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Analysis of Subvisible Particles Linda O. NarhiFormulation and Analytical Resources, R&DAmgen, Inc.
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Outline
Introduction: Protein aggregates
Techniques for analyzing sub micron and micron particles
Generation and characterization of protein aggregates for immunogenicity assessments
Future directions and conclusions
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There are multiple pathways by which proteins can lose their native structure
Krishnan S et al 2002, Biochemistry, 41: 6422-31Chi EY et al 2003, Protein Science, 12: 903-13
Chi EY et al 2003, Pharm Research. 20: 1325-36Thirumangalathu R et al 2009, J Pharm Sci (online)
Bee J et al 2009, J Pharm Sci (online)
Structural Changes
(e.g due to air-water interface)
Silicone oil orNano-particles
Adsorption/Assembly
Assembly
Structural Changes
Particles can have different morphology based on the mechanism of formationThanks to Eva Chi (Univ of New Mexico) and Krishnan Sampath
Conformational or colloidal stability dependant aggregation
Heterogeneous nucleation dependant aggregation
/chemical
/chemical
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Protein aggregates can have widely varying properties, depending on exposure during process and storage
Distribution of particle sizes
Shape
Reversibility
Stability
Total amount of aggregate
Native vs non-native conformation
Chemical ModificationDensityExposed T and B-cell epitopesCovalent vs. non-covalent bondsMorphology (Crystalline structure, amorphous, etc.)
Protein aggregates represent an extremely low fraction by mass of the total protein, are typically dynamic and can be influenced by many factors, including transportation and the drug product container/closure.
Potential effect on safety (immunogenicity) and efficacy.
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Protein aggregates range in size (1 million fold)
Definitions by size (all are aggregates):Oligomers:10 nm – 0.1 µmParticles:- Submicron: 0.1 to 1 µm- Micron: 1 to 125 µm- Visible: ≥125 µm
0.001µm 100µm0.01µm 0.1 µm 1 µm 10µm 1000 µm 10,000 µm
Currently there are no generally accepted definitions for particulates; these can be defined based on either size or mechanism of formation
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Analysis of protein aggregates in solution
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Light Obscuration is currently the common compendial method used for measuring subvisible particles in therapeutics.
Applicable size range: 2 – 102 μm
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Modifications to USP<788> enable more accurate testing of subvisible particles but challenges still exist
Reduced sample volume required for testing (single units ≥1 mL), allows measurement of unit to unit variability
Extended size range for particle measurements: ≥ 2, 5, 10, 15, 20, 25, 50 μmModified sample handling and include degassing step to minimize bubbles (false reading) and improve method performance
Enabled (product specific) method development, qualification and validation
Lack of a protein particle standard still poses challenge to accurate measurements
≥ 10 & 25 μm: required by USP≥ 2 & 5 μm: recommended to USPOthers (e.g. ≥ 15, 20, 50 μm): optional
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Vacuum degassing of protein solution improved method performance
≥ 10 μm particles/mL
Allow sample to equilibrate prior to testing Vacuum (75 Torr) degas
681
459420 444
490 465
592
0
100
200
300
400
500
600
700
800
900
0 1 2 3 4 5 6
Degas Duration (hour)
Part
icle
s/m
L
681
51 71 75 86 87 86
0
100
200
300
400
500
600
700
800
900
0 1 2 3 4 5 6
Degas Duration (hour)
Part
icle
s/m
L
Sample handling is critical to avoid both false positives and false negatives
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Subvisible particle levels vary and are dependent on the container closure and formulation
1
10
100
1000
10000
Sample 1_T1 Sample 1_T2 Sample 1_T3 Sample 2_T1 Sample 2_T2 Sample 2_T3
Samples
Part
icle
s/m
L
2 µm 5 µm 10 µm
1
10
100
1000
10000
Sample 3_T1 Sample 3_T2 Sample 3_T3 Sample 4_T1 Sample 4_T2 Sample 4_T3
Samples
Par
ticle
s/m
L
2 µm 5 µm 10 µm
Type I Glass Vials Pre-filled Syringe
1
10
100
1000
10000
In vials Placebo PFS
Samples
Part
icle
s/m
L
2 µm 5 µm 10 µm
Particles measured in the absence of protein
Light obscuration can only provide information on number and size of particles
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Camera Based TechnologiesMFI Flow cam
A third system, the FPIA, from Malvern, is also availableFor all systems the output is heavily dependent on the analytical algorithms employedby the software.
