Liquid purification or separation – Processes – Chromatography
Reexamination Certificate
1999-09-23
2001-04-24
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Chromatography
C210S101000, C210S198200
Reexamination Certificate
active
06221250
ABSTRACT:
REFERENCE TO RELATED APPLICATIONS
This application is a 371 of PCT/SE/00301 filed Feb. 24, 1997.
1. Technical Field
The present invention relates to a method of generating a liquid mixture of controlled pH, as well as to an apparatus applicable in such a method.
More specifically the invention relates to a method of liquid chromatography using a liquid mixture such as mentioned above, as well as to an apparatus, applicable in such a chromatographic method.
The invention is of special interest in performing ion exchange chromatographic gradient elutions, more specifically for the separation and purification of biomolecules such as proteins.
2. Background and Prior Art
Obtaining liquid media of precisely known composition is in many cases important, as e.g. in the field of liquid chromatography. However, in many chromatographic separations the composition of the liquid media should not only be at each moment precisely known and controlled, but also should vary with time in a precise and controlled manner, as during a gradient elution.
Different liquid chromatographic modes exist, based on the different interactions of the molecules to be separated with the stationary phase on which they are retained and the mobile phase, i.e. the eluent. Especially in the ion exchange chromatographic mode, where the stationary phase is constituted of ion exchange resins, the gradient elution method is widely used; the eluent contains an inert salt and the gradient is performed by varying the concentration of this salt. This is a very important method for the separation and purification of biomolecules such as proteins. See e.g. Warren W., et al. “A new strategy for rapid optimization of protein separations”, American Biotechnology Laboratory, 0749-3223, 34-40, (1989).
The possibility of separating the proteins on ion exchange resins is due to the fact that the protein molecule in general is charged at different sites. It is the presence of these charges that makes the proteins susceptible to separation by the retention on ion exchange columns.
These charges of the protein molecule originate from the acidic/basic character of its amino acid residues, prone to protonations or deprotonations. It is obvious that the charges at the different sites of the molecules, and hence the net charge of the same, will be significantly influenced by the pH of the surrounding liquid media, thereby making protein separation by ionic chromatography highly susceptible to the pH of the liquid medium, i.e. the eluent. See e.g. Diamond P. F., Carberry R., “Systematic Methods of Development for Chromatography of Biomolecules: pH Mapping”, BioConcepts, Technical Newsletter of PerSeptive Biosystems, Sep.-Oct., 1-3, (1990).
In accordance with the above, it has been well documented that the pH and ionic strength of the eluent are the two most important parameters that control selectivity of protein separations on ion exchange resins.
The traditional way of gradient formation has involved the careful preparation of eluents comprising inert salts as well as buffers of predetermined pH to effect the ionic strength gradient at constant pH.
The optimization of the separation of the proteins has been accomplished by changing the slope of the inert salt gradient and/or replacing the buffer system by one with a different pH.
In the prior art, the optimization thus has included the preparation of numerous buffer solutions with predetermined pH and salt concentrations, which have to be meticulously titrated for the separations to be reproducible.
The above described methods, which herein below will be referred to as the “traditional” methods, being very time consuming and awkward, Warren et al. (ibid.) proposed a new procedure, which they called the Waters Auto-Blend™ method, wherein pH, ionic strength and gradient profiles are automatically generated using a single set of stock solutions. These stock solutions are: an acidic buffer, a basic buffer, a salt solution and deionized water (Milli-Q water). By a precise blending of these four solutions, Warren et al. create different gradients, of the ionic strength as well as the pH. In fact, Warren et al. separately blend the two buffer solutions, i.e. the acidic and the basic one, to obtain a certain preselected pH, and then combine this blend with a similar blend of the salt solution and the deionized water with an equally predetermined ionic strength. By varying either the proportion of the salt solution to the water, or of the acidic buffer to the basic one, they create, what they call, independent gradients of either the pH or the ionic strength, or alternatively change the pH from one run to another without having to chose an entirely new buffer system.
(An automatized variety of this method is called the BioCAD™ method, which nevertheless works according to the very same principle, and also belongs to the same society, the PerSeptive Biosystems Inc., Massachusetts, US.) Warren et al. thus reduce the great number of complex buffer solutions which had to be prepared in the traditional methods in optimizing a given protein separation with respect to the pH, to only two different buffer solutions and a salt solution, the latter one being of a much simpler preparation.
A major drawback with the method of Warren et al. is that it in fact fails to provide an eluent of a precisely controlled pH as well as ionic strength. This is due to the fact that they do not take account of the interrelationship of the ionic strength and the pH. However, by considering the Debye-Hückel equation, wellknown from many textbooks in physical or inorganic chemistry, such as “Physical Chemistry” by P. W. Atkins (Oxford university press ISBN 019 855148 7) and “Buffers for pH and Metal Ion Control” by D. D. Perrin, Boyd Dempsey (Chapman and Hall laboratory manuals ISBN 0 412 11700 2), incorporated herein by reference, one realizes that as the ionic strength varies during a salt gradient, the pH will also vary. In view of the foregoing discussion of the importance of the pH to the binding behaviour of the proteins, the failure to control, in a very precise manner, the pH of the eluent appears to be a significant drawback for an efficient optimization of a protein separation: a small variation of pH can entrain an important variation in the retention behaviour of a protein molecule in an unpredictable way.
It appears that the optimization of a protein separation is greatly impaired by the failure to provide an eluent of very precisely known pH in an easy and convenient way. The traditional methods, on the one hand, have been very cumbersome, sometimes making the careful optimization of a protein separation impossible because of a lack of time and/or money. The method of Waters et al., on the other hand, which is said to simplify the optimization, in fact is inherently inaccurate as to the real pH of the eluent in the system during any salt gradient.
SUMMARY OF THE INVENTION
The object of the present invention is to provide a method of preparing a liquid mixture of varying ionic strength wherein the pH can be controlled in a precise and reproducible way.
This object is achieved by a method of preparing a mixture comprising the following components:
(i) one or more buffering species;
(ii) an acid or alternatively a base;
(iii) optionally a salt; and
(iv) a solvent
wherein the proportions of the components (i) to (iv) are concomitantly varied in such a way as to take account of the interrelationship of the pH and the ionic strength of the liquid mixture to obtain at each moment a preselected pH of the mixture.
The inventive method is based on the use of a modified Debye-Hückel equation. It comprises the steps of,
(a) calculating, by the use of the Debye-Hückel equation, the proportions of the different protolytic components of a mixture as defined herein above having a given salt concentration, wherein the ionic strength of said mixture is taken to be due only to the presence of said salt, to obtain a preselected pH of said mixture;
(b) on the basis of the proportions calculated in the preceding step, calculating t
Amersham Pharmacia Biotech AB
Ronning, Jr. Royal N.
Therkorn Ernest G.
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