studies on the potential immunogenicity of …...james thomas david volkin (ku) jette wypych lei...
TRANSCRIPT
Studies on the potential immunogenicity of attributes of protein aggregates
Linda Narhi, Scientific Executive Director
Product Development
Amgen, Thousand Oaks, CA
Acknowledgements
Matthew Baker (Antitope)
Vivian Bi
Christine Bryson (Antitope)
Shawn Cao
Naren Chirmule
Meghana Deshpande
Catherine Eakin
John Ferbas
Greg Flynn
Madeline Fort
Francis Kinderman
Quanzhou Luo
Helen Reynolds (Antitope)
Jilin Sun
Steve Swanson
Srivalli Telikepalli (KU/NIST)
James Thomas
David Volkin (KU)
Jette Wypych
Lei Zhou
Theresa Goletz
Jonathan Herskovitz
Martha Hokom
Vibha Jawa (MERCK)
Wim Jiskoot (Leiden Unv)
Nate Joh
Marisa Jouobert
Arunan Kaliyaperumal
Keith Kelley
Bruce Kerwin (Just)
Related publications:
• Joubert MK, Deshpande M, Yang J, Reynolds H, Bryson C, Fogg M, Baker MP, Herskovitz J, Goletz TJ, Zhou L, Moxness M, Flynn
GC, Narhi LO, Jawa V. “Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics” 2016 PLOS One
• Jiskoot W, Kijanka G, Randolph TW, Carpenter JF, Koulov AAV, Mahler HC, Joubert MK, Jawa V, Narhi, LO Mouse Models for Assessing
Protein Immunogenicity: Lessons and Challenges (2016) J Pharm Sci 105 1567-1575
• Moussa EM, Panchal JP, Balakrishnan SM, Blum JS, Joubert MK, Narhi LO, Topp EM. “Immunogenicity of therapeutic protein aggregates” J
Pharm Sci 2016 (DOI: 10.1016/j.xphs.2015.11.002).
• S Telikepalli, HE Shinogle, PS Thapa, JH Kim, M Deshpande, V Jawa, CR Middaugh, LO Narhi, MK Joubert and DB Volkin. “Physical
characterization and in vitro biological impact of highly aggregated antibodies separated into size enriched populations by FACS” J
Pharm Sci 2015; 104 (5): 1575-91.
▪ V Bi, V Jawa, MK Joubert, A Kaliyaperumal, C Eakin, K Richmond, O Pan, J Sun, M Hokom, TJ Goletz, J Wypych, L Zhou, BA Kerwin,
LO Narhi and T Arora ”Development of a human antibody tolerant mouse model to assess the immunogenicity risk due to aggregated
biotherapeutics” J Pharm Sci 2013 102 (10): 3545-55. Winner of the 2015 American Pharmacists Association Ebert Prize.
▪ MK Joubert, M Hokom, C Eakin, L Zhou, M Deshpande, MP Baker, TJ Goletz, BA Kerwin, N Chirmule, LO Narhi and V Jawa ”Highly
aggregated antibody therapeutics can enhance the in vitro innate and late-stage T-cell immune responses” J Biol Chem. 2012 287 (30):
25266-79.
▪ Q Luo, MK Joubert, R Stevenson, RR Ketchem, LO Narhi and J Wypych ”Chemical modifications in therapeutic protein aggregates
generated under different stress conditions” J Biol Chem 2011 286 (28): 25134-44.
▪ MK Joubert, Q Luo, Y Nashed-Samuel, J Wypych and LO Narhi ”Classification and characterization of therapeutic antibody
aggregates” J Biol Chem. 2011 286 (28): 25118-33.2
Outline
▪Generation and characterization of mAb aggregates
▪ Test systems for Comparative Immunogenicity Assessment
▪ Testing size, oxidation, silicone oil, etc. in the model
systems
3
Characteristics of successful biotherapeutics
▪ Efficacious:
– Achieve desired result at reasonable dose
– With long enough half life (PK) to be effective
▪ Safe:
– No unexpected side effects
– No non-specific binding
– No Toxicity
– Minimized Immunogenicity (both neutralizing and non-neutralizing Abs)
▪ Manufacturable:
– Stable shelf life for up to 2 years,
– Able to make consistently and efficiently
– Fit existing facilities and process platforms as much as possible
4
Rationale for studies on relative immunogenic potential of antibody aggregates
▪ All formulated antibody drug products contain low levels of aggregates.
▪ The clinical immunogenic risk of aggregates is uncertain, resulting in a high risk
factor being assigned to the presence of protein aggregates in biologics.
▪ To reduce this uncertainty, the following should be defined:
− Aggregate attributes that cause a response
− Amount of aggregate required to break the threshold of activation
− Extent and nature of the response
▪ Published reports use a wide range of experimental methods and aggregate
samples and show conflicting results.
