Analysis of Subvisible Particles...Aperture diameter: Nominal diameter 20 – 2000 μm (Dynamic...

Analysis of Subvisible Particles Linda O. NarhiFormulation and Analytical Resources, R&DAmgen, Inc.

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

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

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.

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

Analysis of protein aggregates in solution

Light Obscuration is currently the common compendial method used for measuring subvisible particles in therapeutics.

Applicable size range: 2 – 102 μm

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

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

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

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.

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.

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

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

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

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

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

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


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


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

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

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%

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

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


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


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


FFF-MALS-UV

Detection sensitivity limits FFF, AUC, etc.

Studies with artificially generated subvisible particles

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

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

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

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.

Conclusions and Next Steps

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

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.

Acknowledgements

Shawn Cao

Nancy Jiao

Joey Pollastrini

Yijia Jiang

Marisa Joubert

John Gabrielson

Monica Pallitto

Back-up slides

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

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)

Applications of analytical methods

Information on subvisible particles below 10µm can be useful for process characterization

Sample Particles/mL at ≥ 2 µm

Particles/mL at ≥ 5 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



Control (unfilt., Lot 2) 680 ± 51 36 ± 4 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

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

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

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)

Analysis of Subvisible Particles...Aperture diameter: Nominal diameter 20 – 2000 μm (Dynamic...

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