Low-in-nanoparticulate-impurities sucrose for biopharmaceutical formulations

Purpose

Nanoparticle impurities (NPIs) with a size of 100–300 nm have recently been discovered in pharmaceutical-grade sucrose (Weinbuch et al., 2015). These can lead to false analytical results, as they mimic protein aggregates and can thus cause the potential exclusion of “lead molecules” during early development stages. Studies have also shown that NPIs reduce the stability of final protein formulations by inducing protein aggregation, fragmentation and particle formation (Weinbuch et al., 2017).

Objective

• Investigating the impact of NPIs isolated from beet- or cane-derived sucrose on drug product stability.
• Developing a purification process to reduce the amount of NPIs in sucrose.

Methods

Isolation of NPIs
NPIs were isolated from beet and cane derived sucrose. 50% sucrose solutions (w/v) were prepared in Milli-Q® water and diafiltration was performed against Milli-Q® water (6-fold volume exchange).

NPI Spiking and Forced Degradation Study
Isolated NPIs were spiked into IgG1 antibody mAbC formulation, resulting in a final particle concentration of ~1010 particles/mL. The formulations are summarized in Tab. 1.

Storage and stress conditions as well as time points for sample analysis are described in Tab. 2.

The stability of mAbC was assed by using micro-flow imaging (MFI) using an MFI5200 system (ProteinSimple, Santa Clara, CA, USA) equipped with a 100-μm flow cell.

Particle Size Analysis and Quantification
10% sucrose solutions (w/v) were prepared in Milli-Q® water. The solutions were measured before and after sterile filtration through a 0.22 μm PVDF membrane. For dynamic light scattering (DLS), samples were analyzed with the Zetasizer Nano series (Malvern, Herrenberg, Germany) at 25 °C using automatic attenuation selection and detection via 173° backscatter. Peak size was based on the viscosity of water as dispersant; particle area (%) was based on the intensity. The data processing analysis model was set to general purpose. Nanoparticle tracking analysis (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.

Quantification of β-Glucan contamination
50% sucrose solutions (w/v) were prepared in Milli-Q® water. The (1 3)-β-D-glucan levels were measured by using the Glucatell® Assay (Cape Cod, East Falmout, MA, USA), according to the instructions of the manufacturer.

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Article information: Nelli Erwin (Merck), Markus Greulich (Merck), Tanja Henzler (Merck), Marcus Nagel (Coriolis), Georg Schuster (Coriolis), Andrea Hawe (Coriolis).