Exploring the protein stabilizing capability of surfactants against agitation stress and the underlying mechanisms
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The study investigates the ability of various surfactants to stabilize proteins against agitation-induced stresses, focusing on the impact of these surfactants on protein aggregation during agitation. Results indicate that selected surfactants effectively stabilize a monoclonal antibody compared to controls without surfactants, with mechanisms suggesting competitive surface adsorption as a primary action. This research aids in understanding protein stabilization in biopharmaceutical formulations, particularly the performance of non-ionic surfactants like polysorbate.
![Exploring the protein stabilizing capability
of surfactants against agitation stress and
the underlying mechanisms
Michelle P. Zoellera
, Supriyadi Hafiza
, Andreas Marxa
, Nelli Erwina
, Gert Frickerb
, John F. Carpenterc
© 2022 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.
Merck, the Vibrant M and SAFC 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.
Lit. No. MK_PS11643EN 11/2022
The Life Science business of Merck operates as MilliporeSigma in the U.S. and Canada.
Conclusion
•
The small-volume, rapid agitation stress approach was presented to be a powerful tool to
screen the protein stabilizing capability of surfactants using a minimum of material and time.
•
Studies showed that all four molecules stabilized mAbE compared to the respective
control protein solutions without surfactant.
•
Surface tension results suggest the prevalent presence of surfactants at the newly
created liquid-air interfaces in protein-surfactant mixture solutions during the measurements.
•
Considering the complete stabilization of both model proteins during the agitation studies
by these three surfactants, the competitive surface adsorption mechanism appears
to apply.
1
Koepf, E., et al., The film tells the story: Physical-chemical characteristics of IgG at the liquid-air interface. Eur J Pharm
Biopharm, 2017. 119: p. 396-407.
2
Rudiuk, S., et al., Importance of the dynamics of adsorption and of a transient interfacial stress on the formation of aggregates
of IgG antibodies. Soft Matter, 2012. 8(9).
3
Strickley, R.G. and W.J. Lambert, A review of Formulations of Commercially Available Antibodies. J Pharm Sci, 2021. 110(7):
p. 2590-2608 e56.
4
Donbrow, M., E. Azaz, and A. Pillersdorf, Autoxidation of polysorbates. J Pharm Sci, 1978. 67(12): p. 1676-81.
5
Kishore, R.S., et al., The degradation of polysorbates 20 and 80 and its potential impact on the stability of biotherapeutics.
Pharm Res, 2011. 28(5): p. 1194-210.
6
Kishore, R.S., et al., Degradation of polysorbates 20 and 80: studies on thermal autoxidation and hydrolysis. J Pharm Sci,
2011. 100(2): p. 721-31.
7
Zoeller, MP., Hafiz, S., Marx, A., Erwin, N., Fricker, G., Carpenter, JF., Exploring the protein stabilizing capability of surfactants
against agitation stress and the underlying mechanisms. J Pharm Sci. 2022 Sep 9:S0022-3549(22)00397-5.
doi: 10.1016/j.xphs.2022.09.004. Epub ahead of print. PMID: 36096287.
8
Johann, F., et al., Miniaturized Forced Degradation of Therapeutic Proteins and ADCs by Agitation-Induced Aggregation
Using Orbital Shaking of Microplates. J Pharm Sci, 2021.
a
Merck KGaA, Darmstadt, Germany.
b
Institute of Pharmacy and Molecular Biotechnology, Ruprecht-Karls-University, Heidelberg, Germany.
c
University of Colorado Anschutz Medical Campus, Dept. of Pharmaceutical Sciences,
Aurora, Colorado, USA.
Introduction and Objectives
Agitation of therapeutic protein solutions during manufacturing, shipping and handling is one of the
major initiators for protein aggregation and particle formation during the life history of a protein drug.
