20 February / March 2017
Spheres-on-sphere (SOS) Silica a Real Support for Separation of Large Biomolecules
by Adham Ahmed and Peter Myers,
Department of Chemistry, University of Liverpool, Liverpool L69 7ZQ
Peter.Myers@
liverpool.ac.uk
Review of upcoming challenges
The change in focus for the pharmaceutical industry over recent years to large protein therapeutics has resulted in a growth in the interest in the rapid separation of protein therapeutics, especially monoclonal antibodies and antibody-drug conjugates [1,2]. The changes in the types of analytes being monitored has had a significant impact on new stationary phase inventions where there has been a general drive by manufacturers to reduce particle size to improve the separation capability of the columns. However, there are limitations with the current particle technology, which presents a challenge for in the analysis of proteins [3].
There are two common silica particle types that are employed for the analysis of proteins;
• The first type of silica particle is non-porous and suffers from limited surface area, retentiveness and the ability to load the sample [4].
• The second type is a fully porous media. This has a high surface area, which can overcome the poor retentiveness and sample loading issues associated with the non-porous bead. Unfortunately, when looking at the physical dispersion processes that occur within the column, a larger analyte molecule will diffuse at a slower rate in the porous region of the stationary phase, which results in equilibration issues with the bulk flow [5]. This will result in a difference in retention times of individual analyte molecules and hence broader peaks.
A better compromise between efficiency and loadability is the core-shell technology. This technology overcomes the mass transfer issues while maintaining column performance and low back-pressure [6,7]. These particles were first developed in the late 1970s, and as a consequence several core-shell pellicular sorbents particles were commercialised. These original particles had a typically low surface area in the range of 5 – 15 m2
/g, which results in very low loading
/g and, using a uniform core, produced a very narrow particle size distribution. Despite the impressive progress in the LbL method [8,9], it remains a time-consuming process limited by the need for repeat deposition time, which takes several weeks. The initial offerings for the pore size were limited and did not meet the separation requirements for the increasing number of therapeutic proteins entering the pharmaceutical market. Thus, a particle with a wider pore system is needed due to the size of proteins, but the wider pore also needs to consider the various configurations that proteins can have, which results in an unpredictable behaviour within the porous system. The number of challenges encountered in the development of new biomolecules, has therefore necessitated the need for a new stationary phase to meet these demands.
capacities and poor analyte retention. The new generation of core- shell particles offer better performance due to the many advances in silica sol-gel technology and in particular the control in the layer-by- layer (LbL) addition method used to generate the outer porous layer. This provided an increase in surface area reaching an optimum of 150 m2
5µm
Figure 1. Spheres-on-sphere particles prepared via one-pot synthesis method
Recently, a unique type of core−shell particle, nanospheres-on- microsphere or known as spheres-on-sphere (SOS) silica, has been prepared in a one-pot synthesis from a single precursor 3-mercaptopropyltrimethoxysilane (MPTMS) [10], Figure 1. They offer an interesting alternative to the mainstream approach of producing solid core-shell silica particles which uses time-consuming LbL approach. They are made via a simple and fast one-pot synthesis which is highly advantageous, offering potential benefits on reaction time, easier quality control, materials costs, and process simplicity for facile scale-up. There have been limited reports on the one-pot synthesis of core-shell silica microspheres which are suitable for HPLC [10,11], but these approaches including spheres-on-sphere have not yet been employed for commercial use. The shell thickness, porosity and chemical substituents of the shell can be controlled by using the appropriate reagents and conditions. A time study was carried out to find out how these particles were formed by imaging the particles during the course of the reaction. Microscopic images suggested that a two stage nucleation process occurred. The first stage, not unlike core–shell synthesis, was the formation of the core microsphere. The second stage was nucleation of nanoparticles on the surface of these microspheres. This can be controlled by solution pH and solvent
Development of Spheres-on-Sphere silica microspheres
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