|||||||||||| USOO5167824A United States Patent (19) 11 Patent Number: 5,167,824 Cohen et al. 45 Date of Patent: Dec. 1, 1992 (54) SEPARATION BY CARRIER MEDIATED (56) References Cited TRANSPORT U.S. PATENT DOCUMENTS 75 Inventors: Charles Cohen, Medway; Robert A. 3. E. tal as a - a - a -26% Dishman, Concord; James S. Huston, 4,375,414 3/1983 Strahilevitz ......................... 210/638 Chestnut Hill; Robert L. Bratzler, 4,859,583 8/1989 Heller et al. ...................... 435/28 X Concord; David R. Dodds, Millis; Charles M. Zepp, Berlin, all of Mass. Primary Examiner-Frank Spear Attorney, Agent, or Firm-Testa, Hurwitz & Thibeault 73) Assignees: Creative BioMolecules, Inc., 57 ABSTRACT Hopkinton; Sepracor, Inc., Marlborough, both of Mass. Disclosed are processes and apparatus for separating a desired solute, such as an optically active isomer, from a complex mixture using carrier facilitated transport in (21) Appl. No.: 479,935 an immobilized liquid membrane or carrier facilitated solvent extraction. The carrier is a binding protein se (22 Filed: Feb. 14, 1990 lected and/or engineered to immunochemically revers ibly bind to the solute and to have a significant solubility 51 Int. Cl............................................... B01D 61/28 in the extracting solvent or immobilized liquid men 52 U.S. Cl. ..................................... 210/638; 210/644 brane. 58 Field of Search ....................... 210/638, 644, 632; 435/7.1, 28 12 Claims, 13 Drawing Sheets U.S. Patent Dec. 1, 1992 Sheet 1 of 13 5,167,824 U.S. Patent Dec. 1, 1992 Sheet 8 of 13 5,167,824 550 560 570 580 590 600 GGGTACATTTCCCCATACTCTGGGGTTACCGGCTACAACCAGAAGTTTAAAGGTAAGGCG G Y I S P Y S G W T G Y N Q K F K G K. A Pf M BStEII Dra 610 620 630 640 650 660 ACCCTTACTGTCGACAAATCTTCCTCAACTGCTTACATGGAGCTGCGTTCTTTGACCTCT L T W D K S S S T A Y M. E L R S L T S Sal 670 680 690 700 710 720 GAGGACTCCGCGGTATACTATTGCGCGGGCTCCTCTGGTAACAAATGGGCCATGGATTAT E D S A V Y Y C A G S S G. N. K. W. A. M. D Y SaCII NCOI 730 740 750 760 TGGGGTCATGGTGCTAGCGTTACTGTGAGCTCTTAACTGCAG W G H G A S W T V S S k FIG. 6-2 U.S. Patent Dec. 1, 1992 Sheet 9 of 13 5,167,824 Fig. 7A FIG. 7B FIG.s 7c NH 3. NH3 3 NH3. 3 / y 4 M 4. 4 YN- 2 O 2 O 6 O 2 w--COOT 22 a 'coo- 2 2 2O COO NH 3 14- a 22 M 2O COO U.S. Patent Dec. 1, 1992 Sheet 12 of 13 5,167,824 2 5 2 O 45 O 5 O is is is AOG LWAOL/WD A/6O7W COWCAW/A47/OW/M/ FIG. I.0 O -11 - O -9 -8 -7 -6 AOG LWBOUWD AGO/W COWCAW7A77OW/My FIG. II 5,167,824 1. 2 counts for many of the difficulties encountered in re SEPARATION BY CARRIER MEDIATED solving racenic mixtures. However, these small struc TRANSPORT tural differences may be profound in biological systems, e.g., if the compounds are involved in enzyme-cat BACKGROUND OF THE INVENTION alyzed reactions or bind specifically to cellular recep This invention relates to separation of individual sol tors. Thus, the L-amino acids are metabolized in hu utes from a mixture. More specifically, the invention mans but the corresponding D analogs are not. Only relates to exploitation of carrier mediated transport D-glucose can be phosphorylated and processed into mechanisms in immobilized liquid membrane and re glycogen or degraded by the glycolytic and oxidative lated separation systems. 10 pathways of intermediary metabolism. Similarly, beta There is a continuing need for more efficient methods blockers, pheromones, prostaglandins, steroids, flavor of separating the solutes in mixtures of structurally ing and fragrance agents, pharmaceuticals, pesticides, related bioactive compounds. Synthesis of bioactive herbicides, and many other compounds exhibit critical materials such as pharmaceuticals and pesticides using stereospecificity. In the field of pesticides, for example, organic chemistries often requires separations on a pre 15 Tessier has shown that only two of the eight stereoiso parative scale. The products of expression of engi mers of deltamethrin, a pyrethroid insecticide, have any neered microorganisms and cell lines typically comprise biological activity. (Chemistry and Industry, Mar. 19, a single valuable species in admixture with a host of 1984, p. 199). Other forms of optical isomers are known extraneous proteins, lipids, nucleic acids, and polysac which are of commercial interest. charides. Isolation of pure or substantially pure product 20 Stereochemical purity is also important in the field of on a commercial scale from such mixtures presents a pharmaceuticals, where 12 of the 20 most prescribed significant engineering challenge. Chromatography, drugs exhibit chirality. A case in point is provided by differential precipitation, filtration, and other separation naproxen, or (--) S-2-(6-methoxy-2-naphthyl) propionic technologies are used to remove selectively unwanted acid, used for instance in the management of arthritis. In species. Often, the late-purification stages