Division of Drug Delivery TechnologyLeiden Academic Centre for Drug Research (LACDR)

Biodistribution of monoclonal antibody aggregates upon SC administration

Grzegorz Kijanka

24th February 2015

Table of contents.

1. Introduction

2. Aim of the project

3. Experimental setup

4. Results

5. Conclusions

6. Acknowledgements

1. Protein aggregates - immunogenicity

Immunogenicity

Product related factors:- Origin- Sequence- PTMs- Formulation- Impurities (aggregates)- ...

Patient related factors:- Age- Genetic background- Disease- Immunological state- ...

Therapy related factors:- Dose- Duration- Aplication route- Co-treatment- ...

1. Protein aggregates - immunogenicity

Hermeling et al. J Pharm Sci. 2006 Filipe et al. MAb. 2012

Bertolotto et al. J Neurol Neurosurg Psychiatry 2002

1. Protein aggregates• “Protein aggregate”– assembly of protein molecules with higher

MW than desired species • Protein aggregates characterization:

- size- morphology- secondary/tertiary structure- reversibility- covalent modifications- ...

1. Protein aggregates

0.001 0.01 0.1 1 10 100 1000

Size µm

Monomers

Soluble aggregates

Subvisible particles

Visible particles

Oligomers

Submicron size particles

Micron size particles

Monomers

USP <788>

DLS

HP-SEC

NTA

MFI

GE

1. What happens with aggregates after injection?U

nstr

esse

d

Stre

ssed

Before 0h, 0.5h, 1h, 3h, 8h, 24h, 48h

Filipe et al. Pharm Res. 2014

Kijanka et al. PLOS 2014

Agg Mon

1. What happens with aggregates after injection?

• Questions:

– Which aggregates (dimers, oligomers, sub and/or micron size particles) contribute the most to altered disposition from injection spot?

– How do different aggregates influence the biodistribution of protein?

– Does the origin of protein (self/foreign) influence the biodistribution of aggregates?

– Can (altered) biodistribution of aggegated protein increase the risk of immunogenicity?

– Is it possible, by measuring the biodistribution, to select the most immunogenic size range of protein aggregates?

2. Aim of the project.

• To determine the biodistribution of different IgG’s size species upon SC injection

3. Key materials

• Model proteins:– rhIgG1 (r347)– rmIgG1 (1A7)

• Animal model: SKH1 mice– Hairless strain– Immunecompetent

• Fluorescent dye: IR Dye800 CW– Fluorescence in near infra-red, good penetration through tissues– Very stable in vivo

3. In vivo experiments - overview

Labeling Aggregation

Characterization:1) Degree of labeling2) % of free dye (IRDye 800 CW)

Fractionation

1. Centrifugation2. GPC

Characterization:1) SEC2) SDS-PAGE3) DLS4) NTA5) MFI

Fractions:6) Monomers7) Monomers (stressed)8) Soluble aggregates (oligomers)9) Submicron size particles10) Micron size particles

3. In vivo experiments - overview

Imaging Collecting organs / tissues

1) SC injection2) 50 µg of IgG (in 100 µl

PBS)3) 1A7 and r347

1) 1 hr, 24 hrs, 7 days

BiodistributionEuthanasia

1) Tissues: blood, urine, muscle, skin (hind leg), skin (injection spot)

2) Organs: thymus, lung, heart, liver, kidney, spleen, (lymph nodes)

1) Ex vivo organs imaging

2) Quantitative biodistribution

3. Aggregation and fractionation

• Final aggregation conditions– r347-IR Dye800CW conjugates 1mg/ml, pH=4.6, 63˚C, 1hr +

30min of stirring (700 rpm)– 1A7-IR DyeCW conjugates 1mg/ml, pH=4.6, 55˚C, 1hr + 30min of

stirring (700 rpm)• Fractionation via centrifugation (3000g, 10 min, RT)

– “Pellet”: fraction enriched with micron size particles– “Supernatant”: submicron size particles

• Fractionation via GPC– Monomers subjected to stress conditions: “GPC Monomers ”– Oligomers: “GPC Oligomers”

4. Characterization of 1A7 and r347 fractions – mass balance

4. In vivo biodistribution – 1hrr347 1A7

Unstressed1

2 34

5

1) Liver, 2) Spleen, 3) Kidney, 4) Lung, 5) Heart

3

33

3

3

1

1

1

1

1

2

2

2

2

2

45

45

45

4 5

Dorsal Ventral Dorsal Ventral

5

5

5

5

4

4

4

4

3

3

3

3

54

2

2 2

2

1

1

11

GPC Monomers

GPC Oligomers

Supernatant

Pellet

Unstressed

GPC Monomers

GPC Oligomers

Supernatant

Pellet

4. In vivo biodistribution – 24hrs

3

3

1

1

1

2

2

2

4 5

4 5

3

54

3

3

3

1

1

1

2

2

2

4

4

4

5

5

5

r347 1A7Dorsal Ventral Dorsal Ventral

1

1

1

12

2

2

2

3

3

3

3

5

5

5

5

4

4

4

4

1

1

5

5

4

42

2

3

3

1) Liver, 2) Spleen, 3) Kidney, 4) Lung, 5) Heart

Unstressed

GPC Monomers

GPC Oligomers

Unstressed

GPC Monomers

GPC Oligomers

Supernatant

Pellet

Supernatant

Pellet

4. In vivo biodistribution – 7 days

1

1

1

1

1

1

2

2

2

2

2

2

3

33

3

3

3

5

5

5

5

5

4

4

4

4

4

4

5

11

11

3

3

33

5

5

5

5

4

4

4

42

2

2

2

1) Liver, 2) Spleen, 3) Kidney, 4) Lung, 5) Heart

r347 1A7Dorsal Ventral Dorsal Ventral

Unstressed

GPC Monomers

GPC Oligomers

Supernatant

Pellet

Unstressed

GPC Monomers

GPC Oligomers

Supernatant

Pellet

4. Quantitative biodistributionr347 1A7

5. Conclusions

• Similar biodistribution of murine (1A7) and human (r347) antibody upon SC injection

• Monomeric antibodies, even subjected to stress conditions, nicely distribute within the whole body of animals

• Presence of aggregates (both sub micron size and micron size) alters biodistribution

• There is no specific tissue/organ in which aggregated antibodies accumulate (measurably)

• Fluorescent „dots” were detected in spleens and lymph nodes of some animals injected with „1A7 Oligomers”

6. Acknowladgements• LACDR

– Wim Jiscoot– Stefan Romerijn– Eleni Varypataki

• Med Immune– Jared Bee– Xu Liu– Kirsten Schneider-Ohrum– Robert Kubiak– Melissa Coughlin– Steven Bishop – Richard Remmele– Mark Schenerman– Srilatha Kuntumalla– Maria Andrea Miller– Norman Peterson– Wendy White

• LUMC– Clemens Löwik– Ivo Que

Thank you for your attention!

Imaging control

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2 3 45