EP0000977B1 - Aggregated polyelectrolytes, their preparation and uses in the fractionation of blood and other proteinaceous substances - Google Patents

Aggregated polyelectrolytes, their preparation and uses in the fractionation of blood and other proteinaceous substances Download PDF

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EP0000977B1
EP0000977B1 EP78300175A EP78300175A EP0000977B1 EP 0000977 B1 EP0000977 B1 EP 0000977B1 EP 78300175 A EP78300175 A EP 78300175A EP 78300175 A EP78300175 A EP 78300175A EP 0000977 B1 EP0000977 B1 EP 0000977B1
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polymer
amine
aggregated
imide
polyelectrolyte
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EP0000977A1 (en
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Joseph Edward Fields
Robert Jackson Slocombe
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Monsanto Co
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Monsanto Co
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/12—Powdering or granulating
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
    • C08F8/32—Introducing nitrogen atoms or nitrogen-containing groups by reaction with amines
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L35/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical, and containing at least one other carboxyl radical in the molecule, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Compositions of derivatives of such polymers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2335/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical, and containing at least one other carboxyl radical in the molecule, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Derivatives of such polymers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/827—Proteins from mammals or birds
    • Y10S530/829—Blood
    • Y10S530/83—Plasma; serum

Definitions

  • This invention relates to the production of improved polyelectrolytes which are useful in the fractionation of blood and other proteinaceous substances. More particularly, this invention relates to aggregated water-insoluble, cross-linked polyelectrolyte polymers having amine-imide functional groups.
  • U.S. Patent 3,554,985 describes the preparation of water-insoluble, cross-linked polyelectrolyte polymers having diloweralkylaminoloweralkylimide functional groups, wherein loweralkyl has 1 to 5 carbon atoms.
  • These polyelectrolytes have been found to be useful in the fractionation of blood plasma and serum as described in U.S. Patent 3,555,001 and for the separation of viruses from non-viral proteins as disclosed in U.S. Patents 3,655,509 and 3,846,543.
  • polyelectrolyte polymers also are useful for the immunization of animals against viral diseases as seen from U.S. Patent 3,651,213 and for the purification of water by the removal of contaminating bacteria and viruses as set forth in U.S. Patent 3,398,092.
  • the water-insoluble, cross-linked polyelectrolytes are further described as being copolymers of an (a) unsaturated monomer of 2 to 12 carbon atoms and (b) a monomer selected from the group consisting of (1) a mixture of an unsaturated polycarboxylic acid or anhydride and an unsaturated polycarboxylic acid amine-imide, and (2) an unsaturated polycarboxylic acid amine-imide.
  • the starting copolymer comprises the reaction product of styrene and maleic anhydride cross-linked with divinyl benzene (Example 1, Column 16, which is subsequently converted to the amine-imide derivative by reaction with dimethylaminopropylamine (Example 2, column 16).
  • a preformed polymer such as a copolymer of ethylene and maleic anhydride is cross-linked during the reaction with the dialkylaminoalkylamine by also employing in the reaction a predetermined amount of a difunctional compound such as ethylenediamine (column 12, lines 27-40).
  • the method of the present invention is one of producing a water-insoluble, cross-linked polyelectrolyte containing amine-imide or amine-imide salt functional groups and having protein adsorption capacity from a polymer comprising a copolymer of (a) an unsaturated monomer having from 2 to 18 carbon atoms and (b) a monomer selected from unsaturated polycarboxylic acids or anhydrides having from 4 to 12 carbon atoms, characterised by heating the said polymer in an inert organic solvent or solvent mixture at a temperature ranging from about 115°C. to about 160°C.
  • the said inert solvent or solvent mixture being an organic liquid medium having a boiling point of at least 115°C. at atmospheric pressure that substantially does not dissolve or react with the polymer under the conditons of the aggregation step, and thereafter cross-linking the polymer and substituting it with the major portion of the amine-imide or amine-imide salt functional groups.