▪ The goal of our studies is to:
−Generate and characterize aggregate using stresses to which biotherapeutics
are exposed
−Test the impact of these aggregates in in vitro and in vivo model systems that
could be predictive for relative potential of a clinical impact
−Identify characteristics of aggregate (i.e. size and threshold) that cause a
response
−Rank the potential clinical immunogenic risk of aggregated therapeutics
The biological impact of aggregates is not fully known, so testing their
effect in physiologically relevant model systems can help inform risk
assessment5
Biopharmaceutical aggregates can be generatedduring all steps of the manufacturing process
Steps During the
Manufacturing Process
▪ Cell culture
▪ Purification
▪ Formulation
▪ Storage
▪ Shipping
▪ Administration
Stress Conditions
▪ Heat
▪ Freeze-thaw
▪ Cross-linking
▪ Protein concentration
▪ Formulation change – pH, salt
▪ Addition of
extractables/leachables
▪ Chemical modification
▪ Mechanical Stress
▪ Surface effects
▪ Nano-particles
6
Classification scheme for Mab aggregates
based on their characteristicsClass Stress
Treatment
Particle
Concentration
Particle
Size
(µm)
Folded
Secondary
Structure
Folded
Tertiary
Structure
Surface
Hydro-
phobicity
Reversibility
Rev. (R)
Part Rev. (PR)
Irrev. (I)
Chem.
Mod.
%
Insol.
Agg.
Oligo. Nano. Micro. Vis. Sup Pellet
Class 1
not aggregated
monomer, ft-slow, ft-
fast, store, pH 3.5 to
8.5, pipette, agitate-
4C-so-, agitate-so-
+ + + ND 10 ≥89% - ≤1 ≤12% ND ND ND
Class 2
metal containingmetal - ++ +++ ND 40 92% 24% 2 27% PR ++ 3%
Class 3
small, folded,
partially reversible
H2O2 + + + ND 5 99% - 1 11% PR +++ 3%
pH 11 ++ + ++ ND 25 92% 71% 1 9% R - 4%
syringe-so- + - +++ ND 40 92% - 1 31% PR + 7%
syringe-so+ + ++++ +++ ND 60 96% 32% 1 16% PR + 7%
Class 4
small, folded, irreversiblexlink ++ - ++ ND - 89% - 1 8% - - 3%
Class 5
small, partially folded,
partially reversible
stir-20h ++++ ++++ ++++ ND 25 98% 42% 1 14% R + 28%
agitate-so+ +++ +++ +++ ND 30 89% 50% 1 52% - - 73%
Class 6
medium, mostly
unfolded,
partially reversible
stir-3d ND + +++ ND 65 89% 20% 2 49% R ++ 97%
65C/pH 8.5 ++ ++ ++++ ++ 140 100% 26% 4 95% PR ++ 86%
Class 7
large, unfolded,
irreversible
90C ND + ++ ++ 220 42% 19% 4 100% I +++ 92%
▪ MK Joubert, Q Luo, Y Nashed-Samuel, J Wypych, and LO Narhi J Biol Chem. 2011 286 (28): 25118-33.
▪ Q Luo, MK Joubert, R Stevenson, RR Ketchem, LO Narhi, and J Wypych J Biol Chem 2011 286 (28): 25134-44.
Aggregates have a wide-range of properties that depend on the method of generation.7
mAb1
65C/pH 8.5
≥ 90 µm
mAb3
65C/pH 8.5
≥ 90 µm
mAb2
65C/pH 8.5
≥ 90 µm
IVIG
65C/pH 8.5
≥ 90 µm
mAb2
untreated
≥ 10 µm
mAb3
untreated
≥ 10 µm
IVIG
untreated
≥ 10 µm
mAb1
untreated
≥ 10 µm
mAb1
stir-3d
≥ 80 µm
IVIG
stir-3d
≥ 80 µm
mAb3
stir-3d
≥ 80 µm
mAb2
stir-3d
≥ 80 µm
mAb1
stir-20h
≥ 25 µm
mAb3
stir-20h
≥ 25 µm
mAb2
stir-20h
≥ 25 µm
IVIG
stir-20h
≥ 25 µm
mAb1
syringe-so+
≥ 40 µm
mAb3
syringe-so+
≥ 40 µm
mAb2
syringe-so+
≥ 40 µm
IVIG
syringe-so+
≥ 40 µm
Particle images of aggregates were captured on a liquid-borne particle Micro-Flow Imaging (MFI)
System DPA4100. Representative images of the largest particles detected are shown. The size
threshold indicates the lower size limit of the particles that were used for comparison.
Aggregate generated by the same stress have a
similar morphology
8
10
100
0
25
50
75
2
SI
% d
on
ors
400
10
100
0
25
50
75
2
SI
% d
on
ors
400
10
100
0
25
50
75
2
SI
% d
on
ors
400
IL-1 IL
-6
IL-1
0
MCP-1
MIP
-1
MIP
-1
MM
P-2
TNF-
10
100
0
25
50
75
2
SI
% d
on
ors
400
10
100
0
25
50
75
2
SI
% d
on
ors
400
10
100
0
25
50
75
2
SI
% d
on
ors
400
10
100
0
25
50
75
2
SI
% d
on
ors
400
IL-1 IL
-6
IL-1
0
MCP-1
MIP
-1
MIP
-1
MM
P-2
TNF-
10
100
0
25
50
75
2
SI
% d
on
ors
400
IL-1 IL
-6
IL-1
0
MCP-1
MIP
-1
MIP
-1
MM
P-2
TNF-
10
100
0
25
50
75
2
SI
% d
on
ors
400
10
100
0
25
50
75
2
SI
% d
on
ors
400
10
100
0
25
50
75
2
SI
% d
on
ors
400
10
100
0
25
50
75
2
SI
% d
on
ors
400
Aggregation of mabs enhances the early
response of PBMC from individual donors (n = 22)
mAb1 mAb2 mAb3
syringe-so+
stir-20h
stir-3d
65C/pH 8.5
Stress treatment
SI stir-20h
SI stir-3d
SI 65C/pH8.5
SI syringe-so+
% donors
The extent of the response to aggregates depends on the aggregate and molecule type.
