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, can 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 Resin, 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 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, So 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 (C4-C10)alkanol and the hydrophobic porogen is selected from one or more (C7-C10)aromatic hydrocarbon and (C6-C12)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.
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 throughout the specification, the following terms shall have the following meanings, unless the context clearly indicates otherwise.
The term “alkyl (meth)acrylate” refers to either the corresponding acrylate or methacrylate ester; similarly, the term “(meth)acrylic” refers to either acrylic or methacrylic acid and the corresponding derivatives, such as esters or amides. All percentages referred to will be expressed in weight percent (%), based on total weight of polymer or composition involved, unless specified otherwise. The term “copolymer” refers to polymer compositions containing units of two or more different monomers, including positional isomers. The following abbreviations are used herein: g=grams; ppm=parts per million by weight/volume, cm=centimeter, mm=millimeter, ml=milliliter, L=liter. Unless otherwise specified, ranges listed are to be read as inclusive and combinable and temperatures are in degrees centigrade (°C).
The macroporous polymers useful as RPC stationary phases have increased structural rigidity compared to existing materials, making the polymers suitable for use in commercial-scale manufacturing processes. Increased structural rigidity has been achieved by modification of the structure of polymer matrix by using selected polymerization conditions.
Modifying the polymer matrix porosity is important in preparing the macroporous polymers of the present invention. To be effective in separating large biomolecules, a stationary phase preferably has an open, porous structure that enables rapid diffusion of molecules into and out of the matrix. In addition, a high level of porosity affords a large surface area, which in turn provides a high capacity of the matrix for the target molecule. Most modern, commercial polymeric RPC stationary phases appear to be designed around these criteria, and are used under lower pressure conditions (typically, from 1 bar up to less than 10 bar and preferably from 1 to 5 bar; 1 bar pressure=105 Pascal or 105 Pa). However, at higher pressure conditions (typically from 10 bar to 100 bar) these matrices are compressible. The macroporous polymers of the present invention have increased polymer rigidity, and at the same time have preserved high capacity and rapid intraparticle diffusion for target molecules.
The macroporous polymers of the present invention are useful for purifying biomolecule mixtures dissolved in aqueous solutions by contacting the solution with the macroporous polymer in a liquid chromatography column having internal diameters of 2 to 100, preferably from 5 to 80 and more preferably from 10 to 50 centimeters, where the column is operated at a pressure of 10 to 100 and preferably from 20 to 80 bar. Typically, preparative scale RPC is performed in 10 to 50 centimeter chromatography columns at 20 to 80 bar pressures.
Selected polymerization conditions represent an important factor in preparing macroporous polymers of the present invention. Selected ratios of mixed porogen relative to the monomer phase, as well as the ratio of hydrophilic porogen relative to hydophobic porogen, are the key parameters believed to provide the macroporous polymers of the present invention.
While not wishing to be bound by theory, we believe that, in the case of the present invention, the polymer matrix density is altered to allow more solid polymer per unit volume of total polymer, thereby increasing matrix rigidity, with a consequently increased capacity for target molecules. The particular selection of the amount of porogen relative to monomer and the balance of hydrophobic versus hydrophilic porogen types is believed to affect the pore size distribution in a manner favorable to binding of target molecules (in this case, biomolecules) while at the same time providing improved matrix rigidity.
Crosslinked macroporous copolymers of the present invention are typically spherical copolymer beads having average particle size diameters from 2 to 600 microns (μm). Polymers useful for the separation and purification of biomolecules via high performance reverse phase liquid chromatography (such as in columns from 2 to 100 cm in diameter) typically have average particle size diameters from 2 to 150, preferably from 5 to 100, more preferably from 10 to 75 and most preferably from 10 to 30 μm. Polymers useful for the separation and isolation of biomolecules via large scale adsorption processes (such as in columns up to several meters in diameter or in fermentation broths) typically have average particle size diameters from greater than 150 up to 600, preferably from 200 to 500 and more preferably from 250 to 400 μm.