  • the invention also comprises a water-insoluble, cross-linked polyelectrolyte that is a copolymer of (a) unsaturated monomer having from 2 to 18 carbon atoms and (b) a monomer selected from' unsaturated polycarboxylic acid or anhydride having from 4 to 12 carbon atoms, and in which from 2 to 100% of the reactive sites in said copolymer are substituted with amine-imide or amine-imide salt functional groups, said polyelectrolyte being characterised by being aggregated such that the major proportion thereof has a particle size ranging from 50 to 200 microns prior to cross-linking and substitution with the major portion of said amine-imide or amine-imide salt functional groups.
  • the invention also comprises the use of a polyelectrolyte according to the invention in the fractionation of blood or other proteinaceous substances.
  • polyelectrolytes of the general type described hereinbefore are significantly and substantially improved by an aggregation process whereby the protein adsorption capacity not only is unimpaired but, surprisingly, also is improved in certain blood fractionation systems.
  • the aggregation process comprises treatment of the preformed copolymer, prior to cross-linking and the addition of the functional amine-imide group, with refluxing xylene or other such inert organic solvents. This treatment is carried out at a temperature ranging from about 1 15°C. to about 160°C. but lower than the softening point of the polymer for at least about 15 minutes and until the polymer is substantially aggregated.
  • the product obtained by this treatment is an aggregated polymer which filters rapidly and in which the filter cake breaks apart so easily that ball milling is no longer necessary in most instances. Drying of the filtered material also is faster with the aggregated polymer than with the unaggregated polymer.
  • the protein adsorption capacity of the subsequently prepared cross-linked material containing the amine-imide functional group is substantially undiminished.
  • the albumin adsorption capacity of the aggregated material has been found to be more than three times that of the unaggregated material.
  • the properties of the aggregated polyelectrolyte in which grinding is unnecessary for obtaining suitable handling characteristics differ markedly from those of a ground, unaggregated polyelectrolyte. These differing products have non-equivalent particle structures. It has been found that the protein adsorption characteristics of these products involve both their chemical and physical properties.
  • the desired aggregated polyelectrolyte is prepared with due consideration of difference in the molecular structure of the external shell and the internal core of the particles. When the particles are reduced by grinding, the shell-core relationships are changed.
  • polyelectrolyte particles size reduction of polyelectrolyte particles is preferably avoided and (2) the desired structures for polyelectrolyte particles are achieved by synthesis sequences which develop the surface characteristics and basic core structure preferred for 'selective adsorption and elution of specific proteins.
  • aggregation relates to the overall particle structure and its requirements for protein fractionation as well as providing important process advantages.
  • the initial polymers which are aggregated in accordance with this invention include those disclosed in the aforementioned U.S. Patents 3,554,985 and 3,555,001, said patents being incorporated herein by reference.
  • the initial polymer comprises a copolymer of (a) unsaturated monomer having from 2 to about 18 carbon atoms and (b) a monomer selected from the group consisting of unsaturated polycarboxylic acids and anhydrides having from 4 to about 12 carbon atoms.
  • the aggregated polymer is cross-linked and substituted with an appropriate amine-imide group.
  • Suitable amine-imide groups include not only those specifically described in U.S. Patents 3,554,985 and 3,555,001, but also cyclic amine-imide groups as defined hereinbelow.
  • the desired aggregation process be carried out prior to the cross-linking and substitution with the major proportion of amine-imide groups. It has been found that when the cross-linking and/or the substitution with excessive amounts of the functional amine-imide group is carried out prior to the attempted aggregation process, the desired aggregation is not achieved and the advantages of the invention are not obtained. These results are surprising inasmuch as they are contrary to the expectation that the presence of the functional group would tend to make the polymer softer and thereby more readily susceptible to aggregation by simple particle fusion by the term "major proportion" is meant more than 50% of said groups.
  • the importance of carrying out the aggregation process prior to cross-linking and/or the addition of the major portion of the functional group may be due in part to a bridging reaction to form acylic anhydride groups between carboxyl groups on the backbones of different polymer molecules on adjacent particle surfaces.
  • This bridging differs from the usual anhydride formation by adjacent carboxyl groups on a backbone of a given polymer molecule.
  • the anhydride copolymers normally contain up to 2% moisture, and a portion of this reacts with anhydride groups to form carboxylic acid groups while the remainder is assumed to be present as free water. The latter is rather easily lost on drying, while the former is released by reforming either cyclic or acyclic anhydride groups. This is a slower process; however, it occurs readily under conditions that favour aggregation, e.g., refluxing xylene.
