High-throughput excipient screening using 384-well plates and a pipetting robot: assessing protein stability after freeze-drying to pre-select viable formulations

Abstract

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.

Introduction

In 2023, 15 out of the 25 highest revenue drugs were biopharmaceuticals [Citation1], and this trend is likely to continue. Notwithstanding, process and storage stability remain a major obstacle during development of new products. Some common strategies to improve protein stability include, amongst others, modifying the solution’s pH, the concentration and choice of buffer components, and using surfactants to prevent interfacial adsorption [Citation2], as well as stabilizers such as amino acids, sugars or polymers to increase conservation of the protein’s native state [Citation3,Citation4]. Alternatively, protein stability can be improved by bringing active pharmaceutical ingredients (API) in the solid state, resulting in reduced degradation reaction kinetics due to reduced molecular mobility. To this end, freeze-drying is often used as a relatively gentle drying method. Nevertheless, for freeze-drying, excipients are also often necessary to improve protein stability. As such, over the past few decades, many excipients have been introduced to the market, resulting in virtually limitless combinations when developing a biopharmaceutical formulation. To illustrate, as of June 2020 there were 211 biopharmaceuticals approved by the FDA, represented by 665 different formulations, out of which 397 formulations used a unique combination of excipients [Citation5]. Besides the marketed excipients, there is also active research into new excipients and repurposing existing excipients for new roles, such as the use of amino acids as lyoprotectants [Citation6]. Due to the virtually limitless excipient combinations, sample sizes quickly become hard to manage through classical freeze-drying screening methods.

To minimize costs, time, and efforts spent during the development phase, high-throughput screening of excipients can be used as a strategy to narrow down to a list of viable freeze-drying pre-formulations for subsequent thorough characterization. Firstly, the freeze-drying process can be time-consuming and any method to shorten it increases screening throughput and scalability. Several studies have been published on freeze-drying sample volumes in the microliter range, using 96-well plates as sample containers, which could also be conveniently used during subsequent analyses [Citation7–11]. To take this a step further, Coussot and colleagues [Citation12] were, to the best of our knowledge, the first to freeze-dry using 384-well plates, although their work was focused on preparing freeze-dried well plate assays to detect antigens. To build on these works, in this study, we show that 384-well plates can also be used to screen the effect of excipients on protein stability during freeze-drying, namely the stability of β-galactosidase. To this end, we evaluated the stabilizing capacity of a range of excipient combinations comprising a polysaccharide and low-molecular-weight excipients in various ratios. Such combinations have been shown in prior studies to exhibit strong stabilizing potential [Citation13,Citation14].

To assess β-galactosidase stability, we used its enzymatic activity, before and after freeze-drying, as stability indicating readout, using a straightforward colorimetric assay. In addition to using 384-well plates, we employed a relatively short freeze-drying cycle and an 8-tip pipetting robot to handle all solution processing, as well as preparing and performing the enzymatic activity assays, to further increase throughput. Finally, the results of this proof-of-concept 384-well plate-based screening method were compared to those of a conventional in-vial method, to demonstrate its suitability for a preliminary selection of viable formulations.

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Materials

Bovine serum albumin (BSA), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), magnesium dichloride, arginine hydrochloride (denoted as arginine), L-histidine, L-serine, L-threonine, L-valine, and ortho-nitrophenyl-β-galactoside (ONPG) were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). Sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dihydrate were obtained from Merck (Rahway, NJ, United States). β-Galactosidase was obtained from Sorachim (Lausanne, Switzerland). Sucrose and trehalose dihydrate were gifts from DFE Pharma (Goch, Germany). Pullulan (average molecular weight 200–300 kDa, <10% mono-, di- and oligosaccharides) was a generous gift from Hayashibara (Okayama, Japan).

Zillen, D., van der Ploeg, N. N., van Merkerk, R., Poelarends, G. J., Frijlink, H. W., & Hinrichs, W. L. J. (2025). High-throughput excipient screening using 384-well plates and a pipetting robot: assessing protein stability after freeze-drying to pre-select viable formulations. Drug Development and Industrial Pharmacy, 1–7. https://doi.org/10.1080/03639045.2025.2584353

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