Patent Application
20080203029
The invention relates to the preparation and use of novel chromatographic media, preferably mixed mode polymeric chromatographic media for separation and purification of various biomolecules such as peptides, proteins and antibodies. More particularly, the present invention discloses novel chromatographic media, preferably mixed anion exchangers, mixed anion-cation exchangers and hydrophobic exchangers. The chromatographic media is prepared by modification of polymers with polyethyleneimine and functionalization thereof. It has been unexpectedly discovered that these mixed mode polymeric media offer enhanced separation capability and protein binding capacity.
The analysis of protein mixtures by ion exchange chromatography media is well documented, such as by, Pete Gagnon, “Purification Tools for Monoclonal Antibodies”, Validated Biosystems, Inc., (1996). More recently, the development of protein-based drugs and vaccines has increased the need for larger scale purification of protein mixtures. It is highly desirable that ion exchange chromatographic media be able to be utilized in this area.
One way of preparing such chromatography media materials, particularly anion exchangers containing primary and secondary amine functionality, is by coating the internal surface of porous silica materials with polyethyleneimine (PEI). For example, coating of the internal surface of silica particle with polyethyleneimine followed by immobilization through crosslinking has been disclosed in Alpert and Regnier, J. Chromatogr. 185, 375-392 (1979), and the use of materials prepared in that fashion for the chromatographic separation of synthetic oligonucleotides has been described in Lawson et al., Anal. Biochem. 133, 85-93 (1983).
Similarly, the preparation of PEI coated porous silica particles or controlled pore glass particles obtained by covalent binding of polyethyleneiminopropyl-trimethoxy silane has been disclosed in U.S. Pat. No. 4,540,486 of JT Baker Chemical Co. The same patent discloses the formation of chromatographic resins having mixed weak base/weak acid functionality by reaction of the PEI coated silica with cyclic carboxylic anhydrides. It has been shown that these materials, when used in a chromatographic column, can separate mixtures of cytochrome C, alfa1-acid glycoprotein, ovalbumin and beta-lactoglobulin (weak base media) or ovalbumin, cytochrome C, hemoglobin and lysozyme (mixed weak base/weak acid media). The efficacy of the PEI derivatized porous silica, or its carboxylated version in purifying immunoglobulin G has also been demonstrated in U.S. Pat. No. 4,606,825 of JT Baker Chemical Co.
The acylated form of the PEI coated silica described above can be further converted into a mixed weak acid/strong acid functionality media by the introduction of sulfonic groups, as disclosed in U.S. Pat. No. 4,721,573 of JT Baker Chemical Co. A column packed with this sulfonated media allows the separation of a mixture of cytochrome C, hemoglobin, lysozyme and ovalbumin, as well as the separation of the protein components of hybridoma cell culture medium.
Silica particles to which PEI has been covalently bound can also be converted to hydrophobic chromatographic media by reaction with monoacyl chlorides or linear carboxylic anhydrides where the acyl group can be a linear hydrocarbon chain, a phenyl group or a substituted phenyl group. A silica based weak base/reverse phase chromatographic media prepared in that fashion using butyryl chloride separates cytochrome C, myoglobin, lysozyme ovalbumin and alpha-chympotrypsinogen, as disclosed in U.S. Pat. No. 4,551,245 of JT Baker Chemical Co.
Agarose beads bearing ion exchange groups at the end of a polyvinyl alcohol (PVA) arm has been disclosed in WO 98/58732 to Amersham. However, unlike in the present invention, the PVA spacer arms do not contribute any ion exchange capacity to the product, nor does it in itself provide one of the functionalities in a mixed mode product.
One of the major disadvantages of silica based chromatographic packings is their lack of stability at high pH. This is particularly the case for applications involving the purification of drugs, since treatment of the equipment with 1N sodium hydroxide is a common sterilization practice.