Adsorption of protein molecules to liquid-air interfaces leads to the formation of highly concentrated
protein surface films.1
The rupture of these protein films due to various mechanical processes
can then result in the appearance of protein aggregates and particles in the bulk solution phase.2
One technique to stabilize proteins against stress induced by liquid-air interfaces is the use of
non-ionic surfactants. About 91% of antibody formulations commercially available in 2021 contained
a surfactant. Polysorbate 20 and 80, composed of a hydrophilic polyoxyethylene sorbitan and
hydrophobic fatty acid esters, made up the largest part being employed in 87% of said formulations.3
Despite their frequent use in parenteral drug products, concerns have been raised for decades about
the application of polysorbates as surfactants in biopharmaceutical formulations. Autoxidation of
polysorbate, caused by residual peroxides in polysorbates, can damage the proteins and can further
drive the oxidative degradation of polysorbate.4
Chemical and enzymatic hydrolysis of polysorbate may
lead to the formation of free fatty acid particles, which may become visible; and both mechanisms
eventually lead to the reduction in polysorbate concentration.5,6
Therefore, the purpose of the current study was to compare various molecules for their capabilities
to reduced agitation-induced protein aggregation and particle formation; and furthermore,
investigate their underlying protein stabilizing mechanisms.7
Inhibition of Protein Aggregation and
Particle Formation During Agitation
A small-volume, rapid agitation stress approach was used to investigate the molecules’ abilities
to stabilize a monoclonal antibody (mAbE).8
Formation of protein aggregates and particles was
determined by SEC, turbidity and FlowCam®
measurements.
Figure 1.
SE-HPLC (A), turbidity (B) and flow imaging (C) results of 1 mg/mL mAbE containing 0.1–0.01% (w/v) of surfactants (or 0.035–3.5%
for HPβCD) after high-speed agitation stress. Agitated controls without additive were not measured due to their high turbidity
potentially causing the clogging of the system. Histogram shows mean and standard deviation of three separate samples.
Figure 2.
Representative images of particles detected via FlowCam®
8,000 measurements in a mAbE sample containing 0.035% HPβCD
after agitation stress. Particles were sorted by area-based diameter (ABD).
Figure 3.
STD NMR measurement of a BSA-THDD mixture showing a reference (upper) and the saturation difference spectrum. 10% D2
O
were added to the aqueous protein-excipient (1 mg/mL protein and 2% w/v surfactant) solutions.
•
SEC results (Figure 1A) showed a monomer recovery of about 80% in the control sample agitated
without surfactant and 100% monomer recovery in all tested formulations containing
surfactant.
•
Agitation-induced aggregation in the control sample and the effective inhibition of agitation-induced
aggregation of mAbE was corroborated by turbidity measurements at 350 nm. (Figure 1B)
•
As already indicated by the slightly increased turbidity, the sample containing 0.035% HPβCD
showed higher numbers of protein particles than observed in the other surfactant-containing
solutions (Figure 1C).
1H [ppm]
saturation difference spectrum (on resonance – off resonance spectrum)
reference spectrum (off resonance spectrum)
carbohydrate moiety
2
1
3
4
4.2 3.8 3.4 3.0 2.6 2.2 1.8 1.4 1.0 0.6 0.2 -0
H3
C(H2
C)9
H2
C
O
1 2, 3 4
O
O
O
HO
HO
OH
OH
OH
OH
OH
O
Insights into mechanisms of protein particle formation can be obtained by examining the morphology
of particles shown in FlowCam®
images (Figure 2).
• Highest number of particles was detected in mAbE samples containing 0.035% HPβCD.
•
Thereby, the particles appear to be composed of bits of protein films that have been rolled up
upon themselves during agitation which is consistent with particles formed by protein films
forming and breaking up during agitation.
Protein-Surfactant Interactions and
Mechanisms for Inhibition of Agitation-Induced Aggregation
The first mechanism of protein stabilization investigated was the potential binding of the three
surfactants and HPβCD to the proteins. To this end, saturation transfer difference NMR measurements
were performed. First, the method was established using the model protein BSA as it possesses
hydrophobic binding pockets on its surface, making the binding between BSA and a fatty
acid-containing surfactant likely. Results of a THDD-BSA sample measurement is shown in Figure 3.