▪ Results are shown as the average SI of responding donors (above the monomer) and the percentage of
donors that responded (n = 22).
▪ The level of cytokine secretion induced by the mAb samples was much lower than that induced by the
positive control, LPS, in all cases.
SI
(ab
ove
mo
no
me
r)
MODEL SYSTEMS
10
IFN-g, IL-2, TNF-α,
IL-4, IL-5, IL-13
T-cell proliferationTCR
Antibody
/proteins
APC
Naïve CD4+ cell
B cell
Ig class switch
NAB
CD40-CD40L
MHC-II
Propagation of an immune response
Th17 cell
Th1 cell
Th2 cell
IL-10, IL-1β, IL-6,
MCP-1, MIP-1α,
MIP-1β, IL-1ra,
TNF-α, TNF-β
Aggregated proteins
T-cell
epitope
Early Phase
Late
Phase
11
Human
blood
sample Donor
BankUp to
8 days
~50 donors
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10
Peptide
% D
on
or
Re
sp
on
se
Cytokine Profile
PBMC
50 donors
CD4+ T-cells
CD8+
depletion
T-cell Proliferation
EPISCREENTM – HUMAN T-CELL ASSAYS
PBMC
50 donors
Samples were collected at the early (20 hours) and late-stage (7-8 days).
Experimental design for an in vitro comparative
immunogenicity assessment (IVCIA) assay
12
aggregatemonomer
Samples were analyzed by an assay provided by Antitope (shown)
and in a similar IVCIA assay optimized at Amgen - Wullner, D et al. Clinical Immunol 137, 5-14 (2010); Joubert, MK et al. J Biol Chem 287:30, 25266-79 (2012)
aggregate
/attribute
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10
Peptide
% D
on
or
Resp
on
se
Cytokine Profile
(Early Phase)
Early 11-plex: IL-10,
IL-1ra, IL-1alpha, IL-
1beta, IL-6, IL-8,
MCP-1, MIP-1alpha,
MIP-1beta, TNF-
alpha, TNF-beta
Absorbance
650 nm
Softmax Pro
Seria
l dilu
tion
s (3
X)
Incubate
24 hrs. at
37ºC
5% CO2
Collect sups for
cytokine profile after 6-
24 hrs. store at -70ºC
IIRMIs monomer
Day 2 Day 3 Day 4
▪ THP1-Xblue 24 hracclimatization step added
▪ Biotherapeutics added to cells
▪ 24 hr cell culture incubation
Cell line assay optimized to assess the relative risk of early immune activation of biotherapeuticattributes
RAW-Blue
Ramos-Blue
THP1-Xblue
Selected 3 cell lines
with overlapping PRRs
THP1-
Xblue
acclimatiz
ation in
flask 24 hr
Day 1
Add samples
to cells
Cell line sups (80
µl) + Quanti blue
(120 µl) incubate
for 24 hrs.
XenoMouse
Human IgG2
tolerant
weak immune
response
Wild-type mouse
Human IgG2
non-tolerant
robust immune
response
Model Development
Xeno-het mouse
Human IgG2 tolerant
robust immune response
Test for transient and sustained ADA,
cytokine secretion, etc.
In vivo xeno heterozygous mouse model was
developed and applied
V Bi, V Jawa, MK Joubert, A Kaliyaperumal, C Eakin, K Richmond, O Pan, J Sun, M Hokom, TJ Goletz, J Wypych, L Zhou, BA Kerwin, LO
Narhi and T Arora ”Development of a human antibody tolerant mouse model to assess the immunogenicity risk due to aggregated
biotherapeutics” J Pharm Sci 2013 102 (10): 3545-55.
Winner of the 2015 American Pharmacists Association Ebert Prize.
The XenoMouse and the wild-type mouse were crossbred to generate a strain (Xeno-het)
that is immune tolerant to human IgG2 but can also mount a robust immune response.
Experimental plan to test immunogenic potential of different aggregate (and HCP) species
Attribute Immunization Characterization
IgG2
mAb
1.
Xeno-het
Mouse
• Anti-drug Antibodies
• Cytokine secretion
• B-cell activation
HMW, Si oil,
ox aggregate,
Host Cell
Protein, etc.
Controls
TCE-KLH-mAb
mAb Monomer
Stirred-aggregate
Buffer
• Cytokine secretion
• Early immune activation3. Cell
Line
Assay
2. PBMC-
Based
IVCIA
Assay
TESTING DIFFERENT TYPES OF AGGREGATES
Which attributes correlate to a response?