  • EMA-type polymers ethylene/maleic anhydride or acid
  • the EMA-type polymers have been described previously in U.S. Patents 3,554,985 and 3,555,001 and are further illustrated by the general examples in the following section I:
  • Co-monomers suitable for use with the above poly-carboxylic acid monomers include a-olefins, such as ethylene, 2-methyl-pentene-1, propylene, isobutylene, 1- or 2-butene, 1-hexene, 1-octene, 1- decene, 1-do-decene, 1-octadecene, and other vinyl monomers, such as styrene, a-methyl styrene, vinyltoluene, vinyl acetate, vinyl chloride, vinyl formate, vinyl alkyl ethers, e.g., methyl-vinyl-ether, alkyl acrylates, alkyl methacrylates, acrylamides and alkylacrylamides, or mixtures of these monomers. Reactivity of some functional groups in the copolymers resulting from some of these monomers permits formation of other useful functional groups in the formed copolymer, including hydroxy, lactone, amine and lactam groups.
  • any of the said carboxylic acids or derivatives may be copolymerized with any of the other monomers described above, and any other monomer which forms a copolymer with unsaturated carboxylic acids or derivatives.
  • these copolymers can be prepared by direct polymerization of the various monomers, frequently they are more easily prepared by an after-reaction modification of an existing copolymer. Copolymers are conveniently identified in terms of their monomeric constituents. The names so applied refer to the molecular structure and are not limited to the polymers prepared by the copolymerization of the specified monomers.
  • EMA-type carboxylic acid or anhydride-olefin polymers especially maleic acid or anhydride-olefin polymers of the foregoing type are known, for example, from U.S. Patents 2,378,629; 2,396,785, 3,157,595; and 3,340,680.
  • the copolymers are prepared by reacting ethylene or other unsaturated monomer, or mixtures thereof, with the acid anhydride in the presence of a peroxide catalyst in an aliphatic or aromatic hydrocarbon solvent for the monomers but nonsolvent for the interpolymer formed.
  • Suitable solvents include benzene, toluene, xylene, chlorinated benzene and the like.
  • the copolymer preferably contains substantially equimolar quantities of the olefin residue and the anhydride residue. Generally, it will have a degree of polymerization of about 8 to 100,000, preferably about 100 to 5,000, and a molecular weight of about 1,000 to 1,000,000, preferably about 10,000 to 500,000.
  • the properties of the polymer are regulated by suitable choice of the catalyst and control of one or more of the variables such as ratio of reactants, temperature, and catalyst concentration or the addition of regulating chain transfer agents, such as diisopropyl benzene, propionic acid, alkyl aldehydes, and the like. Numerous of these polymers are commercially available.
  • the aggregation of the foregoing EMA-type polymers and other such polymers as defined herein is carried out preferably by stirring the polymer as a suspension in refluxing or heated organic solvent which is inert to the polymer. This refluxing or heating is carried out at a temperature ranging from about 115°C. to about 160°C. but lower than the softening point of the polymer.
  • a preferred solvent is xylene.
  • Other solvents which can be used are, for example, ethylbenzene, mono- and dichlorobenzene and cumene. Solvents such as benzene and toluene having boiling points below about 115°C. are not practical for purposes of this invention.
  • Heating of the polymer in the refluxing solvent for at least about 15 minutes is desired, and good results have been obtained by heating up to about one hour. Heating for prolonged periods of time substantially in excess of about one hour is unnecessary, but the aggregates remain stable in weak solvents even during such prolonged heat treatment up to 7 hours.
  • the stronger solvents such as chlorobenzene and dichlorobenzene are less preferred solvents because of excessive coagulation that occurs on extended aggregation times.
  • the aggregated polymer is cross-linked and substituted with the desired amine-imide groups in whatever sequence optimizes the properties being sought by tailoring the distribution of specific groups within the particles.
  • These groups are essentially basic groups which can be aliphatic straight chain groups or can be alicyclic or aromatic groups.
  • the aliphatic straight chain groups preferably are diloweralkylaminoloweralkylimide groups or loweralkyliminodi-(loweralkylimide) linkages wherein loweralkyl has 1 to 5 carbon atoms as described previously in U.S. Patents 3,554,985 and 3,555,001.
