Wyatt’s International Light Scattering Colloquium Focuses on the Nano World

The 24th International Light Scattering Colloquium, which attracted about 70 scientists to the Biltmore Hotel on Butterfly Beach in Santa Barbara, Calif., November 3–4, focused on analytical characterization and analysis of nanomaterials, including biologics.

Professor Erica Ollmann Saphire (The Scripps Research Institute, La Jolla, Calif.) opened the lecture sessions with a description of the toolkit encoded by the limited genome of viral hemorrhagic fevers, with a particular focus on Ebola virus. Ebola has only seven genes, and Saphire detailed two of them: GP, which encodes the glycoprotein on the exterior, and VP40, which assembles progeny virons and buds them from cells.

Saphire showed models of Ebola to demonstrate that the complete GP on the viral surface is not the version of GP that binds receptors during infection. During endocytosis, part of the GP coat is cleaved off, exposing the receptor-binding site. Once bound, this transports the virus into the cytoplasm. Other forms of the glycoprotein may act as decoys for immune agents.

The focus turned to what neutralizes the virus in vitro. First, Saphire examined human monoclonal antibody (mAb) KZ52, isolated from a 1995 Ebola survivor. It neutralizes in vitro, but does not protect primates. Although this one mAb was not protective, it was later found that cocktails or mixtures of mAbs were, including one that curiously did not neutralize well in vitro.

Professor Saphire organized a global immunotherapeutic consortium in 2012 to propose and evaluate options, and with the more recent objectives of mapping the epitopes of antibodies in the field and finding predictors of efficacy. This could lead to the design of an effective therapeutic formulation for clinical testing. So far, she concludes that neutralization potency does not necessarily predict the level of protection, nor is it solely sufficient for protection. An early proof of concept of this project is ZMapp therapy (Mapp Biopharmaceutical, San Diego, Calif.), which was used experimentally and successfully during the Ebola outbreak. In this combination, two KZ52-like antibodies neutralize the virus and a third antibody, 13C6, likely recruits immune effector functions.

A poster by Kathryn M. Hastie et al. of Prof. Saphire’s group, “Understanding glycoprotein-antibody interactions using light scattering,” provided more detail on the technology used in the characterization of viral glycoproteins. Size exclusion chromatography-multi-angle light scattering-refractive index (SEC-MALS-RI) detection was used to measure the total molecular weight, protein component weight and glycan weight for a model protein from recombinant technology. The researchers found that the GPs form dynamic dimers. Using composition gradient-MALS (CG-MALS), the Saphire group measured the stoichiometry and kinetics of binding of GP species, including Fab:GP complexes. They concluded that a detailed map of molecular interactions is essential to devising postexposure treatments.

Nanoparticles in the environment

Professor Frank von der Kammer of the University of Vienna (Austria) lectured on “FFF-MALS-ICPMS Methods for the Analysis of Nanomaterials in the Environment, Substances and Products.” Nanoparticles (NPs) are increasingly used as excipients and active materials in products ranging from foods and cosmetics to concrete. Analysis of NPs requires new technology focused on their unique properties. Electron microscopy was the tool of choice in the 1980s. A few years later, light scattering and field flow fractionation (FFF) joined the toolkit. Hyphenated techniques were particularly useful for NP characterization.

Public awareness of NPs in the environment led to demand for regulation, which followed and defined NPs as any particle smaller than 100 nm in diameter. Characterizing NPs in the environment is difficult, as concentrations are low and targets are heterogeneous. FFF, which separates particles according to size, reduces complexity sufficiently for MALS to provide useful information about particle size and uniformity. Since NPs can be of natural and synthetic origin, knowing the elemental composition could be useful, hence an ICP/MS was added to the workflow. Prof. von der Kammer compared various techniques such as sedimentation FFF-MALS (SedFFF-MALS) and asymmetric FFF-MALS (AF4-MALS), along with transmission electron microscopy (TEM), for the characterization of particles to establish different shapes, including branches, rods, plates and spheres.

