CA1292952C - Separation or purification of biomaterials with particulate polymeric adsorbents - Google Patents

Abstract

- i -PATENT APPLICATION OF

John O. Naples and Eric G. Isacoff and Michael J. Byers for Separation Or Purification of Biomaterials With Particulate Polymeric Adsorbents DN85-42A JET/ja Abstract of the Disclosure A biomaterial contained in an impure liquid medium is separated or purified by contacting the medium with a particulate polymeric adsorbent which preferentially and reversibly binds the biomaterial to form a complex, isolating the complex by passing the medium through a membrane filter permeable to impurities but not to the complex, and then liberating the biomaterial from the isolated complex into another liquid medium, which may be membrane-filtered as before, to accumulate the biomaterial but not the particles in the permeate.

Description

SEPARATION OR PURIFICATION OF BIOMATERIALS
WITH PARTICULATE POLYMERIC ADSORBENTS

Technical Field This invention relates to methods for the S separation and/or purification of biomaterials by membrane filtration, and more particularly i5 directed to such methods wherein a biomaterial is adsorbed on a carrier material preliminary to the membrane filtration.

Background of the Invention Ultrafiltration is a method widely used for the separation (including concentration) and/or purification of biomaterials. One such method is described by Geahel and Kula in Biotechnolo~y Letters, Vol. 6(8), 481-6, 1984. In that method, a ~olution of a dehydrogenace is purified by fir~t binding it to a water-soluble dextran of high molecular weight and then 12~Z~Z

subjecting the solution to ultrafiltration. The dehydrogenase-dextran complex is too large to permeate the ultrafiltration membrane, but the impurities in the solution are not, with the result that the complex is retained by the filter and the impurities pass into the permeate. When the impurities have thus been removed from the solution, the dehydrogenase is liberated (desorbed) from the dextran by increasing the ionic strength of the solution and is recovered.
This method has two basic disadvantages. First, the dextran is soluble in the medium, and it may therefore slip through the pores of the ultrafiltration membrane because of its elongated molecular structure.
A membrane with smaller pores could be a remedy: this would reduce the number of dextran molecules slipping through the pores, but would also limit the size of impurity molecules that could be separated from the dextran-product complex, théreby reducing the efficiency of the separation. Secondly, the high molecular weight dextran complex tends to accumulate on the filter membrane to form a secondary membrane or concentration polarization layer, which restricts flow through the filter.
U. S. Patent 4,474,690 and European Patent Application 127,737 filed March 21, 1985 describe lZ~?Z~2 recovery of peptide-containing compounds by combined affinity-chromatographic purification and ultrafiltration wherein a ligand specific to the peptide and bound to a macromolecular solid carrier is S complexed with the peptide and the complex in solution is passed through a membrane filter leaving impurities in the permeate. The peptide is then split off from the ligand and carrier. Carrier materials include synthetic latex polymers of the acrylic derivative type. The technique is complicated by the requirement of a peptide specific ligand and therefore has limited applicability.

5ummary o the Invention It has now been found that the foregoing lS disadvantages and limitations can be overcome or minimizad by using a microsized particulate polymeric adsorbent as the principal complexing agent to selectively adsorb from a mixture a biomaterial which it is desired to purify or to separate from other biomaterials. When this mixture is subjected to membrane filtration, the adsorbent-biomaterial complex, because it is insoluble and because its particles are larger than the membrane filter pores, is retained by the filter, but the impurities and/or other lZ~tZ~Z