  • Such products are further illustrated by the general examples in the following section II: //
  • the initial copolymers of anhydrides and another monomer can be converted to amine or amine salts including quaternary salts by reaction of the carboxyls of their anhydride precursors where applicable with polyfunctional amines such as dimethylaminopropylamine at higher temperatures forming an imide linkage with vicinal carboxyls. Such pendant free amine groups can then be converted, if desired, to their simple or quaternary salts.
  • Partial imides of a starting carboxyl or carboxylic acid anhydride containing polymer e.g., EMA, are produced by:
  • Partial secondary or tertiary aminoloweralkylamides of the starting carboxyl or carboxylic acid anhydride-containing polymer are obtained by contacting the polymer with a limiting amount of the selected amine in suspension in a solvent such as benzene or hexane, resulting in formation of a partial amide-acid anhydride derivative or the polymer, or a corresponding amide- carboxylate derivative thereof.
  • the number of amide groups is dependent upon the quantity of the amine used as compared with the quantity of polymer employed.
  • Such amide-polymer products typically comprise 2-100% amide groups, with remaining carboxyl groups being present as acid or anhydride groups.
  • Suitable blocking and unblocking of the amine moiety of the reactant employed in preparing amines or imides may be effected when required.
  • Residual, non-modified polymer units may optionally be converted to neutral groups or units by attachment to the polymer molecule of compounds including alkylamines, amino-alcohols and alcohols.
  • additional cationic character can be provided in the polymer through incorporation of monomers which impart a basic or cationic character such as C-vinyl pyridines, vinyl amine, the several amino-substituted vinyl benzenes (or toluenes and the like), amine-bearing acrylates (or methacrylates. and the like), vinyl imidazole and similar such monomers.
  • monomers which impart a basic or cationic character such as C-vinyl pyridines, vinyl amine, the several amino-substituted vinyl benzenes (or toluenes and the like), amine-bearing acrylates (or methacrylates. and the like), vinyl imidazole and similar such monomers.
  • the polymer product will have residual active or reactive groups which can be of various types, including mixtures, but these residual active or reactive groups or residual "reactive sites" in the polymer will in one way or another comprise a certain percentage which are of a basic nature, so as to impart the requisite basic nature to the polymer product.
  • Especially preferred polymers subject to the previously referred to requirements are selected from the group consisting of ethylene/maleic acid or anhydride copolymers, styrene/maleic acid or anhydride copolmers, and isobutylene/maleic acid or anhydride copolymers.
  • the preferred basic groups of the polycationic or polyampholytic polyelectrolyte (PE) employed are of an imide nature involving diloweralkylaminoloweralkylimide groupings, e.g., as produced by reacting a diloweralkylaminoloweralkylamine with the carboxyl groups of a pre-formed polymer. According to the invention, such groups are preferred for purposes of the invention.
  • imide groups can be provided by cross-linking the polymer with a loweralkyliminobis(loweralkylamine) which in the process of cross-linking by reaction between the terminal amine groups of the cross-linker and carboxyl groups in the polymer chain is productive of imido groups at both ends of the cross-linking chain with formation of the desired loweralkyliminobis(loweralkylimide) linkages.
  • a loweralkyliminobis(loweralkylamine) which in the process of cross-linking by reaction between the terminal amine groups of the cross-linker and carboxyl groups in the polymer chain is productive of imido groups at both ends of the cross-linking chain with formation of the desired loweralkyliminobis(loweralkylimide) linkages.
  • Other groups such as diloweralkylaminoloweralkylimide groups, from which the desired imide groups can be obtained by heating at elevated temperatures, can also be present.
  • diloweralkylaminoloweralkyl ester groups can be present, as well as other groups, so long as the imide groups of the prescribed type are also present in the polyelectrolyte molecule as well as the residual acid groups of the starting unsaturated acid or anhydride when the polyelectrolyte is a polyampholyte.
  • the imide groups need not necessarily be present in the polyelectrolyte as such, but can be present in the form of their salts, as already indicated.