Joseph Arndt, Ph.D., of Biogen (Weston, Mass.) described the important role of dynamic light scattering (DLS) in the development of a protein biologic, in this case growth-factor-like (GFL) protein. The protein loses efficacy during processing, with colloidal stability one issue, and the viral inactivation step another. The lecture was an impressive tour of biophysical and structural analysis, including hydrogen/deuterium exchange to track changes in protein conformation. Using SEC-MALS, it was determined that the active protein forms noncovalent tetramers, while inactive proteins only form dimers. At the end, it was understood that it is best to monitor and preserve native structure throughout the manufacturing process. Dynamic light scattering (DLS) was cited as an essential tool to rapidly ascertain the key solution properties that correlate to GFL protein activity: size, the diffusion interaction parameter and solution viscosity of concentrated GFL proteins.

David Cistola M.D., Ph.D., of the University of North Texas (Fort Worth) proposed screening for disease susceptibility by profiling nanoparticles in blood. Blood NPs are primarily lipids. Examples include proteins (3–20 nm in diameter); lipoproteins, including low-density lipoprotein (LDL), high-density lipoprotein (HDL) and cholesterol (10–200 nm); and cell-derived microparticles (10–500 nm). The progress of diabetes, which proceeds in stages, can be tracked by marker lipoproteins such as ApoB, very-low-density lipoprotein (VLDL), LDL, HDL and ApoA-1. Prof. Cistola used a Möbiuζ dynamic electrophoretic light scattering instrument (Wyatt Technology Corp., Santa Barbara, Calif.), which was found to provide reliable size distributions corresponding to the marker lipoprotein populations.

More than 70 diagnostic blood assays, including DLS and flow cytometry, were performed on each member of a cohort of 29 healthy individuals. The data were analyzed by correlation analysis in an attempt to discover correlations between DLS and any other set of assay results.

Positive correlations were observed for insulin resistance and the content of triglyceride-rich lipoproteins. Prof. Cistola plans to continue to develop the correlations to improve patient stratification in diabetes.

Biosimilars with SEC-MALS

Biosimilar development is complex since the innovator is not obligated to help the developer. The developer is required to show that the results of a wide range of analytical characterization methods are highly similar to, or interchangeable with, the FDA-licensed product. Dr. Joseph Glajch of Momenta Pharmaceuticals (Cambridge, Mass.) described how SEC-MALS helped characterize a biosimilar candidate to an approved drug for the treatment of relapsing-remitting multiple sclerosis (RRMS). Glatopa, approved by the FDA for the treatment of RRMS, is a heterosynthetic polypeptide containing only four amino acids, with a molecular weight of 5000–9000 Daltons. Momenta had to develop several orthogonal analytical methods comparing and showing equivalence of its candidate product.

Despite the lack of gel permeation chromatography (GPC) calibration and other reference standards, Momenta’s polymerized product was compared to commercial samples from the innovator. Momenta’s team could duplicate the random sequence and size as shown by SEC-MALS and FFF-MALS, which do not require reference standards. They found that z average molecular mass (Mz) was the best discriminator, since slight changes were noticeable.

Dr. Glajch concluded that SEC-MALS was able to provide more accurate and reliable determination of various molecular weight parameters for ingredients and products. In addition, it is useful for showing high similarity in structure since there is no requirement for standards or a standard curve.

For the first time, Dr. Philip Wyatt, founder of Wyatt Technology Corp., gave a lecture in this symposium series. It was no surprise that the presentation was rich in history and stories involving such notables as James Clerk Maxwell (electromagnetic properties of light), Lord Rayleigh (John William Strutt) (interaction of light with solutions of small particles), Albert Einstein (explained how density fluctuations of particles in pure liquids scatter light), Chandrasekhara Venkata Raman (related light scattering to variations in osmotic pressure and refractive index), Gustav Mie (light scattering from suspended metallic spheres, Paul Putzeys (first to relate light scattering to molecular weight), Peter Debye and Paul Florey (application of light scattering to polymers) and Bruno Zimm (light scattering instrumentation and data analysis).

