Watch the presentation of this webinar here: https://bit.ly/36cAQWT Pharmaceutical-grade sucrose – an important stabilizer for biopharmaceuticals – contains nanoparticulate impurities (NPIs) that are a potential threat to protein stability. The scientists behind the discovery explain their impact on drug development and introduce a new sucrose with low NPI content. A main reason for using excipients is to stabilize the active pharmaceutical ingredient and by that, ensuring an effective and safe therapy. Pharmaceutical grade sucrose, an important and commonly used stabilizer, contains nanoparticulate impurities (NPIs) of 100-200 nm size originating from the starting raw material. In this webinar, the scientists behind the discovery discuss the characteristics of these NPIs, explain their impact on aggregate/particle analytics and show, on the example of antibodies, that NPIs are a threat to protein stability. Their research has triggered the development of improved sucrose grades, that are low in NPIs to reduce the risk of unwanted instability. In this webinar, you will learn: • The role of sucrose as a stabilizer in pharmaceuticals • Presence and characteristics of nanoparticulate impurities (NPIs) in pharmaceutical-grade sucrose • Impact of NPIs on protein stability • Succrose with low NPI content

1. The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
Nanoparticulate
impurities in
excipients - a
threat to protein
stability
Dr. Daniel Weinbuch and Dr. Andrea Hawe
Coriolis Pharma

2. The life science business
of Merck KGaA, Darmstadt,
Germany operates as
MilliporeSigma in the U.S.
and Canada

3. Agenda
1
2
3
NPI in sucrose: Where does
it come from and what is it
made of?
Dr. Daniel Weinbuch
Does NPI impact protein
stability?
Dr. Daniel Weinbuch
Impact of NPI concentration
on protein stability
Dr. Daniel Weinbuch
4
Development and scientific
evaluation of novel sucrose
grade
Dr. Andrea Hawe

4. NPI in sucrose: Where
does it come from
and what is it made
of?

5. 5
Sucrose in the pharmaceutical industry
Sucrose is an important stabilizer for biopharmaceuticals:
• Improve conformational stability by preferential exclusion
• Cryo- and lyoprotector for lyophilized formulations
Quality of pharmaceutical-grade sucrose is regulated by Pharmacopoeias

6. 6
A mysterious peak at 100-200 nm
Sucrose:
Mw = 342.3 Da
Size = ~1 nm
?
Sucrose
Fig. 3 Intensity particle size distributions obtained for different
sucrose concentrations measured in a Nano S instrument using
backscatter detection by dynamic light scattering (DLS).
Kaszuba M, et al (2008) Measuring sub
nanometre sizes using dynamic light
scattering. J Nanopart Res 10(5): 823–829.

7. Kaszuba M, et al. 2008:
„probably due to collective diffusion
of the sucrose molecules”
?
Kaszuba M, et al (2008) Measuring sub
nanometre sizes using dynamic light
scattering. J Nanopart Res 10(5): 823–829.
A mysterious peak at 100-200 nm

8. 8
NPI in sucrose
Dynamic light scattering (DLS)
Peaks differ between suppliers and between lots of same supplier
Weinbuch D, et al (2015) Nanoparticulate Impurities in
Pharmaceutical-Grade Sugars and their Interference
with Light Scattering-Based Analysis of Protein
Formulations. Pharm Res 32(7): 2419–2427.

9. 9
NPI in sucrose
• Peaks visible also in NTA
• Concentration dependency
• Up to 109 particles/g sucrose
• Detected also in other sugars
(mannose, fructose, maltose, etc.)
Nanoparticle tracking analysis (NTA)
Weinbuch D, et al (2015) Nanoparticulate Impurities in
Pharmaceutical-Grade Sugars and their Interference
with Light Scattering-Based Analysis of Protein
Formulations. Pharm Res 32(7): 2419–2427.

10. 10
Isolation of NPI in sucrose
Weinbuch D, et al (2015) Nanoparticulate Impurities in
Pharmaceutical-Grade Sugars and their Interference
with Light Scattering-Based Analysis of Protein
Formulations. Pharm Res 32(7): 2419–2427.

11. Isolation of NPI in sucrose
Sucrose G (Ph.Eur.-Grade)
DLS
Isolated NPI maintains size and concentration
DLS, insert NTA
11
Weinbuch D, et al (2015) Nanoparticulate Impurities in
Pharmaceutical-Grade Sugars and their Interference
with Light Scattering-Based Analysis of Protein
Formulations. Pharm Res 32(7): 2419–2427.