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Images of MFI versus FPIA
Silicone oil droplet Protein particlesParticle standards
Protein particles by FPIA
Protein particles by MFI
Size can be expressed in different manners: ferret diameter based on largest diameterECD or equivalent circular diameter, etc.
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FlowCam vs MFI: Placebo from PFSMFI
FlowCam
Placebo PFS_MFI low mag (>15 um feret)
Placebo PFS_MFI high mag (>10 um feret)
16.13 µm
24.88 µm
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The majority of particles measured from product stored in a prefilled syringe are silicone oil
Product in PFS_
Product in vial
Images obtained with MFI
Imaging systems provide information about shape and morphology that can help differentiate between protein and other particles
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Particles suspended in a weak electrolyte solution are drawn through a small aperture. Across the aperture a voltage is applied. This creates the “sensing zone.”A particle passing through the aperture displaces a volume of electrolyte solution equal to its own volume.Displaced electrolyte causes change in resistance across aperture resulting in voltage pulse.Pulse intensity is proportional to the particle volume (converted to Equivalent Spherical Diameter).
How the Technology Works
Overview of Coulter TheoryOverview of Coulter Theory
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What do the pulses depend on?
Particle characteristicsVolumeShapeConductivityPorosity
ApertureCurrentSize
Electrolyte solution
Overview of Coulter TheoryOverview of Coulter Theory
Aperture diameter: Nominal diameter 20 – 2000 μm(Dynamic range 2 - 60%: 0.4 - 1200 μm)
100 μm aperture: dynamic sizing range: 2 – 60 μm
Aperture sizes available (µm)20 70 200 56030 100 280 100050 140 400 2000
Isoton II Electrolyte: 1% salts solution (NaCl, KCl,Phosphate) (1% NaCl = 171 mM)Formulation buffers:100 mM NaOAc: with excipients10 mM NaOAc: with excipients
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Duke PS Standard Linearity on 3 Instruments:Low conductivity buffer
5 µm standard in a formualtion buffer
R2 = 1
R2 = 0.9997
R2 = 0.9987
0
20000
40000
60000
0 20 40 60 80 100% Conc
P/m
L
Coulter
MFI
HIAC
Linear(Coulter)Linear(MFI)Linear(HIAC)
5 µm standard in a formulation bufferCoulter: 100um aperture, current -200A, gain 16 (2.5 - 60 um)
Linearity: PS standard is linear over the dilution range on all 3 instruments.Particle Counts: All 3 instruments have comparable particle counts.
Coulter MFI HIAC
% conc Total P/mL Total P/mL Total P/mL
100 51730 50158 Too
5025768 26000 22418
2010396 10739 9787
105024 5559 4802
21076 1163 1033
0.2141 326 183
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IgG a Linearity on 3 Instruments
The results for this IgG are linear over the dilution range on all 3 instrument.Coulter counter is in agreement with MFI; HIAC gives much lower counts.
IgG a is at 20 mg/mL (100%) in 50mM NaAc and 100 mM NaCl buffer
IgG a in formulation buffer
R2 = 0.9914
R2 = 0.9988
R2 = 0.9784
0
20000
40000