The macroporous polymers of the present invention are typically produced by suspension polymerization, and possess surface areas from 200 to 1500, preferably from 300 to 1200 and more preferably from 400 to 1000 square meters per gram (m2/g). The macroporous polymers are preferably those of the type described in U.S. Pat. No. 4,382,124, for example, in which porosity is introduced into the copolymer beads by suspension-polymerization in the presence of a porogen (also known as “phase extender” or “precipitant”), that is, a solvent for the monomer but a non-solvent for the polymer. Conventional macroporous polymers, such as those prepared according to U.S. Pat. No. 4,382,124, typically encompass the use of a wide range of porogen types, porogen concentrations relative to the monomer phase, monomer types, crosslinking monomer types, crosslinker levels, polymerization initiators and initiator concentrations. The present invention, however, is based on the discovery that macroporous polymers prepared using certain selected porogen types, used in specific concentrations relative to the monomer phase, with specific monomers and selected levels of crosslinking, together with selected polymerization initiator concentrations, have unexpectedly rigid polymer structures corresponding to improved performance in the separation and purification of biomolecules via high performance reverse phase liquid chromatography.
Suitable polyvinylaromatic monomers that may be used in the preparation of the macroporous polymers useful in the present invention include, for example, one or more monomer selected from divinylbenzene, trivinylbenzene, divinyltoluene, divinylnaphthalene, divinylanthracene and divinylxylene; it is understood that any of the various positional isomers of each of the aforementioned crosslinkers is suitable; preferably the polyvinylaromatic monomer is divinylbenzene. Typically the macroporous polymer comprises 50 to 100%, preferably 65 to 100% and more preferably 75 to 100% polyvinylaromatic monomer units.
Optionally, aliphatic crosslinking monomers, such as ethyleneglycol diacrylate, ethyleneglycol dimethacrylate, trimethylolpropane triacrylate, X trimethylolpropane trimethacrylate, glycidyl methacrylate, diethyleneglycol divinyl ether and trivinylcyclohexane, may also be used in addition to the polyvinylaromatic crosslinker. When used, the aliphatic crosslinking monomers typically comprise as polymerized units, from zero to 20%, preferably from zero to 10%, and more preferably from zero to 5% of the macroporous polymer, based on the total monomer weight used to form the macroporous copolymer.
Suitable monounsaturated vinylaromatic monomers that may be used in the preparation of the macroporous copolymers useful in the present invention include, for example, styrene, α-methylstyrene, (C1-C4)alkyl-substituted styrenes, halo-substituted styrenes (such as dibromostyrene and tribromostyrene), vinylnaphthalene and vinylanthracene; preferably the monounsaturated vinylaromatic monomer is selected from one or more of styrene and (C1-C4)alkyl-substituted styrenes. Included among the suitable (C1-C4)alkyl-substituted styrenes are, for example, ethylvinylbenzenes, vinyltoluenes, diethylstyrenes, ethylmethylstyrenes and dimethylstyrenes; it is understood that any of the various positional isomers of each of the aforementioned vinylaromatic monomers is suitable; preferably the monounsaturated vinylaromatic monomer is ethylvinylbenzene. Typically, the macroporous polymer comprises zero to 50%, preferably zero to 35% and more preferably zero to 25%, monounsaturated vinylaromatic monomer units.
Optionally, non-aromatic vinyl monomers, such as aliphatic unsaturated monomers, for example, vinyl chloride, acrylonitrile, (meth)acrylic acids and alkyl esters of (meth)acrylic acids (alkyl (meth)acrylates) may also be used in addition to the vinylaromatic monomer. When used, the non-aromatic vinyl monomers typically comprise as polymerized units, from zero to 20%, preferably from zero to 10%, and more preferably from zero to 5% of the macroporous copolymer, based on the total monomer weight used to form the macroporous copolymer.
Preferred macroporous polymers are selected from one or more of divinylbenzene copolymer, styrene-divinylbenzene copolymer, divinylbenzene-ethylvinylbenzene copolymer and styrene-ethylvinylbenzene-divinylbenzene copolymer; more preferable are divinylbenzene-ethylvinylbenzene and styrene-ethylvinylbenzene-divinylbenzene polymers.