Figure 4.
Dynamic surface tension measurements of mAbE (A–D, black solid line), surfactant solutions (colored solid lines) as well as mAbE-surfactant
mixture solutions (colored, dotted lines) using the maximum bubble pressure method. Surfactant containing solutions were prepared at
concentrations 0.01–0.1% or 0.035–3.5% (w/v) for HPβCD. Graphs represent means of at least duplicated measurements ± SD.
•
PS80-mAbE, PLX188-mAbE and THDD-mAbE surface tension graphs resembled the graphs
of the respective surfactant-only solutions throughout all concentrations, indicating the
surfactants’ ability to replace the protein from the liquid-air interface.
• The HPβCD-mAbE samples consistently showed a higher surface tension than the pure HPβCD
samples suggesting a joint adsorption of the protein and HPβCD at the surface.
•
Signals observed at 2.45 ppm, 1.55 ppm, 1.2 ppm and 0.8 ppm in the difference spectrum suggest
a binding of the alkyl chain of THDD and BSA.
•
Similar observations of potential bindings were obtained for samples containing BSA and
Polysorbate 80.
•
Results of mAbE STD NMR experiments showed no measurable binding between mAbE
and the three tested surfactants or HPβCD.
Surface Competition Experiments by
Maximum Bubble Pressure Measurements
After finding no measurable binding between mAbE and any of the tested surfactants or HPβCD, the
mechanism of competition for the surface was suggested to be the main route of action of inhibiting
agitation-induced aggregation of mAbE by the surfactants or HPβCD.
Recovery
of
Soluble
Monomer
[%]
0
A
120
20
40
60
80
100
HPßCD
THDD
PLX188
PS80
w/o Surf.
Stressed
0.05% Surf. or 0.35% HPßCD
0.01% Surf. or 0.035% HPßCD
0.1% Surf. or 3.5% HPßCD
Particles
[mL]
0
C
45,000
40,000
35,000
30,000
25,000
20,000
15,000
10,000
5,000
0.01% 0.05% 0.1%
PS80
Not
Stressed
≥10 µm
≥2 µm
****
*
****
****
****
****
≥25 µm
0.01% 0.05% 0.1%
PLX188
0.01% 0.05% 0.1%
THDD
0.035% 0.35% 3.5%
HPßCD
****
****
****
Surface
Tension
[mN/m]
C 80
40
50
60
70
1,000
0.01 0.1 1 10 100
THDD 0.05%
THDD 0.01%
THDD 0.1% mAbE + THDD 0.05%
mAbE + THDD 0.01%
mAbE 1 mg/mL
Surface Age [s]
mAbE + THDD 0.1%
Surface
Tension
[mN/m]
D 80
40
50
60
70
1,000
0.01 0.1 1 10 100
HPßCD 0.05%
HPßCD 0.01%
HPßCD 0.1% mAbE + HPßCD 0.35%
mAbE + HPßCD 0.035%
mAbE 1 mg/mL
Surface Age [s]
mAbE + HPßCD 3.5%
Surface
Tension
[mN/m]
B 80
40
50
60
70
1,000
0.01 0.1 1 10 100
PS188 0.05%
PS188 0.01%
PS188 0.1% mAbE + PLX188 0.05%
mAbE + PLX188 0.01%
mAbE 1 mg/mL
Surface Age [s]
mAbE + PLX188 0.1%
Surface
Tension
[mN/m]
A 80
40
50
60
70
1,000
0.01 0.1 1 10 100
PS80 0.05%
PS80 0.01%
PS80 0.1% mAbE + PS80 0.05%
mAbE + PS80 0.01%
mAbE 1 mg/mL
Surface Age [s]
mAbE + PS80 0.1%
Turbidity
[A350]
0.0
B
0.6
0.2
0.4
HPßCD
THDD
PLX188
PS80
w/o Surf.