are most diffi 25 cult and can be achieved only by means of affinity chro this case, the S(--) enantiomer of the drug is known to matography using polyclonal or monoclonal antibodies be 28 times more therapeutically potent than its R(-) which selectively immunochemically bind with the counterpart. Still another example of chiral pharmaceu product. ticals is provided by the family of beta-blockers; the Molecular biology has advanced as a technology 30 L-form of propranolol is known to be 100 times more sufficient to permit production of a variety of valuable potent than the D-enantiomer. biologically active proteins Pending U.S. application Synthesis of chiral compounds by standard organic Ser. Nos. 052,800 and 342,449, and PCT Application synthetic techniques generally leads to a racemic mix US88/01737, disclose biosynthetic multifunctional pro ture which, in the aggregate, may have a relatively low teins comprising plural bioactive domains, one of which 35 specific bioactivity since certain of the stereoisomers in is capable of binding to a preselected compound. The the mixture are likely to be biologically or functionally binding domain is patterned after the antibody binding inactive. As a result, relatively larger quantities of the site and can mimic the binding properties of a light or material must be used to obtain an effective dose, and heavy chain protein, or comprises a single chain con manufacturing costs are increased due to the co-produc struct comprising both light and heavy chains. Such tion of stereochemically "incorrect' and hence, inac constructs may be used in affinity chromatography tive ingredients. Also, the inactive enantiomer may procedures and may have significant cost advantages have unwanted side effects. over monoclonal antibodies. A widely used approach to purifying optical isomers Among the most difficult purification tasks is the is the selective precipitation of the desired compound separation of stereoisomers. Many organic compounds 45 from a racemic mixture. See, for example, U.S. Pat. No. exist in optically active forms, i.e., they have the ability 3,879,451, 4,257,976, 4,151,198, 4,454,344; Harrison et to rotate the plane of plane polarized light. In describing al, J. Med. Chem. 13:203 (1970); Felder et al, UK Patent an optically active compound, the prefixes D and L, or Application No. GB2025968A (1980), and U.S. R and S are used to denote the absolute configuration of 4,285,884. Separation of diastereomers also can be car the molecule about its chiral center(s). The prefixes 50 ried out by chromatography. See, for example, Pollock designate the sign of rotation of plane polarized light by et al, J. Gas Chromatogr. 3:174 (1965); Mikes et al., J. the compound, with (-) or L. meaning that the com Chromatogr. 112:205 (1976); and Hare et al, U.S. Pat. pound is levorotatory. For a given chemical structure, No. 4,290,893. Enzymes have been used for the resolu D and L stereoisomers are mirror images of one an tion of stereoisomers on a preparative scale. For in other. A specific stereoisoner may also be referred to as stance, enzymatic treatment has been applied to the an enantiomer, and a mixture of such isoners is often resolution of racemic mixtures of anino esters. See U.S. called an enantiomeric or racemic mixture. The term Pat. No. 3,963,573, U.S. Pat. No. 4,262,092, Clement "racemic mixture' as used herein, refers to a mixture of and Porter, J. Chen. Ed., 48:695 (1971), and Matta et al at least first and second stereoisomers in any proportion. J. Org. Chem, 39:2291 (1974). Additional examples of Optical activity is typically the result of molecular 60 enzyme-mediated resolution as applied to the produc asymmetry about tetrahedral carbon atoms that are tion of optically purified pharmaceuticals include Sih linked to four different moieties. Where there is only U.S. Pat. No. 4,584,370, Aragozzini et al, Biotechnol one asymmetric carbon atom, or "chiral center', there Letters, 8:95 (1980), Yokozeki et al, EPO No. 0122794 are two possible stereoisomers or enantiomers. Where A2, and Sih, Tetrahedron Letters, 27:1763 (1986). Mul there are n chiral centers, the number of potential ste tiphase and extractive enzyme membrane bioreactors reoisomers increases to 2. The structural differences that selectively produce pure or substantially purified between stereoisomers are subtle and of little conse optically active compounds from achiral precursors or quence in ordinary chemical reactions, and this ac mixtures are disclosed by Matson in U.S. Pat. No. 5,167,824 3 4. 4,800,162. The term "resolution' as used herein, refers as used herein, unless antithetical to its context, refers to separation of a first mixture into second and third broadly to reversible binding of the type that is ubiqui mixtures wherein the proportions of the solutes in the tous in biological systems.