16
Stirring induced aggregates were generated
with varying levels of oxidation
Samples Treatment Purpose
mAb1 monomer - Very low level oxidation
mAb1 stir-N2 stirred for 20 h with
N2/Met
Aggregates with low level of oxidation
mAb1 stir-lowO2 stirred for 20 h with
low aeration
Aggregates with low level of oxidation
mAb1 stir-highO2 stirred for 20 h with
high aeration
Aggregates with moderate level of
oxidation
mAb1-NoMet monomer all 9 Met in mAb1
mutated to Leu
Little to no oxidation. No Met oxidation
mAb1-NoMet stir-lowO2 stirred for 20 h with
low aeration
Aggregated with very low level oxidation.
No Met oxidation
17
Samples included a mAb1 mutant where all Met
residues were mutated to Leu to prohibit Met oxidation
18
Provided October 25, 2017, as part of an oral presentation and is qualified
by such, contains forward-looking statements, actual results may vary
materially; Amgen disclaims any duty to update.
% HMW in
200-μg/ml
mAb1
[mAb1]measure
d (µg/ml)
% [mAb1]
Measured/
Targeted
[Endotoxin]
(EU/mL)
0% 184 92.2 <0.05
5% 178 89.2 0.336
25% 182 91.2 1.868
Successful preparation of enriched mab1 HMW samples
• [mAb] is at ~90% of the target conc.
• [HMW]Targeted agrees with trend in analytical SEC.
• [Endotoxin]target of < 5 EU/ml achieved.
0
0.5
1
12 14 16 18 20N
orm
aliz
ed A
215
Elution Time (min)
SEC-HPLC of Reconstituted mAb1 Samples
9-ug mAb1, 0% Multimer
9-ug mAb1, 5% Multimer
9-ug mAb1, 25% Multimer
HMW
HMW
HMW
Process-related HMW (primarily irreversible) was isolated by SEC and
diluted in a background of monomeric protein for testing at 0%, 5%, and
25%
2012_Sebastien Jouffray
www.ondrugdelivery.com
Formation of silicone oil (SO) droplets in pre-filled syringes (PFS)
# Sample PS80 Transport
1 mAb1 / PS80/ Static Yes No
2 mAb1 / PS80/ Transport Yes Yes
3 Buffer/ PS80/ Static Yes No
4 Buffer/ PS80/ Transport Yes Yes
5 mAb1 / No PS80/ Static No No
6 mAb1 / No PS80/ Transport No Yes
7 Buffer/ No PS80/ Static No No
8 Buffer/ No PS80/ Transport No Yes
9mAb1 Bulk (no Sku) carboy (positive
control)Yes No
Sample plan
21
Provided June 27, 2017, as part of an oral presentation and is qualified by
such, contains forward-looking statements, actual results may vary
materially; Amgen disclaims any duty to update.
Subvisible particles by micro-flow imaging show
differences in spherical vs non-spherical particle counts
PS80 increases amount of spherical particles
Transport increases particles
Inclusions of PS80 protects against formation of non-spherical particles
22
Provided June 27, 2017, as part of an oral presentation and is qualified by
such, contains forward-looking statements, actual results may vary
materially; Amgen disclaims any duty to update.
0 1 2 3 4 5 6 7 8 9 1 0 1 11 2 3 4 5 6 7 8 9 1 0 1 11 2 3 4 5 6 7 8 9 1 0 1 1
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
AD
A S
ign
al
Fo
ld I
nc
re
as
e
(Po
st:
Pre
-In
jec
tio
n)
A n tig e n In je c tio n s
1 2 3 4 5 6 7 8 9 1 0 1 1
Tce-klh-mab1 positive control induces A strong ADA
response in xeno-het mice
Dotted Line: Threshold set at 1.5-Fold Increase
Black Bar: Average Response
Open Marker: Individual Response
Closed Marker: Individual Response\Above
Threshold and Specific
TCE-KLH-mAb1 0.1X TCE-KLH-mAb1 +
0.1X mAb1 DS
in PFS, PS80, transport in PFS, no PS80, transport
( # 3 buffer control was similar)
Buffer
Positive control TCE-KLH-mAb1 induces a strong and persistent ADA response in
contrast to buffer controls, with no masking from high protein concentration
Si oil alone shows no activation
Buffer Positive
Control
High Protein
Concentration Control
Time (weeks)
10 fold dilution
High SO
23
Provided June 27, 2017, as part of an oral presentation and is qualified by
such, contains forward-looking statements, actual results may vary
materially; Amgen disclaims any duty to update.
1 2 3 4 5 6 7 8 9 1 0 1 11 2 3 4 5 6 7 8 9 1 0 1 11 2 3 4 5 6 7 8 9 1 0 1 11 2 3 4 5 6 7 8 9 1 0 1 1
0
2 0
4 0
6 0
8 0
1 0 0
AD
A S
ign
al
Fo
ld I
nc
re
as
e
(Po
st:
Pre
-In
jec
tio
n)
A n tig e n In je c tio n s
No ADA to mab1 with increased so droplets independent of PS80 for mAb1
Dotted Line: Threshold set at 1.5-Fold Increase
Black Bar: Average Response
Open Marker: Individual Response
Closed Marker: Individual Response\Above
Threshold and Specific
mAb1
An increase in mAb1 SO droplets does not induce the generation of ADA in Xeno-
het mice, regardless of the presence or absence of Tween
A 10x dilution yielded the same results, showing signal was not masked by high
protein concentration
Time (weeks)
mAb1 mAb1 mAb1
in PFS, PS80, static in PFS, PS80, transport in PFS, no PS80, static in PFS, no PS80, transport
High SO Low SO
High protein
aggregates
24
Provided June 27, 2017, as part of an oral presentation and is qualified by
such, contains forward-looking statements, actual results may vary
materially; Amgen disclaims any duty to update.