Stressed
0.05% Surf. or 0.35% HPßCD
0.01% Surf. or 0.035% HPßCD
****
****
0.1% Surf. or 3.5% HPßCD](https://image.slidesharecdn.com/exploring-the-protein-stabilizing-capability-of-surfactants-against-agitation-stress-merck-230607075416-5c8d9f2c/85/Exploring-the-protein-stabilizing-capability-of-surfactants-against-agitation-stress-and-the-underlying-mechanisms-1-320.jpg)
Exploring the protein stabilizing capability of surfactants against agitation stress and the underlying mechanisms
- 1. Exploring the protein stabilizing capability of surfactants against agitation stress and the underlying mechanisms Michelle P. Zoellera , Supriyadi Hafiza , Andreas Marxa , Nelli Erwina , Gert Frickerb , John F. Carpenterc © 2022 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. Merck, the Vibrant M and SAFC 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. Lit. No. MK_PS11643EN 11/2022 The Life Science business of Merck operates as MilliporeSigma in the U.S. and Canada. Conclusion • The small-volume, rapid agitation stress approach was presented to be a powerful tool to screen the protein stabilizing capability of surfactants using a minimum of material and time. • Studies showed that all four molecules stabilized mAbE compared to the respective control protein solutions without surfactant. • Surface tension results suggest the prevalent presence of surfactants at the newly created liquid-air interfaces in protein-surfactant mixture solutions during the measurements. • Considering the complete stabilization of both model proteins during the agitation studies by these three surfactants, the competitive surface adsorption mechanism appears to apply. 1 Koepf, E., et al., The film tells the story: Physical-chemical characteristics of IgG at the liquid-air interface. Eur J Pharm Biopharm, 2017. 119: p. 396-407. 2 Rudiuk, S., et al., Importance of the dynamics of adsorption and of a transient interfacial stress on the formation of aggregates of IgG antibodies. Soft Matter, 2012. 8(9). 3 Strickley, R.G. and W.J. Lambert, A review of Formulations of Commercially Available Antibodies. J Pharm Sci, 2021. 110(7): p. 2590-2608 e56. 4 Donbrow, M., E. Azaz, and A. Pillersdorf, Autoxidation of polysorbates. J Pharm Sci, 1978. 67(12): p. 1676-81. 5 Kishore, R.S., et al., The degradation of polysorbates 20 and 80 and its potential impact on the stability of biotherapeutics. Pharm Res, 2011. 28(5): p. 1194-210. 6 Kishore, R.S., et al., Degradation of polysorbates 20 and 80: studies on thermal autoxidation and hydrolysis. J Pharm Sci, 2011. 100(2): p. 721-31. 7 Zoeller, MP., Hafiz, S., Marx, A., Erwin, N., Fricker, G., Carpenter, JF., Exploring the protein stabilizing capability of surfactants against agitation stress and the underlying mechanisms. J Pharm Sci. 2022 Sep 9:S0022-3549(22)00397-5. doi: 10.1016/j.xphs.2022.09.004. Epub ahead of print. PMID: 36096287. 8 Johann, F., et al., Miniaturized Forced Degradation of Therapeutic Proteins and ADCs by Agitation-Induced Aggregation Using Orbital Shaking of Microplates. J Pharm Sci, 2021. a Merck KGaA, Darmstadt, Germany. b Institute of Pharmacy and Molecular Biotechnology, Ruprecht-Karls-University, Heidelberg, Germany. c University of Colorado Anschutz Medical Campus, Dept. of Pharmaceutical Sciences, Aurora, Colorado, USA. Introduction and Objectives Agitation of therapeutic protein solutions during manufacturing, shipping and handling is one of the major initiators for protein aggregation and particle formation during the life history of a protein drug. Adsorption of protein molecules to liquid-air interfaces leads to the formation of highly