How Low-in-Nanoparticulate-Impurities Sucrose Can Enable More Stable Protein Formulations

NANOPARTICULATE IMPURITIES IN PHARMACEUTICAL-GRADE SUCROSE

Nanoparticle impurities (NPIs) with a size of 100–300 nm have 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).

How Low-in-Nanoparticulate-Impurities Sucrose Can Enable More Stable Protein Formulations

This research was conducted to:

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

IMPACT OF NANOPARTICULATE IMPURITIES ON PROTEIN STABILITY

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 ~10¹⁰particles/mL. The formulations are summarized in Table 1.

Table 1. mAbC Formulations for spiking studies. — **Table 1.** mAbC Formulations for spiking studies.

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

Table 2. Experimental conditions for forced degradation studies — **Table 2**. Experimental conditions for forced degradation studies

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

NPIs have a negative impact on protein stability

mAbC Formulations in the presence and absence of NPIs

Spiking with NPIs in a concentration of ~ 1010 particles/mL induced particle formation in mAbC formulation under stress conditions (Figure 1). This indicates that NPIs, independent of the sucrose source, can have a negative impact on protein stability in final drug product.

Figure 1.Results of forced degradation studies using mAbC without or with NPIs spiked that were previously isolated from beet- or cane-derived sucrose. Particle concentration was determined using MFI. — NPIs have a negative impact on protein stability

PURIFICATION OF SUCROSE

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.

Purification process of sucrose

In order to mitigate risks during formulation development, nanoparticulate impurities were removed from sucrose in an improved purification process (Figure 2). This results in a sucrose grade that is low in NPI content, Sucrose Emprove^® Expert. Additionally, the purification leads to a reduction of bioburden and endotoxin contamination.

Reduction of nanoparticulate impurities

Qualitative Analysis by DLS

The first peak at about 1–5 nm is assigned to sucrose (Figure 3). The second peak, with a size distribution of about 100–300 nm, represents the NPIs.
The decrease in the second peak (Figure 3) for Sucrose Emprove^® Expert proves that the purification process successfully reduces NPI contamination in sucrose products.
The remaining signal (Figure 3) around 100-300 nm can result from single larger particles, since the scattering intensity is proportional to diameter⁶ (I ~ d⁶).