Porogens useful for preparing the macroporous polymers of the present invention include hydrophobic porogens, such as (C7-C10)aromatic hydrocarbons and (C6-C12)saturated hydrocarbons; and hydrophilic porogens, such as (C4-C10)alkanols and polyalkylene glycols. Suitable (C7-C10)aromatic hydro-carbons include, for example, one or more of toluene, ethylbenzene, ortho-xylene, meta-xylene and para-xylene; it is understood that any of the various positional isomers of each of the aforementioned hydrocarbons is suitable. Preferably the aromatic hydrocarbon is toluene or xylene or a mixture of xylenes or a mixture of toluene and xylene. Suitable (C6-C12)saturated hydrocarbons include, for example, one or more of hexane, heptane and isooctane; preferably, the saturated hydrocarbon is isooctane. Suitable (C4-C10)alkanols include, for example, one or more of isobutyl alcohol, tert-amyl alcohol, n-amyl alcohol, isoamyl alcohol, methyl isobutyl carbinol (4-methyl-2-pentanol), hexanols and octanols; preferably, the alkanol is selected from one or more (C5-C8)alkanols, such as, methyl isobutyl carbinol and octanol. Preferably, the porogen mixture comprises a hydrophilic porogen selected from one or more (C5-C8)alkanol and a hydrophobic porogen selected from one or more (C7-C10)aromatic hydrocarbon.
Typically, the total amount of porogen used to prepare the polymers of the present invention is from 100 to 170%, preferably from 110 to 160%, more preferably from 115 to 150% and most preferably from 120 to 140%, based on weight of the monomers. At porogen levels above 170%, the polymers have poor flow resistance values (high compressibility) at high pressure conditions in packed columns; at porogen levels below 100%, the polymers have poor chromatographic properties (as measured by insulin capacity in column flow-through tests). In addition, the porogens used to prepare the polymers of the present invention are based on a mixed solvent system, comprising a hydrophobic solvent (“hydrophobic” porogen) and a less hydrophobic solvent (“hydrophilic” porogen). It is understood that the hydrophilic porogen has some limited water solubility (for example, 0.5 to 5%) and is more water soluble than the hydrophobic porogen (typical water Solubility of 10 to 100 ppm, or less).
Typically, the ratio of hydrophilic porogen to hydrophobic porogen is from greater than 1.2/1 up to 3/1, preferably from 1.3/1 to 2.7/1, more preferably from 1.4/1 to 2.5/1 and most preferably from 1.6/1 to 2.4/1. At hydrophilic/hydrophobic porogen ratios of about 1.2/1 and lower, the polymers having acceptable flow resistance performance also have decreased chromatographic performance (measured by restricted mass transfer access in the insulin capacity test). At hydrophilic/hydrophobic porogen ratios above 3/1, the polymers would have decreased overall capacity performance (measured by decreased amounts of insulin sorbed during the column flow test). Typically, the mixed porogens comprise a (C7-C10)aromatic hydrocarbon and a (C4-C10)alkanol; preferably, the mixed porogens comprise xylenes and methyl isobutyl carbinol.
Polymerization initiators useful in preparing polymers of the present invention include monomer-soluble initiators such as peroxides, hydroperoxides and related initiators; for example benzoyl peroxide, tert-butyl hydroperoxide, cumene peroxide, tetralin peroxide, acetyl peroxide, caproyl peroxide, tert-butyl peroctoate (also known as tert-butylperoxy-2-ethylhexanoate), tert-amyl peroctoate, tert-butyl perbenzoate, tert-butyl diperphthalate, dicyclohexyl peroxydicarbonate, di(4-tert-butylcyclohexyl)peroxydicarbonate and methyl ethyl ketone peroxide. Also useful are azo initiators such as azodiisobutyronitrile, azodiisobutyramide, 2,2′-azo-bis(2,4-dimethylvaleronitrile), azo- bis(α-methyl-butyronitrile) and dimethyl-, diethyl- or dibutyl azo-bis(methylvalerate). Preferred peroxide initiators are diacyl peroxides, such as benzoyl peroxide, and peroxyesters, such as tert-butyl peroctoate and tert-butyl perbenzoate; more preferably, the initiator is benzoyl peroxide. Suitable use levels of peroxide initiator are 0.5% to 10%, preferably from 1 to 9%, more preferably from 2 to 7% and most preferably from 3 to 5%, based on the total weight of vinyl monomers. Most preferably, the free radical initiator is present at 2 to 7 percent, based on total weight of monomer, and is selected from one or more diacyl peroxide and peroxyester.
