A new technology that encapsulates polymers within mass-produced glass beads shows promising use in chromatography, peptide synthesis and biocatalysis. Dr Donald Wellings, chief scientific officer of Chromatide, explains how the technology saves costs and offers improved efficiency
Polymeric matrices are used in many scientific applications. The more obvious of these include stationary phases for separation technologies through to solid supports for the synthesis of peptides and oligonucleotides. The use of organic and inorganic polymers as supports in biocatalysis, chemo catalysis, cell culture, plasma-phoresis, diagnostics and slow release is also well established.
Even though polymeric media have a widespread acceptance in many fields, the physical form or particle morphology of the polymers remains, in many instances, seriously under-developed.
Polymeric particles are often used in chromatographic separations where they form the stationary phases. In certain types of chromatography, the cost of stationary phases is restrictive while in others, the physical nature of the stationary phase can reduce the effectiveness of the separation.
For instance, the soft microporous polymers that are often used for affinity, ion-exchange and gel permeation chromatography cannot be used at high flow rates or in very large columns because of the deformable nature of the particles. Conversely, the rigid macroporous polymers can often be mechanically friable and subsequently suffer from a short lifespan.
The application of solid supports or stationary phases in chromatographic separations is extensive: from the complex, high-tech separations used in the pharma and biotech industry through to the mining industry. Some of the pharma industry's most valuable drugs are purified by preparative chromatography. The use of polymeric particles in solid phase extraction and in the preparation of solid phase reagents is also common in the chemical, pharma and biotech industry.
For ease of use, polymer particles are often spherical and have a defined particle size distribution. The spherical nature of the particles improves the flow and filtration characteristics of the polymer.
Although the use of solid supports has operational benefits, there are disadvantages to the solid phase approach. For example, commercially available supports commonly used for solid phase synthesis of peptides and oligonucleotides are relatively expensive.
The high cost of the supports arises from the manufacturing processes. Microporous and macroporous polymers are generally used. Micro-porous polymers have a relatively low level of cross-linker, which allows the polymer particles to solvate and consequently swell in suitable solvents. Macroporous polymers have a high level of cross-linker in the polymer matrix and contain large pores. These polymer particles are generally rigid and have good flow characteristics in packed columns.
Polymeric particles are typically made by a dispersion or emulsion polymerisation process in which a solution of monomers is dispersed in an immiscible solvent (continuous phase) prior to initiation of the polymerisation. The polymer particles formed are then filtered, washed and classified.
These processes have some drawbacks such as monomer loss to the continuous phase, generation of a range of particle sizes and generation of fine particles during the polymerisation.
Loss of monomers to the continuous phase is inefficient in terms of both raw material and environmental costs. Classification of the polymer particles to isolate the particle size required for a particular application may be a laborious and complex process, typically involving sieving and air classification which may lead to losses in yield. Fine particles are often produced which can be problematic in isolation of the polymer beads and are often removed by settling and decantation.
Besides these drawbacks, certain other disadvantages arise with the physical properties of the known polymeric particles. Microporous polymeric particles are generally soft and unsuitable for use at a high flow rate in a packed column bed. Furthermore, the soft particles may be compressed and cause fouling, for example during filtration, often leading to compressive intrusion into the sinter or mesh being used. Rigid macroporous and macro-reticular particles are more suited to high flow rates in packed column beds. However, due to their rigid nature the particles may be fragile and fragment under physical stress.
The problems associated with production wastage, physical integrity of the support and poor product performance can be ameliorated by encapsulating the polymer in a preformed exoskeleton, which is effectively used as a container for the polymer particle.
Chromatide has developed a novel technology for encapsulating polymers within rigid and uniform mass-produced glass beads. These glass beads, commonly used in the jewellery and textile industry, are particularly useful for this application (Figure 1).
Normally referred to as seed beads, they are inexpensive and made on a large scale in excess of 30 tons per day so eventual scale of operations will not be an issue.
The process used to manufacture the beads involves drawing out lengths of capillary tubing, cutting these into precise, small lengths then annealing these at temperatures just below the melting point of the glass. The surface tension of the near-molten glass causes the capillary ends to collapse, effectively forming a hollow glass sphere with a hole at either end.
Ideally, the beads are spherical or almost spherical. Their uniform nature is advantageous in many applications and facilitates, for example, packing in columns and improved flow characteristics over a bed during filtration.
Although the smallest glass beads used in the jewellery and textile trade are in the region of 1mm diameter, Chromatide has workedg with a major Japanese jewellery bead manufacturer to miniaturise and optimise the beads as exo-skeletal supports for polymers. The bead size has been reduced to less than 1mm diameter and more importantly the thickness of the glass wall has also been reduced to a point where the ratio of polymer to glass volume is maximised without compromising mechanical robustness (Figures 2 & 3).