The present invention provides chromatographic media that is able to minimize or avoid this lack of stability at high pH. The invention also provides mixed mode chromatographic media. It has been discovered that one possible way to avoid this instability problem is to use chromatographic packings based on a polymeric support derivatized on the surface of the polymer (i.e., not cross-linked) with PEI, which surface derivatized media can be further functionalized by reaction of a functionalization reagent with terminal amino groups of the polyethyleneimine on the surface of the polymeric resin. In accordance with this invention there preferably can be provided mixed mode media, such as for example, media with mixed primary, secondary and tertiary amine exchange sites, media with both weak anion and weak cation exchange sites, media with weak anion, weak cation and strong cation exchange sites, media with weak anion and hydrophobic (reverse phase) exchange sites, and media with weak anion and strong anion. Furthermore, it is unexpectedly discovered that such polyethyleneimine derivatized polymeric and functionalized derivatives thereof-based chromatographic media provide different and unique separation characteristics. In one aspect of the present invention there is provided polymeric chromatographic media for bioseparations. This invention differs from current polymeric chromatographic media by method of preparation and characteristics of the media as these current chromatographic media are simple ion exchangers. Also, the media of the present invention differs from current silica based media in terms of method of preparation, composition, use and performance. Surprisingly, polymeric mixed mode media prepared according to this invention have enhanced separation and binding capacities.
The present invention is illustrated by, but not limited by, the accompanying drawings in which:
a and 3b are graphs of the elution profile, as recorded by a UV detector, of the separation of proteins using media prepared according to Example 19 and silica media according to the procedure Example 37;
This invention concerns the preparation and use of novel polymeric chromatographic media and preferably mixed mode polymeric chromatographic media. In accordance with the present invention, polymeric media is prepared using polymeric particles derivatized with polyethyleneimine, and preferably such polyethyleneimine derivatized polymeric particles further functionalized with appropriate reactants.
Polymeric materials used for the chromatographic separation of proteins will preferably have certain properties, such as,
The polymeric resin particles to be surface derivatized with polyethyleneimine can be any suitable polymeric resin particles capable of being derivatized with polyethyleneimine and, with such polyethyleneimine derivatized polymers or further functionalized derivatives thereof being useful as chromatographic separation media. Examples of polymeric resin particle suitable for derivatization with polyethyleneimine in accordance with this invention include, but are not limited to cellulose, agarose, epoxidized or halogenated polystyrenes, epoxidized or halogenated polyacrylates or polymethacrylates, and epoxidized or halogenated polydivinylbenzenes. For examples, porous poly(meth)acrylates, highly cross-linked resins based on (meth)acrylic monomers bearing multiple polymerizable double bonds, such as ethylene glycol dimethacrylate (U.S. Pat. No. 4,118,347 to Showa Denko, pentaerythritol trimethacrylate (U.S. Pat. No. 4,256,842 to Toyo Soda), trimethylol propane trimethacrylate (U.S. Pat. No. 4,582,860 to Rohm and Haas) or glycerol dimethacrylate (U.S. Pat. No. 2,254,634 to Mitsubishi) prepared in the presence of a pore forming agent, have been shown to provide materials with the desired properties. Furthermore, the addition of functional monomers to the polymerization mixture affords final product with functional groups to which other molecules can be bound through the formation of covalent bonds. Example of such monomers are glycerol methacrylate (—OH groups) (U.S. Pat. No. 2,254,634 to Mitsubishi, 1993), dimethylaminoethyl methacrylate (tertiary amines) or glycidyl methacrylate (U.S. Pat. No. 4,118,347 to Showa Denko, U.S. Pat. No. 4,256,842 to Toyo Soda, U.S. Pat. No. 4,582,860 to Rohm and Haas). As examples of suitable functionalization reagents suitable for reaction with the PEI surface-derivatized polymeric resin particles are, for example, acid anhydrides such as cyclic carboxylic anhydrides such as glutaric and succinic anhydrides, unsaturated carboxylic anhydrides such as maleic anhydride, sulfonation agent such as bisulfites such as sodium meta-bisulfite, alkyl chlorides or anhydrides such as butryl chloride and acetic or butyric anhydride, and alkyl chlorides containing quaternary ammonium functionality such as (3-chloro-2-hydroxypropyl)trimethylammonium chloride, and mixtures of these functionalization reagents.