  • Alicyclic or aromatic groups which can be substituted on the aggregated EMA-type polymers are for example, aminoloweralkyl-pyridine, piperidine, piperazine, picoline, pyrrolidine, morpholine and imidazole. These groups can be substituted on the aggregated polymer in a manner analogous to the aliphatic chain amines but by using, instead, cyclic amines such as, for example:
  • Figure 1 of the drawings shows a photomicrograph of the aggregated polymer prepared in Example 3 at a magnification of 200 X. A similar polymer was prepared as in this example but without the aggregation process.
  • Figure 2 of the drawings shows a photomicrograph of this unaggregated polymer prepared in Example 9 also at a magnification of 200 X. The striking differences in physical structure are readily apparent from these comparative photomicrographs.
  • the polymer aggregates are a multiplicity of small individual particles held together in clusters without fusion.
  • the major portion of the unaggregated polymer has a particle size ranging from about 0.1 microns to about 10 microns whereas the major portion of the aggregated polymer has a particle size ranging from about 50 to about 200 microns.
  • a 5-liter reaction flask, equipped with reflux condenser, Dean-Stark water take-off, stirrer, reagent addition vessel, thermometer and nitrogen-purge attachments is charged with 193.05 g. ethylene/maleic anhydride copolymer (EMA) (1.5 moles, anhydride basis) and 2700 ml. xylene.
  • EMA ethylene/maleic anhydride copolymer
  • the charge is stirred at 200 r.p.m. with a 6.5 inch (0.165 metre) blade-type stirrer while it is heated to the reflux temperature.
  • This reflux temperature will vary from 135 to 139°C. depending on the water content of the EMA and upon whether this water is azeotropically removed during the ensuing reflux period.
  • the slurry was maintained at total reflux for 60 minutes under total reflux return at a temperature of 135°C.
  • the reactor was cooled to 125°C. under nitrogen and a solution mixture of 10.89 g. MIBPA (0.075 mole) and 1.5 ml. water was added.
  • the mixture was heated to reflux (134°C.) and maintained at reflux for 1 hour while continuously removing water azeotrop (final temperature was 137°C.).
  • the reaction mixture was again lowered to 125°C. under nitrogen and a solution mixture of 153.3 g. DMAPA (1.5 moles) and 4.5 ml. water was added.
  • the slurry was then heated to 133°C.
  • the above slurry was filtered hot and the product cake was reslurried in 2700 ml. of a 3:1 mixture of xylene and ethanol, stirred at reflux temperature for one hour and then filtered hot. This was repeated a second time for a two hour period and a third time for a three hour refluxing period, in each case followed by hot filtration.
  • the resulting extracted product cake was then reslurried in 2700 ml. hexane for 1 hour at room temperature and filtered. The hexane extraction was repeated a total of four times.
  • the final product was airdried for 30 minutes and finally dried in a vacuum oven at 55°C.
  • the final reaction slurry was filtered hot and the product cake reslurried at reflux in 3:1 xylene-alcohol three times, as above, followed by two 1-hour room temperature extractions with 2700 ml. acetone.
  • the filtered product was converted to the hydrochloride by reslurrying in either 2700 ml. alcohol or acetone and gradually adding with stirring (over 10 min.) 112 ml. conc. (12N) hydrochloric acid and stirring at room temperature for two hours.
  • the filtered product was subsequently washed (slurry with stirring) three consecutive times with 10 lites of water (deionized) for 2 hours each time and finally filtered.
  • the filtered salt cake was reslurried four times in 2700 ml. acetone (1 hour each time) to remove the water, filtered, air dried for 30 minutes and vacuum oven dried at 55°C.
  • the final dried product either as free amine or as salt, was screened without grinding with 95% of the product going through a 100 mesh screen before bottling for use.
  • the aggregated diethylaminoethyl derivative was prepared using the identical procedure of Example 1 except that 174.32 g. DEAEA (1.5 mole) was substituted for the DMAPA in Example 1.
  • the final product was obtained as the free amine form using the work-up procedure of Example 1 wherein the reaction product was consecutively extracted with three 3:1 xylene-alcohol extractions followed by four hexane extractions.
  • the product was sieved unground through a 100 mesh screen to yield 229 g. of material finer than 100 mesh and 13.0 g. coarser than 100 mesh.
  • This example utilized the same equipment, the same aggregation procedure and the same initial charge (EMA and xylene) as described in Example 1.