The encapsulation process is as simple as the process for seed bead manufacture. The glass seed beads are mixed with sufficient monomer solution to fill the holes of the beads and the interstitial spaces. Capillary action helps to retain the monomer solutions within the beads and the excess is drained off before initiating polymerisation by traditional means. Any small amounts of polymer that remain adhered to the outer walls of the glass beads where they were in contact during the polymerisation process are removed by an abrasion and decantation washing process.
Figure 4a shows a photograph of an amino functional polydimethyl-acrylamide encapsulated in the hole of a bead. For clarity, Figure 4b shows the same beads where the polymer is stained with ninhydrin. In theory, virtually any polymer can be encapsulated and to date we have worked with polyacrylamide, polymethacrylate, polystyrene, agarose, polyurea, polyethylene glycol and silica sol-gels, to name just a few.
At one end of the scale polymers have been encapsulated with extremely low levels of cross-linking that would render them useless outside the glass skeleton. These soft gels exhibit extremely fast mass transfer combined with high mechanical strength.
At the other end of the scale we have encapsulated macroporous polystyrene-divinylbenzene based polyHIPES and silica sol-gels that are rigid but would be extremely fragile outside glass seed beads.
This technology can be applied in many arenas and we are currently working on solid phase peptide synthesis, oligonucleotide synthesis, affinity purification, enzyme immobilisation, immobilisation of transition metal catalysts, slow release of drugs and stem cell culture.
It is important to realise that Chromatide is not necessarily developing new polymers for these applications but simply encapsulating well-developed polymers within a rigid exo-skeleton. This has allowed the company to cut cross-linking to a minimum in microporous gels. The importance of doing this has been demonstrated clearly for peptide synthesis (Figure 5).
In addition, the results observed for the synthesis of a 20 base oligonucleotide show a dramatic improvement over traditional solid supports. Figure 6 shows the ion-exchange HPLC trace of the crude oligonucleotide assembled on controlled pore glass overlaid upon the trace of the crude oligonucleotide assembled on a soft gel encapsulated using Chromatide technology. The level of deletions and in particular n-1 impurities is greatly reduced.
The potential benefits to biopharmaceutical production are dramatic. Encapsulation of much softer gels for ion-exchange, gel filtration, hydrophobic interaction and affinity based separations will bring increased accessibility of the active sites on the polymer. This increases the capacity of the polymers used and allows them to be run at high flow rates compared with traditional stationary phases. The uniform and rigid nature of the exo-skeleton means that there is no resistance to flow and no fouling of column frits.
For expanded-bed based separations the high density of the glass brings benefits. The overall result will be a much higher productivity with less plant down time. The chromatographic characteristics are illustrated in Figure 7, which shows a typical trace for separation of Human IgG on a Protein A medium prepared using this technology.
For this stationary phase the cross-linking was reduced to 0.25 mole per cent, a small fraction of that used in traditional stationary phases, which are known to have poor diffusional characteristics. The soft gel particles normally used in the protein purification industry typically have 4-6 mole per cent cross-linking and often only 10-15% of the outer layer of the particles is accessible for binding.
For many years chemists and biologists have used enzymes to perform chemical reactions outside their natural origin. The applications range from the addition to detergents to aid in the breakdown of "oil and dirt" in washing machines through to their use in complex organic chemical reactions in the pharmaceutical industry. The immobilised enzymes come in many forms but in general the enzymes are commonly supported on polymer particles. Existing immobilised enzymes are relatively expensive due to inefficient enzyme usage and poor accessibility once immobilised.
In addition to the use of isolated enzymes it is common to use whole cells that contain the enzyme to perform the organic chemical reaction required. The process in which an enzyme, protein or whole cell is used to perform a chemical reaction is termed biotransformation. The overall concept is also referred to as bio-catalysis. A large proportion of commercially available pharmaceuticals involve at least one biotransformation in the manufacturing process.
It is clear that there is a massive industry developing the greener chemistry of enzymes for numerous applications and, therefore, efficient immobilisation processes will be essential in achieving efficacious use of an enzyme.
Chromatide has successfully immobilised a number of enzymes for biocatalysis. For example, immobilised Penicillin G amidase is used for the hydrolysis of benzylpenicillin in the manufacture of Amoxicillin1. Penicillin G amidase immobilised using this technology (Figure 8) shows an activity of 680U/g of polymer for the immobilised enzyme. This is more than five times that observed for the commercially available version of the immobilised enzyme used routinely at industrial scale. This not only demonstrates a more efficient use of the enzyme but provides the immobilised catalyst in a more user-friendly and mechanically robust form. The growing trend to use continuous systems for biocatalysis and other processes will benefit from this polymer encapsulation technology.
The company is currently working in conjunction with the Stem Cell Consortium at Liverpool University to develop encapsulated polymers for stem cell culture and differentiation. The results of this work will no doubt stimulate development of the technology for more generic cell culture in biocatalytic and recombinant processes.
The potential for this technology in controlled release of drugs is also in its early stages. Proof of principle studies for a veterinary slow release application is starting to show promise.