Learn about a purification method to reduce nanoparticle impurities in sucrose.

1. Objective
•
Development of a purification process to reduce the amount of NPIs in sucrose.
•
A comparative analysis of isolated NPIs (beet- or cane-derived) to provide
information on possible variations in NPIs’ impact on drug product stability
caused by the different sucrose sources.
Methods
10% sucrose solutions (w/w) were prepared in Milli-Q®
water. The solutions were
measured before and after sterile filtration through a 0.22 μm PVDF membrane. For
DLS, samples were analyzed with the Zetasizer Nano series (Malvern, Herrenberg,
Germany) at 25 °C using automatic attenuation selection and detection via 173°
back­
scatter. 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.
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.
Isolation of NPIs
50% sucrose solutions (w/w) were prepared in Milli-Q®
water and diafiltration was
performed against Milli-Q®
water (6-fold volume exchange). The zeta potential of
isolated NPIs was determined, and a forced degradation study with IgG1 antibody
mAbC was initiated.
Results
NPI Spiking and Forced Degradation Study
mAbC formulations were prepared at a concentration of 2 mg/mL and spiked with
cane- or beet-derived NPIs.
Sucrose Low in Nanoparticulate Impurities for
Biopharmaceutical Formulations
Christoph Korpus*
, Michelle Zöller*
, Tanja Henzler*
, Markus Greulich*
, Georg Schuster**
, Olimpia Popko**
, Marina Gühlke**
, Tim Menzen**
, Andrea Hawe**
Figure 1:
Results of forced degradation studies using mAbC or control samples spiked with previously isola-
ted beet- or cane-derived nanoparticles. Particle concentration was determined using ­
Micro-Flow
Imaging (MFI).
Contact information: Tanja.Henzler@merckgroup.com
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Zeta Potential
•
Beet-derived NPIs show a more negative zeta potential; cane-derived NPIs a slightly
negative to neutral potential.
•
A clear pH effect was observed for the shape and count rate of the zeta potential
distribution of the beet-derived NPIs.
The sucrose source appears to have an impact on the NPIs’ properties. Different
NPI surface charges may govern different aggregation pathways, and may thus
have different effects on drug product stability.
Figure 2: Zeta potentials of beet- and cane-derived sucrose samples.
Figure 4:
Total particle concentration of non-purified raw material sucrose compared to the means of
batches 1–4 of purified, low-NPI Sucrose Emprove®
Expert. Error bars indicate std. dev. from two
(raw material) or three (batches) replicate measurements
Dynamic Light Scattering (DLS)
•
The first peak in the qualitative DLS measurements (Figure 3), observed at about
1–5 nm, can be identified as sucrose. The second peak (Figure 3, left), with a size
distribution of about 100–300 nm, represents the NPIs.
•
The decrease in the second peak with Sucrose Emprove®
Expert proves that the
developmentofapurificationsteptodecreaseNPIcontaminationinsucrose
products was successful.
•
For all four batches of purified Sucrose Emprove®
Expert, the sucrose peak had a
significantly higher relative area (%) than for the non-purified raw material.
Nanoparticle Tracking Analysis (NTA)
•
The DLS results were confirmed by quantitative NTA measurements, which showed a
significantly lower particle concentration in the four purified Sucrose Emprove®
Expert
batches compared to the non-purified raw material sucrose (Fig. 4).
•
In Sucrose Emprove®
Expert, NPIs were reduced to levels around the NTA detection
limit.
Conclusions
Common sucrose, including pharmaceutical-grade products, contains nano­particle
impurities (NPIs) in the 100–300 nm size range that have been implicated in causing
false analytical results and API damage (Weinbuch et al., 2015 and 2017).
In this study, we were able to confirm the presence of these NPIs in several sucrose
products and show that they have a negative effect on the stability of therapeutic
proteins.
The sucrose source used to isolate the NPIs was also shown to play a role in the NPIs’
effect on drug stability.
A purification method to reduce nanoparticle impurities in sucrose was
successfully developed, enabling the launch of Sucrose Emprove®
Expert Ph
Eur, ChP, JP, NF.
The results of this study are consistent with the data previously published by Weinbuch
et al. 2015 and 2017.