biomaterials pass through the filter into the permeate, where they can be collected and removed. The adsorbed biomaterial can then be liberated rom the adsorbent into the medium passed through the filter and S recovered, while the regenerated adsorbent is retained by the filter and can be reused.
The invention thus provides a new form of chromatographic separation or purification of biomaterials, particularly with respect to proteinaceous materials. In contrast to standard chromatography, the microsized polymeric adsorbents flow like liquids and ars not packed in a column as are conventional ad~orbent~. Therefore, there ~ no problem re~ulting from weight o adsorbent particles:
there is no column to collapse of it~ own welght nor wlll packing occur, causing re~tricted flow and undesirable pressure drop. Con~equently, the adsorbents can carry biomaterial easily through the lumens of the fine hollow fibers of ultrafiltration cartridges (where impurities or non-adsorbed biomaterials pas~ into the permeate) for recirculation to and concentratlon of effluent in an adsorption ve~sel. The ad~orbent i9 thu~ indefinitely reu~able over many cycle~.
The present invention, therefore, provides a method of separating or purifying a biomaterial contained in a first liquid medium with other components selected from 12~952 _ 4(a) ~

impurities, other biomaterials and mixtures of said impurities and other biomaterials, which comprises:
(A) contacting the first liguid medium with a particulate polymeric adsorbent capable of preferentially adsorbing a biomaterial therefrom, the adsorbent having an average particle size in the range of 0.01 to 5 microns and selected from the group consisting of non-porous ion exchange resin bearing up to about 1.5 functional groups per monomer unit and solid, uncharged, non-porous polymer particles not yet functionalized with ion exchange functional groups, whereby the adsorbent reversibly binds the biomaterial to form a complex; and ~B) subjecting the medium containlng the complex and said other components to membrane filtration wherein the membrane is permeable to the said other components but not to the complex, whereby the complex and said other components are separated.

f~

Detailed Description As used in this specification, the term "bio-material" means any water soluble or water dispersible bio-affecting substance, whether of biological or non-S biological origin. Biomaterials include compounds, molecules, polymers, particles and simple as well as complex substances which exist in nature or which may be synthesized by biological or non-biological processes. The term thus includes proteinaceous substances ~such as albumins and enzymes~, amino acids, nucleic acids, peptides and polypeptides, antibodies, antigens, steroids, hormones, antibiotics, vitamins, polymeric carbohydrates, and the like, which reveraibly complex with or otherwi3e bind to or are adsorbed or carried by the particulate polymeric adsorbents. The binding may be due to electrostatic attraction, hydrophilic/hydrophobic interactions caused by van der Waals forces, specific affinity, or any combination thereof.
Most often, the biomaterials are produced in liquid media, either by chemical synthesis or by fermentation, or a combination of these methods, and are separated as solutions or fine disper~ions from cell, cellular debris or other ~olids of a ~ermentation broth or cell culture medium. In any case, the -6- 12~2952 resulting biomaterial-containing medium is generally accompanied by impurities and/or other biomaterials which must ultimately be separated if the biomaterial is to meet commercial or medical standards of purity.

For convenience and clarity of understanding, the term "bioproduct" is sometimes used in this specification to identify the biomaterial resulting from the purification or separation process of the invention.
The polymeric adsorbents used in the method of the invention are particulate, of such size that the particles will not permeate the filter membrane but will allow good flow through hollow ~ibers. For most application~, the particles will have diameters in the range of 0.01-5 micrometers, preerably 0.1-1.5 micrometers. The particles are ~ubstantially water-insoluble; consequently, the adsorbents and adsorbent-biomaterial complex will not pass through the pores of a membrane filter nor form a polarization layer on the ~ membrane.

