Light scattering provides the most effective technology to characterize large molecules and subvisible particles. The 23rd International Light Scattering Colloquium (ILSC) attracted almost 100 scientists to the historic Biltmore Hotel in Santa Barbara, CA, October 22–24, 2012. Although there were reports on many topics, the program focused on exploratory nanotechnology, protein structures, and biomedical research.
Advances in nanotechnology
Prof. Andre Nel (Professor of Nanomedicine, University of California, Los Angeles) opened the meeting with a plenary lecture reviewing the development of nanotechnology accompanied with predictions on the future. He noted that major advancements in technology occur about twice each century and lead to massive wealth creation. Cited examples include textiles, railroads, automobiles, and recently computers. He expects nanotechnology to be the fifth revolution in the series with the exponential growth phase beginning in 2025.
Prof. Nel’s view of the potential provided an interesting segue into vignettes of his own research, which explored new functionality that arises due to size and huge surface area. He introduced a 3-D nanomaterial “genome”—one edge was the periodic table, another edge was 16 shapes, and the third was size range. His point was that one material could have many shapes and a range of sizes. Carbon is one example, with carbon black, buckyballs, single-wall nanotubes, multiple-wall nanotubes, diamond…not to mention carbon compounds and composites.
This tremendous range in possible materials is associated with corresponding risks. Silica rods are taken up by some cells preferentially to spherical particles. In one case, this was cancer cells, but in another, normal cells. Nano ZnO particles inhibit development of zebrafish. Carbon nanotubes lead to fiber formation in the lung similar to asbestosis. In response to these and similar reports, a 2007 report from the National Academy of Sciences recognizes the need for improved toxicity testing strategies for nanomaterials. Prof. Nel started to study the effects on a case-by-case basis, but this is expensive (~$70 K/material) and slow (several years/material).
Since the traditional approach was too slow and unaffordable, he investigated high-throughput (HT) screening where various cells (liver, kidney, heart, lung, etc.) in thousands of wells are challenged with aliquots of the nanomaterials from libraries. From this the dose–response curves are created for each cell and material. This approach is also useful in evaluating formulations. One study of the oxidative stress of metal oxide nanoparticles showed a correlation with band gap energy. The HT approach increased throughput of toxicology studies from ~100/year to ~100,000/day. This and other studies are using systems biology approaches to support an effort in predictive toxicology based on correlations of biological responses to properties of the nanomaterials.
Prof. Axel H.E. Müller (Johannes Gutenberg University, Mainz, Germany) lectured on “Self-Organized Multicompartment Nanostructures from New Triblock Terpolymers.” The variety of products produced seems endless. Triblock polymers connect three distinct polymer units into one larger covalent assembly (ABC). Terpolymers present opportunities to form superstructures or to create oriented interfaces, which could be useful in stabilizing emulsions.
Terpolymers of methacrylate, butadiene, and styrene (MBS) are a particularly interesting combination. Depending on the composition and ratio, a variety of structures self-assemble, including cubes, rods, spheres, sandwich, and rod–cube composites. One of the most interesting structures was MBS treated with S2Cl2 to cross-link the butadiene into a core cross-linked micelle. Animations showed that the styrene and methacrylate extended arms are located in specific regions of the composite.
Under other conditions, the styrene regions can form ordered solids, creating supermicelles and giant particles up to 300 nm. I asked if these had been evaluated for chromatography. Prof. Müller replied not yet, but this could/would be explored. The particle size would be in the range that Prof. Mary Wirth (Purdue University, Lafayette, IN) is exploiting with slip flow chromatography.
Several lectures showed that light scattering technology is essential in elucidating the structure of large proteins (>10 kDa). According to Prof. Stephen G. Harding (University of Nottingham, U.K.), multiangle light scattering (MALS) coupled with steric exclusion chromatography (SEC) is essential for determining molecular mass and mass distribution of glycol proteins and other polymers. This information can be extended to provide information on the conformation if a flow-through viscometer and quasi-elastic light scattering (QELS) photometer (Wyatt Technology Corp., Santa Barbara, CA) are added in series. If the polymers are too large, Harding recommends replacing the SEC with field flow fractionation (FFF). These are useful in characterizing lightly glycosylated proteins such as antibodies to polysaccharides. Harding also coined the title “crystal hydrodynamics,” where small-angle X-ray scattering (SAXS) is added to the HPLC string. This is useful for estimating the radius of gyration of large proteins, even in the presence of aggregates.
For very large proteins (1 megadalton and larger) SEC-MALS has been limited by the column packing. At the meeting, Shodex (New York, NY) introduced three SEC columns—OHPak SB-807 HQ, SB-806 HQ, and SB-806M HQ. The SB-807 column has an estimated exclusion limit of 500,000, 000 Da. This is the largest exclusion limit column that I am aware of.
Dr. Tapan Das of Pfizer (St. Louis, MO) lectured on biophysical physical methods for predicting formation of protein aggregates, with a particular focus on antibody analytes. In general, one cannot look at a structure and predict aggregation behavior in detail. Certain events such as heat stress and freeze–thaw cycles will cause aggregation, but one needs to run the experiments. Results from freeze–thaw depend on how the process is carried out. Slow freezing concentrates the analyte as ice forms, increasing the concentration in the water by as much as a factor of ten. This can induce aggregation. Also, ice can serve as a nucleation surface. The devil is in the details.
