Pharmaceutical Chemistry Department

Tertiary Structure and Stability

Stability in the tertiary structure of macromolecules under various formulation conditions, such as pH, temperature, ionic strength and concentration, is usually studied with spectroscopic techniques. Among these, fluorescence (intrinsic and extrinsic), UV-Visible absorption, and near-UV-CD are the most powerful and commonly used tools. The global thermal stability of biopharmaceuticals is also generally studied by differential scanning calorimetry.

We are capable of performing all of these studies over a large temperature range (typically 10 to 95 °C), with high-throughput capabilities available for some of the techniques.


  • OLIS Protein Machine
  • Avacta Optim 1000
  • Applied Photophysics Chirascan Plus
  • PTI T-format fluorometer
  • PTI L-format fluorometer
  • Jasco CD
  • Agilent HP 8453 UV-Vis spectrophotometer
  • Microcal high throughput differential scanning calorimeter
  • MicroCal VP-DSC

Fluorescence (Intrinsic and Extrinsic) Spectroscopy

Fluorescence emission of fluorophores is usually sensitive to the changes (such as polarity) of their immediate environment, and reflects alterations in protein folding. Natural fluorophores in proteins, such as the indole ring of Trp residue, and extrinsic fluorophores, such as ANS, Congo Red, thioflavine S and thioflavine T, can be used to probe conformational changes of proteins under various conditions. Similar studies can be performed with DNA and RNA using compatible fluorescent dyes, including YOYO-1, acridine orange, propidium iodide, and the Hoechst dye.

UV-Vis Absorption

Using high-resolution derivative UV absorption, spectra of a protein are resolved into five to seven peaks originating from Trp, Tyr and Phe residues. This method can simultaneously monitor changes in the microenvironment of all three aromatic residues, thus providing a more global picture of the behavior of protein tertiary structure than the fluorescence-based techniques. In addition, this method can be used to detect significant protein aggregation by measuring changes in optical density (turbidity) in the near-UV region (320 - 400 nm). 

Near-UV-CD Spectroscopy

CD signals in the near-UV region (250-350 nm) arise from the Coulomb interaction between the asymmetrically positioned transition dipoles of chromophores, which are aromatic residues and disulfide bonds in proteins. Since changes in the surrounding environment of the chromophores affect the dipole moments, the Coulomb interaction near-UV CD signals are sensitive to alterations in protein tertiary structure. Near-UV CD signals are usually weak, therefore higher (10-fold) sample concentrations are typically required than for far-UV CD experiments. With the advanced instrumentation at MVSC, we have developed a method to simultaneously acquire near- and far-UV CD spectra of proteins, and this method improves efficiency and reduces sample consumption.

Differential Scanning Calorimetry (DSC)

DSC is widely used to study thermal stability of biopharmaceuticals. It directly measures changes in sample heat capacity as a function of temperature, and the Gibbs free energy change associated with a denaturation process can be obtained from these results. In the MVSC, screening of excipients and other formulation conditions can be performed in high-throughput mode since the two MicroCal DSC instruments are capable of continuously analyzing samples from six 96-well plates.

Selected Publications

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. Multidimensional Methods for the Formulation of Biopharmaceuticals and Vaccines. J. Pharm. Sci. in press, 2011.
PMID 21647886: