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High-throughput excipient screening using 384-well plates and a pipetting robot: assessing protein stability after
Daan Zillen1, Noa N van der Ploeg1, Ronald van Merkerk2
1Department of Pharmaceutical Technology and Biopharmacy, Rijksuniversiteit Groningen, Groningen Research Institute of Pharmacy, Groningen, the Netherlands.
Introduction:
Formulation research benefits from high-throughput excipient screening methods, considering the ever-growing excipient space. We investigate the use of 384-well plates as freeze-drying containers and for subsequent analyses, to screen the effect of excipients on the stability of proteins during freeze-drying.
Methods:
For both the preparation and analysis methods of a range of β-galactosidase formulations, an 8-tip pipetting robot was used. Formulations were lyophilized in 384-well plates, which were also used for subsequent enzymatic activity assessment, serving as an indication of protein stability.
Results:
Excipient screening revealed that threonine, histidine, arginine, sucrose, and trehalose enhance the recovery of the enzymatic activity of β-galactosidase compared to the protein freeze-dried in buffer without other excipients. Moreover, pullulan only showed a stabilizing effect when it was combined with low-molecular-weight excipients that by themselves were poor stabilizers, which was especially the case for serine and to some extent for valine.
Discussion:
There were no significant differences in enzymatic activity when comparing the automated 384-well plate freeze-drying method with a common in-vial method, while offering the added sustainability benefits of increased throughput, reduced workload, and lower protein and reagent usage. This approach might be suitable for the pre-selection of viable formulations.