concentrated protein surface films.1 The rupture of these protein films due to various mechanical processes can then result in the appearance of protein aggregates and particles in the bulk solution phase.2 One technique to stabilize proteins against stress induced by liquid-air interfaces is the use of non-ionic surfactants. About 91% of antibody formulations commercially available in 2021 contained a surfactant. Polysorbate 20 and 80, composed of a hydrophilic polyoxyethylene sorbitan and hydrophobic fatty acid esters, made up the largest part being employed in 87% of said formulations.3 Despite their frequent use in parenteral drug products, concerns have been raised for decades about the application of polysorbates as surfactants in biopharmaceutical formulations. Autoxidation of polysorbate, caused by residual peroxides in polysorbates, can damage the proteins and can further drive the oxidative degradation of polysorbate.4 Chemical and enzymatic hydrolysis of polysorbate may lead to the formation of free fatty acid particles, which may become visible; and both mechanisms eventually lead to the reduction in polysorbate concentration.5,6 Therefore, the purpose of the current study was to compare various molecules for their capabilities to reduced agitation-induced protein aggregation and particle formation; and furthermore, investigate their underlying protein stabilizing mechanisms.7 Inhibition of Protein Aggregation and Particle Formation During Agitation A small-volume, rapid agitation stress approach was used to investigate the molecules’ abilities to stabilize a monoclonal antibody (mAbE).8 Formation of protein aggregates and particles was determined by SEC, turbidity and FlowCam® measurements. Figure 1. SE-HPLC (A), turbidity (B) and flow imaging (C) results of 1 mg/mL mAbE containing 0.1–0.01% (w/v) of surfactants (or 0.035–3.5% for HPβCD) after high-speed agitation stress. Agitated controls without additive were not measured due to their high turbidity potentially causing the clogging of the system. Histogram shows mean and standard deviation of three separate samples. Figure 2. Representative images of particles detected via FlowCam® 8,000 measurements in a mAbE sample containing 0.035% HPβCD after agitation stress. Particles were sorted by area-based diameter (ABD). Figure 3. STD NMR measurement of a BSA-THDD mixture showing a reference (upper) and the saturation difference spectrum. 10% D2 O were added to the aqueous protein-excipient (1 mg/mL protein and 2% w/v surfactant) solutions. • SEC results (Figure 1A) showed a monomer recovery of about 80% in the control sample agitated without surfactant and 100% monomer recovery in all tested formulations containing surfactant. • Agitation-induced aggregation in the control sample and the effective inhibition of agitation-induced aggregation of mAbE was corroborated by turbidity measurements at 350 nm. (Figure 1B) • As already indicated by the slightly increased turbidity, the sample containing 0.035% HPβCD showed higher numbers of protein particles than observed in the other surfactant-containing solutions (Figure 1C). 