Dispersants and suspending agents useful for preparing the macroporous polymers of the present invention are nonionic surfactants having a hydroxyalkylcellulose backbone, a hydrophobic alkyl side chain containing from 1 to 24 carbon atoms, and an average of from 1 to 8, preferably from 1 to 5, ethylene oxide groups substituting each repeating unit of the hydroxyalkyl-cellulose backbone, the alkyl side chains being present at a level of 0.1 to 10 alkyl groups per 100 repeating units in the hydroxyalkylcellulose backbone. The alkyl group in the hydroxyalkylcellulose may contain from 1 to 24 carbons, and may be linear, branched or cyclic. More preferred is a hydroxyethylcellulose containing from 0.1 to 10 (C16)alkyl side chains per 100 anhydroglucose units and from about 2.5 to 4 ethylene oxide groups substituting each anhydroglucose unit. Typical use levels of dispersants are from about 0.01 to about 4%, based upon the total aqueous-phase weight.
Other dispersants and suspending agents useful for making the macroporous polymers of the present invention are polymers containing hydrophilic backbones, which can orient their lipophilic portions to the monomer phase and their hydrophilic portions to the aqueous phase at the interface of the two phases. These polymeric dispersants include celluloses, polyvinyl pyrrolidones, polyvinyl alcohols, starches and the like. Mixtures of dispersants may also be used. These other dispersants tend to be less preferred, as they tend to produce a somewhat greater amount of agglomerated or otherwise undesirable material.
A typical macroporous copolymer preparation, for example, may include preparation of a continuous aqueous phase solution containing suspension aids (such as dispersants, protective colloids and buffers) followed by mixing with a monomer mixture containing 50 to 100% polyvinylaromatic monomer, free-radical initiator and 1 to 1.7 parts mixed porogen (hydrophobic and hydrophilic porogen) per one part monomer mixture. The monomer/mixed porogen combination is then polymerized at elevated temperature (typically at 40 to 120° C., preferably 60 to 100° C.; for 1 to 20 hours, preferably 3 to 15 hours, for example) and the porogens are subsequently removed from the resulting polymer beads by various means; for example, toluene, xylene and (C4-C10)alcohols may be removed by distillation or solvent washing, and polyalkylene glycols by water washing. The resulting macroporous copolymer is then isolated by conventional means, such as dewatering followed by drying.
Optionally, the preparation of the macroporous polymers may include an enzyme treatment to cleanse the polymer surface of residues of dispersants and suspending agents used during the polymerization. The enzyme treatment typically involves contacting the macroporous polymer with the enzymatic material (selected from one or more of cellulose-decomposing enzyme and proteolytic enzyme) during polymerization, following polymerization or after isolation of the polymer. Japanese Patent Applications No. 61-141704 and No. 57-98504 may be consulted for further general and specific details on the use of enzymes during the preparation of polymer resins. Suitable enzymes include, for example, cellulose-decomposing enzymes, such as β-1,4-glucan-4-glucano-hydrase, β-1,4-glucan-4-glucanhydrolase, β-1,4-glucan-4-glucohydrase and β-1,4-glucan-4-cellobiohydrase, for cellulose-based dispersant systems; and proteolytic enzymes, such as urokinase, elastase and enterokinase, for gelatin-based dispersant systems. Typically, the amount of enzyme used relative to the polymer is from 2 to 35%, preferably from 5 to 25% and more preferably from 10 to 20%, based on total weight of polymer.
The macroporous polymers of the present invention are especially useful in packed chromatography column applications where porosity and mechanical strength of the polymer allows for high performance separation and purification of biomolecules at high throughput rates without pressure buildup on prolonged use.