Any particulate polymeric adsorbent meeting the foregoing standards can be used if it is also capable of selectively adsorbing a bioproduct but not the impurities or other biomaterials in the medium, and _7_ 1Z929S2 will not denature the bioproduct, or alternatively if it is capable of adsorbing the impurities or other biomaterials but not adsorbing or denaturing the bioproduct. The adsorbent particles may be charged or S uncharged. A charged state may be achieved by the presence of functional groups which modify the hydrophobic/hydrophilic character of an adsorbent relative to an aqueous system containing a biomaterial.
Typical of a charged adsorbent is a particulate ion exchange resin wherein the ion exchange functionality imparts a surface charge to a polymer. Typical uncharged polymers are ion exchange resin precursors, i.e., a crosslinked or uncrosslinked solid polymer not yet functionalized.
If the particulate polymeric adsorbent is a charged material, the mechanism by which a biomaterial is bound into the complex may primarily be electro-static attraction. If the adsorbent is uncharged, and even to some extent when the adsorbent i~ charged, the binding mechanism may be understood in terms of one or more of hydrophobic/hydrophilic attraction, specific affinity intaraction, and other effects.
Preferably, the particulate polymeric adsorbent is characterized by the presence of ionogenic groups and comprises an ion exchange resin of micrometer or submicrometer particle size, carrying a charge opposite the charge on the bioproduct. As ion exchange resins, the adsorbents may comprise single resins containing anion exchange functionality, single resins containing S both anionic and cationic functionality (amphoteric resins), or may be mixed, hybrid, chelating or composite resins having anionic or both anionic and cationic exchange character. Furthermore, both gel and maeroretieular resins are useful.
Typieal of the ion exehange resins useful in the invention are the maeroretieular vinyl aromatie or aerylie resin adsorbents and exehangers deseribed in U. S. Patents 3,037,052; 3,637,535; 3,843,566;
3,791,86~; 3,275,548 and 3,357,158; the hybrid resins deseribed in U. S. Patent 3,991,017; the eomposite resins deseribed in U- S. Patent 3,645,922; the amphoterie resin~ de-~eribed in U. S. Patent 4,202,737;
and others deseribed in Ion Exehanqe, J. Marinsky, ed., Vol. II, Chap. 6 (New York, 1969).
5peeifie adsorbents are homopolymers and eopolymers formed from vinylidene monomers sueh as aerylie and methaerylie aeids and esters, and other monoethylenieally unsaturated monomers or mixtures thereof, such as monoeyelie and polycyclie aromatic monomers, e.g., styrene and substituted styrenes, and lZ92952 the like. The monoethylenically unsaturated monomers may be polymerized without crosslinking or may be crossl~nked ln situ with a polyethylenically unsaturated monomer such as a polyvinyl aromatic S hydrocarbon (divinylbenzene, divinyltoluene, etc.), a glycol dimethacrylate such as ethylene glycol dimethacrylate, or a polyvinyl ether of a polyhydric alcohol, such as divinoxyethane and trivinoxypropane.
The polymeric adsorbents are prepared in a conventional manner including bulk, solution, su~pension and emulsion polymer~zation. If the polymerlzat~on process i5 an emulsion polymerization, the des~red small particle ~lze range can be obtained directly, as shown ~n U. S. Patents 4,359,537 and 4,380,590 to Chong, U. S. Patent 4,200,695 to Chong, I~acoff and Neely, and U. S. Patent 4,537,683 to Isacoff and Neely. If the polymerization is suspension or other form, the particulate product polymers can be reduced in size by grinding techniques well known in the art. Irregularly shaped particles (e.g., ground resins) are assumed, for purposes of this invention, to have longest dimensions with the diameter limitations set forth above.