  • the slurry temperature was lowered to 125°C. and 10.89 g. (0.075 moles) MIBPA was added.
  • the slurry was stirred at 120-125°C for one hour without reflux.
  • 7.66 g. (0.075 moles) DMAPA was added and the slurry was again stirred at 120-125°C. for one hour without reflux.
  • the slurry was heated to reflux and the total water of the condensation reaction was removed by distillation as the azeotrope.
  • the final temperature was 139°C.
  • the reaction mixture was then cooled to 120°C, 87.05 g. of HOEtA was added, and the slurry maintained at 120° for 1 hour. The temperature was then raised to reflux and the water from this final condensation reaction completely removed over a 6 hour period by distillation as the azeotrope. The final temperature was 140°C.
  • the product was worked up as the free amine as described in Example 1 for free amine work-up procedure. 230 g. of product was obtained which passed through a 100 mesh screen unground; 17 g. of product was retained on the screen.
  • Example 3 The same amounts of amines and other raw materials of Example 3 were used. The procedure was identical through the aggregation step. After cooling the aggregated slurry to 125°C., 7.66 g. of DMAPA was added and the slurry was held at 120-125°C. for one hour. Then 10.89 g. MIBPA was added and the slurry was again held at 120-125°C. for one hour. From this point on the procedure was exactly the same as described in Example 3. The final product was worked-up as the free amine form.
  • Example 4 The identical procedure of Example 4 was repeated except that the final product was worked-up as the hydrochloric acid salt by the procedure described in Example 1. For this purpose only 14 ml. concentrated hydrochloric acid (12N) was used instead of the 112 ml. used in Example 1. After drying, 240 grams of product was obtained.
  • Example 3 The procedure of Example 3 was repeated except that water of the condensation reaction was removed by azeotropic distillation after each of the amine reactions and holding times, i.e., after MIBPA, after DMAPA and after HOEtA reaction instead of as in Example 3.
  • the product was obtained as the hydrochloride salt in 240 g. yield.
  • Example 1 The same equipment and the same initial charge of EMA and xylene was used as in Example 1. Aggregation, as obtained in Example 1, was precluded by one of two methods: (a) heat slurry of EMA at 200 r.p.m. to 90°C and add 10.89 g. MIBPA plus 1.5 ml. water, continue stirring at 90°C. for one hour, raise temperature to reflux (136°C.) and take off total water of reaction in the Dean-Stark trap continued reflux (final temperature 139°C.); or (b) heat slurry of EMA at 200 r.p.m. to 125°C. and add the MIBPA and water and immediately raise to reflux temperature of 136°C.
  • the filtered product was worked up as either the free amine or as the hydrochloride salt by procedures described in Example 1. Again, during work-up, filtering times were long (30 minutes to 2 hours) as contrasted to work-up filtering times associated with aggregated products of Examples 1 through 7 where these times varied from 5 to 10 minutes. Finally, non-aggregated products, prepared by this procedure and others to follow, dried poorly and had to be ground or ball-milled prior to sieving through a 100 mesh screen in contrast to aggregated products from Examples 1 through 7 which required no grinding or ball-milling prior to screening through 100 mesh screens after drying.
  • This example utilized the same equipment and the same EMA and xylene charge as in Example 8.
  • the slurry was heated to 90-95°C. and 10.89 g. (0.075 mole) MIBPA was added and stirred at 95°C. for 1 hour.
  • 7.66 g. (0.0075 mole) DMAPA was added and stirred at 95°C. for 1 hour.
  • the slurry was heated to reflux (134°C.) and water of reaction was completely removed by azeotropic distillation to a final temperature of 139°C.
  • the slurry was then cooled to 95°C. and 87.05 g. of hydroxy- ethylamine was added and the slurry stirred at 95°C. for 1 hour.
  • the slurry temperature was then raised to 134°C. and the total water of reaction was completely removed by azeotropic distillation to a final temperature of 139 to 140°C.
  • the final slurry was filtered hot (30 minutes) and worked up as the free amine by the procedure of Example 1, dried, ground by extensive ball milling and screened through a 100 mesh screen.
  • the recovered yields over 12 runs varied from 219 to 244 grams depending on the ball-milling efficiency prior to screening.
