Secondary Structure and Stability
The secondary structure of macromolecules can be affected by formulation conditions, including pH, temperature, buffer identity, ionic strength, and excipients. Maintaining the native secondary structure is an important aspect of the development of stable formulations.
We have multiple circular dichroism and Fourier transform infrared (FTIR) spectrometers that are equipped with peltier temperature controllers and 4- or 6-position sample holders. Thus, studies on the secondary structure of macromolecules can be performed in high-throughput mode over a wide temperature range (typically 5 to 95 °C).
- OLIS Protein Machine
- Applied Photophysics Chirascan Plus
- Jasco J-815 circular dichroism spectropolarimeter
- Jasco J-810 circular dichroism spectropolarimeter
- Jasco J-720 circular dichroism spectropolarimeter
- Bruker Tensor 27 FTIR spectrometer
- Bruker Vertex FTIR spectrometer, HYPERION FTIR Microscope
Circular Dichroism (CD)
The most frequently used method to estimate changes in secondary structure of macromolecules is far-UV CD. The shape and intensity of the signal as a function of wavelength can usually be related to the particular type of secondary structure in proteins and nucleic acids.
The far-UV CD spectrum of proteins (180-260 nm) and nucleic acids (200-300 nm) is very sensitive to their conformation, and therefore can be used to monitor very minor changes in structure as a function of factors such as pH, ionic strength and temperature. Thus, the stability of the secondary structure of biopharmaceuticals and vaccines can be defined by this simple approach.
Fourier Transform Infrared Spectroscopy (FTIR)
A second method used to examine secondary structure in proteins and nucleic acids is infrared spectroscopy. This method is always used in a Fourier transform mode, and it is thus referred to by the abbreviation FTIR.
For proteins, the amide I, II and III bands near 1700-1600, 1575-1475 and 1300-1220
cm-1, due primarily to C=O stretching, NH stretching, and a combination of C-N stretching and N-H bending, are the most commonly employed signals to monitor secondary structure.
For nucleic acids, vibrational bands from the bases (C=O and N-H stretching vibrations) and phosphate groups, as well as OH absorptions from the sugars are most often examined.
Joshi, SB., Bhambhani, A., Zeng, Y. and Middaugh, C. R. (2010) An Empirical Phase Diagram/High Throughput Screening Approach to the Characterization and Formulation of Biopharmaceuticals in “Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals”, Jameel, F and Hershenson, S (editors), Wiley & Sons publication, p173-204.
Maddux, N.R., Joshi, S.B., Volkin, D.B., Ralston, J.R. and Middaugh, C.R. (2011) Multidimensional Methods for the Formulation of Biopharmaceuticals and Vaccines. J. Pharm. Sci. in press.
PMID 21647886: http://www.ncbi.nlm.nih.gov/pubmed/21647886