Water-insoluble, uncrosslinked or partially functionalized materials may also be suitable for use in the invention. Eor instance, ion exchange polymers functionalized with an ionogenic group near the particle surface, e.g., a monolayer of ion exchange groups about the periphery of the bead, are useful.
Lightly crosslinked or surface-crosslinked beads having low water solubility are also effective.
The adsorbents of the foregoing Chong, Chong et al. and Isacoff et al. patents are, generally speaking, ion exchange resins composed of crosslinked polymers in the shape of spherical beads, bearing up to about 1.5 unctional groups per monomer unit, e.g., 0.1 to 1.5.
These groups can be strongly acidic (e.g., -HSO3 groups), weakly acidic (e.g., -COOH groups), strongly basic (e.g., quaternary ammonium groups), or weakly baRic (e.g., tertiary amine groups).
As indicated, the preferred polymeric adsorbents are ion exchange resins, the selection of which in any given situation depends primarily on the electrical charge of the biomaterial to be purified or separated. If the charge of the biomaterial i8 negative, a positively charged (basic) resin is used;
if the charge is positive, a negatively charged (acidic) resin is used. In either case, the selection can be easily made by one skilled in this art using the princlples of ion exchange chemistry.
The amount of polymeric adsorbent used must be enough to adsorb most of the bioproduct in the system, but not so much that the medium becomes viscous and difficult to handle and filter. In most cases, the adsorbent is used in an amount about equivalent to the weight of bioproduct in the medium, for example about 0.01 to lO wt.% in a medium containing the same weight of bioproduct.
The process of the invention is carried out by first removing most of the particulate matter from the bioproduct-containing medium by centrifugation, conventional filtration or other solid material separation technique, if the medium contains such solids. The adsorbent of choice, in the proper amount, is then added to the filtrate or to a liquid medium containing the biomaterial and the other components to be separated from the biomaterial. The resulting mixture is then stirred for a time sufficient to permit adsorption of the bioproduct to the adsorbent particles, ordinarily a matter of several minutes. The mixture is then passed through a filtration membrane, preferably by recirculating it through a module of the hollow fiber type. The membrane is selected so that it -12- lZ9Zgsz passes impurities, biomaterials other than bioproduct and non-adsorbed bioproduct, but not the adsorbent particles carrying bioproduct. Typically, the membrane filters are those semipermeable membranes known in the art for their ability to remove dissolved or dispersed matter by ultrafiltration or microfiltration, but excluding separation of dissolved salts by the technique known as reverse osmosis.
The adsorbent-bioproduct complex is retained by the filter, and the permeate containing impurities or other biomaterials is collected and removed from the unit. The bioproduct is then liberated from the adsorbent by known desorption methods. Depending on the nature o the bioproduct, this is achieved by changing the pH of the medium, by changing the electrolyte balance, or by any other suitable method known in affinity chromatography or related fields.
The medium containing desorbed bioproduct is then passed through the same membrane filter and collected. Regenerated absorbent is retained and can - be reused.
Those skilled in this art will be able to efficiently practice the invention after referring to the following illustra~ive examples. These artisans will be able to compose numerous variations on the themes disclo~ed, such as changing the amounts of ingredients slightly but insignificantly from those ~hown, adding innocuous substances, or substituting equivalent or nearly equivalent components for those shown. All these variations are considered to be part of the inventive concept.
In the examples, all parts and percentages are by weight unless indicated otherwise.

Example 1 This example demon~trates the efficacy of a negatively charged adsorbent for separating a po~itively charged biomaterlal (Cytochrome C) from a negatively charged biomaterial ~bovine serum albumin).

lS (A) Adsorbent Loading:
Bovine serum albumin ("~SA"), 200 mg, was di~solved in 200 ml of 0.01M potassium phosphate buffer in filtered, deionized water (~PPB solution"). The re~ulting solutlon was then filtered through a 0.22 micrometer Millipore filter. Total volume in the system at thi~ po~nt was 175 ml. One-half gram of strong acid, emul~ion polymerized, styrene * Trademark A~

divinylbenzene gellular copolymer t7.3~ crosslinker) resin beads of 0.26 i 0.02 micrometer average diameter and cation exchange capacity ~ 5.1 meq/g dry, was then added to i95 ml of the BSA/PPB ~olution. To the resulting suspension was added 146.2 mg of Cytochrome C, Type III.

(B) Ultrafiltration:
The BSA and Cytochrome C preparations were then mixed and circulated for 15 minutes through the membrane of an Amicon HlMPO1-43 hollow fiber ultrafiltration system, cartridge height 20 cm in which the fiber~ have 1.1 mm I.D, w~th 0.1 mlcrometer pore diameter and a 280 cm~ effectlve ~urface area. The permeate was collected and removed. The retained resin suspension wa~ diafiltered for five volume replacements with PPB solution and then reconstituted to a volume of 200 ml. Analy~l~ showed that 97.4 mg of the Cytochrome C remained on the resin and that 48.8 mg of the Cytochrome C and 88% of the BSA passed into the permeate.