IL-1
IL-1
IL-1
rIL
-6IL
-8
IL-1
0
MC
P-1
MIP
-1
MIP
-1
TN
F-
TN
F-
0
25
50
75
100
% D
on
or w
ith
2
-Fo
ld R
es
po
ns
e
IL-1
IL-1
IL-1
rIL
-6IL
-8
IL-1
0
MC
P-1
MIP
-1
MIP
-1
TN
F-
TN
F-
1
10
100
1000
Re
sp
on
se
Fo
ld I
nc
re
as
e
IL-1
IL-1
IL-1
rIL
-6IL
-8
IL-1
0
MC
P-1
MIP
-1
MIP
-1
TN
F-
TN
F-
IL-1
IL-1
IL-1
rIL
-6IL
-8
IL-1
0
MC
P-1
MIP
-1
MIP
-1
TN
F-
TN
F-
Symbols: individual donor responseDotted line: 2.0 thresholdLines: % donors above thresholdBars: ave response above threshold
TCE-KLH-mAb1
Cytokines
Positive control
Early phase response in the IVCIA assay to
increased levels of SO droplets with and without
tween for MAb1
Increased numbers of SO droplets, in the presence or absence of tween,
induced little to no increase in cytokine secretion at the early phase in PBMC
N=5 donors
mAb1
in PFS, PS80, transport
mAb1
in PFS, no PS80, transport
Buffer
in PFS, PS80, transport
High SO High SOLow SO
High protein
aggregates
Summary of studies on impact of silicone oil
Attributes Xeno-het
mice
(anti-drug
antibodies)
Xeno-het
mice
(cytokines)
IVCIA Assay
(Early phase
cytokines in
PBMC)
IVCIA Assay
(Late phase
cytokines in
PBMC)
OCLA Assay
(PRR
Activation
Signals in
Cell Lines)
mAb1 SOD↑, PS80+ - - - + -mAb1 SOD↑, PS80- - - - - -Buffer SOD↑, PS80+ - - - + -mAb1 5% HMW - + - + -mAb1 25% HMW - + + + -Positive Ctrls
(KLH-TCE-mAb1 w/ or w/o ↑[mAb1],
LPS/PHA, Tri-Dap, or
CL075)
++++ ++++ ++++ ++++ ++++
▪ The IVCIA assay was found to be highly sensitive (more sensitive than the in vivo model) to
low numbers of aggregates making it a useful tool for probing the relative risk of
immunogenicity of aggregates attributes.
▪ Highly aggregated therapeutic antibodies (orders of magnitude more particles than found in
product) can enhance the early and late-stage responses in the IVCIA Assay and induce a low
and transient ADA response in Xeno-het mice.
▪ Protein aggregates are a heterogeneous population, depending at least in part on the method
by which they were generated. Attributes that can contribute to a signal are size, amount,
structure, and for some populations, chemical modification. The signal is weak and transient
▪ The effect of silicone oil droplets was also tested and found to elicit no immune response
▪ In contrast, the oxidation of methionine residues in the monomeric and/or aggregated protein
do not appear to enhance the risk of immunogenicity in the assays employed.
▪ Samples enriched in HMW species induced a low level (less than even stirred aggregate) of cytokine
secretion in Xeno-het mice and the IVCIA assay, but no response in the cell line assay and no ADA in
vivo.
▪ The response seen with even the most potent aggregate is transient and low compared to that
seen with proteins that are potent immune system activators.
26
Conclusions
Conclusions II
▪ This approach of testing attributes across structurally similar proteins in model systems can be used to help determine criticality of attributes
▪ Cross lab experiment to test aggregates generated with defined stress of a few MAbs in vitro and in vivo (after characterization) is being initiated to try and further identify attributes with potential risk of immunogenicity being initiated
▪ Immune response in preclinical studies is not currently predictive of a clinical outcome, efforts to tie clinical response to attributes of lots can help bridge studies with model to clinical results
27
28
Thank you!