In one embodiment of this invention, an anion exchanger with mixed primary and secondary and tertiary amine sites is prepared by reacting polyethyleneimine with a polymeric support bearing epoxy or halo groups such as chloro, bromo, iodo groups. Such polymeric support can be any suitable synthetic polymer or natural polymer resin such as poly(meth)acrylate, cellulose, polystyrene-divinylbenzene, and agarose. Examples of such commercially available resins are Tosoh Biosciences Toyopearl AF-epoxy 650M, epoxy activated Sepharose 6B. These materials can be reacted with one of the terminal amino group of polyethyleneimine of various molecular weights through the formation of chemically stable alpha-hydroxy amino groups. Some of the properties of materials prepared in that manner, according to following examples 1 through 5, are summarized in Table 1.
In a second embodiment, mixed mode media with weak anion and weak cation exchange sites are prepared by reacting the PEI functionalized polymeric beads with cyclic carboxylic acid anhydrides, such as glutaric or succinic anhydride in the following examples 6 to 10.
In a third embodiment, mixed mode media having weak anion, weak cation and strong cation sites were prepared by reacting the PEI derivatized polymer with an unsaturated carboxylic acid anhydride followed by sulfonation, as described in examples 11 through 13.
In a fourth embodiment, mixed mode media having weak anion and hydrophobic (reverse phase) sites were prepared by reacting the PEI derivatized polymer with alkyl chloride (examples 14 through 18) or monobasic acids anhydrides Examples 19 through 24). Reactions with butyryl chloride were performed for 2 hr. at room temperature using either toluene or dioxane as the solvent. Triethylamine was used to scavenge the hydrochloric acid produced as a by-product of the reaction.
In a fifth embodiment, mixed mode media are obtained by reaction of the PEI coated resin with butyric anhydride conducted in 1-methoxy-2-propanol for 3 hr. at 60° C. as set forth in following Examples 20-22.
Mixed mode weak base/reverse phase resins where the hydrophobic moiety is a methyl group or a combination of methyl and butyl groups can be prepared by using acetic anhydride or sequential addition of butyric and acetic anhydride, as shown in Example 24 (acetic anhydride) or in Examples 20 and 23 (mixed anhydrides) respectively. As such, it is possible to vary and select proper hydrophobicicty by using one to a combination of reagents.
In a sixth embodiment, mixed mode media having weak anion, and strong anion (quaternary ammonium) sites were prepared by reacting the PEI derivatized polymer with alkyl chlorides containing terminal quaternary ammonium functionality. More particularly, the PEI derivatized polymer is reacted with (3-chloro-2-hydroxypropyl) trimethylammonium chloride in aqueous sodium hydroxide at 70-80° C. as shown in following Examples 25 to 30.
In accordance to the present invention, it has been unexpectedly discovered that these polymeric mixed mode chromatographic media offer enhanced separation capability as well as high capacity and stability. For example, the polymeric mixed mode anion exchange media can separate lysozyme, immunoglobulin G, bovine serum albumin, beta- lactoglobulin A and beta-lactoglobulin B while PEI-silica based media cannot separate all of these proteins as shown in
12 g epoxy bearing methacryate polymer with average particle size of 35 micron diameter and 250 ml dioxane are placed in a 1 L round bottom flask equipped with a funnel, agitator, reflux condenser and positive nitrogen pressure inlet and stirred for 30 min to allow the resin to swell. 20 g polyethyleneimine (PEI, average molecular weight 600 Daltons) is added and the funnel rinsed with an additional 150 ml dioxane. The stirred mixture is refluxed overnight. After allowing the mixture to cool, the mixture is transferred to a filter flask, drained, washed once with dioxane, three times with methanol and dried under vacuum at 60° C. Elemental analysis: 57.8% C, 7.4% H and 5.6% N.