12. Weinbuch D, et al. 2015:
„the interference is caused by
particulate matter rather than by
monomeric sucrose molecules.”
NPI in sucrose
Weinbuch D, et al (2015) Nanoparticulate Impurities in
Pharmaceutical-Grade Sugars and their Interference
with Light Scattering-Based Analysis of Protein
Formulations. Pharm Res 32(7): 2419–2427.

13. Characterization of NPI in sucrose
SEM-EDX
The elemental composition (Si, Al, Ca, Mg, ...)
matches the description of ash, a commonly
encountered inorganic contaminant in unprocessed
cane or beet juice.
13
Weinbuch D, et al (2015) Nanoparticulate Impurities in
Pharmaceutical-Grade Sugars and their Interference
with Light Scattering-Based Analysis of Protein
Formulations. Pharm Res 32(7): 2419–2427.

14. 14
Characterization of NPI in sucrose
FTIR microscopy and Mass-spectrometry
Dextran (Mw varying from a few kDa – several
MDa) is a well-known impurity in the sugar
industry; produced by Leuconostoc bacteria, which
mainly enter the sugar cane or beet during
harvesting, cutting and grinding, but can also be
introduced in later production steps.
Weinbuch D, et al (2015) Nanoparticulate Impurities in
Pharmaceutical-Grade Sugars and their Interference
with Light Scattering-Based Analysis of Protein
Formulations. Pharm Res 32(7): 2419–2427.

15. 15
Characterization of NPI in sucrose
Fluorescence spectroscopy
Diverse amounts of fluorescent impurities of
different compositions have been found in various
sugar products; these include catechols and
species similar to Trp and Tyr, formed by base-
catalyzed sugar degradation and again suggested
being Maillard reaction polymers.
Weinbuch D, et al (2015) Nanoparticulate Impurities in
Pharmaceutical-Grade Sugars and their Interference
with Light Scattering-Based Analysis of Protein
Formulations. Pharm Res 32(7): 2419–2427.

16. 16
Disturbance of DLS analysis of proteins by NPI
IgG and lysozyme at different
concentrations in presence of
7% sucrose after sterile filtration.
NPI peak may or may not appear,
depending on the sample type and
concentration
The impact of NPI on sample analysis also depends on the sample
IgG
Lysozyme
*
*
*NPI peak
Weinbuch D, et al (2015) Nanoparticulate Impurities in
Pharmaceutical-Grade Sugars and their Interference
with Light Scattering-Based Analysis of Protein
Formulations. Pharm Res 32(7): 2419–2427.

17. • A nanoparticulate impurity (NPI) is present in
pharmaceutical grade sucrose
• The NPI originates form raw materials and the
production, and are not (entirely) removed during
sugar refinement
• The pharmacopoeias do not require
testing for NPI
Interim
conclusions

18. Does NPI
impact
protein
stability?

19. 19
Experimental setup
Weinbuch D, et al (2017) Nanoparticulate
Impurities Isolated from Pharmaceutical-Grade
Sucrose Are a Potential Threat to Protein
Stability. Pharm Res 34(12): 2910–2921.
Final NPI concentration:
3.5x1010 particles/mL
based on NTA
• Storage conditions: 2-8°C, 25°C and 40°C
• Sampling time points: T0, 2 weeks, 8 weeks and 14 weeks
• Analytical methods: SEC, NTA, MFI, visual inspection and cIEF
+ NPI + Control + NPI
formulation buffer2 mg/ml mAbmAb
Trastuzumab
Rituximab
Infliximab
Cetuximab
mAb
Trastuzumab
Rituximab
Infliximab
Cetuximab

20. 20
Trastuzumab at T0
+ control + NPI
Size by MFI Particles per ml
> 1 µm 1,395 2,654,460
> 2 µm 546 28,942
Weinbuch D, et al (2017) Nanoparticulate
Impurities Isolated from Pharmaceutical-Grade
Sucrose Are a Potential Threat to Protein
Stability. Pharm Res 34(12): 2910–2921.

21. 21
Trastuzumab
NPIs →
more
µm-sized
particles
Weinbuch D, et al (2017) Nanoparticulate
Impurities Isolated from Pharmaceutical-Grade
Sucrose Are a Potential Threat to Protein
Stability. Pharm Res 34(12): 2910–2921.

22. 22
Infliximab at T0
Weinbuch D, et al (2017) Nanoparticulate
Impurities Isolated from Pharmaceutical-Grade
Sucrose Are a Potential Threat to Protein
Stability. Pharm Res 34(12): 2910–2921.

