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![Page 1: Division of Drug Delivery Technology Leiden Academic Centre for Drug Research (LACDR) Biodistribution of monoclonal antibody aggregates upon SC administration.](https://reader035.fdocuments.net/reader035/viewer/2022062308/56649cce5503460f9499a0a5/html5/thumbnails/1.jpg)
Division of Drug Delivery TechnologyLeiden Academic Centre for Drug Research (LACDR)
Biodistribution of monoclonal antibody aggregates upon SC administration
Grzegorz Kijanka
24th February 2015
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Table of contents.
1. Introduction
2. Aim of the project
3. Experimental setup
4. Results
5. Conclusions
6. Acknowledgements
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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- ...
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1. Protein aggregates - immunogenicity
Hermeling et al. J Pharm Sci. 2006 Filipe et al. MAb. 2012
Bertolotto et al. J Neurol Neurosurg Psychiatry 2002
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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- ...
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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
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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
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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?
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2. Aim of the project.
• To determine the biodistribution of different IgG’s size species upon SC injection
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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
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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
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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
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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”
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4. Characterization of 1A7 and r347 fractions – mass balance
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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
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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
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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
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4. Quantitative biodistributionr347 1A7
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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”
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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
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Thank you for your attention!
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Imaging control
1
2 3 45