5 g epoxy bearing methacryate polymer and 150 ml of dioxane are placed in a 500 ml round bottom flask equipped with an agitator and reflux condenser. 30 g of PEI (average molecular weight 1200 Daltons) are added and the mixture is refluxed overnight. After allowing to cool, the mixture is transferred to a filter flask, drained, washed twice with dioxane, three times with methanol and dried. Elemental analysis: 55.7% C, 7.8% H, 5.5% N
5 g epoxy bearing methacrylate polymer and 150 ml of a 50/50 mixture of dioxane and water are placed in a 500 ml round bottom flask equipped with an agitator and reflux. 30 g of PEI (average molecular weight 1200 Daltons) are added and the mixture is refluxed overnight (89° C.). After allowing to cool, the mixture is transferred to a filter flask, drained, washed twice with dioxane, three times with methanol and dried. Elemental analysis indicates that the final product contains: 57.5% C, 7.4% H, 2.5% N
5 g epoxy bearing methacrylate polymer and 100 ml of a 30 wt % solution of 10,000 daltons average molecular weight PEI in dioxane are placed in a 250 ml round bottom flask equipped with an agitator, nitrogen inlet and reflux condenser and the stirred mixture is refluxed overnight. After allowing to cool, the mixture is transferred to a filter flask, drained, washed three times with 60° C. water, three times with methanol and dried overnight in a vacuum oven at 60° C. Elemental analysis indicates that the final product contains: 56.5% C, 7.8% H, 6.4% N
5 g epoxy bearing methacrylic resin and 100 ml of a 30 wt % solution of 10,000 daltons average molecular weight PEI in water are placed in a 250 ml round bottom flask equipped with an agitator and reflux condenser and the stirred mixture is refluxed for 17½ hr. After allowing to cool, the mixture is transferred to a filter flask, drained, washed three times with water, three times with methanol and dried overnight in a vacuum oven at 60° C. Elemental analysis indicates that the final product contains 56.9% C, 7.5% H and 3.2% N.
As the results in Table 1 indicate, the presence of water in the system has a detrimental effect on nitrogen incorporation, while similar nitrogen contents are obtained when PEI of various molecular weight are reacted with the resin in dry is dioxane (albeit at a lower PEI to polymer ratio then in example 1).
As the results in Table 1 further indicate, the reactions performed in pure dioxane, as opposed to dioxane/water mixture or pure water, give a higher degree of functionalization, as indicated by the nitrogen content of the final product. When pure dioxane is used as the solvent (first row of Table 1), there does not appear to be a significantly higher level of nitrogen introduction as the molecular weight of the PEI is increased from 600 to 10,000 Daltons.
30 g PEI polymer prepared as in example 1 is washed with twice its volume of 100% ethanol and twice with its volume of 1-methoxy-2-propanol to remove any residual moisture. The polymer is then slurred in 450 ml 1-methoxy-2-propanol, transferred to a flask equipped with overhead agitator, reflux condenser and positive nitrogen pressure inlet, and heated to 60° C. After adding a solution of 19.43 g of 95% glutaric anhydride (1 eq, based on nitrogen content of the PEI resin) the temperature is maintained at 60 +/−2° C./ for 2½ hr. The reaction mixture is then transferred to a filter flask, drained, and the residual solid washed once with 100 ml of 1-methoxy-2-propanol, twice with 100 ml methanol and twice with 100 ml storage solution (ethanol:water 20:80 v/v, or 100 mM sodium acetate pH 4.5 with 2% benzyl alcohol). The product was stored in the respective storage solution for characterization and for chromatographic separation of proteins. Elemental analysis indicates that the final product contains: % C=55.1, % N=4.6.
73.7 g PEI coated polymer prepared as in example 1, 225 ml 1-methoxy-2-propanol and 14.4 g succinic anhydride are heated to 60 +/−2° C. and maintained at that temperature for 2½ hr. The reaction mixture is then transferred to a filter flask, drained, and the residual solid washed once with 100 ml of 1-methoxy-2-propanol, twice with 100 ml methanol and twice with 100 ml of storage buffer solution as described in example 5. Elemental analysis indicates that the final product contains: 53.08% C, 7.13% H and 4.86% N
The reaction was conducted as in Example 7 except the succinic anhydride charge is 17.34 g. Elemental analysis indicates that the final product contains: 53.18% C, 7.97% H and 5.18% N
11 g PEI coated polymer prepared as in Example 1 is washed with twice its volume of 100% ethanol and twice with its volume of dioxane to remove any residual moisture. The resin is then slurred in about 200 ml dioxane, transferred to a three flask equipped with a nitrogen inlet tube and heated to 50-60° C. After one hour, 7 g glutaric anhydride are added and the temperature maintained at 60 +/−2° C. for 2½ hr. The reaction mixture is then transferred to a filter flask, drained, and the residual solid washed three times with dioxane, three times with methanol and dried under vacuum. Elemental analysis indicates that the final product contains: 56.64% C, 7.54% H and 4.36% N.
