-1 - CONJUGATES OF A CHOLINESTERASE MOIETY AND A POLYMER CROSS REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Serial No. 61/127,928, filed 16 May 2008, the disclosure of which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION [0002] Among other things, one or more embodiments of the present invention relate generally to conjugates comprising a cholinesterase moiety (i.e., a moiety having at least some activity similar to human chohnesterase) and a polymer, hi addition, the invention relates to (among other things) compositions comprising conjugates, methods for synthesizing conjugates, and methods of administering a composition. BACKGROUND OF THE INVENTION [0003] The himian nervous system controls bodily fimctions through the transmission of electrical signals over specialized nerve cells. With respect to the gaps between nerve cells (or between a nerve cell and an effector cell), however, continuation of the signal is typically achieved via chemical means. Chemical transmission through the nerve gap or "synapse" takes place via the release of a substance known as a "neurotransmitter" (alternatively known as a "neuromediator"). Upon release, the neurotransmitter crosses the synapse by diffusion and activates (or inhibits, depending on the system) the postsynaptic cell by binding to a receptor located on the postsynaptic celL The signal having thus been passed to the postsynaptic cell, enzymes in the synapse degrade the neurotransmitter so as to prevent repeated signal transfer to the postsynaptic cell. In this way, signals within the nervous system are successfully transmitted. [0004] Nerve cells that release acetylcholine as the neurotransmitter are called cholinergic nerves and are located in both the peripheral and central nervous systems in humans. Acetylcholine is involved with the transmission of signals fi^om specialized motor nerves to the skeletal muscle as well as much of the autonomic nervous system, which controls - 2 - the smooth muscles and glands associated with (for example) respiration, circulation, digestion, sweating and metabolism. In the body, acetylcholine is degraded — and therefore its effects controlled — by acetylcholinesterase located in the synaptic cleft. Given the ubiquity of the acetylcholine/acetylcholinesterase system in the central nervous system, the proper balance and fimctioning of this system is critical to normal functioning and health. [0005] The balance of the acetylcholine/acetylcholinesterase system in the central nervous system can be disrupted through exposure an acetylcholinesterase inhibitor, which results in the accumulation of acetylcholine in the synaptic cleft This accumulation, in turn, results in continuous signal propagation (typically via persistent depolarization) and concomitant disruption of effective neural transmission. Such a disruption, if allowed to continue, can cause any number of deleterious conditions and ~ if severe — even death. [0006] O-Isopropyl methylphosphonofluoridate (also known as "sarin") and other organophosphates in its class are irreversible cholinesterase inhibitors. These organophosphates inhibit the activity of cholinesterase by covalently binding to a serine residue in the enzyme which forms the site where acetylcholine normally undergoes hydrolysis. Sarin is such a potent and effective inhibitor of cholinesterase enzymes that it has been developed and used in the military context. Other cholinesterase inhibitors have been used as insecticides and pesticides in the agricultural context. [0007] Exposure to cholinesterase inhibitors can be remedied by the administration of cholinesterase itself. By effectively "saturating" the biological system with cholinesterase, overall normal cholinesterase functioning would remain substantially unaffected insomuch as even though some cholinesterase activity would be inhibited by the cholinesterase inhibitor, the presence of excess cholinesterase activity would minimize the effects of cholinesterase inhibitor exposure. Such an approach would be advantageous for accidental exposure to organophosphates as well as in the defense of a military attack in which seirin or similar chemical agent is used. A recombinant version of human butyiylcholinesterase (BChE), a naturally occiuring protein, is bemg developed under the name PROTEXIA® as a pre- and post-exposure therapy for casualties on the battlefield or civilian victims of nerve agent attacks. [0008] One problem associated with administering an excess of molecules having cholinesterase activity is that these protein-based enzymes themselves degrade relatively quickly in vivo. PEGylation, or the attachment of a poly(ethylene glycol) derivative to a - 3 - protein, has been described as a means to prolong a protein's in vivo half-life, thereby resulting in prolonged pharmacologic activity. For example, U.S. Patent Application No. 2004/0147002 describes uses of chemically modified cholinesterases for detoxification of organophosphorus compounds. [0009] Notwithstanding these conjugates, however, there remains a need for other conjugates of cholinesterase. Among other things, one or more embodiments of the present invention is therefore directed to such conjugates as well as compositions comprising the conjugates and related methods as described herein, which are believed to be new and completely unsuggested by the art. SUMMARY OF THE INVENTION [0010] Accordingly, in one or more embodiments of the invention, a conjugate is provided, the conjugate comprising a residue of a cholinesterase moiety covalently attached to a water-soluble polymer. [0011] In one or more embodiments of the invention, a conjugate is provided, the conjugate comprising a residue of a cholinesterase