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