  • Example 8 This procedure was the same as Example 8 except that the water of reaction was not removed after addtion of MIBPA but was allowed to remain in the reaction slurry until after the DMAPA addition and then the total water of reaction from both amine reactions was removed in a single final azeotropic distillation. Final slurry temperature was 140°C. The product was worked-up as the free amine.
  • Example 2 This procedure was identical to that of Example 1 except that the water of reaction was not removed after addition of MIBPA, following aggregation, but was allowed to remain in the reaction mixture slurry until after the DMAPA addition and then the total water of reaction from both amine reactions was removed in a single final azeotropic distillation. The finaly slurry was filtered hot in less than 5 minutes and the product was worked up as the free amine by the procedure of Example 1.
  • Example 8 A series of comparable runs were made using the procedure of Example 1 wherein the aggregation time and stirring speed were varied. The products were all finished as the free amine following the Example 1 procedures. The results are shown in the following table as compared with a non-aggregated product prepared by Example 8.
  • a one-liter flask was used and the charge was 700 ml. solvent and 50 g. of EMA.
  • a 20 ml. aliquot of the slurry was removed and placed in vials. After cooling, the vials were shaken and the time for settling of the polymer from the solvent was measured with a stop watch as an indication of aggregate development. The results are recorded below.
  • a measure of particles size in dispersion, whether aggregated or non-aggregated, is the swelling index defined as the grams of aqueous or other dispersant which is absorbed at equilibrium per gram of polymer derivative.
  • a suitable sized sample is dispersed in excess dispersant and adjusted to pH 4 or any other desired pH value. The dispersion is allowed to reach equilibrium over a 1 hour period and is then centrifuged at 750 x g. for thirty minutes in a preweighed centrifuge bottle. The supernate is decanted and the weight of centrifuged swollen gel is determined. All of the values are reported using 0.04 Molar saline as the dispersant and a pH of 4.0. The swelling index number is thus the weight of 0.04M saline absorbed by one gram of polymer.
  • Swelling is known to be inversely proportional to the crosslink density for crosslinked insoluble resins. For a series of derivatives with increasing MIBPA, the swelling decreases as expected, all other parameters being equal.
  • Protein adsorption capacity was measured by the following method. 40 mg. human albumin and 10 mg. of polymer product in the amine or salt form were dispersed in 1.0 ml. of 0.04 molar saline and the pH adjusted to 7.0. The slurry was shaken for 30 minutes while keeping the pH at 7.0. After the 30 minute adsorption period the resin-albumin complex was centrifuged while saving the supernate. The solids were washed 3 times with 1.0 ml. water (5-min. shaking, centrifuging) and the combined supernates were assayed for protein by the method of Miller-Lowry, Analytical Chemistry, 31, 964 (1959). The albumin capacity values are given in terms of mg. albumin adsorbed per mg. polymer product.
  • Example 14 The preparations listed in Example 14 are summarized in the following table wherein their albumin capacity is given as a result of preparation variation.
  • Aggregated products of the type described not only have improved filtration characteristics during synthesis processes but have been found to give high flow characteristics during processes of plasma fractionation.
  • non-aggregated polymers used for plasma fractionation by adsorbing desired proteins from plasma solutions had to be separated from the mother liquor by centrifugation processes because of immediate clogging of filter papers and cloths.
  • the present aggregated products with their improved and stable filtration character, were able to be used in plasma fractionation processes and separated from mother liquors by conventional vacuum filtration processes with fast filter times and little or no filter plugging in the presence of proteinaceous material, thus avoiding the need for expensive centrifugation-type apparatus.
  • Flow rates of 0.04 molar saline were measured for both aggregated and non-aggregated polyelectrolyte types.
  • the expressed value of "relative flow rate” is given in cubic cm. per hour per unit volume of resin bed in the column under gravity flow and maintaining a 20 cm. saline head on top of the resin during the test.
  • the DMAPA used in several of the previous Examples illustrates an example of a dialkylaminoalkylimide substituent on the polyelectrolyte.