60000
80000
100000
0 20 40 60 80 100% Conc
P/m
L
Coulter
MFI
HIAC
Linear(Coulter)Linear(MFI)Linear(HIAC)
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IgG b Linearity on 3 Instruments: 100 mMNaAcetate buffer
This IgG is linear over the dilution range on all 3 instruments.The counts of SbVPs: Coulter > MFI > HIAC
IgG b is at 150 mg/mL (100%) in 100mM NaAc buffer
IgG b in 100 mM NaAc
R2 = 0.9854
R2 = 0.9744
R2 = 0.9891
0
20000
40000
60000
80000
0 20 40 60 80 100% Conc
P/m
L
Coulter
MFI
HIAC
Linear(Coulter)Linear(MFI)Linear(HIAC)
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The absolute quantity of particles varies between techniques
1
10
100
1000
10000
100000
2.00 5.00 10.00 15.00 20.00 25.00 50.00
ECD (µm)
Part
icle
s/m
L
HIAC MFI
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Conclusions: Electrazone, LO, Imaging systems
Coulter/Elzone HIAC Flow Cam MFI
Results Concentration, Size, MassConcentration, Size Concentration, Size,
Shape, TransparencyConcentration, Size, Shape, Transparency
Principle Electrical sensing zone Light obscuration Flow microscopy Flow microscopy
Size 0.4 µm to 1.2 mm 1 - 400 µm 1 µm to 3 mm 0.75 - 400 µm
Shape No No Yes Yes
Transparency No No Yes Yes
Volume required 10 mL 2 mL 1 mL 1 mL
Sampling Efficiency 10% (1/10) 60% (1.5/2.0) 30% (0.3/1.0) 50% (0.5/1.0)
Calibration yes yes no no
Interference by shape no yes yes yes
Interference by RI no yes yes yes
Electrolyte required yes No no no
Sampling efficiency = 100 x volume analyzed/total volume required
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Mab Monomer/Oligomer Separation by AF4: Satisfactory
time (min)0.0 5.0 10.0 15.0 20.0 25.0
UV
220
nm (r
elat
ive
scal
e)
0.0
0.5
1.0
5.0 10.0 15.0 20.00.00
0.05
0.10SEC
Monomer: 70.2%
HMWS: 29.8%
FFF
Monomer: 74.0%
HMWS: 26.0%
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Sub-μm Particle Quantification by FFF-MALS is Possible in Some Cases (enriched in SbVP)
time (min)10.0 15.0 20.0 25.0 30.0 35.0
rela
tive
scal
e
0.0
50.0
100.0
time (min)10.0 15.0 20.0 25.0 30.0 35.0
radi
us (n
m)
0.0
50.0
100.0
sin²(theta/2)0.0 0.2 0.4 0.6 0.8
R(t
heta
)
-73.0x10
-73.1x10
-73.2x10
-73.3x10Angular Dependence
T = 15 minr = 23 nm
time (min)10.0 15.0 20.0 25.0 30.0 35.0
rela
tive
scal
e
0.0
50.0
100.0
time (min)10.0 15.0 20.0 25.0 30.0 35.0
radi
us (n
m)
0.0
50.0
100.0
sin²(theta/2)0.0 0.2 0.4 0.6 0.8
R(t
heta
)
-73.0x10
-73.1x10
-73.2x10
-73.3x10Angular Dependence
T = 15 minr = 23 nm
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FFF-MALS detection is not sensitive enough to detect levels of SbVP present in product samples
time (min)0.0 10.0 20.0 30.0 40.0
rela
tive
scal
e
0.0
0.2
0.4
0.6
0.8
1.0
sub-μm particles would be here
FFF-MALS-UV
time (min)0.0 10.0 20.0 30.0 40.0
rela
tive
scal
e
0.0
0.2
0.4
0.6
0.8
1.0
sub-μm particles would be here
FFF-MALS-UV
time (min)0.0 10.0 20.0 30.0 40.0
rela
tive
scal
e
0.0
0.2
0.4
0.6
0.8
1.0
sub-μm particles would be here
FFF-MALS-UV
time (min)0.0 10.0 20.0 30.0 40.0
rela
tive
scal
e
0.0
0.2
0.4
0.6
0.8
1.0
sub-μm particles would be here
FFF-MALS-UV
Detection sensitivity limits FFF, AUC, etc.