Joint development
Sucrose Emprove®
Expert was developed in cooperation with Coriolis Pharma, Munich,
Germany.
pH Buffer Tonicity agent NPI source Amount of NPIs
used for spiking
Surfactant
Placebo control
7.2 ± 0.1 5 mM phosphate 50 mg/mL sucrose Beet 3.5x1010
/mL 0.005 % (w/v) PS80
7.2 ± 0.1 5 mM phosphate 50 mg/mL sucrose Cane 3.5x1010
/mL 0.005 % (w/v) PS80
Formulations containing 2 mg/mL mAbC
7.2 ± 0.1 5 mM phosphate 50 mg/mL sucrose Beet 3.5x1010
/mL 0.005 % (w/v) PS80
7.2 ± 0.1 5 mM phosphate 50 mg/mL sucrose Cane 3.5x1010
/mL 0.005 % (w/v) PS80
Sample Conditions
T0 Directly after production
2w 25 °C 2 weeks storage at 25 °C
2w 25 °C mech. 2 weeks shaking at 400 rpm
and 25 °C
4w 40 °C 4 weeks storage at 40 °C
•
A negative impact on protein stability after
spiking with NPIs in concentrations ≥1x1010
/mL
was observed.
•
NPIs isolated from beet-derived sucrose have
a more severe effect on mAbC stability than
from cane derived sucrose.
References
1.
Weinbuch, D., Cheung, J. K., Ketelaars, J., Filipe, V., Hawe, A., den Engelsman, J., Jiskoot, W. (2015).
Nanoparticulate Impurities in Pharmaceutical-Grade Sugars and their Interference with Light Scattering-
Based Analysis of Protein Formulations. Pharm Res, 32(7), 2419–2427. doi:10.1007/s 11095-015-1634-1.
2.
Weinbuch, D., Ruigrok, M., Jiskoot, W., Hawe, A. (2017). Nanoparticulate Impurities Isolated from
Pharmaceutical- Grade Sucrose Are a Potential Threat to Protein Stability. Pharm Res, 34(12), 2910–2921.
doi:10.1007/s11095-017-2274-4.
* MilliporeSigma, Frankfurter Straße 250, 64293 Darmstadt, Germany
** Coriolis Pharma Research GmbH, Fraunhoferstraße 18 b, 82152 Martinsried, Germany
0
2
4
6
8
10
12
14
Before purification Batch 1 Batch 2 Batch 3 Batch 4
Total
concentration
(*10^8
particles/mL)
After purification
Figure 3:
DLS measurement results for the beet-derived, non-purified sucrose raw material (left), and
triplicate measurements of one representative batch of purified Sucrose Emprove®
Expert (right).
0
2
4
6
8
10
12
14
16
10
Intensity
in
%
Size in nm (log)
Sucrose raw materia
non-purified (1)
Sucrose raw materia
non-purified (2)
0.1 1,000
0
2
4
6
8
10
12
14
16
10
Intensity
in
%
Size in nm (log)
Batch 1
Batch 2
Batch 3
Batch 4
0.1 1,000
!#$%'(
Total
Counts
0
-100 0 100 200
A Zeta Potential Distribution
A
Apparent Zeta Potential (mV)
0.5*106
1*106
1.5*106
2*106
0
-100 0 100 200
B
Apparent Zeta Potential (mV)
-100 0 100 200
Zeta Potential Distribution
Total
Counts
0
0.2*106
0.4*106
0.6*106
0.8*106
1*106
Particles/mL
2,000
4,000
6,000
8,000
10,000
12,000
14,000
= 2 m = 5 m = 10 m = 25 m
T0
0
2w
25 °C
2w
25 °C
mech.
4w
40 °C
Placebo +
beet-derived NPIs
T0 2w
25 °C
2w
25 °C
mech.
4w
40 °C
Placebo +
cane-derived NPIs
T0 2w
25 °C
2w
25 °C
mech.
4w
40 °C
mAbC +
beet-derived NPIs
T0 2w
25 °C
2w
25 °C
mech.
4w
40 °C
mAbC +
cane-derived NPIs
Purpose
Nanoparticle impurities (NPIs) with a
size of 100–300 nm have recently been
discovered in pharmaceutical-grade
sucrose (Weinbuch et al., 2015)1
. 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 aggre-
gation, frag­
mentation and particle
formation (Weinbuch et al., 2017)2
.
0
2
4
6
8
10
12
14
16
!' 10 !!!
Intensity
in
%
Size in nm (log)
0.1 1,000
23!%
0
2
4
6
8
10
12
14
16
!' 10 !!!
Intensity
in
%
Size in nm (log)
0.1 1,000
23!%
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Lit. No. MS_PS4993EN
10/2019