moiety covalently attached to a water-soluble polymer, wherein the residue of the cholinesterase moiety is covalently attached to the water-soluble polymer through a cysteine residue within the residue of the cholinesterease moiety. [0012] In one or more embodiments of the invention, a conjugate is provided, the conjugate comprising a residue of a cholinesterase moiety covalently attached to a water-soluble polymer, wherein the water-soluble polymer, prior to being covalently attached, is a polymeric reagent bearing a maleimide group. [0013] In one or more embodiments of the invention, a conjugate is provided, the conjugate comprising a residue of a cholinesterase moiety covalently attached, either directly or through a spacer moiety comprised of one or more atoms, to a water-soluble polymer, wherein the cholinesterase moiety is attached to the water-soluble polymer or spacer moiety via a disulfide linkage. - 4 - [0014] In one or more embodiments of the invention, a conjugate is provided, the conjugate comprising a residue of a cholinesterase moiety covalently attached to a water-soluble polymer, wherein the cholinesterase moiety is a precursor cholinesterase moiety. [0015] In one or more embodiments of the invention, a conjugate is provided, the conjugate comprising a residue of a cholinesterase moiety covalently attached to a water-soluble polymer, wherein the cholinesterase moiety is a mature cholinesterase moiety. [0016] In one or more embodiments of the invention, a method for delivering a conjugate is provided, the method comprising the step of subcutaneously administering to the patient a composition comprised of a conjugate of a residue of a cholinesterase and a water-soluble polymer. BRIEF DESCRIPTION OF THE DRAWINGS [0017] FIG. 1 depicts an SDS-PAGE analysis of conjugate solutions of rBChE prepared in accordance with Examples 4, 5 and 6 and explained more fuUy in each of these examples. The lane marked C is the rBChE protein control (not PEGylated). rBChE was PEGylated with different activated PEG regents as indicated above the lanes. Three mol equivalent concentrations (10,25 and 50) of PEG were tested using the described methods. For each reagent 10 mol equivalents of PEG reagent resulted in low to medium levels of PEGylation, 25 mol equivalents of PEG reagent resulted in medium to high levels of PEGylation and 50 mol equivalents of PEG reagent in very high levels of PEGylation. DETAILED DESCRIPTION OF THE INVENTION [0018] Before describing one or more embodiments of the present invention in detail, it is to be understood that this invention is not limited to the particular polymers, synthetic techniques, cholinesterase moieties, and the like, as such may vary. [0019] It must be noted that, as used in this specification and the intended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polymer" includes a single polymer as well as two or more of the same or different polymers, reference to "an optional excipient" refers to a - 5 - • single optional excipient as well as two or more of the same or different optional excipients, and the like. [0020] In describing and claiming one or more embodiments of the present invention, the following terminology will be used in accordance with the definitions described below. [0021] "PEG," "polyethylene glycol" and "poly(ethylene glycol)" as used herein, are interchangeable and encompass any nonpeptidic water-soluble poly(ethylene oxide). Typically, PEGs for use in accordance with the invention comprise the following structure "-(OCH2CH2)„-" where (n) is 2 to 4000. As used herein, PEG also includes "-CH2CH2-0(CH2CH20)n-CH2CH2-" and "-(OCH2CH2)„0-," depending upon whether or not the terminal oxygens have been displaced, e.g., during a synthetic transformation. Throughout the specification and claims, it should be remembered that the term "PEG" includes structures having various terminal or "end capping" groups and so forth. The term "PEG" also means a polymer that contains a majority, that is to say, greater than 50%, of-OCH2CH2- repeating subunits. With respect to specific forms, the PEG can take any mmiber of a variety of molecular weights, as well as structures or geometries such as "branched," "linear," "forked," "multifimctional," and the like, to be described in greater detail below. [0022] The terms "end-capped" and "terminally capped" are interchangeably used herein to refer to a terminal or endpoint of a polymer having an end-capping moiety. Typically, although not necessarily, the end-capping moiety comprises a hydroxy or C1.20 alkoxy group, more preferably a Cj.io alkoxy group, emd still more preferably a Ci-s alkoxy group. Thus, examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxy and benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and the like. It must be remembered that the end-capping moiety may include one or more atoms of the tennuial monomer in the polymer [e.g., the end-capping moiety "methoxy" in CH30(CH2CH20)„- and CH3(OCH2CH2)„-]. hi addition, saturated, unsaturated, substituted and unsubstituted forms of each of the foregoing are envisioned. Moreover, the end-capping group can also be a silane. The end-capping group can also advantageously comprise a detectable label. When the polymer has an end-capping group comprising a detectable label, the amoimt or location of the polymer and/or the moiety (e.g., active agent) to which the polymer is coupled can be determined by using a suitable detector. Such labels iaclude, without limitation, fluorescers, chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g., dyes), metal ions, radioactive moieties, and the like. - 6 - Suitable detectors include photometers, films, spectrometers, and the like. The end-capping