29
backup
Xeno-het study 1: modified mAbs were tested in xeno-het mice for
their ability to break tolerance to the monomeric mAb
Immunized mice were evaluated for the release of cytokines from T-cells, mAb-
specific antibody-secreting B-cells, and the presence of ADA in circulation.30
The oxidation level of aggregates was confirmed by
peptide mapping
31
mAb1
monomer
mAb1
stir-N2
mAb1
stir-lowO2
mAb1
stir-highO2
mAb1-NoMet
monomer
mAb1-NoMet
stir-lowO2
M34+Oxidation 0.4% 2.6% 1.7% 4.0% - -
M48+Oxidation 0.2% 1.8% 1.1% 3.1% - -
M70+Oxidation 0.2% 3.3% 2.2% 5.9% - -
M81+Oxidation 0.2% 3.1% 2.0% 4.9% - -
M114+Oxidation 0.3% 3.9% 2.6% 6.6% - -
M258+Oxidation 1.9% 6.5% 5.4% 11.4% - -
M364+Oxidation 0.4% 3.1% 2.1% 4.9% - -
M403+Oxidation 0.7% 4.3% 3.3% 7.2% - -
M434+Oxidation 0.3% 0.7% 0.9% 2.7% - -
W36+Oxidation 0.0% 0.6% 0.4% 1.0% 0.0% 0.0%
W50+Oxidation 0.1% 0.6% 0.5% 1.2% 0.0% 0.0%
W168+Oxidation 0.3% 0.5% 0.3% 0.9% 0.0% 0.0%
W319+Oxidation 0.1% 2.1% 1.4% 3.0% 0.2% 2.6%
W387+Oxidation 0.0% 1.4% 1.0% 2.6% 0.2% 1.9%
W152+Oxidation 0.0% 0.3% 0.3% 1.2% 0.0% 0.6%
W189+Oxidation 0.1% 1.8% 1.2% 3.2% 0.2% 2.1%- not applicable
The mAb1-NoMet stirred sample contains aggregates that
have no Met oxidation and only a low level of Trp oxidation
mA
b1 m
on
om
er
mA
b1 s
t ir -
N 2
mA
b1 s
t ir -
low
O 2
mA
b1 s
t ir -
hig
hO
2
mA
b1-N
oM
et
mo
no
mer
mA
b1-N
oM
et
st i
r -lo
wO
2
11 02
11 03
11 04
11 05
11 06
1 5 - 2 0 m
2 0 - 2 5 m
2 5 -1 5 0 m
5 -1 0 m
1 0 - 1 5 m
2 - 5 m
Pa
rtic
le n
um
be
rs
/ m
l
Size distribution and morphology of aggregate samples
with different levels of oxidation
Stirring induced aggregates (with different levels of oxidation)
show similar particle number trends and morphology
mAb1
monomer
≥ 10 µm
mAb1
stir-lowO2
≥ 25 µm
mAb1
stir-N2
≥ 25 µm
mAb1
stir-highO2
≥ 25 µm
mAb1-NoMet
monomer
≥ 10 µm
mAb1-NoMet
stir-lowO2
≥ 25 µm
HIAC Aggregate Numbers
Micro-flow Imaging Aggregate Images
mA
b1 m
on
om
er
mA
b1 s
t ir -
N 2
mA
b1 s
t ir -
low
O 2
mA
b1 s
t ir -
hig
hO
2
mA
b1-N
oM
et
mo
no
mer
mA
b1-N
oM
et
st i
r -lo
wO
20
2 0
4 0
6 0
8 0
2
SI
(ab
ov
e b
ac
kg
ro
un
d) %
do
no
rs
re
sp
on
din
g
1 0
7 0
Healthy donor T-cell proliferative responses induced by
aggregates with different levels of oxidation
Aggregates that contain Met oxidation and aggregates without Met
oxidation caused a similar T-cell proliferative response at the late phase
▪ The average SI of all donors from 5-8 days (blue bars) and the percentage of donors that responded
(grey bars) are shown (SI ≥ 1.9, p < 0.05).
Degree of Chemical Modification+ ++ ++ +++ - -
- ++ + ++ - ++
Key
+ > 1%
++ > 2%
+++ >10%
Met
Trp
33
Time course of T-cell proliferative responses induced by
aggregates with different levels of oxidation
▪ The % of donors that responded up to 8 days (n=48) is shown (SI ≥ 1.9, p < 0.05).
Aggregates that contain Met oxidation and aggregates without Met
oxidation caused a similar T-cell response over time at the late phase
0
10
20
30
40
Nu
mb
er
of
Res
po
nses
mAb1 monomer
Day 5 Day 6 Day 7 Day 8
mAb1 stir-lowO2
mAb1 stir-highO2
mAb1 stir-N2
0
10
20
30
40
Nu
mb
er
of
Re
sp
on
se
s
mAb1-NoMet monomer
mAb1-NoMet stir-lowO2
Day 5 Day 6 Day 7 Day 8
34
IFN
-g
IL-2
IL-4
IL-1
0
IL-1
3
0
2 5
5 0
7 5
2
1 0
2 0
SI
% d
on
ors
Cytokine secretion profile of donors in response to
aggregates with different levels of oxidation
Aggregates both with and without Met oxidation caused
the secretion of T-cell specific cytokines at the late phase
▪ The average SI of responding donors above the monomer (light blue bars) and % of donors that
responded (grey bars) is shown (n=12). A positive response is SI ≥ 1.9.
0
2 5
5 0
7 5
2
1 0
2 0
SI
% d
on
ors
mAb1 stir-lowO2
mAb1-NoMet stir-lowO2
0
2 5
5 0
7 5
2
1 0
2 0
SI
% d
on
ors
mAb1 stir-N2
0
2 5
5 0
7 5
2
1 0
2 0
SI
% d
on
ors
mAb1 stir-highO2
Degree of
Chemical Modification
Met Trp
++ ++
++ +
+++ ++
- ++
Key
+ > 1%
++ > 2%
+++ >10%
This data shows 12 of the 48
donors. The remaining
donors are being tested.