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Studies with artificially generated subvisible particles
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Known treatments and techniques can induce aggregates of various properties/populations for further evaluation
Treatment Targeted Properties
Heat (80C)
Crosslink
Hydrogen peroxide oxidation
Metal catalyzed oxidation
pH change
Mechanical manipulation
Protein-coated nanosphere
Separate by Size (2 µm to > 10 µm)
Non-native like conformation
Native-like conformation; Irreversible repeatability
Chemical modification
Chemical modification; Covalently bound molecules
Non-native like conformation; Native disulfide bonds
Native and non-native conformation
Regular array; Higher order structure
Distribution of sizes
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Treatment
# Particles(+ = 101)
HIAC
Particle Size(µm)
MFI
Amorphous(A)
Structured(S)
MFI/FlowCAM
Reversible (R)Partially
Reversible (PR)
Irreversible (I)
Secondary Structure
(CC)
FTIRSup pellet
Surface Hydrophobicity
% ANS Binding
Oxidation of Amino Acids
Peptide Mapping
% Protein Collected by
Spinning
Absorbance
1 untreated + < 10 A
A
A
A
A
A
A
A
A
-
A
A
A
A
A
A
A
A
- 100 - 11% None < 1%
2 untreated + <30 - 100 - 8% None < 1%
1 H2O2 ++ < 10 R 99 - 11% Met < 3%
2 CuSO4 + <5 R 45 - - Met,Trp,His < 1%
2 syringe SO (+) +++ <25 - 96 67 14% - ~17%
2 stir 1d +++++ <25 PR 98 77 99% None ~45%
2 stir 3d +++++ <80 PR 55 - 52% Met,Trp,Cys ~89%
1 80C +++ < 220 I 42 20 100% - ~ 90%
1 CuSO4 +++ < 40 R 92 - 27% Trp,Met,His < 5%
1 pH 11 ++ < 20 R 92 40 9% - ~ 4%
1 syringe SO (+) ++++ < 50 PR 96 76 16% Met < 10%
1 syringe SO (-) +++ < 40 PR 92 - 31% Met < 10%
1 x-link ++ < 15 - 89 - 8% - < 3%
1 stir 1d +++++ < 20 PR 98 34 14% None ~ 25%
1 agitate SO (+) +++ < 10 - 89 76 52% - ~75%
1 stir 3d +++++ < 60 PR 89 20 49% Met,Trp ~ 100%
1 65C pH 8.5 ++++ < 130 PR 100 26 95% - ~ 85%
2 80C +++ < 100 I - - 100% - ~86%
Class Number
Class 1 –not aggregated
Class 2 –chemically modified
Class 3 –small, native, reversible
Class 4 –small, native, irreversible
Class 5 -small, partially native, partially reversible
Class 6 –medium, partially native, partially reversible
Class 7 –large, unfolded, irreversible
Classification of Aggregates from 2 IgG2 Molecules
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0
20000
40000
60000
80000
100000
untrea
tedsto
re 37
C 24hr
store
37C 8dpH 3.
5pH 4.
3pH 8.
5pH 11
pipette
agita
te 4C
8d
agita
te wSO 4C
8dag
itate
22C 3d
agita
te wSO 22
C 3dsy
ringe
no SO
syrin
ge w
SOsti
r 1d
stir 3
d
Cross
link -
EDC
H2O2
CuSO4 80
C
65C an
d pH 8.5
Aggregate Treatment
Diff
eren
tial P
artic
le C
ount
s
251015202550100
141,000 930,000 119,000 252,000
storagepH
x-link heat
oxidation
Particles generated under different forced conditions have different population distributions
mechanical stress
≥ size (µm)
untreate
dsto
re 37
C 1dsto
re 37
C 8dpH 3.
5pH 4.
3pH 8.
5pH 11pipett
e ag
itate
4C 8d
agita
te wSO
4C 8d
agita
te 22
C 3d
agita
te wSO
22C 3d
syrin
ge no SO
syrin
ge wSO
stir 1
dsti
r 3d
Cross
link -
EDCH2O
2 CuSO4
80C
65C an
d pH 8.5
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Aggregate Sets have Distinct Morphological Features
80Cdiameter ≥ 220µm
H2O2diameter ≥ 5µm
agitate 22C SO (+)diameter ≥ 5µm
syringe SO (-)diameter ≥ 40µm
stir 3ddiameter ≥ 60µm
CuSO4 diameter ≥ 40µm
stir 1ddiameter ≥ 20µm
syringe SO (+)diameter ≥ 50µm
pH 11diameter ≥ 20µm
untreateddiameter ≥ 10µm
65C pH 8.5diameter ≥ 130µm
IgG2 1 images taken by MFI: 1mg/ml aggregates diluted 20-220X in A5, 0.5 ml analyzed.
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Conclusions and Next Steps
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ConclusionsLimitations with current technology include:– Lack of specificity to protein particles– Lack of protein aggregate standards– Differences in absolute quantitation between analytical methods– Lack of technology to quantitate and characterize below 2µm– Lack of validated predictive models to assess immunogenicity
Limitations of the current technology and inherent variability of aggregate population confound establishing meaningful acceptancecriteria for release testing and comparability
Additional testing and characterization of subvisible particles is necessary to better understand the nature and utility of monitoring subvisible particles
Additional testing is necessary to understand limits and utility of assays for immunogenicity, and ability to predict clinical outcome
Results and correlations will likely be specific to the product and the aggregate involved, and need to be evaluated on a case by case basis
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Next StepsContinue to explore the relationship between amounts of protein particles of different sizes (what species are on pathway to SbVP?)
