BRTT Analytical Core Facility

1. BRTT Analytical Core Facility Mark J. Cooper, M.D.

2. CMC Section of IND Core Objectives • Nanoparticle physical characterization • size, shape, charge, payload stability • population homogeneity (EM) • reproducibility of formulation • Stability • water, saline, serum • aggregation at physiologic ionic strength • long term stability at various temperatures • route of delivery: aerosols • Pharmaceutically-acceptable process • endotoxin, sterility (bioburden)

3. Nanoparticle Analytical Testing Property CTI Solvent • Transmission electron microscopy • Dynamic light scattering • Zeta potential • Turbidity parameter • Sedimentation analysis • Gel analysis +/- serum incubation • Endotoxin • Bioburden

4. DNA Nanoparticle:Component Formulation CK30 PEG maleimide DNA CK30PEG10k

5. Optimization of DNA Nanoparticle Formulation • Ratio of +/- charges • Relative volume of polycation and DNA • Mixing method • Ionic strength • DNA concentration • pH • Buffers • Kinetics

6. Matrix of DNA Nanoparticle Formulations • Polycation Composition • Polycation Counterion • Length of Polycation • +/- PEG • PEG Size • Percent PEG Substitution • Pre Vs Post Compaction Addition of PEG • Targeting Ligands

7. Characterization Flow Chart of DNA Nanoparticles TANGENTIAL FLOW FILTRATION EM A260/A280 removal of excess polycation EM A260/A280 GEL ANALYSIS TURBIDITY ANALYSIS SALT STABILITY SERUM STABILITY OSMOLALITY, pH ENDOTOXIN BIOBURDEN STERILITY CONCENTRATION DIAFILTRATION only detectable component is compacted DNA

8. 100 nm 100 nm Copernicus Formulation ofPEG-Substituted DNA Nanoparticles • PEG-CK30 and plasmid DNA • lysine counterion determines shape • single DNA molecule • reproducible formulation • homogeneous population • no aggregation in saline • [DNA]  12 mg/ml • nuclease resistant • stable > 3 years • consistent in vivo results • transfect post-mitotic cells • PHASE I TRIAL CF TFA Acetate

9. Sigma Scan Analysis of Electron Micrographs of Compacted DNA

10. Sigma Scan Analysis of Electron Micrographs of Compacted DNA

11. Calculated and Measured Volumes of Compacted DNA Nanoparticles J. Biol. Chem. 278:32578-32586, 2003

12. Nanoparticle Shape is Determined by Polycation Counterion at the Time of DNA Mixing • Trifluoroacetate (TFA) ellipsoids • Acetate rods • Bicarbonate rods, toroids • Chloride rods (partial) • Bromide ellipsoids IMPORTANT FUNCTIONAL CORRELATES FOLLOWING IN VIVO GENE TRANSFER

13. Counterion of PEG-substituted Polycation Influences Shape of DNA Nanoparticles HCO3 Acetate 100 nm 100 nm Chloride TFA 100 nm 100 nm

14. Dynamic Light Scattering Analysis of Compacted DNA Nanoparticles

15. Zeta-Potential of PEG-Substituted DNA Nanoparticles

16. Turbidity Parameter • Rayleigh’s law predicts that particles having a diameter << l will scatter light in proportion to l-4 • Optical densities 330-420 nm (no absorbance by either DNA or polylysine)

17. RT96 -0.50 -4.058 ± 0.08747 -0.75 log(Abs) -1.00 -1.25 -1.50 2.6 log(Wavelength) Turbidity Parameter • 69 unique compactions • -4.29 +/- 0.40 (SD) • 95% CI: -4.191 to -4.383 • Aggregated compacted particles scatter light with a smaller slope value (-3.0 to -3.4)

18. Turbidity Parameter Vs. Sedimentation

19. Salt Stability of PEG-SubstitutedDNA Nanoparticles 10 kD

20. Gel Electrophoresis TRYPSIN TRYPSIN TRYPSIN SERUM SERUM SERUM --- --- ---

22. Stability of Compacted DNA

23. Sample Submission and Costs • Call (231-0227)/email Mark or Tom to review nanoparticle composition and properties • Mark Cooper mcooper@cgsys.com • Tom Kowalczyk tkowalczyk@cgsys.com • Darren Lucas (new research assistant) • Copernicus Therapeutics (BioEnterprise incubator, 1st floor) • 11000 Cedar Avenue, Suite 110 (prior University West bldg) • Volume requirements: ~500 ul at adequate concentration to permit payload assays • No cost to BRTT labs for first 3 samples • $39 per sample for QC tests, $25 additional for endotoxin • Typically 1-2 weeks to obtain results