92%
35
mo
no
mer
st i
r -20h
to
tal
st i
r -20h
su
pern
ata
nt
st i
r -20h
pellet
nan
osp
here
s 4
0 n
m
nan
osp
here
s 5
00 n
m
mic
rosp
here
s 5
m
mic
rosp
here
s 2
1
m
TC
E-K
LH
-mA
b
1
1 0
1 0 0
1 0 0 0
0
2 5
5 0
7 5
1 0 0
AD
A (
sig
na
l /
no
ise
)
% A
DA
Po
sitiv
e M
ice
4 0 0 0
The importance of size as an attribute of aggregates
was evaluated in xeno-het mice
▪ The average ADA of all mice (colored bars) and the % of mice that were ADA positive and showed
depletion (grey bars) is shown (n=11).
▪ mAb4 stir-20h induces a low and transient ADA response (5/11 mice positive), lacks a memory
response, and requires high particle numbers (orders of magnitude above that found in product).
All size enriched fractions had lower particle numbers than the stir-20h
total sample and produced only a low incidence of ADA in the mice
ADA Levels of Xeno-het Mice
36
IFN
- g
IL-1
0
IL-1
3
IL-2
IL-7
1 0
1 0 0
0
2 5
5 0
7 5
1 0 0
2
IL-1
0
IL-1
MC
P-1
MIP
-1
TN
F-
1 0
1 0 0
1 0 0 0
0
2 5
5 0
7 5
1 0 0
IL-1
0
IL-1
MC
P-1
MIP
-1
TN
F-
1 0
1 0 0
1 0 0 0
0
2 5
5 0
7 5
1 0 0
▪ Four methionine residues were oxidized at 70-100%
▪ The average SI of responding donors (colored bars) and % of
donors that responded (grey bars, n = 11) is shown.
▪ The average ADA of all mice (colored bars) and the % of mice
that were ADA positive and showed depletion (grey bars) is
shown (n=11).
Early Response
Late Stage Response
mAb1 Oxidized
SI (a
bo
ve
mo
no
me
r)
% d
on
ors
mAb4 Oxidized
IFN
- g
IL-1
0
IL-1
3
IL-2
IL-7
1 0
1 0 0
0
2 5
5 0
7 5
1 0 0
2
mAb1 Oxidized
mAb4 Oxidized
IVCIA Assay Xeno-het Mouse
The potential biological impact of met oxidation was
evaluated in the IVCIA assay and xeno-het mice
Oxidized mAbs caused little to no response in the IVCIA assay
and no increase in immunogenicity in the Xeno-het mouse37
mA
b1 m
on
om
er
mA
b1 o
xid
ized
mA
b4 m
on
om
er
mA
b4 o
xid
ized
mA
b4 s
t ir -
20h
200
g/m
l
TC
E-K
LH
-mA
b
1
1 0
1 0 0
1 0 0 0
0
2 5
5 0
7 5
1 0 0
AD
A (
sig
na
l /
no
ise
)
% A
DA
Po
sitiv
e M
ice
4 0 0 0
Overview of xeno-het mouse study
Blood Collection for ADA and cytokine analysis
Sacrifice 3-4 mice for SPC & BMC Harvesting
(in vitro B-cell activation studies)
30 1 2 4wk 75 6 8
Dosage Injection mAb Monomer boost
9 10
10-11 mice/group
Sacrifice
4 mice
7-8 mice
11
Sacrifice
4 mice
Sacrifice
3 mice
Xeno-het mice produce a robust initial cytokine response to tce-klh-mab1 in contrast to buffer only
G-C
SF
Eo
taxin
GM
-CS
F
IFN
-g
IL-1
IL-1
IL-2
IL-4
IL-3
IL-5
IL-6
IL-7
IL-9
IL-1
0
IL-1
2p
40
IL12-p
70
LIF
IL-1
3L
IX
IL-1
5
IL-1
7
IP-1
0K
C
MC
P1
MIP
-1
MIP
-1
MC
SF
MIP
2
MIG
RA
NT
ES
VE
GF
TN
F-
1
1 0
1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
G-C
SF
Eo
taxin
GM
-CS
F
IFN
-g
IL-1
IL-1
IL-2
IL-4
IL-3
IL-5
IL-6
IL-7
IL-9
IL-1
0
IL-1
2p
40
IL12-p
70
LIF
IL-1
3L
IX
IL-1
5
IL-1
7
IP-1
0K
C
MC
P1
MIP
-1
MIP
-1
MC
SF
MIP
2
MIG
RA
NT
ES
VE
GF
TN
F-
1
1 0
1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
%M
ice
res
po
nd
ed
(ab
ove
2-fo
ld)
Ind
ivid
ua
l P
os
t-b
lee
d:P
reb
lee
dra
tio
TCE-KLH-mAb1
buffer, PS80, transport
Monitoring the relative change of cytokine secretion in
Xeno-het mice can be used to detect immune responses.
