Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...
Reexamination Certificate
1999-10-15
2001-06-12
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Liquid/liquid solvent or colloidal extraction or diffusing...
C210S656000, C210S198200, C530S413000, C530S417000
Reexamination Certificate
active
06245238
ABSTRACT:
REFERENCE TO RELATED APPLICATIONS
This application is a 371 of PCT/GB98/01109 filed Apr. 16, 1998.
This invention is concerned with chromatographic separation, more particularly with a new and improved method of sample displacement chromatography and applications thereof, as well as with apparatus useful in such methods.
During the last 10-15 years displacement chromatography has been suggested as a useful alternative to liquid chromatography using elution techniques. In elution chromatography, components of a sample are transported along stationary phase material, e.g. in a column, by a mobile solvent phase. The various components interact at different levels with the stationary phase material and are therefore separated into bands. Displacement chromatography, on the other hands, utilises as mobile phase a displacer solution which has higher affinity for the stationary phase material than do the sample components. In the case of column chromatography the sample components are thereby displaced and driven down the column ahead of the displacer front, competing for adsorption sites and separating into individual component bands as they proceed.
Whereas elution chromatography normally results in substantial dilution of the sample material, displacement chromatography permits recovery of sample components at significantly higher concentrations and generally makes more efficient use of the stationary phase material. Furthermore, the boundaries between individual component bands tend to be self-sharpening as a result of being driven by the sharp displacer front; the “tailing” of bands observed in elution chromatography is thus avoided. However, displacement chromatography does suffer the disadvantage that, to achieve optimum results, operating conditions such as the composition, concentration and flow rate of the displacer solution must be tailored specifically to individual sample types.
Sample displacement chromatography is a self-displacement technique which was first proposed by Hodges et al. [
J. Chromatogr
. 444 (1988), pp. 349-362] for preparative purification of peptides by reversed phase HPLC, and which does away with the need for an extraneous displacer solution. The peptide components are applied to the column input and themselves compete for binding sites on the stationary phase as they are washed through the column or series of columns by an appropriate solvent. The more strongly binding components bind first and displace less strongly binding components to further along the column(s). The components are therefore separated according to their different degrees of hydrophobicity/hydrophilicity and thus their affinity for the stationary phase material. In a representative example a short pre-column is used to trap impurities which are more hydrophobic than the desired sample component, this being retained in and saturating the main column, while the more hydrophilic impurities are further displaced and so are washed out of the main column. It is suggested that the size of the pre-column may be adjusted to match the amount of hydrophobic impurities present in a particular sample, whilst the size of the main column may be adjusted to ensure maximum product retention and outflow of hydrophilic impurities.
Curiously Hodges et al. subsequently use gradient elution to recover the desired product from the main column, so that the advantageous potential of displacement chromatography for yielding relatively high concentration product solutions is lost. This would suggest that the initial component separation was incomplete.
Veeraragavan et al. [
J. Chromatogr
. 541 (1991), pp. 207-220] report application of the Hodges technique to purification of proteins using high performance anion exchange chromatography columns. The apparatus used was a low pressure fast protein liquid chromatographic system and again gradient elution was employed; this was presumably felt to be necessary in light of the observation that peak overlaps were a problem in the primary separation procedure. One and two column systems are specifically described, the former being applicable on what are said to be the rare occasions where the desired product is either the most or least strongly binding component. The possibility of using a multi-column system “in which theoretically every component of the protein sample could be fixed to a column of the proper dimensions” is noted.
Multi-column HPLC systems for sample displacement chromatography have in fact been described by Hodges, inter alia in CA-A-2059114. A representative illustration shows the use of ten reversed phase HPLC columns or column segments connected in series for the purification of peptide samples; after the sample material has been loaded and distributed/separated over the train of columns, individual columns or segments may be eluted separately, without resort to gradient elution, the desired product component being recovered in substantially pure form from at least one such column or segment. Advantages of this process are said to be that (i) it allows ten-fold greater loading than comparable gradient elution separations; (ii) it involves minimal use of costly HPLC solvents; (iii) it requires minimal use of fraction analyses; (iv) it avoids the need to use displacer solutions during the actual separation; and (v) operating costs in terms of solvents, column packings and machine usage are much lower than typical gradient elutions.
The Hodges multi-column procedure does not appear to have been widely adopted and has been found in practice to give products with insufficient purity as a result of inadequate resolution of the product from closely related impurities. Moreover, by virtue of the need to operate HPLC procedures at high pressure, typically 80-200 bar, the apparatus required is necessarily complicated and expensive.
The present invention is based on the finding that efficient and reproducible separation of closely related sample components may be achieved using sample displacement chromatography at low operating pressures and recovering the desired product in a non-gradient manner. The use of low operating pressures greatly simplifies apparatus requirements, permitting the use of simpler and less expensive pumps, taps, connectors and the like than are required for HPLC systems; the consequential low mobile phase flow rates have been found to give rise to good separation of the sample components. By avoiding use of gradient elution the separation procedure also minimises solvent requirements and facilitates recovery of the desired product in an advantageously high concentration.
The procedure also makes optimum use of the stationary phase chromatographic material, since at the end of a sample displacement chromatography separation the entire length of the chromatography bed will typically be in active use. In HPLC separations, on the other hand, only a small part of the stationary phase participates in the separation process at any given time. Sample displacement chromatography therefore permits a 10- to 100-fold increase in capacity for a given stationary phase material compared to HPLC.
According to one aspect of the present invention there is provided a method of sample displacement chromatography which comprises (i) applying a multi-component sample to one end of a chromatography bed comprising stationary phase material having affinity for components of the sample, causing components of the sample to become distributed along the chromatography bed by passage over the bed of a non-eluting mobile solvent phase under an operating pressure not exceeding 30 bar, and (ii) recovering a desired component of the sample from at least a portion of the chromatography bed under steady state (i.e. non-gradient) processing conditions.
In general, it is preferred that the desired product should be the major component (e.g. at least 50%) of its type within the sample so that it will give rise to adequate displacement effects in respect of less strongly bound impurities, although less closely related products having substantially d
Sage G. Patrick
The Firm of Hueschen and Sage
Therkorn Ernest G.
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