Liquid purification or separation – Processes – Chromatography
Reexamination Certificate
1999-03-22
2003-09-02
Walker, W. L. (Department: 1723)
Liquid purification or separation
Processes
Chromatography
C210S198200, C502S400000, C502S402000, C502S404000
Reexamination Certificate
active
06613234
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates to beads which are useful as packed bed and fluidized bed support materials for adsorption and chromatography, and methods of making these support materials.
2. BACKGROUND OF THE INVENTION
Modern preparative and analytical solid phase adsorption and chromatography techniques call for improved stationary phases exhibiting high selectivity, large capacity, high mechanical resistance, and high chemical compatibility. These properties, defined by the characteristics of the solid matrix, have evolved with the development of adsorbent media, from soft organic material to semi rigid packing and then to rigid mineral solid phases.
Large capacity, and the ability to control pore size as well as chemical functionalization, has led to the development of many types of soft organic sorbents, based on polysaccharides (dextran, agarose, cellulose) or on weakly crosslinked synthetic materials (dilute polyacrylates, dilute polymethacrylates, dilute polyacrylamide derivatives). These materials have been employed in many applications, such as ion-exchange, gel filtration, and affinity chromatography, but they have always suffered from limited mechanical stability unfavorable for utilization at large scale or high velocity.
Additionally, when mixed in solvents (e.g. 95% ethanol) or in high salt concentration solutions, or when submitted to moderate temperatures (e.g. 35-50° C.) or to mechanical stresses (e.g. pressures of 1.5-3 bar), the properties of these soft organic adsorbents are altered such that their specificity or the efficacy of the separation is reduced or even lost. These undesirable mechanical and functional modifications include pore size reduction, network shrinkage, alteration of bead sphericity and chemical degradation. Additionally, the low density of soft organic polymers makes it difficult to use them in situations where the solid phase must be separated from the liquid phase. This is particularly the case for stirred tank solid phase adsorption, in which the beads must be collected by sedimentation. These organic particles are also difficult to handle in fluid and expanded beds due to the low density difference between the beads and the liquid in which they are suspended.
Semi-rigid organic materials, such as synthetic organic polymers (e.g. crosslinked polyacrylamide derivatives, crosslinked polystyrene, or crosslinked polystyrene-divinylbenzene copolymers) as well as crosslinked natural polymers (e.g. crosslinked agarose) have also been used as sorbents for liquid chromatographic applications. These polymers possess improved mechanical resistance over soft hydrophilic organic materials, but their use is restricted to the low to medium pressure range, which is prejudicial to maximum process velocity and bed height. Operating at high velocity is often advantageous to improve the column productivity or, in some instances, to reduce the processing time of labile molecule. Semi-rigid packing materials subjected to a low or medium hydrostatic pressure can be deformed to such an extent that the packed bed interstitial volume is contracted. This reduction in the bed permeability induces a further increase in the pressure drop, followed by clogging of the column.
Similarly, the low density differential between the aqueous solutions usually used in liquid chromatography and organic polymer based chromatographic media precludes their use for fluidized bed applications. In fluidized bed applications, upward liquid speeds for a given bed expansion depend on particle density and particle diameter. There is little benefit to compensating for low density by increasing the particle diameter, because there is a concomitant increase in the characteristic diffusion length within the bead that constrains the mass transfer efficiency, and hence the productivity of the media.
Therefore, there is a need to provide relatively small porous particles which retain their shape, their chemical and mechanical properties in specific environments useful for biomolecule separation in column as well as in suspension, and which offer a substantial density difference with liquids used in adsorption and chromatography.
To circumvent the compressibility and related drawbacks of organic materials, mineral based sorbents have been developed. These sorbents are based on porous mineral materials, on the surface of which chemical functions are grafted for chromatographic application. Porous silica material, the most popular mineral chromatographic solid phase, is relatively easily modified to a desired surface area, pore volume and pore size.
The binding capacity of conventional mineral grafted silica is directly related to the internal surface area available for chemical modification. The trend, therefore, has been to select high specific surface area material to obtain the highest chemical grafting ratio (see, Unger, K.,
Porous Silica,
Elsevier, Amsterdam-Oxford-New York (1979)). However, due to the inverse relationship between specific surface area and pore diameter, a compromise between pore size and specific surface area must be reached, especially for large solute adsorption applications. A silica with a large surface area yields a low pore diameter, which hinders or even prevents the diffusion of large solutes into the pores and causes incomplete surface utilization for binding (Mohan, S. et al.,
Biotechnology and Bioengineering,
40, 549-563 (1992)). On the other hand, a silica bead having a pore diameter large enough for unhindered large molecule diffusion possesses a reduced surface area and therefore a low grafting ratio and a low binding capacity (Kopaciewicz, W. et al.,
Journal of Chromatography,
409, 111-124 (1987)).
To solve the specific problem related to the separation of large molecules, particularly proteins and nucleic acids, silicas with the pore volume filled by weakly crosslinked natural and synthetic hydrogels have been described (U.S. Pat. Nos. 4,673,734 and 5,268,097). With such media, large pore silica with low surface area is converted to a high capacity media by intraparticle polymerization of functionalized monomers and a crosslinking agent or by introducing polysaccharides that are crosslinked in place. It has been demonstrated that the sorptive capacity of this type of packing material is only dependent on the mineral matrix pore volume. Unlike surface grafted or polymer coated silica, the surface area of this media does not impact directly the binding capacity. The sorptive capacity is a function only of the amount of hydrogel present within the pores, and therefore the pore volume plays a primary role. The bead porosity must therefore be as large as possible, to increase the volume of the hydrogel on which the sorption of macromolecules occurs. However, like classical silica based material, a diminution of capacity is observed for low pore diameter matrixes, due to steric hindrance for large molecular weight solutes, which are unable to access the totality of the gel filled pore volume. For example, U.S. Pat. No. 5,268,097 discloses that at a constant pore volume of 1 mL/g, a 40% decrease in bovine serum albumin binding capacity was observed when the pore diameter was reduced from 3000 to 300 Å.
In this composite stationary phase, the rigid skeleton provides mechanical strength enabling operation at high flow rates without compression, while the soft gel provides the adsorption sites. This approach has been successful in providing a variety of large protein binding capacity media, he functionality of which depend on the hydrogel composition (Boschetti, E.,
Journal of Chromatography A,
658, 207-236 (1994); Horvath, J. et al.,
Journal of Chromatography A,
679, 11-22 (1994)). However, silica shows a high sensitivity to alkaline conditions that precludes its utilization in applications requiring the use of a base. However, basic conditions are required for the large majority of biomolecule separation processes, as they require an alkaline pH treatment either for compound elution, or for sorbent cleaning. As a result,
Boschetti Egisto
Girot Pierre
Perez Marc
Viguie Jean-Claude
Voute Nicolas
Ciphergen Biosystems Inc.
Foley & Lardner
Sorkin David
Walker W. L.
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