group can also advantageously comprise a phospholipid. When the polymer has an end-capping group comprising a phospholipid, unique properties are imparted to the polymer and the resulting conjugate. Exemplary phospholipids include, without limitation, those selected from the class of phospholipids called phosphatidylcholines. Specific phospholipids include, without limitation, those selected from the group consisting of dilauroylphosphatidylcholine, dioleylphosphatidylcholine, dipahnitoylphosphatidylcholine, disteroylphosphatidylcholine, behenoylphosphatidylcholine, arachidoylphosphatidylcholine, and lecithin. (00231 "Non-naturally occurring" with respect to a polymer as described herein, means a polymer that in its entirety is not found in nature. A non-naturally occurring polymer may, however, contain one or more monomers or segments of monomers that are naturally occurring, so long as the overall polymer structure is not foimd in nature. [0024] The term "water soluble" as in a "water-soluble polymer" polymer is any polymer that is soluble in water at room temperature. Typically, a water-soluble polymer will transmit at least about 75%, more preferably at least about 95%, of light transmitted by the same solution after filtering. On a weight basis, a water-soluble polymer will preferably be at least about 35% (by weight) soluble in water, more preferably at least about 50% (by weight) soluble in water, still more preferably about 70% (by weight) soluble in water, and still more preferably about 85% (by weight) soluble in water. It is most preferred, however, that the water-soluble polymer is about 95% (by weight) soluble in water or completely soluble in water. [0025] Molecular weight in the context of a water-soluble polymer, such as PEG, can be expressed as either a number average molecular weight or a weight average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the weight average molecular weight. Both molecular weight determinations, number average and weight average, can be measured using gel permeation chromatography or other liquid chromatography techniques. Other methods for measuring molecular weight values can also be used, such as the use of end-group analysis or the measurement of coUigative properties (e.g., freezing-point depression, boiling-point elevation, or osmotic pressure) to determine number average molecular weight or the use of light scattering techniques, ultracentrifugation or - 7 - viscometry to determine weight average molecular weight. The polymers of the invention are typically polydisperse (i.e., number average molecular weight and weight average molecular weight of the polymers are not equal), possessing low polydispersity values of preferably less than about 1.2, more preferably less than about 1.15, still more preferably less than about 1.10, yet still more preferably less than about 1.05, and most preferably less than about 1.03. [0026] The terms "active," "reactive" or "activated" when used in conjimction with a particular fimctional group, refers to a reactive functional group that reacts readily with an electrophile or a nucleophile on another molecule. This is in contrast to those groups that require strong catalysts or highly impractical reaction conditions in order to react (i.e., a "nonreactive" or "inert" group). [0027] As used herein, the term "functional group" or any synonym thereof is meant to encompass protected fonns thereof as well as unprotected forms. [0028] The terms "spacer moiety," "linkage" and "linker" are used herein to refer to a bond or an atom or a collection of atoms optionally used to link intercoimecting moieties such as a terminus of a polymer segment and a cholinesterase moiety or an electrophile or nucleophile of a cholinesterase moiety. The spacer moiety may be hydrolytically stable or may include a physiologically hydrolyzable or enzymatically degradable linkage. Unless the context clearly dictates otherwise, a spacer moiety optionally exists between any two elements of a compound (e.g., the provided conjugates comprising a residue of cholinesterase moiety and water-soluble polymer can attached directly or indirectly through a spacer moiety). [0029] "Alkyl" refers to a hydrocarbon chain, typically ranging from about 1 to 15 atoms in length. Such hydrocarbon chains are preferably but not necessarily saturated and may be branched or straight chain, although typically straight chain is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, and the like. As used herein, "alkyl" includes cycloaUcyl as well as cycloalkylene-containing alkyl. [0030] "Lower alkyl" refers to an alkyl group containing from 1 to 6 carbon atoms, and may be straight chain or branched, as exemplified by methyl, ethyl, /i-butyl, j-butyl, and f-butyl. [0031] "CycloaUcyl" refers to a saturated or unsaturated cychc hydrocarbon chain, including bridged, fused, or spiro cyclic compounds, preferably made up of 3 to about 12 carbon atoms, more preferably 3 to about 8 carbon atoms. "CycloaUcylene" refers to a - 8 - cycloalkyl group that is inserted into an alkyl chain by bonding of the chain at any two carbons in the cyclic ring system. 