High-throughput 96-well cocrystal screening workflow for active pharmaceutical ingredients

Significance of the topic

The identification of stable and soluble solid forms of active pharmaceutical ingredients (APIs) is critical for enhancing in vivo absorption and drug development success. Cocrystals offer advantages over traditional salts by improving solubility, dissolution rate, bioavailability and solid-state stability while providing intellectual property protection and structural diversity.

Objectives and study overview

This work aimed to develop and validate a high-throughput (HT) 96-well cocrystal screening workflow using solvent-mediated sonic blending. Initial development employed two well-characterized model systems (caffeine–oxalic acid and theophylline–oxalic acid) to optimize solvent selection, volume and processing parameters. Validation was performed on a poorly soluble Amgen compound (AMG517) screened against 15 coformers.

Methodology and instrumentation

A 96-well crystallization assembly fitted with a glass universal substrate was mounted on an Unchained Labs Big Kahuna platform configured for small-molecule preformulation. Key workflow steps included:

• Dispensing 1 mg coformer and API at a 2:1 molar ratio into each well
• Adding 10 µL screening solvent (water, ethanol, water/ethanol 50:50, 1,4-dioxane, dioxane/water 50:50, ethyl acetate)
• Vortex mixing at 3 000 rpm for 30 min then sonicating (130 W, 40 kHz) in a water bath for 60 min (temperature ≤ 40 °C)
• Centrifugation at 1 650 rpm for 10 min, overnight standing, wick-drying and direct on-plate XRPD analysis
• Hydration stability testing at 98 % relative humidity for up to 14 days
• High-throughput solubility measurements via addition of excess API/coformer, sonication, centrifugation and gravimetric analysis of dried supernatant

The main instruments included:

• Unchained Labs Big Kahuna HT platform
• On-deck vortex mixer
• Ultrasonic water bath
• Benchtop centrifuge
• X-ray powder diffractometer (XRPD)

Main results and discussion

For caffeine–oxalic acid (CO) cocrystals, pure CO formed quantitatively across all six solvents and volumes. In theophylline–oxalic acid (TO) trials, pure TO cocrystals predominated under anhydrous conditions, while mixed TO and theophylline monohydrate (TM) appeared in water-containing solvents. Ten microliters was identified as the optimal solvent volume to maximize oxalic acid saturation and pure cocrystal yield.

Solubility screening showed API and coformer saturation levels ranging from 3.9 mg/mL to > 100 mg/mL depending on solvent, supporting solvent selection for cocrystallization.

Validation with AMG517 and 15 coformers recovered all ten known cocrystals and uncovered two new forms (with nicotinamide and fumaric acid). Of twelve cocrystals formed, nine remained stable at 98 % relative humidity for two weeks.

Benefits and practical applications

The integrated HT workflow enables rapid and material-efficient exploration of large solvent and coformer spaces. Direct on-plate XRPD avoids delicate crystal handling, reducing analysis time and risk of sample disruption. This approach accelerates API solid-form selection, minimizes compound consumption and supports simultaneous screening of multiple variables.

Future trends and opportunities

Emerging directions include:

• Integration with in situ spectroscopic techniques (Raman, IR) for real-time cocrystal monitoring
• Coupling HT crystallization with automated solubility and dissolution assays
• Application of machine learning for predictive coformer and solvent selection
• Expansion to multicomponent systems (polymorphs, salts, hydrates) and challenging APIs
• Miniaturization and continuous flow adaptations for even higher throughput

Conclusion

A robust 96-well solvent-mediated sonic blending workflow was developed and validated for HT cocrystal screening. Leveraging a combination of automated mixing, sonication, on-plate XRPD and hydration testing, this method accelerates discovery of stable cocrystals while conserving material and enabling extensive design-space coverage.

References

Van Luu J., Jona M., Stanton M.K., Peterson M.L., Morrison H.G., Nagapudi K., Tan H. High-throughput 96-well solvent mediated sonic blending synthesis and on-plate solid/solution stability characterization of pharmaceutical cocrystals. International Journal of Pharmaceutics. 2012;441(1-2):356–367.