23. 23
Infliximab at T0
buffer + NPI
infliximab + NPI
Weinbuch D, et al (2017) Nanoparticulate
Impurities Isolated from Pharmaceutical-Grade
Sucrose Are a Potential Threat to Protein
Stability. Pharm Res 34(12): 2910–2921.

24. 24
Infliximab at T0
+ control + NPI
Size by MFI Particles per ml
> 1 µm 7,926 2,433,650
> 2 µm 1,488 247,657
> 5 µm 217 1,181
Weinbuch D, et al (2017) Nanoparticulate
Impurities Isolated from Pharmaceutical-Grade
Sucrose Are a Potential Threat to Protein
Stability. Pharm Res 34(12): 2910–2921.

25. 25
Infliximab
Weinbuch D, et al (2017) Nanoparticulate
Impurities Isolated from Pharmaceutical-Grade
Sucrose Are a Potential Threat to Protein
Stability. Pharm Res 34(12): 2910–2921.

26. 26
Infliximab
T0 T2w T8w T14w
Weinbuch D, et al (2017) Nanoparticulate
Impurities Isolated from Pharmaceutical-Grade
Sucrose Are a Potential Threat to Protein
Stability. Pharm Res 34(12): 2910–2921.
NPIs → more
nm- & µm-
sized particles
and LMW-
species

27. Interim
conclusions
• NPI impact the stability of all tested monoclonal antibodies
(under elevated stress conditions)
• Degradation profiles differ between the tested mAbs

28. Impact of NPI
concentration on
protein stability

29. • Final NPI concentrations:
3.5x1010, 3.5x109, 3.5x108, and 3.5x107 particles/mL
• Sampling time points: T0 and 4 weeks at 40 C
• Analytical methods: SEC, NTA, MFI and visual inspection
reflect a high, medium, and low
potential contamination of NPIs, in a
hypothetical formulation containing
10% w/v sucrose
29
Experimental setup
mAb
Trastuzumab
Rituximab
Infliximab
mAb
Trastuzumab
Rituximab
Infliximab
Weinbuch D, et al (2017) Nanoparticulate
Impurities Isolated from Pharmaceutical-Grade
Sucrose Are a Potential Threat to Protein
Stability. Pharm Res 34(12): 2910–2921.
+ NPI + Control + NPI
formulation buffer2 mg/ml mAb

30. 30
Concentration-dependency
Rituximab and infliximab
are destabilized by NPIs in
concentrations potentially
present in drug products.
rituximabinfliximab
Weinbuch D, et al (2017) Nanoparticulate
Impurities Isolated from Pharmaceutical-Grade
Sucrose Are a Potential Threat to Protein
Stability. Pharm Res 34(12): 2910–2921.
reflect a high, medium, and low
potential contamination of NPIs, in a
hypothetical formulation containing
10% w/v sucrose

31. 31
Attribute Method trastuzumab rituximab infliximab cetuximab
Visible particles
Visual
inspection
+ - + -
Turbidity
Visual
inspection
++ - ++ +
µm-particles MFI ++ + ++ +
nm-particles NTA - ++ ++ ++
HMW species SEC - - - +
Sample recovery SEC + - + -
LMW species SEC - + + -
Conformational
Instability
nDSF - - - -
Charge variants cIEF - - - -
- was not affected
(relative to control)
+ was affected, but only at high
concentrations of NPIs and/or
not immediately (relative
control)
++ was highly and immediately
affected and/or affected even
at NPIs concentrations
potentially present in drug
products (relative to control)
Summary: Impact of NPI on protein stability
Weinbuch D, et al (2017) Nanoparticulate
Impurities Isolated from Pharmaceutical-Grade
Sucrose Are a Potential Threat to Protein
Stability. Pharm Res 34(12): 2910–2921.