Optionally, the macroporous polymers may be coated or post-functionalized with various conventional ionizable functional groups (weak-acid functional group, such as a carboxylic acid group; weak-base functional group, such as a primary, secondary or tertiary amine functional group; strong-acid functional group, such as sulfonic acid group; strong base functional group, such as quaternary ammonium chloride or hydroxide group) by known methods, such as conventional sulfonation, chloromethylation and amination.
The macroporous polymers of the present invention are characterized by improved permeability (low flow resistance) that is the result of the enhanced rigid polymer structure and the selected porosity introduced into the polymer during polymerization. The permeability (K) is related to the backpressure generated in a column through Darcy's Law (Equation 1):
ΔP=μV/[K(d p)2] Equation 1
μ=viscosity (milliPascal·second or centipoise)
V linear velocity (cm/hr)
ΔP=pressure drop (bars)
dp=mean particle size of the polymer (microns)
The units of the above variables are expressed in their common form; it is understood that unit conversion is required to render Equation 1 dimensionless. The more rigid (that is, less compressible) the polymer, the greater the permeability of the polymer, translating to lower backpressure for any given combination of solvent viscosity, linear velocity and particle size. Under laminar flow conditions, which are typical for chromatographic separation and purification applications, the backpressure in a column can also be expressed by the Carman-Kozeny Equation (Equation 2):
ΔP=150·[(1−ε)2/ε3 ]·μV/(d p)2 Equation 2
ε=interparticle void volume (cm3/cm3)
References, such as Fundamentals of Preparative and Nonlinear Chromatography, G. Guiochon, S. Goshan Shirazi and A. Katti; Academic Press (1994) and Unit Operations in Chemical Engineering, W. L. McCabe, J. C. Smith and P. Harriott; McGraw Hill (1985), may be consulted for further general and specific details on Darcy's Law and the Carman-Kozeny Equation (Equations 1 and 2).
By combining Equations 1 and 2, it can be seen that permeability (or flow resistance) in the chromatography column is related to the interparticle void volume of the polymer resin bed (that is, the volume between polymer particles); ε is expressed as volume of voids per unit volume of polymer bed. This relationship is expressed by Equation 3:
1/K=150·[(1−ε)2/ε3] Equation 3
For the purposes of the present invention, we define the characteristic “flow resistance” value of a polymer to be the inverse of the permeability. The characteristics “flow resistance” value is an indication of how well the polymer will perform under medium to high pressure conditions: low flow resistance values represent low compressibility and high flow resistance values represent poor compressibility.
Additionally, according to Darcy's Law, expressions for either the permeability or the flow resistance commonly include the particle size effect (dp in Equation 1). One objective of the present invention is to provide improved flow resistance via increased polymer rigidity, independent of particle size effects. It is understood that reduced particle size alone would, for a given polymer, generate higher backpressures as given by Equations 1 and 2.
Typically, macroporous polymers of the present invention have flow resistance values (that is, 1/K) from 700 to less than 1,800, preferably from 700 to less than 1,500 and more preferably less than 1,300 at operating pressures of 10 bar (medium pressure). At high pressure operation (represented by 60 bar), the macroporous polymers have flow resistance values from 1,500 to less than 7,000, preferably from 1,500 to less than 5,000 and more preferably less than 4,500. Macroporous polymers suitable for use in RPC have flow resistance values of (i) less than 1,800 at a pressure of 10 bar pressure and (ii) less than 7,000 at 60 bar pressure. Polymers having flow resistance values greater than the limits indicated above do not provide sufficient resistance to compression at the medium to high pressures found in commercial RPC columns and consequently suffer from reduced throughput and column pressure buildup during operation.
