440

6
MeOHµL

7Total materialloading400

Row360C1010 mg

20 mg

D2010 mg

20 mg

320280#Steelballs

Steelball size

3Small(2.4 mm)

D7240
D6200

C7160
120

C680
Bis-pyridylethane

400Glutaric acid

510152025303540

Figure 11: Comparison of total material loading and volume of grinding solvent. The original coformer peaks are less intense indicating
cocrystal formation. Of the four wells compared, the conditions in well D6 (10 mg of material and 20 μl of grinding solvent) provides themost complete cocrystal formation.

400

6Original confomer peaks

360

MeOHµL

Total materialloading#Steelballs

B2010 mg2

D2010 mg

3F

2010 mg1

H2010 mg

2Row

320
280
H6240
200

F6160

D6120

Steelball size

Small(2.4 mm)

Medium(3.9 mm)

B680Bis-pyridylethane

400Glutaric acid

510152025303540Figure 12: Evaluation of the effect of the number and size of steel balls on cocrystal formation with 20 μl of grinding solvent and 10 mg ofmaterial. H6 has the least amount of starting material indicating more complete cocrystal formation demonstrating that more and larger
size steel balls lead to more effective cocrystal formation.

7AN AUTOMATED, HIGH-THROUGHPUT APPROACH TO GENERATING COCRYSTALS

260240220200180160140120
H61008060

Bis-pyridylethane

40200Glutaric acid510152025303540Figure 13: Comparison of optimized method conditions with starting materials. The optimized condition (10 mg total loading, 20 μL of MeOH, with two medium steel balls) has
little to no peaks from the original starting materials indicating excellent conversion to thedesired cocrystal.

This automated method can now be used for fullplate screening in­volving novel APIs and a broad
spectrum of coformers and grinding solvents.
By automating SDG, it is possible to eliminate
the labor intensive steps, reduce material andsolvent usage and quickly implement a truly
high-throughput approach to the engineering ofpharmaceutical cocrystals.

experiments can be completed on a single
platform. Data from birefringence, X-ray powder
diffraction and Raman spectroscopy can be easily
linked to reaction conditions and solvent systems
resulting in rapid under­standing that can inform
sound scientific decision making.

Using a freeslate system, a scientist can ac­celerate
polymorph screens, finding suitable crystalline
forms faster while reducing the risk of late stage
failure from emergence of new metastable forms.
With automated heating, cooling and mixing,
SDG, slurry, precipitation, cooling and evaporation

1 High-throughput 96-well solvent mediated sonicblending synthesis and on-plate solid/solution
stability characterization of pharmaceuticalcocrystals, Luu et al., International Journal of
Pharmaceutics, 441, (2013), 356–364.

References

Unchained Labs
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Pleasanton, CA 94566
Phone: 1.925.587.9800
Toll-free: 1.800.815.6384
Email: info@unchainedlabs.com© 2016 Unchained Labs. All rights reserved. UnchainedLabs is a registered trademark. All other brands orproduct names mentioned are trademarks owned bytheir respective organizations.

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