32. Interim
conclusions
• NPI impact the stability of all tested monoclonal antibodies
(at concentrations potentially encountered in “real-life”)
• Degradation profiles differ between the tested mAbs
• No effect on conformational stability or charge variants
observed

33. Development
and scientific
evaluation of
novel sucrose
grade

34. A novel sucrose
grade
Sucrose Emprove® Expert
(Ph Eur, ChP, JP, NF)
Prod.-No: 103789
co-developed
with Coriolis

35. mAbC
• The impact of NPIs isolated from
beet and cane-derived sucrose
after spiking on protein stability
(mAbC) was studied.
• A negative impact on protein
stability after spiking of NPIs in
concentrations ≥ 1x1010/10 ml was
observed.
35
Micro Flow
Imaging
Negative Impact of NPIs on Protein Stability
NPI Spiking Studies

36. NPI Characterization
NPIs Vary in Zeta Potential Depending on the Source
Zeta potential pH 3.0
(1 mM citric acid b.)
pH 7.4
(1 mM phosphate b.)
Beet derived NPIs -12.0 ± 0.2 mV -16.8 ± 0.0 mV
Cane derived NPIs -3.1 ± 0.1 mV -4.3 ± 0.4 mV
Beet derived NPIs
• Beet derived NPIs show a more negative zeta potential; cane derived NPIs a slightly negative to neutral potential
• Clear pH effect was observed for the shape and count rate of the zeta potential distribution of NPIs isolated from beets
• Sucrose source appears to have an impact on the NPIs’ properties.
• Different surface charge of NPIs may govern different NPI-induced protein aggregation
pathways and may contribute differently to drug product stability.
Apparent Zeta Potential (mV)
TotalCounts
0
0.6*106
1*106
0.4*106
0.2*106
-100 0 100 200
0.8*106
Zeta Potential Distribution
Apparent Zeta Potential (mV)
TotalCounts
0
1.5*106
2*106
1*106
0.5*106
-100 0 100 200
Zeta Potential Distribution
Cane derived NPIs
36

37. 0
2
4
6
8
10
12
14
16
0,1 10 1000
Intensityin%
Size [nm]
After purificationMERCK purified
sucrose
Purification & Ultrafiltration
Nanoparticulate Impurities
• Joint development project:
Together with formulation
development experts in the field
• Process development with
dedicated purification strategy
• High purity sucrose, low in
nanoparticulate impurities
(NPIs)
00
02
04
06
08
10
12
14
16
0.1 10 1000
Intensity[%]
Size [nm]
Emprove (1)
Emprove (2)
Sucrose A (1)
Sucrose A (2)
37
Purified
Sucrose
Product was launched in August 2019
Sucrose low in NPIs

38. Protein
Aggregation
Crystalli-
zation
Filtration
(TFF)Solution PackagingCentrifugation
Drying
Sucrose
(Beet-
derived)
Emprove®
Expert
product• Endotoxins: ≤ 0.3 I.U./g
• TAMC: ≤ 10² CFU/g
• TYMC: ≤ 101 CFU/g
Resulting specification:
Controlled production environment for high-risk application
• Reduction of bioburden/endotoxin level
• Reduction of nanoparticulate impurities
38
Reduction of bioburden, endotoxin levels and NPIs
Risk mitigation in manufacturing

39. Qualitative NPI Analytics
NPI Analytics via Dynamic Light Scattering (DLS)
Sucrose before purification
• Reduction in NPIs is visible via qualitative DLS measurement.
• Note: single larger particles result in a large intensity, because
the scattering intensity is proportional to diameter6 (I ~ d6).
Sucrose after purification
39
0
2
4
6
8
10
12
14
16
0,1 1 10 100 1000 10000
Intensityin%
Size in nm (log)
Before Purification (1)
Before Purification (2)
0
2
4
6
8
10
12
14
0,1 1 10 100 1000 10000
Intensityin%
Size in nm (log)
Batch 1
Batch 2
Batch 3
Batch 4

40. NPI Analytics via Nanoparticle Tracking Analysis (NTA)
Sucrose before vs. after purification
NTA proves successful reduction of NPIs
40
0,0E+00
2,0E+08
4,0E+08
6,0E+08
8,0E+08
1,0E+09
1,2E+09
1,4E+09
Before
purification
Batch 1 Batch 2 Batch 3 Batch 4
Particles/ml
After purification
Quantitative NPI Analytics

41. 41
• NPI are present in pharmaceutical-grade sucrose
• NPI are not considered by pharmacopeia quality criteria
• NPI concentration can vary between
0 – 109 particles/gram (supplier- and batch-dependent)
• NPI disturb light scattering based analytics
e.g., DLS, NTA, MALLS …
• NPI can negatively affect the stability of monoclonal
antibodies in pharmaceutically relevant concentrations
• Novel sucrose grade now available with low NPI content:
Sucrose Emprove® Expert (Ph Eur, ChP, JP, NF)
Prod.-No: 103789
Conclusions

42. The vibrant M, SAFC®, Emprove® Expert are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of
their respective owners. Detailed information on trademarks is available via publicly accessible resources.
© 2019 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.