The reaction was conducted as in Example 9 except 10 g PEI polymer prepared according to Example 1 and 6.6 g succinic anhydride are used. Elemental analysis indicates that the final product contains: 54.46% C, 7.96% H and 4.6% N.
86.5 g PEI polymer prepared according to Example 1 and 865 ml m are methoxy-2-propanol placed in a 2 Liter round bottom flask equipped with an agitator and reflux condenser and stirred for 30 min. 23.7 g maleic anhydride are added and the mixture stirred for 2.5 hr. at 60° C. After allowing the mixture to cool, the mixture is transferred to a filter flask, drained, washed once with methoxy-2-propanol, three times with water, three times with methanol. Elemental analysis indicates that the final product contains: % C 55.6, % N 4.9. % H 7.1, % S 0.
86.5 g of a resin maleated as in Example 11 is heated in 900 ml of 0.01 N sodium hydroxide in the presence of 190 g of sodium meta-bisulfite for 6 hr at 80 +/−2° C. After allowing the reaction mixture to cool, the mixture is transferred to a filter flask, drained, washed once with methoxy-2-propanol, three times with water, three times with methanol and stored in storage buffer for further use. Elemental analysis indicates that the final product contains: % C 49.8, % N 4.6. % H 6.8, % S 2.1.
The reaction was conducted as in Example 12 except the reaction is run for 20 hr at 80 +/−2° C. Elemental analysis indicates that the final product contains: % C 50.2, % N 4.5, % H 6.8, % S 2.1.
Comparison of the sulfur content of samples obtained after 8 hrs of reaction (3.8% S, Example 12) and 20 hrs. of reaction (3.40% S, example 13) indicates that the reaction is complete after 8 hrs.
30 g PEI functionalized prepared according to Example 1 are washed twice with 120 ml 100% ethanol and twice with 120 ml toluene to remove residual moisture. The material was then slurred in 300 ml of toluene, 9.29 g butyryl chloride (1 eq, based on nitrogen content of the resin) and 9.24 g of triethylamine were added and the reaction is allowed to proceed for 2 hr at 25 +/−2° C. The resin was then transferred to a filter flask and washed with 300 ml of the toluene, 300 ml of methanol, twice with 300 ml of DI water, three times with 300 ml methanol and twice with 300 ml of pH 5 storage buffer (10 mM sodium acetate, pH 4.5). Elemental analysis indicates that the final product contains: 53.7% C, 7.5% H and 5.6% N.
The reaction was conducted as in Example 14, except solvent is 450 ml dioxane. Elemental analysis indicates that the final product contains: 54.7% C, 8.0% H and 5.5% N.
The reaction was conducted as in Example 14, except 13.3 g butyryl chloride and 13.2 g triethylamine were used. Elemental analysis indicates that the final product contains: 53.6% C, 7.9% H and 4.5% N.
The reaction was conducted as in Example 14, except 19 g butyryl chloride and 18 g triethylamine were used. Elemental analysis indicates that the final product contains: 55.4% C, 7.8% H and 4.2% N.
The reaction was conducted as in Example 14, except 25.3 g butyryl chloride and 24 g triethylamine were used. Elemental analysis indicates that the final product contains: 55.0% C, 7.9% H and 4.5% N.
Some of the results obtained using toluene as the solvent are summarized in Table 3
As the results in Table 3 indicate, increasing amounts of reagent result in an increased level of functionalization (as indicated by the corresponding decrease in nitrogen content of the product).