(C) Adsorbent Unloading ~elution):
Aqueou~ l.OM KCl solution, 200 ml r was added to the suspension, which was then recirculated through the * Trademark .~

~29Z9S2 ultrafiltration membrane for 15 minutes. The permeate was collected, diafiltered for seven volume replacements with 1.0M KCl, and reconstituted to 200 ml. Analysis indicated that 102.0 mg of the Cytochrome C had eluted. Elution efficiency was calculated as follows:

102.0 mg eluted x 100 = 105%
97.4 mg loaded Example 2 This example demonstrates efficient separation of Cytochrome C (positively charged biomaterial) and Penicillin G Ipositively charged biomaterial) with a negatively charged adsorbent.

lS ~A) Adsorbent Loadinq:

Penicillin G (nPGn), 200 mg, was dissolved in 200 ml of PPB solution (Example 1). Total volume at this point was 200 ml. One-half gram of the strong acid ion exchange resin used in Example 1 was then added to 195 ml of the PPB solution containing the PG. To the resulting suspension was added 146.2 mg of Cytochrome C, Type III.

lZ9Z952 (B) Ultrafiltration:
The PG and Cytochrome C preparations were mixed and circulated for 15 minutes through the membrane of the same ultrafiltration system used in Example 1. The permeate was collected and removed. The retained resin suspension was diafiltered for five volume replacements with PPB solution and then reconstituted to a volume of 200 ml. Analysis showed that 105.6 mg of the Cytochrome C remained on the resin and that 40.6 mg of the Cytochrome C and 92% of the PG passed into the permeate.

(C) Adsorbent Unloading lelution) Aqueous l.OM KCl solution, 200 ml, was added to the resin suspension, which was then recirculated lS through the ultrafiltration mem~rane for 15 minutes.
The permeate was collected, diafiltered for seven volume replacements with l.OM KCl, and reconstituted to 200 ml. Analysis showed that 109.8 mg of the Cytochrome C had eluted to give an elution efficiency calculated as follows:
109.8 mg eluted x 100 = 104%
105.6 mg loaded -17- ~Z929Sz Example 3 This example illustrates the use of an uncharged adsorbent for separation of a biomaterial, and also demonstrates that increasing the electrolyte level in S the medium containing the biomaterial to be puri~ied or separated reduces the loading capacity of the adsorbent, i.e., its ability to bond to the biomaterial.
a) To 195 ml. of O.lM PPB solution (described in Example 1) was added KCl to bring the KCl concentration to O.lM, and 2.0 g (dry basis) of an emulsion copolymer of 50% hydroxyethyl acrylate and 50% trimethylolpropane trimethacrylate. To thi~ 150 mg of Cytochrome C
~described in Example 1) in 200 ml of the O.lM PPB/O.lM
KCl buffer solution was added, and the loading capacity of the copolymer was determined as in Example 1, using lM KCl/O.OlM PPB solution as the eluent. 1.6 mg Cytochrome C per gram of copolymer was bound under these conditions, and 96.9% of that was eluted.
b) To 195 ml of distilled water was added 2.0 g (dry basis) of the emulsion copolymer described in (a) above. To this was added 150 mg of Cytochrome C and 200 ml distilled water, and the loading capacity was determined by ultrafiltration as in Example 1. 67.2 mg Cytochrome C per gram of copolymer was bound, and 99.5%

~z9z9sz of that was eluted by lM KCl/O.OlM PPB solution.