10032] "Alkoxy" refers to an -OR group, wherein R is alkyl or substituted allcyl, preferably Cj-e alkyl (e.g., methoxy, ethoxy, propyloxy, and so forth). [0033] The term "substituted" as m, for example, "substituted alkyl," refers to a moiety (e.g., an alkyl group) substituted with one or more noninterfering substituents, such as, but not limited to: alkyl, C3-8 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g., fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl; substituted phenyl; and the like. "Substituted aryl" is aryl having one or more noninterfering groups as a substituent. For substitutions on a phenyl ring, the substituents may be in any orientation (i.e., ortho, meta, or para). [0034] "Noninterfering substituents" are those groups that, when present in a molecule, are typically nonreactive with other ftmctional groups contained within the molecule. [0035] "Aryl" means one or more aromatic rings, each of 5 or 6 core carbon atoms. Aiyl includes multiple aryl rings that may be fused, as in naphthyl or unflised, as in biphenyl. Aryl rings may also be fused or unflised with one or more cyclic hydrocarbon, heteroaiyl, or heterocyclic rings. As used herein, "aryl" includes heteroaryl. [0036] "Heteroaryl" is an aryl group containing from one to four heteroatoms, preferably sulfur, oxygen, or nitrogen, or a combination thereof. Heteroaryl rings may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings. [0037] "Heterocycle" or "heterocyclic" means one or more rings of 5-12 atoms, preferably 5-7 atoms, with or without unsaturation or aromatic character and having at least one ring atom that is not a carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen. [0038] "Substituted heteroaryl" is heteroaryl having one or more noninterfering groups as substituents. [0039] "Substituted heterocycle" is a heterocycle having one or more side chains formed from noninterfering substituents. [0040] An "organic radical" as used herein shall include akyl, substituted alkyl, aryl, and substituted aryl. - 9 - [0041] "Electrophile" and "electrophilic group" refer to an ion or atom or collection of atoms, that may be ionic, having an electrophilic center, i.e., a center that is electron seeking, capable of reacting with a nucleophile. [0042] "Nucleophile" and "nucleophilic group" refers to an ion or atom or collection of atoms that may be ionic having a nucleophilic center, i.e., a center that is seeking an electrophilic center or with an electrophile. [0043] A "physiologically cleavable" or "hydrolyzable" or "degradable" bond is a bond that reacts with water (i.e., is hydrolyzed) under physiological conditions. The tendency of a bond to hydrolyze in water will depend not only on the general type of linkage connecting two central atoms but also on the substituents attached to these central atoms. Appropriate hydrolytically unstable or weak linkages include but are not limited to carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides. [0044} An "enzymatically degradable linkage" means a linkage that is subject to degradation by one or more enzymes. [0045] A "hydrolytically stable" linkage or bond refers to a chemical bond, typically a covalent bond, that is substantially stable in water, that is to say, does not xmdergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time. Examples of hydrolytically stable linkages include, but are not limited to, the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, urethanes, and the like. Generally, a hydrolytically stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks. [0046] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient. "Pharmacologically effective amount," "physiologically effective amount," and "therapeutically effective amount" are used interchangeably herein to mean the amount of a polymer-(choIinesterase) moiety conjugate that is needed to provide a desired level of the conjugate (or corresponding imconjugated cholinesterase moiety) in the bloodstream or in the target tissue. The precise amount will depend upon numerous factors, e.g., the particular cholinesterase moiety, the components and physical - 1 0 - characteristics of the therapeutic composition, intended patient popiilation, individual patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein. [0047] "Multi-functional" means a polymer having three or more functional groups contained therein, where the functional groups may be the same or different. Multi-functional polymeric reagents of the invention will typically contain from about 3-100 functional groups, or from 3-50 fimctional groups, or from 3-25 fimctional groups, or from 3-15 functional groups, or from 3 to 10 functional groups, or will contain 3,4, 5, 6, 7, 8, 9 or 10 functional groups within the polymer backbone. [0048] The term "cholinesterase moiety," as used herein, refers to a moiety having human cholinesterase activity. The cholinesterase moiety will also have at least one electrophilic group or nucleophilic group suitable for reaction with a polymeric reagent. In addition, the term "cholinesterase moiety" encompasses both the cholinesterase moiety prior to conjugation as well as the cholinesterase moiety residue following conjugation. As will be explained in fiirther detail below, one of ordinary skill in the art can determine whether any given moiety has cholinesterase activity. Proteins comprising an amino acid sequence corresponding to any one of SEQ ID NOs: 1 through 2 is a cholinesterase moiety, as well as any protein or polypeptide substantially homologous thereto, that can act as a substrate for a cholinesterase inhibitor. As used herein, the term "cholinesterase moiety" includes such proteins modified deliberately, as for example, by site directed mutagenesis or accidentally through mutations. These terms also include analogs having from 1 to 6 additional glycosylation sites, analogs having at least one additional amino acid at the carboxy terminal end of the protein wherein the additional amino acid(s) includes at least one glycosylation site, and analogs having an amino acid sequence which includes at least one glycosylation site. The term includes both natural and recombinantly produced moieties. [0049] The term "substantially homologous" means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. For purposes