Synthetic resins or natural rubbers -- part of the class 520 ser – Synthetic resins – Cellular products or processes of preparing a cellular...
Reexamination Certificate
2001-08-07
2002-05-14
Foelak, Morton (Department: 1711)
Synthetic resins or natural rubbers -- part of the class 520 ser
Synthetic resins
Cellular products or processes of preparing a cellular...
C210S645000, C210S656000, C424S486000, C521S146000, C521S149000, C530S815000
Reexamination Certificate
active
06387974
ABSTRACT:
BACKGROUND
This invention relates to novel macroporous polymers having selected porosity and permeability characteristics that provide rigid polymer matrices suitable for use in medium and high pressure reversed phase liquid chromatography (RPC). The polymers are especially useful as stationary phases in large scale chromatography columns without developing increased pressures during prolonged use.
Stationary phases useful in RPC, especially in high performance preparative mode (such as required in the separation and purification of biomolecules), must be mechanically rigid to withstand the high operating pressures generated within the chromatography columns. Silica matrices, which have been commonly used for these applications in the past, have satisfactory mechanical rigidity; however, silica matrices cannot be operated under high pH conditions, which severely limits their use in a wide range of biomolecule separations. This factor limits the purification options available to the process chromatographer, and adversely affects the production lifetime of silica media (they degrade faster because they cannot be cleaned under aggressive conditions), resulting in poorer overall economics of commercial manufacturing processes.
Stationary phases based on organic polymers, on the other hand, call typically be operated over a very wide range of pH conditions, providing greater utility in biomolecule separations. The chromatographer has the option to develop a high pH process, which may offer such benefits as improved solubility, selectivity and capacity characteristics of the separation media for certain molecules. In addition, polymeric resins may be cleaned aggressively, under high pH conditions, thus improving the column lifetime and, consequently, process economics. However, current polymeric stationary phases are somewhat compressible at the medium to high pressure conditions used in high-performance biomolecule separations. This compressibility is detrimental to separation processes because it limits the range of operable flowrates, and it can degrade the integrity of the polymer bed in the column. For example, the following references disclose polymers used at column conditions representative of high-pressure analytical operations (less than 0.5 cm internal diameter columns), where “wall effects” are known to minimize polymer compressibility; however, these references do not disclose operations in larger scale, high-pressure commercial chromatography columns where one would expect additional pressure buildup from polymer compressibility due to the absence of wall effects: Lloyd, L. L. and Warner, F. P.,
Preparative High Performance Liquid Chromatography on a Unique High
-
Speed Macroporous Resiv, J. Chromatography,
Vol. 512, pp 365-376 (1990); and Lloyd, L. L.,
Rigid Macroporous Copolymers as Stationary Phases in High Performance Liquid Chromatography, Review, J. Chromatography,
Vol. 544, pp 201-217 (1991).
Conventional macroporous copolymers produced from the suspension polymerization of divinylbenzene (DVB)-containing monomer mixtures in the a presence of a nonsolvent represent polymers having a wide range of pore size distributions and surface areas. For example, U.S. Pat. No. 4,686,269 discloses a method to prepare polymers for use in analytical scale liquid chromatography columns (internal diameter of 0.8 centimeter) having average particle diameters from 0.5 to 50 microns and containing at least 60% polyvinylaromatic monomer, by polymerizing the monomers with 50 to 300% of organic cosolvents, based on total weight of monomer; the reference does not disclose a method to prepare polymers having selected porosity and permeability characteristics that provide rigid polymer matrices that do not compress under the high pressure use conditions common in production scale chromatography columns, that is, those having internal diameters of 2 to 100 centimeters, typically from 5 to 80 centimeters.
Polymer compressibility translates to restricted flow through the separation media, producing additional backpressure in the chromatography system, and ultimately, longer cycle times. There is a need for a polymeric stationary phase that can withstand, without significant compression, the medium to high operating pressures generated under typical RPC process conditions. In addition, it is essential that the stationary phase also have satisfactory mass transfer and capacity characteristics for certain targeted classes of biomolecules in order to yield the desired chromatographic performance.
The problem addressed by the present invention is to provide a macroporous polymer stationary phase suitable for biomolecule separation and purification, while at the same providing satisfactory pressure and flow characteristics during RPC.
SUMMARY OF INVENTION
The present invention provides a macroporous polymer comprising polymerized monomer units of (a) 50 to 100 percent by weight of one or more polyvinylaromatic monomer, and (b) zero to 50 percent by weight of one or more monounsaturated vinylaromatic monomer; wherein the polymer has (i) a total porosity of 0.7 to 2 cubic centimeter per gram; (ii) an operational mesoporosity of 0.7 to 1.9 cubic centimeter per gram; (iii) an average particle size diameter of 2 to 600 microns; (iv) a surface area of 200 to 1500 square meters per gram; (v) a flow resistance value from 700 to less than 1,800 at 10 bar pressure and from 1,500 to less than 7,000 at 60 bar pressure; and (vi) a total insulin capacity of 75 to 150 grams insulin/liter of polymer and a dynamic insulin capacity of 60 to 150 grams insulin/liter of polymer.
In a preferred embodiment, the present invention provides the aforementioned macroporous polymer having (a) a surface area of 400 to 1000 square meters per gram; (b) an operational mesoporosity of 0.9 to 1.4 cubic centimeter per gram; (c) an average particle size diameter of 10 to 75 microns; (d) a flow resistance value from 700 to less than 1,500 at 10 bar pressure and from 1,500 to less than 5,000 at 60 bar pressure; and (e) a total insulin capacity of 90 to 150 grams insulin/liter of polymer and a dynamic insulin capacity of 75 to 150 grams insulin/liter of polymer.
The present invention also provides a process for preparing a macroporous polymer comprising polymerizing zero to 50 percent monovinylaromatic monomer and 50 to 100 percent polyvinylaromatic monomer, in the presence of 100 to 170 percent of a porogen mixture comprising a hydrophobic porogen and a hydrophilic porogen, and 0.5 to 10 percent free radical polymerization initiator, in an aqueous suspension; wherein all percent amounts are based on total weight of monomer; and wherein: (a) the hydrophilic porogen is present in a weight ratio of greater than 1.2/1 up to 3/1 relative to the hydrophobic porogen; and (b) the hydrophilic porogen is selected from one or more (C
4
-C
10
)alkanol and the hydrophobic porogen is selected from one or more (C
7
-C
10
)aromatic hydrocarbon and (C
6
-C
12
)saturated hydrocarbon.
The present invention further provides a method for purifying aqueous solutions of mixed biomolecules, comprising contacting the aqueous solution with the aforementioned macroporous polymer in a liquid chromatography column having an internal diameter of 2 to 100 centimeters, wherein the column is operated at a pressure of 10 to 100 bar.
DETAILED DESCRIPTION
We have discovered that novel macroporous polymers useful for large scale separation and purification of biomolecules by high pressure liquid reverse phase chromatography can be prepared having selected porosity and permeability characteristics. In particular, we have discovered that using specific porogen solvents in specific proportions relative to the monomer phase under specific polymerization conditions unexpectedly provides the rigid polymer matrices of the present invention. The novel macroporous polymers may be used in high pressure RPC without significant compressibility and pressure buildup while maintaining good throughput and capacities (dynamic and equilibrium) for targeted biomolecules.
As used throu
Deissler Karl Chaplin
Kinzey Marlin Kenneth
Maikner John Joseph
Rosen Robert E.
Foelak Morton
Howell Thomas J.
Rohm and Haas Company
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