Category: Formulation and Quality
Purpose: The disaccharide sucrose, alongside with trehalose, is a commonly employed excipient in liquid protein formulations which stabilizes proteins by preferential exclusion. However, despite the positive effect of sucrose on the protein stability, nanoparticle impurities (NPIs) have been recently discovered in pharmaceutical-grade sucrose. These NPIs, which are present in an amount of up to 109 particles/gram, can lead to false analytical results as they mimic protein aggregates, potentially leading to exclusion of lead molecules during early phase development. In addition, recent studies have suggested that NPIs can also cause damages of the API and an increased formation of protein aggregates. Therefore, the establishment of a purification process to eliminate NPIs from sucrose was the main goal of this study. To this end, nanoparticle impurities in various pharmaceutical‑grade sucrose products were evaluated, the comparative analysis of NPIs isolated from different sucrose sources was performed and the NPIs’ impact on protein stability was to be determined.
Methods: Sucrose products from various suppliers and the purified EMPROVE® sucrose were analyzed using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). For this, 10% sucrose solutions (w/w) were prepared in Milli-Q water. Solutions were measured before and after sterile filtration through a 0.22-µm PVDF membrane. For DLS, samples were measured three times with the Zetasizer Nano Series (Malvern, Herrenberg, Germany) at 25°C using the automatic attenuation selection and were detected via 173° backscatter detection. Peak size was based on viscosity of water as dispersant; particle area % was based on intensity. Data processing analysis model was set to general purpose. NTA was performed with a NanoSight LM20 (NanoSight, Amesbury, UK) using a pre-run volume of 0.5 ml and triplicate measurements with 0.1 ml sample volume. For the isolation of the NPIs, 50% sucrose solutions (w/w) were prepared in Milli-Q water. Diafiltration was done against Milli-Q water (6-fold volume exchange). Afterwards, morphology analyses via the Glucatell® assay, FT-IR, fluorescence and energy-dispersive X-ray (EDX) spectroscopy as well as the determination of the zeta potential were performed to characterize the NPIs and to potentially identify the NPIs’ composition.
Results: Figure 1 shows comparative DLS measurements of sucrose samples from two different suppliers alongside with the purified EMPROVE® sucrose. The first peak, observed at about 1-5 nm, can be identified as sucrose. The second peaks in figures 1A and 1B represent the NPIs with a size distribution of about 100‑500 nm. The reduction of the second peak in the EMPROVE® sucrose high purity samples suggests the successful establishment of a purification step to decrease the NPI contaminations in sucrose products which was furthermore confirmed by NTA results (data not shown).
DLS and NTA measurements both showed comparable NPI size distributions in cane and beet derived sucrose. However, differences in sucrose source could be observed in the number of particles as well as in their zeta potential. Beet derived sucrose showed a more negative zeta potential compared to cane derived sucrose with a rather neutral zeta potential. A variation in the NPIs’ surface charge can lead to various mechanisms of protein aggregation and may affect the API’s stability differently.
Additionally, the DLS results in figure 1 indicate a higher number of NPIs in cane derived sucrose (figure 1B) compared to the beet derived sucrose sample (figure 1A). Therefore, it can be suggested that the sucrose source does have an impact on the chemical composition and on the properties of the present NPIs. Fluorescence spectroscopy indicated two distinct patterns that may be caused from a mixture of different fluorophores including tryptophan, tyrosine or from the group of catechols. Furthermore, the presence of the two polysaccharides (dextran, observed via FT-IR measurements and β‑glucans obtained by the Glucatell® assay) was shown. Furthermore, it was described in literature that these NPIs also contain non-organic elemental impurities.
Conclusion: In the current study, the presence of NPIs in pharmaceutical-grade sucrose was confirmed and a successful purification of the sucrose to eliminate nanoparticle impurities was established, enabling the launch of the new product EMPROVE® Sucrose. The generated results of this study were consistent with the previously published data from Weinbuch et al. [Weinbuch et al. (2015) Pharm Res, 32: 2419-2427]; [Weinbuch et al. (2017) Pharm Res, 34: 2910-2921].
Christoph Korpus– Darmstadt, Hessen, Germany
Tanja Henzler– Head of Liquid Formulation R&D, MilliporeSigma, Darmstadt, Hessen, Germany
Markus Greulich– Darmstadt, Hessen, Germany
Georg Schuster– Martinsried, Bayern, Germany
Olimipa Popko– Martinsried, Bayern, Germany
Marina Guehlke– Martinsried, Bayern, Germany
Tim Menzen– CTO, Coriolis Pharma, Martinsried, Bayern, Germany
Andrea Hawe– Chief Scientific Officer, Coriolis Pharma, Martinsried, Bayern, Germany