of the present invention, sequences having greater than 95 percent homology, equivalent biological properties, and equivalent expression characteristics are considered substantially homologous. - 1 1 - For purposes of detennining homology, truncation of the mature sequence should be disregarded. Sequences havuig lesser degrees of identity, comparable bioactivity, and equivalent expression characteristics are considered substantial equivalents. Exemplary cholinesterase moieties for use herein include those sequences that are substantially homologous SEQ ID NO: 1. [0050] The term "fragment" means any protein or polypeptide havuig the amino acid sequence of a portion or fragment of a cholinesterase moiety, and which has the biological activity of P-cholinesterase. Fragments include proteins or polypeptides produced by proteolytic degradation of a cholinesterase moiety as vi'ell as proteins or polypeptides produced by chemical synthesis by methods routine in the art. Enzymatic activity is typically measured, e.g., by enzymatic or inhibitory activity using cultured cell lines or tissue culture based methods. [0051] The term "patient," refers to a living organism suffering from or prone to a condition that can be prevented or treated by administration of an active agent (e.g., conjugate), and includes both humans and animals. [0052] "Optional" or "optionally" means that tiie subsequently described circumstance may or may not occiu", so that the description includes instances where the circimistance occurs and instances where it does not, [0053] "Substantially" means nearly totally or completely, for instance, satisfying one or more of the following: greater than 50%, 51% or greater, 75% or greater, 80% or greater, 90% or greater, and 95% or greater of the condition. [0054] Amino acid residues in peptides are abbreviated as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is lie or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidiue is His or H; Glutamine is Gin or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Argtnine is Arg or R; and Glycine is Gly or G. [0055] Turning to one or more embodiments of the invention, a conjugate is provided, the conjugate comprising a residue of cholinesterase moiety covalently attached (either directly or through a spacer moiety) to a water-soluble polymer. The conjugates of the invention will have one or more of the following features. - 1 2 - [0056] The Cholinesterase Moiety [0057] As previously stated, the conjugate generically comprises a residue of cholinesterase moiety covalently attached, either directly or through a spacer moiety, to a water-soluble polymer. As used herein, the term "cholinesterase moiety" shall refer to the cholinesterase moiety prior to conjugation as well as to the cholinesterase moiety following attachment to a nonpeptidic water-soluble polymer. It will be understood, however, that when the original cholinesterase moiety is attached to a nonpeptidic water-soluble polymer, the cholinesterase moiety is slightly altered due to the presence of one or more covalent bonds associated with linkage to the polymer. Often, this slightly altered form of the cholinesterase moiety attached to another molecule is referred to a "residue" of the cholinesterase moiety. [0058] The cholinesterase moiety can be derived from non-recombinant methods and from recombinant methods and the invention is not limited in this regard. In addition, the cholinesterase moiety can be derived from himian sources, animal sources, and plant sources. [0059] The cholinesterase moiety can be derived non-recombinantly. For example, it is possible to isolate butyrylcholinesterase from biological systems. As explained in U.S. Patent No. 5,272,080, for example, butyrylcholinesterase can be produced in a purity of at least 90% by subjecting plasma fraction rV-4 alone or in admixture with fraction IV-1 to both anion exchange chromatography and affraity chromatography. [0060] The cholinesterase moiety can be derived from recombinant methods. For example, U.S. Patent Nos. 5,248,604 and 5,595,903 describe recombinant-based methods for producing enzymatically active human cholinesterase. A cholinesterase moiety obtained through the approaches described in these references can be used as a cholinesterase moiety in preparing the conjugates described herein. [0061] The cholinesterase moiety can be expressed in bacterial [e.g., E. coli, see, for example, Fischer et al. (1995) Biotechnol. Appl. Biochem. 21(3):295-311], mammalian [see, for example, Kronman et al. (1992) Gene 121:295-3041. yeast [e.g., Pichia pastoris, see, for example, Morel et al. (1997) Biochem. J. 328(1):121-129], and plant [see, for example, Mor et al. (2001) Biotechnol. Bioeng. 75(3):259-266] expression systems. The expression can occur via exogenous expression (when the host cell naturally contains the desired genetic coding) or via endogenous expression. The production of butyrylcholinesterase in transgenic mammals has been described. See, for example, U.S. Patent Application Publication No. 2004/0016005. - 1 3 - [0062] Although recombinant-based methods for preparing proteins can differ, recombinant methods typically involve constructing the nucleic acid encoding the desired polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming a host cell (e.g., plant, bacteria, yeast, transgenic animal cell, or mammalian cell such as Chinese hamster ovary cell or baby hamster kidney cell), and expressing the nucleic acid to produce the desired polypeptide or fragment. Methods for producing and expressing recombinant polypeptides in vitro and in prokaryotic and eukaryotic host cells are known to those of ordinary skill in the art, [0063] To facilitate identification