Monitor 2 and 5 µm sizes during process and formulation development
Work with vendors to develop technology to distinguish between silicone oil droplets, foreign particles and proteins
Develop methods to characterize small amounts of particles with minimal sample manipulation
Develop and verify in vivo and/or in vitro models to evaluate the significance of subvisible particles and immunogenicity.
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Acknowledgements
Shawn Cao
Nancy Jiao
Joey Pollastrini
Yijia Jiang
Marisa Joubert
John Gabrielson
Monica Pallitto
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Back-up slides
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Analysis of isolated particles can be used to compare biochemical and biophysical properties
FT-IR Spectra show presence of Amide I and II bands
Second Derivatives of FT-IR Spectra indicate presence of β-sheet structures
Ref
eren
ce S
tand
ard
Part
icle
A
B
C
SE-HPLCrCE-SDS
Slight increase in HMWC due to dissociable
aggregates
Subvisible particles isolated from a product sample retain full potency and similar quality profile
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The absolute number of particles varies between techniques although relative number relationships are consistent
1.E-01
1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
0.1 1 10 100
Diameter (um)
Part
icle
s/m
L
FFFPMSHIAC
Note: For FFF there is dilution during the analysis; Error bars represent STD for triplicate measurements; PMS = Particle Measuring System (based on static light scattering)
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Applications of analytical methods
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Information on subvisible particles below 10µm can be useful for process characterization
Sample Particles/mL at ≥ 2 µm
Particles/mL at ≥ 5 µm
Particles/mL at ≥ 10 µm
Particles/mL at ≥ 25 µm
Control (unfilt., Lot 1) 339 ± 78 29 ± 2 Below LOQ Below LOQ
1x filtration Lot 1 29 ± 6 11 ± 5 Below LOQ Below LOQ
2x filtration Lot 1 19 ± 9 14 ± 9 Below LOQ Below LOQ
5x filtration Lot 1 50 ± 14 12 ± 9 Below LOQ Below LOQ
Control (unfilt., Lot 2) 680 ± 51 36 ± 4 Below LOQ Below LOQ
1x filtration Lot 2 20 ± 3 5 ± 2 Below LOQ Below LOQ
Filtration by 0.2 µm filter has removed particles at ≥ 2 μm1x filtration is effective in removing the particles
Particle concentrations are below LOQ at ≥ 10 and 25 μm
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A relationship exists between particles of different sizes, with the slope being product-dependent
1
10
100
1000
10000
0 5 10 15 20 25 30Particle Size (um)
Part
icle
s/m
LNative Protein Light & Heat Stressed Protein
1
10
100
1000
1 10 100
Sizes (µm)
Part
icle
s/m
L
No visible particles observed
Visible particles observed in 80% units
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FTIR Spectra of IgG2 1 Aggregate Groups
Wavenumber cm-1
Treatment CC Total CC Sup CC Pellet
untreated 100 100
89
97
92
89
89
92
96
98
89
99
92
-
100
42
-
freeze-thaw 98 -
LN 94 -
pH 11 99 40
x-link EDC 94 -
agitate SO (+) 95 76
syringe SO (-) 96 9
syringe SO(+) 96 32
stir 1d 93 34
stir 3d 56 20
H2O2 99 -
CuSO4 78 25
pH 3.5 98 -
65C pH 8.5 98 26
80C 16 20
Correlation Coefficient
160016201640166016801700
0.00
0040
0.00
0045
0.00
0050
0.00
0055
0.00
0060
Abs
orba
nce
Uni
ts
Pellet
- = not tested
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Glutaraldehyde Crosslinking of IgG2
0
500
1000
1500
2000
0 0.5 1 2 10 0 0.5
Time (minutes)
Diff
eren
tial P
artic
le C
ount
s
251015202550100
EDC Crosslinking of IgG2
0500
100015002000250030003500400045005000
0 0.5 1 2 5 10 30 60 90
Time (minutes)
Diff
eren
tial P
artic
le C
ount
s
251015202550100
Particles can also be generated by Crosslinking in Solution
HIAC Particle Counts
Mechanism of EDC Mechanism of Glutaraldehyde
low pH high pHMore EDC added
2 µm ~ 163,000 5 µm ~ 28,000
size(µm)