60 g PEI functionalized polymer prepared as in Example 1 and swollen in 200 ml of 1-methoxy-2-propanol, 600 ml 1-methoxy-2-propanol, 56.3 g butyric anhydride (1.5 molar excess) and 36 g triethylamine are reacted for 3 hr at 60 +/−2 ° C. The reaction mixture is transferred to a filter flask, drained, washed once with 500 ml 1-methoxy-2-propanopl, once with 500 ml methanol, twice with 500 ml DI water and twice with 500 ml storage buffer. Elemental analysis indicates that the final product contains: 54.6% C, 7.9% H and 4.8% N.
60 g PEI functionalized polymer prepared as in Example 1 and swollen in 200 ml of 1-methoxy-2-propanol, 600 ml 1-methoxy-2-propanol, 6.2 g butyric anhydride and 4.0 g triethylamine are reacted for 3 hr at 60 +/−2° C. At that point, 24.2 g acetic anhydride were added and the reaction allowed to proceed at the same temperature for an additional 3 hr. The reaction mixture is transferred to a filter flask, drained, washed once with 500 ml 1-methoxy-2-propanopl, once with 500 ml methanol, twice with 500 ml DI water and twice with 500 ml storage buffer. Elemental analysis indicates that the final product contains: 53.7% C, 7.7% H and 5 4.7% N.
The reaction was conducted as in Example 19 except 6.26 g butyric anhydride and 4.0 g triethylamine are used. Elemental analysis indicates that the final product contains: 53.7% C, 7.9% H and 4.8% N.
The reaction was conducted as in Example 19 except 1.52 g butyric anhydride and 1.0 g triethylamine are used. Elemental analysis of the final product is is 54.6% C, 8.3% H and 5.0% N.
Some of the results are summarized in Table 4.
Comparison of the results in Tables 3 and 4 shows that levels of substitutions similar to those obtained with butyryl chloride can be achieved with butyric anhydride using considerably lower stoichiometric amounts of reagent.
The reaction was conducted as in Example 20 except 1.52 g butyric anhydride and 1.0 g. triethylamine and 10.7 g acetic anhydride are used. Elemental analysis indicates that the final product contains: 55.4% C, 7.9% H and 4.9% N.
60 g PEI functionalized polymer prepared as in Example 1 swollen in 20 ml of 1-methoxy-2-propanol, 500 ml 1-methoxy-2-propanol and 14.07 g acetic anhydride are reacted for 6 hr. at 60 +/−2° C. The reaction mixture is transferred to a filter flask, drained, washed once with 500 ml 1-methoxy-2-propanol, twice with 500 ml 0.1N NaOH, twice with 500 ml DI water and twice with 500 ml storage buffer. Elemental analysis indicates that the final product contains: 53.8% C, 7.5% H and 5.3% N.
80 g PEI functionalized polymer prepared as in Example 1 is reacted for 8 hr at 80° C. with a 59 g of a 60% solution of (3-chloro-2-hydroxypropyl) trimethyl ammonium chloride in 500 ml 0.5 N sodium hydroxide. The reaction mixture is transferred to a filter flask and washed twice with 0.1N sodium hydroxide, twice with DI water and once with storage buffer. After washing and drying, the resin has the following elemental analysis: 54.2% C, 8.4% H, and 5.9% N.
The reaction was conducted as in Example 25 except the sodium hydroxide solution is 0.05N. After washing and drying, the resin has the following elemental analysis: 51.9% C, 7.6% H, and 5.3% N.
The reaction was conducted as in Example 25 except the reaction is continued for 16 hr. After washing and drying, the resin has the following elemental analysis: 53.4% C, 8.4% H, and 5.7% N.
The reaction was conducted as in Example 26 except the reaction is continued for 16 hr. After washing and drying, the resin has the following elemental analysis: 52.8% C, 8.1% H, and 5.7% N.
The reaction was conducted as in Example 25 except the ratio of (3-chloro-2-hydroxypropyl) trimethyl ammonium chloride 60% solution to resin is 0.4 instead of 0.73. After washing and drying, the resin has the following elemental analysis: 53.72% C, 7.32% H, and 6.04% N.
