22 February / March 2017
dimension [19]. However, the random nature of the pore morphology means that there is no scale dilation associated with traditional silica’s. In such complex separations, the high kinetic performance dramatically effects the mass transfer of large molecules into and out of the pores, where the diffusion affects the rate of equilibration of the analyte concentration between the pressure driven regime and the diffusional regime. By using particulate (packed) columns, the efficiency of large molecule separations can be improved significantly by reducing the intra-particle mass transfer resistance. Since large molecules possess slow diffusivity, they spend more time in the intra-particle pores, therefore their bands tend to broaden. To lessen this contribution, non-porous materials or partially porous materials can be applied. However, non-porous materials suffer from limited loading capacity and retention; therefore they have not become widespread in routine analytical labs. It has been proposed that for large molecules, larger partially porous materials coupled with the reduction of the shell thickness can be advantageous, due to the shorter diffusion distance and greater access to the surface area of the material [20,21].
sometimes superior) to other wide pore stationary phases, regardless of the gradient time and flow rate, when analysing the largest model protein, such as BSA. These benefits may be attributed to the SOS particle porous shell morphology, minimising the intra-particle mass transfer resistance [10].
The SOS column was also applied for the analytical characterisation of commercial mono-clonal antibody (mAb) and antibody-drug conjugate (ADC) samples [23] (Figure 5). Characterisation of a bio-pharmaceutical product, performed with appropriate analytical techniques, provides useful information on purity and/or protein stability in its formulation. Antibody heterogeneity is related to conformational isoforms. The reduction of the disulfide bonds of IgGs and then the RPLC analysis of the reduced fragments is a commonly used test to determine whether the conformational variants are disulfide-related or not. With these classes of proteins, the performance of SOS column was similar to the best wide pore stationary phases available on the market. There is still some way to go with the concept of SOS particles, as only a few examples have been investigated and substantial more work has to be done in applying the technology for more complex separations
Figure 4. h–ʋ plots, observed on sphere-on-sphere column with butylparaben, decapaptide (CH-869) and glucagon. Mobile phase consisted of 83/17 (v/v%) water/acetonitrile for butylparaben. Mixtures of water (0.1% TFA)/acetonitrile (0.1%TFA) 83/17 (v/v%) and 75/25 (v/v%) were used as mobile phases for the deca-peptide and glucagon, respectively. Temperature was set to 30°C.
The SOS particles have been used for the successful separation of complex proteins mixtures under gradient elution with good peak shapes [10,12,22]. Further studies have shown that the prototype particles with standard C4 bonding have similar chromatographic performance to commercial core–shell materials (2.6 µm) when separating standard peptides and proteins of various sizes (e.g., lysozyme, myoglobin,
ovalbumin...etc), whilst reducing the operating time and pressure which is advantageous for high flow chromatography [22,23]. The kinetic performance of this material was also evaluated in both isocratic and gradient modes using various model analytes. The data were compared to those obtained on other wide pore state-of-the-art core–shell and fully porous materials commonly employed to separate proteins moreover to a reference 5 µm wide pore material that is still often used in QC labs. In isocratic mode, minimum reduced plate height values of hmin= 2.6, 3.3 and 3.3 were observed for butylparaben, deca-peptide and glucagon, respectively (Figure 4). In gradient elution mode, the SOS column performed with very high peak capacity when working with fast gradients. This prototype column was also comparable (and
Figure 5. Representative chromatogram of reduced ADC (brentuxi- mab-vedotin). Columns: Prototype SOS (sphere-on-sphere) C4 (100 mm × 2.1 mm, ~2.5 µm), HaloProtein C4 (150 × 2.1 mm, 3.4 µm), BE- H300C18 (150 × 2.1 mm, 1.7 µm) and Aeris Widepore C18 (150 × 2.1 mm, 3.6 µm). Mobile phase A: 0.1% TFA, mobile phase B: 0.1% TFA in acetonitrile. Flow-rate of 0.4 mL/min, gradient: 27 – 42%B in 12 min on the SOS column and 30 – 45% B on the other columns, temperature: 80°C, UV detection was carried out at 280 nm.
Conclusion and outlook
Spheres-on-Spheres particles can have different structures and morphologies depending on the reaction conditions. It has been widely exploited for a very wide range of applications. However, for chromatography, SOS microspheres are utilised for protein separation. The relevant preparation methods are explained and its advantages over the current LbL approach. We have focused on the method of synthesis and explained the route the particles take to form the desired morphology via a one-pot synthesis method to form a type of core–shell structure. The method was utilised to produce mesoporous shell with high surface area and more complex structures that are more analogous to the structure of a fractal.
One type of SOS particles was assessed for small molecules separation and entrapment of new crystalline phases. This demonstrated the potential of this material as a new stationary phase for chromatographic application. With the unique core-shell property and the superficial macroporosity of the shell, the packed
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