and purification of the recombinant polypeptide, nucleic acid sequences that encode for an epitope tag or other affinity binding sequence can be inserted or added in-frame with the coding sequence, thereby producing a fusion protein comprised of the desired polypeptide and a polypeptide suited for binding. Fusion proteins can be identified and purified by first running a mixture containing the fusion protein through an affinity column bearing binding moieties (e.g., antibodies) directed against the epitope tag or other binding sequence in the fusion proteins, thereby binding the fusion protein within the column. Thereafter, the fiision protein can be recovered by washing the column with the appropriate solution (e.g., acid) to release the bound fusion protein. The recombinant polypeptide can also be identified and purified by lysing the host cells, separating the polypeptide, e.g., by size exclusion chromatography, and collecting the polypeptide. These and other methods for identifying and purifying recombinant polypeptides are known to those of ordinary skill in the art. hi one or more embodiments of the invention, however, it is preferred that the cholinesterase moiety is not in the form of a fiision protein. [0064] Depending on the system used to express proteins having cholinesterase activity, the cholinesterase moiety can be unglycosylated or glycosylated and either may be used. That is, the cholinesterase moiety can be unglycosylated or the cholinesterase moiety can be glycosylated. In one or more embodiments of the invention, it is preferred that the cholinesterase moiety is glycosylated, preferably at four glycosylation sites. For example, it is also preferred to have the oligosaccharide chain at each glycosylation site terminate in a mannose sugar. [0065] The cholinesterase moiety can advantageously be modified to include and/or substitute one or more amino acid residues such as, for example, lysine, cysteine and/or - 1 4 - arginine, in order to provide facile attachment of the polymer to an atom within the side chain of the amino acid. An example of substitution of a cholinesterase moiety is described in Fischer et al. (1995) Biotechnol. Appl Biochem. 2i(3):295-311. In addition, the cholinesterase moiety can be modified to include a non-naturally occuning amino acid residue. Techniques for adding amino acid residues and non-naturally occmring amino acid residues are well known to those of ordinary skill in the art. Reference is made to J. March, Advanced Organic Chemistry: Reactions Mechanisms and Structure, 4th Ed. (New York: Wiley-Interscience, 1992). [0066] In addition, the cholinesterase moiety can advantageously be modified to include attachment of a functional group (other than through addition of a functional group-containing amino acid residue). For example, the cholinesterase moiety can be modified to include a thiol group. In addition, the cholinesterase moiety can be modified to include an N-terminal alpha carbon. In addition, the cholinesterase moiety can be modified to include one or more carbohydrate moieties. In some embodiments of the invention, it is preferred that the cholinesterase moiety is not modified to include a thiol group and/or an N-terminal alpha carbon. [0067] Exemplary cholinesterase moieties Eire described in the literature and in, for example, US. Patent Application Publication Nos. 2002/0119489, 2006/0263345 and 2008/0213281. Preferred cholinesterase moieties include those having an amino acid sequence comprising sequences selected firom the group consisting of SEQ ID NOs: 1 through 2, and sequences substantially homologous thereto. A preferred cholinesterase moiety has the amino acid sequence corresponding to himian acetylcholinesterase. Another preferred cholinesterase has the amino acid sequence corresponding to human butyrylcholinesterase, e.g., the recombinant version of human butyrylcholinesterase being developed under the PROTEXIA name (PharmAthene Inc., Annapolis, MD). It is recognized that both acetylcholinesterase and butyrylcholinesterase exist in multiple molecular forms composed of different numbers of catalytic and non-catalytic subunits. In hmnans, however, both enzymes are composed of subunits of about 600 amino acids each, and both are glycosylated. Acetylcholinesterase may be distinguished from the closely related butyrylcholinesterase by its high specificity for the acetylcholine substrate and sensitivity to selective inhibitors. While acetylcholinesterase is primarily used in the body to hydrolyze acetylcholine, the specific fimction of butyrylcholinesterase is not as clear. In any event, the terms "acetylcholinesterase" and - 1 5 - "butyrylcholinesterase" encompass all of the molecular forms within each enzyme. In some instances, the choHnesterase moiety will be in a "monomer" form, wherein a single expression of the corresponding peptide is organized into a discrete unit. In other instances, the cholinesterase moiety will be in the form of a "dinaer" (e.g., a dimer of recombinant human butyrylcholinesterase) wherein two monomer forms of the protein are associated (e.g., by disulfide bonding) to each other. For example, in the context of a dimer of recombinant human butyrylcholinesterase, the dimer maybe in the form of two monomers associated to each other by a disulfide bond formed fi-om each monomer's Cys571 residue. [0068] In addition, precursor forms of a protein that has cholinesterase activity can be used. [0069] Truncated versions, hybrid variants, and peptide mimetics of any of the foregoing sequences can also serve as the cholinesterase moiety. Biologically active fiagments, deletion variEints, substitution variants or addition variants of any of the foregoing that maintain at least some degree of cholinesterase activity can also serve as a cholinesterase moiety. [0070] For any given peptide or protein moiety, it is possible to determine whether that moiety has cholinesterase activity. Various methods for in vitro cholinesterase enzymatic activity assays are described in the art. See, for example, Lockridge et al. (1978) J. Biol. Chetn. 