The reaction was conducted as in Example 25 except the ratio of (3-chloro-2-hydroxypropyl) trimethyl ammonium chloride 60% solution to resin is 1.2 instead of 0.73. After washing and drying, the resin has the following elemental analysis: 53.72% C, 7.32% H, and 6.04% N.
160 g of PEI functionalized polymer prepared according to Example 1 is reacted for 5 hours@ 40° C. with 111.8 g of formaldehyde, 400 ml of acetonitrile and 25.9 g of sodium cyanoborohydride. After reaction, the polymer was washed with 480 ml of acetonitrile, 480 ml of DI water and 3 times 480 ml of methanol and stored in storage buffer for further use. (Elemental analysis % C=55.2, % N=5.8)
25 mg of polymer prepared according to Example 31 and 200 ml of acetonitrile was charged to an autoclave (Parr series 4500 bench mounted pressure reactor with Parr 4840 temperature controller). While stirring the polymer at 120 RPM, methyl chloride gas was charged and pressurized to 60 PSI. The reaction was continued for 5 hours at 80 C. After reaction, the product was washed with 60 ml actonitrile, 120 ml 0.1 N sodium hydroxide, 120 ml DI water, 240 ml of storage buffer and stored in storage buffer.
71 g of PEI modified polymer prepared according to Example 1 was mixed with 500 ml of 0.5N sodium hydroxide and reacted with 49 g of 3-(dimethylamino) propyl chloride while stirring at 50-100 RPM at 80° C. for 8 hours. After reaction, the product was washed with twice with 500 ml sodium hydroxide, twice with 500 ml DI water, twice with 500 ml of storage buffer and stored in the buffer for further use. Elemental analysis—% C: 55.4, % N: 6.2
A chromatographic media prepared as in Example 1 and PEI silica media from JT Baker (Product number 7264, lot N16084) packed in a 4.6×100 mm chromatographic column. 200 micro liters of a solution of 1 mg/ml lysozyme, 2 mg/ml rabbit immunoglobulin G, 2 mg/ml bovine serum albumin and 2 mg/ml each of beta-lactolobulin A and B in a 20 mM sodium acetate buffer at pH 6.2 is injected in the column and eluted using a flow rate of 1 ml/min and a 30 min gradient from 100% of 20 mM sodium acetate buffer at pH 6.2 to 100% 1.0M sodium acetate buffer at pH 6.2. Elution of the proteins is recorded by a UV detector at 280 nm (
A chromatographic media prepared as in Example 9 is packed in a 4.6×100 mm chromatographic column. 200 micro liters of a solution of 4 mg/ml BSA, 2 mg/ml Rabbit IgG, 2 mg/ml of cytochrome-c and 2 mg/ml solution of lysozyme in 20 mM sodium acetate buffer at pH 6.2 is injected in the column and eluted using a flow rate of 1 ml/min and a 40 min gradient from 100% of 20 mM sodium acetate buffer at pH 6.2 to 100% of the same eluent but containing 1 mole per liter of sodium acetate, pH 6.2. Elution of the proteins by the media of this Example and for a comparative silica based mixed mode media (JT Baker Product no. 7269) is recorded by a UV detector at 280 nm (
The capacity of chromatographic media prepared according to Example 9 and silica based products were measured and compared. A column (4.6 mm×50 mm) was packed with the media. IgG solution (1 mg/ml) in 20 mM sodium acetate at pH 5.6, 6.2 and 6.9 was applied to the column at varying linear velocity(cm/hr). The breakthrough was determined by monitoring UV at 280 nm. The breakthrough capacity of the media in the column was determined at 10% breakthrough (Table 7).