Differential scanning fluorimetry provides a profile of the melting process for protein interaction. One starts with a quantitative PCR instrument such as the iCycler5 from Bio-Rad (Hercules, CA). Typically the ligand or excipient is added to a well in a 96- or 384-well plate, followed by the dye solution. SYPRO® Orange (Sigma-Aldrich Corp., St. Louis, MO) is often selected since it fluoresces when it accesses the hydrophobic regions of the protein. The temperature is increased slowly. The readout is the fluorescence. Relative thermal stability is compared by measuring the beginning or half-height point in the melting curve. Throughput is one plate per hour (see http://thermofluor.org/resources/Oxford-SGC-P-Niesen-DCF.pdf).
Dr. Stefan Fisher of Late Stage Pharmaceutical Development at Roche (Basel, Switzerland) described the essential role of light scattering in the development of formulations for protein therapeutics. Admonishing “large molecules are not just big small molecules,” he described their fickle character. They are fragile; activity depends on chemical and structural integrity. Most require parenteral administration, often in high doses. His lecture presented short case histories tracing problems to cryoconcentration, pumping, shaking, and pH excursions. He also described a novel method for measuring the second virial coefficient (A2) with dynamic light scattering that is much faster and uses less sample than other methods.
Immunoprophylaxis tries to train the new host to provide an immune response to an antagonist. This is the challenge of the Crucell Vaccine Institute (Leiden, Holland). A lecture by Dr. Adrian Apetri discussed “Life in Biopharma Before and After Light Scattering.” According to Dr. Apetri, vaccine development prior to light scattering technology was part inspiration and visionary. Amazingly, vaccines worked as well as they did, but with proper instruments, vaccine developers could start to see that phenomena such as aggregation were tractable and potentially useful. Large aggregates were useful in initiating immune responses, as in the adjuvant effect. Instrumentation has improved, and so has the regulatory oversight. Potential immune therapeutics can be characterized for homogeneity and stability with asymmetric-flow field flow fractionation-multiangle light scattering-quasi-elastic light scattering-differential refractometry-ultraviolet (AF4-MALS-QELS-dRI–UV). Protein interactions are measurable with the Wyatt DynaPro Plate Reader, which evolved into the NanoStar. Two case histories showed that apparent aggregates were really different compounds, which saved the projects. Hydrogen–deuterium exchange was also used to map out the binding epitopes. Regions with significant reduction in hydrogen-to-deuterium exchange usually indicate that the sites are stabilized by intermolecular interaction, and are thus classed as in the binding epitope.
Protein refractive index and crystallins
The plain titles at ILSC usually camouflage a brilliant diamond. The lecture by Peter Shuck (NIH, Bethesda, MD), “Protein Refractive Index (RI) Increments and the Structure and Function of γ-Crystallins,” first impressed me as probably much todo about nothing. Wrong again! It turns out that the refractive index (RI) of a protein can be calculated quite accurately from the amino acid (AA) composition. However, a study of the RI of proteins in the human genome showed that the range in RI, measured by dn/dc (change in RI with concentration) is very narrow and nearly independent of the AA sequence, except for the crystallin family, which have a very high dn/dc.
Crystallins are found only in the eye, where they are partly responsible for focusing. The exact mechanism depends on the species. Crystallins are formed about two months prior to gestation and are not regenerated, and thus must last a lifetime. They are present in a tight assembly of compact, dense spheres with no crosslinking. Crosslinking produces cataracts. Crystallins must be transparent, even when the concentration is 700–1000 mg/mL, as found in fish. This is the highest protein concentration of any tissue. The peculiar RI is due to the relatively high frequency of high RI AA (methionine and cysteine) and low frequency/low content of alanine, valine, leucine, and threonine.
Alzheimer’s is one of the most debilitating and feared diseases. The cost of treatment in the U.S.A. is over $330 billion/year, making it one of the most expensive, according to Prof. Annelise Barron of Stanford University (CA). Amyloid β-sheets are associated with Alzheimer’s disease, but the cause or effect is still debated. Barron noted that LL-37 appears to disrupt the β-sheet, perhaps even marking it for destruction. LL-37 is extremely toxic and hence well-regulated in the body. However, one can modulate the production with vitamins A and D, which are found in fish oil. She flashed back on cod liver oil, which was administered to young children decades ago. I remember the dose of cod liver oil as the worst event of the morning. I’m sure that this topic will soon be in the popular press.
The 23rd ILSC lived up to the expectation of excellent speakers presenting state-of-the-art reports on a wide range of topics. Attendees now have a new appreciation of the potential of nanotechnology and the construction of organs such as the eye. The immense investment of the biopharm industry in characterizing candidates and approved therapeutics builds confidence that we have the tools to assure that products are safe and effective.
The team at Wyatt Technology Corp. deserves special thanks for organizing the colloquium, including selecting superb speakers. The location was an outstanding choice that provided an atmosphere that was complementary to the program.
Robert L. Stevenson, Ph.D., is a Consultant and Editor of Separation Science for American Laboratory/Labcompare; e-mail: firstname.lastname@example.org.