ABSTRACT
Recent advances in optical and spectroscopic technologies have enabled a plethora of Raman spectrometers that are suitable for studies of protein pharmaceuticals. Highly sensitive Raman spectrometers have overcome the handicap of the fundamentally weak Raman effect that hampered their applications to protein pharmaceuticals in the past. These Raman spectrometers can now routinely measure protein therapeutics at the low concentration of 1 mg/mL, which is on par with other spectroscopic methods such as CD, fluorescence and FTIR spectroscopies. In this article, various Raman techniques that can be used for protein pharmaceutical studies are reviewed. Novel Raman marker of proteins discovered from fundamental studies of protein complexes are examined along with established Raman spectra and structure correlations. Examples of Raman spectroscopic studies of protein pharmaceuticals are demonstrated. Future applications of Raman spectroscopy to protein pharmaceuticals are discussed. © 2007 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 2861–2878, 2007
INTRODUCTION
Raman spectroscopy was born nearly 80 years ago when Sir C.V. Raman discovered the inelastic light scattering phenomena of molecules (Raman effect, named after him), with K.S. Krishnan in 1928 using their eyes as the detector.1 It measures changes in the scattered light frequencies or vibrational energy differences between the ground and excited vibrational states of molecules when they are interacting with a beam of light. The result of the measurement is a Raman spectrum of the molecules
ADVANCE IN RAMAN SPECTROMETERS
The renaissance of Raman spectroscopy has resulted in a plethora of Raman spectrometers that each offer unique features for protein studies.18., 19., 20., 21., 22., 23., 24., 25. In the following section, we will briefly describe the various Raman spectrometers available for protein pharmaceutical applications. There are numerous other exotic Raman techniques, which are beyond the scope of this article and further details can be found in the relevant literatures.2 The salient feature of
SPECTRAL AND STRUCTURE CORRELATIONS
The correlation between the Raman band frequencies, intensities and protein structure was necessary to allow the analysis of protein pharmaceuticals by Raman spectroscopy. These correlations are the result of extensive research in both theoretical calculation of model compounds of N‐methylacetamide and small peptides42, 43 as well as experimental studies of proteins in the last four decades.5., 6., 7., 42., 43., 44., 45., 46. The main contributors to the Raman spectrum of a protein are the
Conformation Determination
One of the most important applications of Raman spectroscopy to protein pharmaceuticals studies is to determine the protein conformation along with CD, fluorescence and FTIR spectroscopies in solution43, 44 and in solid states.75 The majority of the early studies of protein conformation by visible Raman spectroscopy took the advantage of the sensitivity of the Amide I band frequency to the protein conformation. The Amide I band profiles were deconvoluted into individual components and the
CONCLUDING REMARKS
In the last two decades Raman spectroscopy has been transformed from the exclusive tool of academic research to a turn‐on key technique that is equivalent to other spectroscopic methods such as FTIR, CD and fluorescence spectroscopy that are routinely employed for protein studies. It is anticipated that its application in the characterization of protein pharmaceuticals will thrive as the potential to solve real problems is realized in biopharmaceutical science and industry. The future