Chromatographic media prepared according to Example 19 and silica media (J T Baker product no. 7285) were packed in 7.75 mm×100 mm column. 200 ul solution of 2 mg/ml solution of Cytochrome C, ribonuclease, Lysozyme and Ovibumin in 25 mM sodium phosphate pH 7.0+1.7M ammonium sulfate was injected. The protein was eluted from the column by 60 minute linear gradient of 100% 25 mm sodium phosphate+1.7M ammonium sulfate to 100% of 25 mM sodium phosphate pH 7.0. The elution profile was monitored by UV @ 280 nm (
Chromatographic media prepared according to Example 20 was packed in 7.75 mm×100 mm column. 200 ul solution of 2 mg/ml solution of Cytochrome C, ribonuclease, Lysozyme and Ovibumin in 25 mM sodium phosphate pH 7.0+1.7M ammonium sulfate was injected. The protein was eluted from the column by 60 minute linear gradient of 100% 25 mm sodium phosphate+1.7M ammonium sulfate to 100% of 25 mM sodium phosphate pH 7.0. The elution profile was monitored by UV @ 280 nm (
Chromatographic media prepared according to Example 22 was packed in 7.75 mm×100 mm column. 200 ul solution of 2 mg/ml solution of Cytochrome C, ribonuclease, Lysozyme and Ovibumin in 25 mM sodium phosphate pH 7.0+1.7M ammonium sulfate was injected. The protein was eluted from the column by 60 minute linear gradient of 100% 25 mm sodium phosphate+1.7M ammonium sulfate to 100% of 25 mM sodium phosphate pH 7.0. The elution profile was monitored by UV @ 280 nm (
Chromatographic media prepared according to Example 23 was packed in 7.75 mm×100 mm column. 200 ul solution of 2 mg/ml solution of Cytochrome C, ribonuclease, Lysozyme and Ovalbumin in 25 mM sodium phosphate pH 7.0+1.7M ammonium sulfate was injected. The protein was eluted from the column by 60 minute linear gradient of 100% 25 mm sodium phosphate+1.7M ammonium sulfate to 100% of 25 mM sodium phosphate pH 7.0. The elution profile was monitored by UV @ 280 nm (
The polymer media prepared according to Example 7 was packed in 1.0 cm×10 cm column. First the column was equilibrated by passing 10 column volume of buffer A (0.05M MES, pH 5.6). After equilibration 0.5 ml of protein solution containing rabbit globulin (0.5 mg/ml) and lysozyme (0.25 mg/ml) was injected. The proteins were eluted from the column by running 40 minute linear gradient of 100% buffer A to 100% buffer B (1M Sodium chloride in buffer A). After the first protein separation run, the column was washed by circulating 500 ml of 10 mM phosphoric acid at 1 ml/min for 48 hours. After washing the protein separation was carried out again to determine the acid stability of the column (
The polymer media prepared according to Example 7 was packed in 1.0 cm×10 cm column. First the column was equilibrated by passing 10 column volume of buffer A (0.05M MES, pH 5.6). After equilibration 0.5 ml of protein solution containing rabbit globulin (0.5 mg/ml) and lysozyme (0.25 mg/ml) was injected. The proteins were eluted from the column by running 40 minute linear gradient of 100% buffer A to 100% buffer B (1M Sodium chloride in buffer A). After the first protein separation run, the column was washed by circulating 500 ml of 0.1 M sodium hydroxide at 1 ml/min for 24 and 48 hours. After washing the protein separation was carried out again to determine the base stability of the column (
The capacity of chromatographic media prepared according to Example 1, 25, 26, 27, 28, 29, 30 and 32 were measured and compared. A column (4.6 mm×50 mm) was packed with the media. BSA solution (1 mg/ml) in 20 mM CAPS (3-[cyclohexylamino]-1-1propane sulfonic acid) pH 11 was applied to the column at a flow rate of 1 ml/min. The breakthrough was determined by monitoring UV at 280 nm. The breakthrough capacity of the media in the column was determined at 10% breakthrough. After loading BSA on the columns, the columns were washed with CAPS buffer and then adsorbed BSA was eluted using 1M sodium chloride to calculate saturation capacity (Table 8).
The invention has been described herein with reference to the specific embodiments thereof. It will be appreciated that changes, modification and variations can be made without departing from the spirit and scope of the inventive concept disclosed herein. Accordingly, it is intended to embrace all such changes, modification and variations that fall with the spirit and scope of the appended claims.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US05/45710 | 12/16/2005 | WO | 00 | 7/6/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60646780 | Jan 2005 | US |
