operation mode in Contract Preparations of targets.
Almost every difficult application at the AECS- QuikPrep Ltd laboratory uses L-LC to polarity fractionate highly complex matrices (that would poison an HPLC column with one injection), into narrow polarity bands. We would stress the mutual benefit of Sequential L-LC and HPLC, and never view L-LC and HPLC as mutually exclusive. The L-LC fractions are so extremely restricted in polarity, that they only require isocratic HPLC to complete our standard Sequential L-LC to HPLC runs. 95 to 99+% pure target can be obtained after a single Generic Gradient L-LC run and Sequential HPLC Prep column (see below). This Sequential L-LC and HPLC principle can be repeatedly successful, even when starting from unknown, and totally uncharacterised, raw natural product or crude synthesis materials. Sequential L-LC to L-LC, utilising different biphasic solvents for the second L-LC run, is only used if targets irreversibly adsorb or degrade on 5um, end capped C18 etc HPLC phases.
Context
Figures 1 & 2 show the chassis and coil/volume options of the Quattro CCC™ model range. (‘J’ Type Planetary Centrifuge, open, constant id tubing, wound on a planetary bobbin, with no rotating seals). The bobbins (planetary rotating body, holds the coiled columns) can have tubing with different material choice. Options include PTFE, Stainless Steel or Titanium. Tubing bore for id can vary from 0.5 to 12.5 mm, and volumes from 7 to 3000ml for a single rotor assembly. A single bobbin can have two coils. All models except the entry IntroPrep™ have two dynamically balanced bobbins, with up to 4 coils as an option. Each coil can be used independently for same or different preparations, or used in any combination, in series with any coil or multiple of coils of the same id. Uniquely for hydrodynamic L-LC model ranges, all models share the same key L-LC design parameters, inclusive of the same sun & planet radii, speed ranges, beta values, winding techniques and only tubing bore is varied. This model range is also the only one that allows even the largest bore to be tested on a laboratory based unit, prior to introduction to process based preparation. Hybrid coil winding, that is multiple id.’s in the same instrument or bobbin, can be manufactured produced. Multiple bobbin sets for a single chassis are available. In this way the major 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.
The Partitron CPC™ is shown in Figure 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, proteins and enzymes, 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 (Figure 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.
Figure 4 shows an industrial sub & super-critical extraction plant which is used in conjunction with L-LC production and research
Figure 1
Figure 4
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 Figure 5 were obtained. Two HPLC gradient traces are shown. 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 2 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.
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. Figure 6 shows collaborative research with the University of Vicosa, Brasil. Previous to installing the Quattro L-LC, Professor 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 150mg per 1000ml CPC capacity. On upgrade to a modern manufactured 1000 ml CPC they doubled loadings to 300mg per 1000ml CPC capacity. Their method failed on the Quattro CCC. A method developed in less than a day increased loading to 1625mg per 1000ml Quattro L-LC capacity; over ten times that of historic CPC. The client subsequently increased the initial loading to closer to a typical 5 to 40g loading per 1000ml.
Figure 3 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 250mm, 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 four weeks for first preparation and was
C ase Study Wine Research Figures 7, 8, 9 & 10 by wine researchers [5] shows the worth of gradients in L-LC, and of L-LC in unravelling difficult identification issues. This research led to the targets sensory properties being determined and tentative structural elucidation of new unknown oligomeric anthocyanins. The chromatogram on HPLC revealed an absence of standard baseline hump seen once the monomers etc were separated by L-LC. This highlights the value of doing a L-L chromatography sample polarity screening. L-LC helped these wine researchers to identify a new class of compounds (oligomeric anthocyanin species), and to study their influence to the colour and sensory properties in wine.
Case Study 7 HTPrep/Combinatorial. In 2007 we custom designed the World’s first Quattro HTPrep™ for a Pharmaceutical Company in the USA. The research was presented at CCC2008 and published in the proceedings [6].
Figure 5
Chromatography Focus
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