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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
1
2 3 45