1H [ppm] saturation difference spectrum (on resonance – off resonance spectrum) reference spectrum (off resonance spectrum) carbohydrate moiety 2 1 3 4 4.2 3.8 3.4 3.0 2.6 2.2 1.8 1.4 1.0 0.6 0.2 -0 H3 C(H2 C)9 H2 C O 1 2, 3 4 O O O HO HO OH OH OH OH OH O Insights into mechanisms of protein particle formation can be obtained by examining the morphology of particles shown in FlowCam® images (Figure 2). • Highest number of particles was detected in mAbE samples containing 0.035% HPβCD. • Thereby, the particles appear to be composed of bits of protein films that have been rolled up upon themselves during agitation which is consistent with particles formed by protein films forming and breaking up during agitation. Protein-Surfactant Interactions and Mechanisms for Inhibition of Agitation-Induced Aggregation The first mechanism of protein stabilization investigated was the potential binding of the three surfactants and HPβCD to the proteins. To this end, saturation transfer difference NMR measurements were performed. First, the method was established using the model protein BSA as it possesses hydrophobic binding pockets on its surface, making the binding between BSA and a fatty acid-containing surfactant likely. Results of a THDD-BSA sample measurement is shown in Figure 3. Figure 4. Dynamic surface tension measurements of mAbE (A–D, black solid line), surfactant solutions (colored solid lines) as well as mAbE-surfactant mixture solutions (colored, dotted lines) using the maximum bubble pressure method. Surfactant containing solutions were prepared at concentrations 0.01–0.1% or 0.035–3.5% (w/v) for HPβCD. Graphs represent means of at least duplicated measurements ± SD. • PS80-mAbE, PLX188-mAbE and THDD-mAbE surface tension graphs resembled the graphs of the respective surfactant-only solutions throughout all concentrations, indicating the surfactants’ ability to replace the protein from the liquid-air interface. • The HPβCD-mAbE samples consistently showed a higher surface tension than the pure HPβCD samples suggesting a joint adsorption of the protein and HPβCD at the surface. • Signals observed at 2.45 ppm, 1.55 ppm, 1.2 ppm and 0.8 ppm in the difference spectrum suggest a binding of the alkyl chain of THDD and BSA. • Similar observations of potential bindings were obtained for samples containing BSA and Polysorbate 80. • Results of mAbE STD NMR experiments showed no measurable binding between mAbE and the three tested surfactants or HPβCD. Surface Competition Experiments by Maximum Bubble Pressure Measurements After finding no measurable binding between mAbE and any of the tested surfactants or HPβCD, the mechanism of competition for the surface was suggested to be the main route of action of inhibiting agitation-induced aggregation of mAbE by the surfactants or HPβCD. Recovery of Soluble Monomer [%] 0 A 120 20 40 60 80 100 HPßCD THDD PLX188 PS80 w/o Surf. Stressed 0.05% Surf. or 0.35% HPßCD 0.01% Surf. or 0.035% HPßCD 0.1% Surf. or 3.5% HPßCD Particles [mL] 0 C 45,000 40,000 35,000 30,000 25,000 20,000 15,000 10,000 5,000 0.01% 0.05% 0.1% PS80 Not Stressed ≥10 µm ≥2 µm **** * **** **** **** **** ≥25 µm 0.01% 0.05% 0.1% PLX188 0.01% 0.05% 0.1% THDD 0.035% 0.35% 3.5% HPßCD **** **** **** Surface Tension [mN/m] C 80 40 50 60 70 1,000 0.01 0.1 1 10 100 THDD 0.05% THDD 0.01% THDD 0.1% mAbE + THDD 0.05% mAbE + THDD 0.01% mAbE 1 mg/mL Surface Age [s] mAbE + THDD 0.1% Surface Tension [mN/m] D 80 40 50 60 70 1,000 0.01 0.1 1 10 100 HPßCD 0.05% HPßCD 0.01% HPßCD 0.1% mAbE + HPßCD 0.35% mAbE + HPßCD 0.035% mAbE 1 mg/mL Surface Age [s] mAbE + HPßCD 3.5% Surface Tension [mN/m] B 80 40 50 60 70 1,000 0.01 0.1 1 10 100 PS188 0.05% PS188 0.01% PS188 0.1% mAbE + PLX188 0.05% mAbE + PLX188 0.01% mAbE 1 mg/mL Surface Age [s] mAbE + PLX188 0.1% Surface Tension [mN/m] A 80 40 50 60 70 1,000 0.01 0.1 1 10 100 PS80 0.05% PS80 0.01% PS80 0.1% mAbE + PS80 0.05% mAbE + PS80 0.01% mAbE 1 mg/mL Surface Age [s] mAbE + PS80 0.1% Turbidity [A350] 0.0 B 0.6 0.2 0.4 HPßCD THDD PLX188 PS80 w/o Surf. Stressed 0.05% Surf. or 0.35% HPßCD 0.01% Surf. or 0.035% HPßCD **** **** 0.1% Surf. or 3.5% HPßCD