253:361-366, Lockridge et al. (1997) Biochemistry 36:786-795, Plattborze et al. (2000) Biotechnol. Appl. Biochem. 31:226-229, and Blong et al. (1997) Biochem. J. 121:1^1-151. Samples can be tested for the presence of enzymatically active cholinesterase activity by using the activity assay of Ellman [EUman et al. (1961) Biochem. Pharmacol. 7:88]. Levels of cholinesterase activity can be estimated by staining non-denaturing 4-30% polyacrylamide gradient gels with 2 mM echothiophate iodide as substrate (as described in Lockridge et al., supra), where this method is a modification of the same assays using 2 mM butrylythiocholine as substrate [from Kamovsky et al. (1964) J. Histochem. Cytochem. 12:219]. Using these methods, the catalytic properties of a moiety of interest, including Km, Vmax, and kcat values, can be determined using butyrylthiocholine or acetylthiocholine as substrate. Other methodologies known in the art can also be used to assess cholinesterase function, including electrometry, spectrophotometry, chromatography, and radiometric methodologies. - 1 6 - 10071] The Water-Soluble Polymer [0072] As previously discussed, each conjugate comprises a cholinesterase moiety attached to a water-soluble polymer. With respect to the water-soluble polymer, the water-soluble polymer is nonpeptidic, noiitoxic, non-naturally occurring and biocompatible. With respect to biocompatibility, a substance is considered biocompatible if the beneficial effects associated with use of the substance alone or with another substance (e.g., an active agent such as an cholinesterase moiety) in connection with living tissues (e.g., administration to a patient) outweighs any deleterious effects as evaluated by a clinician, e.g., a physician. With respect to non-immunogenicity, a substance is considered non-immunogenic if the intended use of the substance in vivo does not produce an imdesired immune response (e.g., the formation of antibodies) or, if an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician. It is particularly preferred that the nonpeptidic water-soluble polymer is biocompatible and non-inmnmogenic. [0073] Further, the polymer is typically characterized as having fi^m 2 to about 300 termini. Examples of such polymers include, but are not limited to, pol3^alkylene glycols) such as polyethylene glycol ("PEG"), poly(propyIene glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrroUdone), poly(hydroxyatkylmethacrylamide), poly(hydroxyaIkyImethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines ("POZ") (which are described in WO 2008/106186), poly(N-acryloylmorpholine), and combinations of any of the foregoing. [0074] The water-soluble polymer is not limited to a particular structure and can be linear (e.g., an end capped, e.g., alkoxy PEG or a bifunctional PEG), branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core), a dendritic (or star) architecture, each with or without one or more degradable linkages. Moreover, the internal structure of the water-soluble poljoner can be organized in any number of different repeat patterns and can be selected from the group consisting of homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer. [0075] Typically, activated PEG and other activated water-soluble polymers (i.e., polymeric reagents) are activated with a suitable activating group appropriate for coupling to a desired site on the cholinesterase moiety. Thus, a polymeric reagent will possess a reactive - 17- group for reaction with the choHnesterase moiety. Representative polymeric reagents and methods for conjugating these polymers to an active moiety are knov/n in the art and further described in Zalipsky, S., et al., "Use of FunctionalizedPoly(Ethylene Glycols) for Modification of Polypeptides" in Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, Plenus Press, New York (1992), and in Zalipsky (1995) Advanced Drug Reviews 16:157-182. Exemplary activating groups suitable for coupling to a cholinesterase moiety include hydroxyl, maleimide, ester, acetal, ketal, amine, carboxyl, aldehyde, aldehyde hydrate, ketone, vinyl ketone, thione, thiol, vinyl sulfone, hydrazine, among others. [0076] Typically, the weight-average molecular weight of the water-soluble polymer in the conjugate is from about 100 Daltons to about 150,000 Daltons. Exemplary ranges, however, include weight-average molecular weights in the range of greater than 5,000 Daltons to about 100,000 Daltons, in the range of from about 6,000 Daltons to about 90,000 Daltons, in the range of from about 10,000 Daltons to about 85,000 Daltons, in the range of greater than 10,000 Daltons to about 85,000 Daltons, in the range of from about 20,000 Daltons to about 85,000 Daltons, m the range of from about 53,000 Daltons to about 85,000 Daltons, in the range of from about 25,000 Daltons to about 120,000 Daltons, in the range of from about 29,000 Daltons to about 120,000 Daltons, in the range of from about 35,000 Daltons to about 120,000 Daltons, and in the range of from about 40,000 Daltons to about 120,000 Daltons. For any given water-soluble polymer, PEGs having a molecular weight in one or more of these ranges are preferred. [0077] Exemplary weight-average molecular weights for the water-soluble polymer include about 100 Daltons, about 200 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1,000 Daltons, about 1,500 