Example 4 This example illustrates ultrafiltration chromatography of yeast-cell hexokinase using the process of the present invention.
Dried yeast cells (Sigma Chemical Co., Cat. No.
YSC-l) were ly~ed by su~pending them in 0.12M ammonium hydroxide. When lysis was complete the pH of the suspension wa~ reduced to 4.5 by adding acetic acid.
The final volume was 1.6 liter and the solids content was 9%. The suspension was further diluted to 2.6 liters with deionized water and microfiltered in a flltratlon unit equlpped with a"Romlcon"HPl-47MPOl cartridge, to ~e~arate the cell debri~ from the ~oluble enzymes, including hexokinase ~HK). The suspension was concentrated to 500 ml and then diafiltered again~t 10 volume replacements of 0.14M ~odium acetate, pH 5Ø
Recovery of hexokina~e in the permeate wa~ 73% of the lnltial en~yme content of the lysed suspen~ion~
Hexokina~e from 2 liters of the microfiltration filtrate containing 1 unit hexokinase and 0.96 mg protein/ml was concentrated by ad~orption onto a strong acid, emulsion polymerized, ~tyrene-7.3S divinylbenzene ~ Trademark -19- lZ9Z952 gellular resin of 0.26 micrometer average particle diameter and cation exchange capacity of 5.1 meq/g dry resin. The resin was then added to the suspension at a final concentration of 2100 ppm. The suspension of enzyme and resin was stirred slowly for 15 minutes at which time analysis of a filtered aliquot indicated no evidence of enzyme activity in the supernatant. When stirring was stopped, the resin settled rapidly to the bottom of the beaker and could be recovered in various ways. In this case the entire resin suspension was placed in the recirculation tank of the filtration unit equipped with the microfiltration cartridge indicated abova. After recirculation for lS minutes analysis of the filtrat~ indicated that 50% of the enzyme initially lS bound to the resin was present in the filtrate. The suspension was concentrated to 500 ml and diafiltered with 2 liters of the adsorption buffer. No enzyme activity was detected in the final diafiltrate. 500 ml of 4M NaCl was added to the retentate tank and recirculated for 15 minutes. The suspension was then concentrated to 500 ml. This procedure was repeated two additional times. Analysis of the 2M NaCl filtrate indicated that 60% of the adsorbed enzyme was recovered at this step.

Claims (17)

1. A method of separating or purifying a biomaterial contained in a first liquid medium with other components selected from impurities, other biomaterials and mixtures of said impurities and other biomaterials, which comprises:
(A) contacting the first liquid medium with a particulate polymeric adsorbent capable of preferentially adsorbing a biomaterial therefrom, the adsorbent having an average particle size in the range of 0.01 to 5 microns and selected from the group consisting of non-porous ion exchange resin bearing up to about 1.5 functional groups per monomer unit and solid, un-charged, non-porous polymer particles not yet functionalized with ion exchange functional groups, whereby the adsorbent reversibly binds the biomaterial to form a complex; and (B) subjecting the medium containing the complex and said other components to membrane filtration wherein the membrane is permeable to the said other components but not to the complex, whereby the complex and said other components are separated.

2. The process of claim 1 including the additional step of (C) liberating the biomaterial from the polymer particles of the complex into a second liquid medium.

3. The process of claim 2 including the additional step of (D) subjecting the second liquid medium containing bio-material and polymer particles to membrane filtration as in step (B), whereby the biomaterial but not the polymer particles accumulates in the permeate.

4. The method of claim 1 wherein the particles of the ion exchange resin comprise substantially spherical beads.

5. The method of claim 1 wherein the particles of the ion exchange resin comprise ground particles.

6. The method of claim 1 wherein the resin is acidic.

7. The method of claim 1 wherein the resin is basic.

8. The method of claim 1 wherein the biomaterial is positively charged and the ion exchange resin is acidic.

9. The method of claim 1 wherein the biomaterial is negatively charged and the ion exchange resin is basic.

10. The method of claim 1 wherein the adsorbent for the biomaterial comprises an uncharged polymer.

11. The method of claim 10 wherein the polymer and the biomaterial are hydrophobic.

12. The method of claim 10 wherein the uncharged polymer comprises an ion exchange resin precursor.

13. The method of claim 1 wherein the biomaterial is a proteinaceous substance.

14. The method of claim 1 wherein the biomaterial is a protein, an amino acid, a nucleic acid, an antibiotic or a vitamin.

15. The method of claim 2 wherein, in step (C), the bio-material is liberated by treatment of the complex with an electrolyte.

16. The method of claim 1 wherein the biomaterial and other components are suspended or dissolved in the media.

17. The method of claim 1 wherein the other biomaterials include one or more of a protein, an amino acid, a nucleic acid, an antibiotic or a vitamin.