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November/December 2009
difficulty of needing several different instruments, to validate scale-up is avoided.
For Process Chromatography, the base module is of 3 litres. Bobbins are interchangeable, and can be exchanged for re-winding if PTFE tubing chosen and cGMP requires virgin material. Most would use stainless steel or titanium tubing and appropriate cleaning techniques, but renewing PTFE coils is an option. If different bore sizes are required, different bobbin sets may be used. Bobbins can be used in series, in parallel or in simulated moving bed operations. Clutches and switching valves allow operating mode changes.
Figure 4 Figure 3
The Partitron CPC™ is shown in Fig 3 (sun centrifuge, with separation chambers and id restricted links between chambers, with 2 rotating seals). This model range has a single process-scale chassis unit. Most hydrostatic CPC are manufactured as chambers created by a sandwich of machined or etched chambers formed into a stainless steel disc, with layers of PTFE sealing the individual disc layers from other layers. The whole assembly is bolted together, but can be prone to leaks and blockages. Machining and in particular etching of any surface radically increases the surface area exposed. Viewed under a microscope the machined/ etched surfaces will appear as mountains and valleys relative to the same material before machining/etching. As CPC units are particularly suited to aqueous/aqueous chromatography of peptides and proteins, enzymes etc, all of which are prone to degradation, the choice of machined/etched stainless steel for most hydrostatic L-LC is suspect.
The Partitron CPC™ was specifically designed for large scale, GMP process chromatography. A totally different construction is utilised. The whole rotor assembly (Fig 3) is machined from a single titanium block. Titanium is well recognised in chromatography for its inertness. A variety of titanium rotors, with volumes from 5 to 25 litres, with either one or two volumes per rotor, may be fitted to this versatile, uniquely modular hydrostatic L-LC.
Fig 4 shows an industrial sub & super-critical extraction plant which is used in conjunction with L-LC production and research
Results & Discission
All experiments in Case Studies completed with a Quattro CCC™.
Discussions regarding Confusions 1 to 2 Unpublished Grant funded research (“The
Industrial Scale up of Countercurrent Chromatography”. BBSRC/DTI LINK Award Ref: 100/BCE08803. Feb 98 - Jan 00 (£322,668), a collaboration of AECS, Brunel University, University College of Swansea, GSK, Astra Zeneca & Shell Research) supported comments by CCC experts, that CCC of different designs or even a single concept, if one varies key parameters this can, on occasions, prohibit scale-up. Keeping all parameters the same, only changing tubing bore, certain scale-ups failed. AECS & Brunel University interpreted the implication of these results in radically different ways in their subsequent independent commercialisation of L-LC. AECS rationalised design to minimise variability and has spent 9 years increasing its understanding scale- up failures. Brunel University and staff developed a range of CCC with radically different sun & planet radii, speed ranges etc. and formed their own spin-off company (DE Ltd) six years ago to exploit their research.
Discussions regarding Confusions 3 to 7 Non-confidential research is detailed below along with confidential research (concept only), plus on our website
www.ccc4labprep.com and in publications.
Case Study 1. Client had a complex extract, when target mix prepared by reverse phase HPLC, had desired bioactivity. When process transferred to industrial non-HPLC manufacture, target mix exhibited extreme cytotoxicity. L-LC was used in direct cross correlation to gradient prep HPLC (a single multi gram injection of same matrix onto a custom packed 50 x 250 mm, 15um C18 column, poisoned column, yet multiple L-LC preparations could be run) showed that laboratory studies with end capped, C18 HPLC prep columns, removed the then unknown cytotoxic compounds, which L-LC methods found.
Case Study 2. Client had complex mixture, which had taken Sequential Flash, MPLC & HPLC 3 different International Labs each 6 months to prepare target. Two contract laboratories refused to do repeat preparations. By Sequential L-LC and HPLC, target was prepared in 4 weeks for first preparation and was completed in less than one week in repeat separation. There was a massive; over ten-fold reduction in solvent usage, as well as the obvious huge time saving.
Case Study 3. During LINK Grant project working with GSK the results shown in Fig 5 were obtained. Two HPLC gradient traces are shown.
Figure 5
Top is original gradient HPLC. Below is the HPLC of a single 4 ml fraction from a 200+ ml gradient Quattro L-LC run. The insert shows the amount of target in fractions before and after the main fraction. Over 90% of target was in one single 4 ml fraction. The bars labelled F above top chromatograph show polarity range of L-LC fractions. Apart from solvent front, all show the very small polarity range of OT HPL-LC fractions. In addition an unknown bioactive was found.
Figure 6
Case Study 4. Sequential L-LC plus HPLC. The NEEM tree is the Holy Tree of India; it produces such a variety of bioactive targets, that villages in India define it as their Pharmacy. Fig 6 shows collaborative research with the University of Vicosa, Brasil. Previous to installing the Quattro L-LC, Prof Gulab Jham took months to prepare just the required amounts of AzA, by Sequential L-LC & HPLC, AzA and six other key related compounds, never prepared in that laboratory before, were prepared in weeks with better than 95% recovery and better than 95% purity (4). An injection/recovery mass balance was conducted, by weighing the dried residue in each L-LC fraction. Within the scope of the method, a full mass balance was obtained. That would be an extreme rarity in S-LC for a raw natural product injection.
Case Study 5. Deguelin obtained from an Amazonian plant is very valuable (~$20,000 g), the contaminant rotenone is of little value, but contaminates extracts. Researchers with decades of historic Japanese CPC 1000 ml instrument experience for this separation achieved loading of 150 mg per 1000ml CPC capacity. On upgrade to a modern manufactured 1000 ml CPC they doubled loadings to 300 mg per 1000ml CPC capacity. Their method failed on the Quattro CCC. A method developed in less than a day increased loading to 1625 mg per 1000ml Quattro L-LC capacity; over ten times that of historic CPC. The client subsequently increased
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