Presence of Si oil alone does not elicit signal
G-C
SF
Eo
taxin
GM
-CS
F
IFN
-g
IL-1
a
IL-1
bIL
-2IL
-4IL
-3IL
-5IL
-6IL
-7IL
-9
IL-1
0
IL-1
2p
40
IL12-p
70
LIF
IL-1
3L
IX
IL-1
5
IL-1
7
IP10
KC
MC
P1
MIP
1a
MIP
1b
MC
SF
MIP
2
MIG
RA
NT
ES
VE
GF
TN
F-a
1
1 0
1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
L e g e n d
L e g e n d
L e g e n d
W e e k 1
W e e k 6
W e e k 1 1
Wk 1 Individual Response
Wk 6 Individual Response
Wk 11 Individual Response
G-C
SF
Eo
taxin
GM
-CS
F
IFN
-g
IL-1
a
IL-1
bIL
-2IL
-4IL
-3IL
-5IL
-6IL
-7IL
-9
IL-1
0
IL-1
2p
40
IL12-p
70
LIF
IL-1
3L
IX
IL-1
5
IL-1
7
IP10
KC
MC
P1
MIP
1a
MIP
1b
MC
SF
MIP
2
MIG
RA
NT
ES
VE
GF
TN
F-a
1
1 0
1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
L e g e n d
L e g e n d
L e g e n d
W e e k 1
W e e k 6
W e e k 1 1
Wk 1, % Responders
Wk 6, % Responders
Wk 11, % Responders
High SO
G-C
SF
Eo
taxin
GM
-CS
F
IFN
-g
IL-1
IL-1
IL-2
IL-4
IL-3
IL-5
IL-6
IL-7
IL-9
IL-1
0
IL-1
2p
40
IL12-p
70
LIF
IL-1
3L
IX
IL-1
5
IL-1
7
IP-1
0K
C
MC
P1
MIP
-1
MIP
-1
MC
SF
MIP
2
MIG
RA
NT
ES
VE
GF
TN
F-
1
1 0
1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
G-C
SF
Eo
taxin
GM
-CS
F
IFN
-g
IL-1
IL-1
IL-2
IL-4
IL-3
IL-5
IL-6
IL-7
IL-9
IL-1
0
IL-1
2p
40
IL12-p
70
LIF
IL-1
3L
IX
IL-1
5
IL-1
7
IP-1
0K
C
MC
P1
MIP
-1
MIP
-1
MC
SF
MIP
2
MIG
RA
NT
ES
VE
GF
TN
F-
1
1 0
1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
No increase in cytokine secretion in mice was observed in response to samples with PS80 +\- transport
%M
ice
res
po
nd
ed
(ab
ove
2-fo
ld)
Ind
ivid
ua
l P
os
t-b
lee
d:P
reb
lee
dra
tio
mAb1, PS80, static
mAb1, PS80, transport
G-C
SF
Eo
taxin
GM
-CS
F
IFN
-g
IL-1
a
IL-1
bIL
-2IL
-4IL
-3IL
-5IL
-6IL
-7IL
-9
IL-1
0
IL-1
2p
40
IL12-p
70
LIF
IL-1
3L
IX
IL-1
5
IL-1
7
IP10
KC
MC
P1
MIP
1a
MIP
1b
MC
SF
MIP
2
MIG
RA
NT
ES
VE
GF
TN
F-a
1
1 0
1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
L e g e n d
L e g e n d
L e g e n d
W e e k 1
W e e k 6
W e e k 1 1
Wk 1 Individual Response
Wk 6 Individual Response
Wk 11 Individual Response
G-C
SF
Eo
taxin
GM
-CS
F
IFN
-g
IL-1
a
IL-1
bIL
-2IL
-4IL
-3IL
-5IL
-6IL
-7IL
-9
IL-1
0
IL-1
2p
40
IL12-p
70
LIF
IL-1
3L
IX
IL-1
5
IL-1
7
IP10
KC
MC
P1
MIP
1a
MIP
1b
MC
SF
MIP
2
MIG
RA
NT
ES
VE
GF
TN
F-a
1
1 0
1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
L e g e n d
L e g e n d
L e g e n d
W e e k 1
W e e k 6
W e e k 1 1
Wk 1, % Responders
Wk 6, % Responders
Wk 11, % Responders
Increased numbers of SO droplets in the presence of tween
induced little to no cytokine secretion in Xeno-het mice
High SO
mo
no
mer
st i
r -20h
200g
/ml
st i
r -20h
2g
/ml
TC
E-K
LH
-mA
b
1
1 0
1 0 0
1 0 0 0
0
2 5
5 0
7 5
1 0 0
AD
A (
sig
na
l /
no
ise
)
% A
DA
Po
sitiv
e M
ice
4 0 0 0
mAb
Stir-20h
protein
conc.
(μg/mL)
Particle concentration (#/mL) Response
in
PBMC
Response
in
Xeno-het
Mouse2-10 μm ≥ 10 µm 2-150 μm
mAb4
stir-20h
0.1 954 61 1,015 - NT
1 7,649 191 7,841 + NT
2 * 15,298 382 15,682 + -
5 38,247 957 39,203 + NT
10 76,493 1,913 78,407 + NT
40 305,973 7,653 313,626 + NT
200 * 1,529,865 38,265 1,568,130 NT +
Particle Number and Size
Distribution of Samples Tested
The importance of particle number as an attribute of
aggregates was evaluated in xeno-het mice
ADA Levels of Xeno-het Mice
▪ The average ADA of all mice (colored bars) and the % of mice that were ADA positive and showed
depletion (grey bars) is shown (n=11).
▪ The mAb4 stir-20h 2 μg/ml sample, which had 100 fold less aggregates but was diluted in the
sample total protein concentration, induced only a low incidence of ADA similar to monomer.
* samples had 200 μg/ml total protein
High numbers of particles in the 2-150 μm size range appear to be
a dominant factor for determining if an immune response occurs
41