Transmission Electron Microscopy (TEM) analysis is complementary to other classical biophysical methods (X-Ray, NMR, Analytical Ultracentrifugation, Mass spectrometry, Chromatography, Isothermal Titration Calorimetry, Surface Plasmon Resonance, Differential Scanning Fluorimetry) and enables a thorough characterization of the molecular architecture of biologics and their population distributions. TEM can also provide a high resolution structural model showing molecular interactions between the members of a biological complex.
Negative staining TEM
Negative staining TEM is a well established method in structural biology, applicable to most samples even when their concentrations are low (typically below 0.1 mg/ml). 2D Images or 3D tomograms of the complex of interest can be obtained. Classification and averaging of the particles, both in 2D (Single Particles Analysis – SPA) as well as in 3D (volumetric averaging), can be performed within few hours depending on the nature of the sample. For SPA, a gallery of “class averages” is obtained. If this gallery contains enough classes displaying different orientation, a 3D model can be constructed. In volumetric averaging 3D tomograms are recorded, after which the different 3D classes are averaged to improve the model. Thanks to the ability of TEM to directly image the distribution of particles in an aqueous solution, it is often the only technique that can confirm various conformational models in a formulation. Hence, the structural architecture of a population of complexes can be related to its potency, target affinity and bioactivity measured by other methods. When combined with other classical techniques, TEM can decipher the role of supramolecular complexes (which may be classified as aggregates otherwise) in immunogenicity studies.
Relating the function to the population of complexes distribution provides reliable guidelines during drug development. For example, during the optimization of biologics formulation, requirements relating manufacturing, handling and storage can be devised. Negative staining TEM can also play a decisive role during patent interpretation.
The sample structure can be preserved from the hard electron beam radiation when flash frozen in a cryogenic fluid and analyzed at low temperature in a Cryo TEM. In addition, NovAlix offers services to optimize the sample for high resolution TEM analysis (ProteoPlex). Under these conditions, 2D projections of the quaternary structure of protein complexes at high resolution (better than 1 nm) can be obtained. This process can take a few days or more depending on the desired outcome. Higher resolution structures require the acquisition of larger data sets, which require longer microscopy and processing times. The usual required concentration of the sample is 1 mg/ml. The same purification techniques used by crystallographers are applied, although re-concentration steps are banned. Also, as crystallization is not required, the TEM analysis can be performed in the “native buffer” in contrast to the “crystallization buffer”. Hence, membrane proteins in detergent or reconstituted in lipids can be directly analyzed.
Near atomic structures have been solved recently by Cryo TEM in the 150 kDa to the multi-megadalton range, and more structures are about to be released. This has been achieved by the new generation of electron microscopes, which are available to NovAlix. They display higher optical and mechanical stability than their predecessors, and they preserve the biological cryo sample during its insertion in the instrument and during the data acquisition step that can last for days thanks to their advanced contamination-free environment. Another significant recent improvement is the introduction of direct electron detectors, which currently offer a higher sensitivity than traditional film, with the added benefit of digital recording allowing automation and “movie-mode” allowing for motion correction. This recent development in detectors has supplied the last piece of the puzzle to generate high resolution structures also for non-symmetrical small protein complexes, which used to be only possible for highly symmetrical virus structures. It also significantly improved throughput from several months of data collection and processing to several days or weeks. Hence, cryo TEM is consolidating its position as an alternative tool for characterizing biopharmaceuticals as complexes unsolved by XRD and NMR can be eventually addressed.