Analytical Core

Core Director:
Mark Cooper, M.D.

An Analytical Core Facility will be set up at Copernicus Therapeutics Inc. to establish the structural determinants of formulation reproducibility, define stability criteria in relevant biologic fluids, determine storage stability parameters, and to provide a pharmaceutical framework for subsequent regulatory document submissions. Various nanoparticle formulations will be characterized using quality control assays to monitor complex shape, size, charge density, colloidal stability in saline, and integrity of the complex and constituent components after incubation in serum.

The following panel of quality control tests are already established and validated for Copernicus’ DNA nanoparticles. Assays are conducted according to formal SOPs and end-release specifications for DNA nanoparticles are in place for each assay. All required equipment is in place and operational.

Quality Control Assays.

1. transmission electron microscopy (nanoparticle size and shape)
   
2. static light scattering (colloidal stability in saline; turbidity parameter is determined based on fitting scattering data to Rayleigh’s law)
   
3. dynamic light scattering (size, colloidal stability in saline)
   
4. zeta potential (surface charge)
   saline stability (colloidal stability assessed using sedimentation assay)
   
5. serum stability (2 hours in 75% murine serum at 37oC, followed by payload integrity assay (e.g. for DNA nanoparticles, structural integrity can be assess using agarose gel electrophoresis)
   
6. payload integrity (gel analysis for DNA; for other compounds, assays based on the specific chemistry of the payload moiety will be developed in concert with submitting laboratory)

As an example, Table I lists the end-release specifications for Copernicus’ DNA nanoparticles manufactured for our recent cystic fibrosis clinical trial. Establishment of these specifications have been critical in ensuring batch to batch reproducibility, and is likely responsible for the highly reproducible animal efficacy data that we have generated. Moreover, without this quality control program in place, it is doubtful that we would have been able to rapidly optimize DNA nanoparticle formulation parameters for in vivo intrapulmonary gene transfer, as successfully accomplished over a ~6 month period in 2001. It is our expectation that application of these quality control assays to other types of nanoparticle formulations, including payloads of proteins or small molecule drugs, will establish production reproducibility and thereby form the basis for pharmaceutical optimization in definitive animal tests.

Table I : DNA Nanoparticle Certificate of Analysis: Quality Control End-Release Specification

¹ Gel electrophoresis expected result: samples not treated by trypsin are fully retarded or migrates up slightly; no smear after serum and trypsin treatment.

Assays not considered release specifications for DNA nanoparticles include surface charge determination (zeta potential) and size by dynamic light scattering (EM has replaced DLS because EMs provide shape as well as size information, and volume can be derived with image analysis software (1)).  As new formulations of nanoparticles are initially characterized, zeta potential and DLS measurements can be made to establish important baseline characteristics, and if they provide unique characteristics, may be included as formal release specifications.  The Analytical Core will also offer evaluation of endotoxin levels using a QCL Kinetic Chromogenic Assay (Cambrex), since endotoxin contamination may be a source of toxicity in vivo.  For experimental testing, nanoparticles must be stable at a defined temperature and solvent, so the Core will offer formal storage stability testing. Samples of nanoparticles will be stored in temperature- and humidity-controlled chambers at 4^oC, 22^oC, and 37^oC, and aliquots removed every 2 wks for 4 mos to assess stability. Baseline stability characteristics are necessary for regulatory submissions, and provide a basis for batch-specific IND-directed efficacy and toxicology studies.