  • Another such substituent was the DEAEA(diethylaminoethylamine) used in Examples 2 and 7 (run 3). These two amines were used to represent aggregated dialkylaminoalkylimide polyelectrolyte substitution with 5 mole percent MIBPA in a 5/90 composition and with 5 mole percent HMDA and 85 mole percent HOEtA in a 5/5/85 composition.
  • non-aggregated dialkylaminoalkylimide preparations were made using the Example 9 procedure for the 5/5/85 compositions and the Example 8 procedure for the 5/90 compositions except that in no case was water added with any of the amines.
  • the amines used were dimethylaminoethylamine, diethylaminoethylamine, diethylaminopropylamine, dimethylaminopropylamine, di-n-butylaminopropylamine, di-hydroxyethylaminopropylamine and 2 - amino - 5 - diethyl- aminopentane.
  • N-phenylethylenediamine was also used to prepare the above non-aggregated resins.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
  • Emulsifying, Dispersing, Foam-Producing Or Wetting Agents (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • External Artificial Organs (AREA)
EP78300175A 1977-07-25 1978-07-21 Aggregated polyelectrolytes, their preparation and uses in the fractionation of blood and other proteinaceous substances Expired EP0000977B1 (en)

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US05/818,919 US4118554A (en) 1977-07-25 1977-07-25 Aggregated polyelectrolytes
US818919 1997-03-17

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EP0000977A1 EP0000977A1 (en) 1979-03-07
EP0000977B1 true EP0000977B1 (en) 1981-09-02

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EP (1) EP0000977B1 (hu)
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IL (1) IL55193A (hu)
IT (1) IT1097313B (hu)
PT (1) PT68335B (hu)
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US4411795A (en) * 1980-03-10 1983-10-25 Baxter Travenol Laboratories, Inc. Particle adsorption
US6986902B1 (en) 1998-04-28 2006-01-17 Inex Pharmaceuticals Corporation Polyanionic polymers which enhance fusogenicity
AU3764099A (en) * 1998-04-28 1999-11-16 Tao Chen Polyanionic polymers which enhance fusogenicity
US6740633B2 (en) * 2000-05-09 2004-05-25 Basf Aktiengesellschaft Polyelectrolyte complexes and a method for production thereof
JP2003535438A (ja) * 2000-05-09 2003-11-25 ビーエーエスエフ アクチェンゲゼルシャフト 高分子電解質複合体およびその製造法

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US3157595A (en) * 1959-09-16 1964-11-17 Monsanto Co Clarification of water with copolymers containing half-amides of olefinic anhydrides
US3554985A (en) * 1963-01-02 1971-01-12 Monsanto Co Cross-linked copolymer polyelectrolytes based on alpha,beta-ethylenically unsaturated acids
GB1057827A (en) * 1963-01-02 1967-02-08 Monsanto Co Purification of water and air
US3340680A (en) * 1966-02-01 1967-09-12 Monsanto Co Air purification process
US3651213A (en) * 1969-05-29 1972-03-21 Monsanto Co Method for the immunization of a living animal body against viral disease
US3655509A (en) * 1969-05-29 1972-04-11 Monsanto Co Process for the separation of virus from non-viral proteins
US3555001A (en) * 1969-05-29 1971-01-12 Monsanto Co Process for the fractionation of plasma and serum using water-insoluble polyelectrolytes containing diloweralkylaminoloweralkylimide groups

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IL55193A (en) 1981-06-29
JPS6330325B2 (hu) 1988-06-17
CA1129145A (en) 1982-08-03
RO85538B (ro) 1984-11-30
ES471955A1 (es) 1979-10-16
IL55193A0 (en) 1978-09-29
DE2861005D1 (en) 1981-11-26
RO85538A (ro) 1984-10-31
PT68335A (en) 1978-08-01
BR7804723A (pt) 1979-04-03
HU180881B (en) 1983-05-30
ATA531878A (de) 1981-06-15
US4118554A (en) 1978-10-03
SU795490A3 (ru) 1981-01-07
AU520096B2 (en) 1982-01-14
EP0000977A1 (en) 1979-03-07
PT68335B (pt) 1994-02-25
IT7826004A0 (it) 1978-07-21
AU3824178A (en) 1980-01-24
AT365612B (de) 1982-02-10
JPS5460392A (en) 1979-05-15
IT1097313B (it) 1985-08-31

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