Circular Dichroism

Circular Dichroism (CD) is an excellent method for analyzing protein and nucleic acid secondary structure in solution. It can be used to track changes in folding as a function of temperature, and is also useful for measuring protein-ligand and nucleic acid-ligand interactions.

CD measures the difference in absorption between left- and right-circularly polarized light as a function of wavelength. When light passes through an optically active chromophore, differential absorption results in light with an electronic vector that is elliptical rather than circular.

Electron Microscopy

Electrons have a shorter wavelength than light allowing magnifications up to many thousand times. The theme in this older technology is automation, resolution, and speed. FEI Company has published a useful, amusing instructional booklet on this topic.⁹

On April 18, 2005 FEI reported that the Titan 80-300, its newest scanning electron microscope, achieved atomic scale imaging with a resolution below 0.7 Angstrom.10 FEI claims this as a new record for a commercial tool. On August 1, 2005 this firm released new software and hardware for the Tecnai G2 transmission electron microscope.¹¹ Technai 3.0 software runs under Windows XP and takes full digital control of the microscope and detectors.

Electrophoresis

The separation of proteins, DNA, RNA, and peptides in an electric field is based on their relative electric charge. Greater charges migrate faster. BioPharm International published an application article in April 2005; which discussed a case study of developing analytical methods for an API.¹² One that was improved is SDS-PAGE. The excerpt from the article follows:

"GEL ELECTROPHORESIS Formulation development could not progress until an initial method to evaluate product quality was available. Although not required as a release method, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was the first technique established because of the simplicity of method development. SDS-PAGE separates proteins based on size and can be used to detect impurities, truncation, or aggregation. All materials used for SDS-PAGE were purchased from Invitrogen.

SDS-PAGE was used to analyze all early formulation development samples and was invaluable in identifying formulations prone to aggregation. In particular, a significant dimer band was observed in samples freeze-dried with only mannitol and stored at 37°C for two weeks. Dimer was not observed in frozen controls or frozen solutions of the same formulation. Also, dimer was not observed in samples freeze-dried with only sucrose, or with mixtures of sucrose and mannitol.

SDS-PAGE required little development time, but it was not an ideal product release method. The technique was useful for comparing the purity of samples within one analysis, but intermediate precision was generally poor. An analysis of the purity results by densitometry was subjective because streaks, bubbles, and background must be excluded from the purity calculation. In addition, the limit of quantitation (LOQ) was high. While not experimentally determined, the LOQ can be estimated from linearity data to be approximately 60 μg/mL or 10 percent aggregates (proteins stuck together but still soluble). Aggregates are undesirable because there are concerns that they can cause immunogenic reactions in patients.

SDS-PAGE was optimized during initial method development. We loaded samples at 0.5, 1, 2, 3, 4, and 5 μg to determine optimum loading conditions. The gel was scanned with a desktop scanner and analyzed by gel analysis software. We then plotted the amount of protein loaded against optical density to determine the linear range of staining, which was from 0.5 to 3μg with a 0.999 coefficient of determination."