Good formulation attributes can be achieved only when informative analytical methods are available. Results obtained from analytical methods specifically developed for the interested protein will be also used to present the quality of the formulation. It is important to wisely select analytical methods that are both relevant and sensitive to the specific issues. Listed below are examples of analytical methods that Integrity Bio is using for formulation development of protein therapeutics.
Structural analyses: Measure the secondary, tertiary, and quaternarystructure of biologically active proteins,
- Circular Dichroism (CD)
- Fourier Transformed Infrared Spectroscopy (FTIR)
- Fluorescence Spectroscopy
- Differential Scanning Calorimetry (DSC)
HPLC methods: HPLC methods with variety of high resolution analytical columns represent the most effective and efficient means of monitoring various degradation of protein therapeutics.
- Size-exclusion chromatography
- Ion-exchange (cation-exchange, anion-exchange) chromatography
- Reversed phase chromatography
- Hydrophobic interaction chromatography
- Various detectors: UV, fluorescence, reflective Index, evaporative light scattering, mass spectrometry
Electrophoresis: Separation of impurities based on both charge and mass makes electrophoresis an excellent orthogonal method
- Capillary Electrophoresis (CE)
- Sodium dodecyl sulfate electrophoresis (SDS-PAGE)
- Protein Analyzer
Particle Analysis: Particles represent a unique challenge in the stability of protein therapeutics as only few micrograms of degraded protein is sufficient to generate unacceptably large number of particulates. As a result,a formulation with good purity profile by HPLC methods can be rejected by visual inspection if it contains particulates. Various analytical methods are available dependent on the size of particulates.
- Microflow Imaging (MFI): excellent for sub-visible particles
- Light scattering (dynamic and static)
- Analytical Ultrafiltration (AUC)
- HIAC Liquid Particle Counter
Viscosity: Good measurement of viscosity may be required for manufacturing process as well as injection through narrow bore needles.
- Instron – Syringeability study
Biological activity assays
Other useful analytical methods:
- Subambient differential scanning calorimetry (characterization of frozen formulations)
- DSC for the glass transition temperature of dry powder
- Karl Fisher moisture titrator