Next, Wyatt turned to the benefits of having exact equations governing light scattering: 1) The equations can be related to the physical features of particles, and 2) chemists can confirm that modifications of the particles, such as coatings, exist. He examined the math in detail, with several equations per slide. Of importance was that particles measured for purposes of deduction of properties must be monodisperse, hence the need for some means of generating monodispersity, either in the origin of the particle or by sizing. Another slide showed the root-mean-square radius (a.k.a. radius of gyration, rg ) for aggregates of a sphere. Surprisingly, a pentamer may have rg identical to a trimer. Wyatt advised that the results need to be confirmed with other techniques—in their sweet spot, not on their fringe.

A new product and experimental method for characterization of nanomaterials, the μDAWN from Wyatt, extends MALS technology to be compatible with the narrow peaks produced by SEC with low-dispersion, ultrahigh-performance liquid chromatography (UHPLC) instruments. Conventional MALS flow cells are >60 μL, which is too large for narrow UHPLC peaks. In contrast, the μDAWN features a 6-μL cell volume, which decreases band broad ening by 25 times. Reducing the response time of the electronics was also necessary. Like standard Wyatt MALS detectors, the flow cell uses a Wyatt COMET ultrasonic flow cell cleaner. The μDAWN MALS detector can be combined with the Optilab UT-rEX high-sensitivity differential refractive index (dRI) detector to provide a complete μSEC-MALS detector set.

Beyond reducing the flow cell volume, the optical bench incorporates a new design that improves stability and reduces stray light, as required for the tighter focus of the laser beam in the smaller cell. DLS and MALS are measured in the same cell, which facilitates evaluation of molecular conformation by combining the hydrodynamic radius (Rh) determined by DLS with molar mass and the rg determined by MALS.

Several applications were offered to demonstrate the utility of the new detector. One reported the molar mass and retention time in process-scale SEC separation. The results were obtained fast enough to be useful in selecting cut points. Another example showed the determination of molecular weight of drug/protein conjugates. Others included protein aggregation and protein conformation in solution, and polymer characterization using μSEC-MALS detectors downstream of an Advanced Polymer Characterization (APC) chromatography system (Waters Corp., Milford, Mass.).

A new technology on its way to commercialization by Wyatt is electrical AF4, or EAF4, in which an electrical field is applied across the planar AF4 separation channel in order to modify the separation power based on the charge or zeta potential of the particle. Dr. Christoph Johann, managing director of Wyatt Technology Europe (Dembach, Germany), presented very promising results showing that EAF4 provides reliable charge-based separation and analysis of proteins and nanoparticles. In particular, EAF4 offers the opportunity to analyze the electrophoretic mobility of mixtures of charged analytes, whether charge variants with the same size, size variants with the same mobility or combinations of these.

Related to this development, a poster by Vincent Hsieh et al., “Enhanced assessment of nanoparticle colloidal stability via FFF-PALS” (phase analysis light scattering) showed how the Möbiuζ electrophoretic mobility/DLS detector can be placed online and downstream of a separation device such as AF4 or SEC to obtain high-resolution simultaneous size and charge or zeta potential distributions of proteins and nanoparticles. This is possible because the Möbiuζ, unlike any other mobility detector on the market, is equipped with a flow cell for online operation. The combination of EAF4 and online mobility is expected to become a uniquely powerful approach to protein and nanoparticle characterization.

Conclusion

The 24th International Light Scattering Colloquium easily exceeded the high bar set by prior meetings. The Wyatt team is commended for organizing an outstanding program. Visit www.wyatt.com/events/ilsc. html for more information.

Robert L. Stevenson, Ph.D., is Editor Emeritus, American Laboratory/Labcompare; e-mail: [email protected].

 


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