The macroporous polymers of the present invention are characterized by selected porosities and pore size distributions produced by the porogen types and ratios used to prepare the polymers. Porosities were determined using a Micromeretics™ ASAP-2400 nitrogen Porosimeter. Porosities according to IUPAC nomenclature are as follows:
Microporosity=pores less than 20 Ångstrom units
Mesoporosity=pores between 20 and 500 Ångstrom units
Macroporosity=pores greater than 500 Ångstrom units
For the purposes of the present invention, “operational” microporosity is defined as pores having a diameter of less than 50 Ångstrom units and “operational” mesoporosity is defined as pores having diameters between 50 and 500 Ångstrom units. The slight difference between “operational” porosity, as used herein, and porosity defined according to IUPAC nomenclature is due to the fact that 50 Ångstrom units is a more suitable and appropriate cutoff point (compared to 20 Ångstrom units) in order to accommodate the sorption of biomolecules of interest in the macroporous polymers of the present invention.
The macroporous polymers of the present invention typically have a total porosity of 0.7 to 2, preferably from 0.9 to 1.8 and more preferably from 1.0 to 1.7 cm3/g. Typically, the macroporous polymers have an operational mesoporosity of 0.7 to 1.9, preferably from 0.8 to 1.7 and more preferably 0.9 to 1.4 cm3/g. Typically, the macroporous polymers have an operational microporosity from zero to 0.5, preferably from zero to 0.3, more preferably from zero to 0.2 and most preferably from zero to less than 0.1 cm3/g. Typically, the macroporous polymers have a macroporosity from zero to 0.6, preferably from zero to 0.5 and more preferably from zero to 0.3 cm3/g. Macroporosity values above about 0.6 cm3/g decrease the working capacity of the polymer for biomolecules of the targeted molecular size and shape, in terms of total capacity.
Insulin capacity is used as an indicator of the capability of a polymer matrix as a suitable medium for large scale separation and purification of biomolecules of similar size and molecular configuration. The macroporous polymers of the present invention typically have a dynamic insulin capacity of 60 to 150 g/L, preferably from 70 to 150 g/L and more preferably from 75 to 150 g/L. Typically, the macroporous polymers have a total insulin capacity of 75 to 150 g/L, preferably from 80 to 150 g/L and more preferably from 90 to 150 g/L. Dynamic capacity is a measure of how quickly the polymer matrix is able to take up the biomolecule and is defined as the insulin capacity at the breakthrough point where 1% leakage (relative to total insulin sorbed on the polymer) occurs. Insulin capacity is defined in g/L, that is, grams insulin/liter of polymer resin. Macroporous polymers of the present invention suitable for use in high performance, large scale preparative chromatography of biomolecules, typically have a combination of dynamic and total insulin capacity values of 60 and 75 g/L, respectively; preferably, 70 and 80 g/L, respectively; and more preferably 75 and 90 g/L, respectively.
The total capacity of the polymer resin for a given biomolecule (for example, insulin) is significant because it is related to the mass of the particular molecule that can be loaded on the column during the purification procedure. The primary economic factors important to the purification process (column throughput, solvent use, labor or time cycle) are directly related to the quantity of the mass loaded onto the column.
The dynamic capacity of the polymer resin is important since it is related to the mass transfer efficiency of the polymer for the particular molecule, and it dictates the time-scale under which the purification can occur. A low dynamic capacity indicates that the polymer matrix is not suitable for high-speed purification processes.
Some embodiments of the invention are described in detail in the following Examples. All ratios, parts and percentages are expressed by weight unless lo otherwise specified, and all reagents used are of good commercial quality unless otherwise specified. Abbreviations used in the Examples and Tables are listed below with the corresponding descriptions:
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| ||MIBC = ||methyl isobutyl carbinol (4-methyl-2-pentanol) |
| ||DVB = ||divinylbenzene (mixture of meta/para isomers) |
| ||EVB = ||ethylvinylbenzene (mixture of meta/para isomers) |
| ||BPO = ||benzoyl peroxide |
| ||rpm = ||revolutions per minute |
| ||v/v = ||volume/volume |
| ||w/v = ||weight/volume |
| ||μm = ||micron |
| ||nm = ||nanometer |
| ||g/L = ||grams/Liter |
| ||cm3/g = ||cubic centimeter per gram |
| ||μl = ||microliter |
| ||NA = ||not analyzed |
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