Daltons, about 2,000 Daltons, about 2,200 Daltons, about 2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons, about 4,400 Daltons, about 4,500 Daltons, about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 55,000 Daltons, about 60,000 Daltons, about - 1 8 - 65,000 Daltons, about 70,000 Daltons, and about 75,000 Daltons. Branched versions of the water-soluble polymer (e.g., a branched 40,000 Dalton water-soluble polymer comprised of two 20,000 Dalton polymers) having a total molecular weight of any of the foregoing can also be used. In one or more embodiments, the conjugate will not have any PEG moieties attached, either directly or indirectly, with a PEG having a weight average molecular weight of less than about 6,000 Daltons. [0078] When used as the polymer, PEGs will typically comprise a nimiber of (OCH2CH2) monomers [or (CH2CH2O) monomers, depending on how the PEG is defined]. As used throughout the description, the number of repeating units is identified by the subscript "n" in "(OCH2CH2)n." Thus, the value of (n) typically falls within one or more of the following ranges: from 2 to about 3400, from about 100 to about 2300, from about 100 to about 2270, from about 136 to about 2050, from about 225 to about 1930, from about 450 to about 1930, from about 1200 to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to about 2730, from about 795 to about 2730, from about 909 to about 2730, and from about 1,200 to about 1,900. For any given polymer in which the molecular weight is known, it is possible to determine the number of repeating units (i.e., "n") by dividing the total weight-average molecular weight of the polymer by the molecular weight of the repeating monomer. [0079] One particularly preferred polymer for use in the invention is an end-capped polymer, that is, a polymer having at least one terminus capped with a relatively inert group, such as a lower Ci-6 alkoxy group, although a hydroxyl group can also be used. When the polymer is PEG, for example, it is preferred to use a methoxy-PEG (commonly referred to as mPEG), which is a linear form of PEG wherein one terminus of the polymer is a methoxy (-OCH3) group, while the other terminus is a hydroxyl or other functional group that can be optionally chemically modified. [0080] In one form usefiil in one or more embodiments of the present invention, free or unboimd PEG is a linear polymer terminated at each end with hydroxyl groups: HO-CH2CH20-(CH2CH20)„-CH2CH2-OH, wherein (n) typically ranges from zero to about 4,000. - 1 9 - (0081) The above polymer, alpha-, omega-dihydroxylpoly(ethylene glycol), can be represented in brief form as HO-PEG-OH where it is understood that the -PEG- symbol can represent the following stmctviral imit: -CH2CH20-(CH2CH20)„-CH2CH2-, wherein (n) is as defined as above. [0082] Another type of PEG useful in one or more embodiments of the present invention is methoxy-PEG-OH, or mPEG in brief, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group. The structure of mPEG is given below. CH30-CH2CH20-(CH2CH20)n-CH2CH2-OH wherein (n) is as described above. [0083J Multi-armed or branched PEG molecules, such as those described in U.S. Patent No. 5,932,462, can also be used as the PEG polymer. For example, PEG can have the structure: polya—P R"—C ( pofyb—Q wherein: polya and polyb are PEG backbones (either the same or different), such as methoxy poly(ethylene glycol); R" is a nonreactive moiety, such as H, methyl or a PEG backbone; and P and Q are nonreactive linkages. In a preferred embodiment, the branched PEG polymer is methoxy poly(eihylene glycol) disubstituted lysine. Depending on the specific cholinesterase moiety used, the reactive ester fimctional group of the disubstituted lysine may be fiirther modified to form a fimctional group suitable for reaction with the target group within the cholinesterase moiety. [0084] In addition, the PEG can comprise a forked PEG. An example of a forked PEG is represented by the following structure: Z / PEG-X-CH Z - 2 0 - wherein: X is a spacer moiety of one or more atoms and each Z is an activated terminal group linked to CH by a chain of atoms of defined length. International Patent Application Publication WO 99/45964 discloses various forked PEG structures capable of use in one or more embodiments of the present invention. The chain of atoms linking the Z functional groups to the branching carbon atom serve as a tethering group and may comprise, for example, alkyl chains, ether chains, ester chains, amide chains and combinations thereof. [0085] The PEG polymer may comprise a pendant PEG molecule having reactive groups, such as carboxyl, covalently attached along the length of the PEG rather than at the end of the PEG chain. The pendant reactive groups can be attached to the PEG directly or through a spacer moiety, such as an alkylene group. [0086] In addition to the above-described forms of PEG, the polymer can also be prepared with one or more weak or degradable linkages in the polymer, including any of the above-described polymers. For example, PEG can be prepared with ester linkages in the polymer that eire subject to hydrolysis. As shown below, this hydrolysis results in cleavage of the polymer into fragments of lower molecular weight: -PEG-CO2-PEG-+ H2O *" -PEG-CO2H + HO-PEG- [0087] Other hydrolytically degradable linkages, useful as a degradable linkage within a polymer backbone and/or as a degradable linkage to a cholinesterase moiety, include: carbonate linkages; imine linkages resulting, for example, from reaction of an amine and an aldehyde (see, e.g., Ouchi et al. (1997) Polymer Preprints 38(l)"-582-3); phosphate ester linkages formed, for example, by reacting an alcohol with a phosphate group; hydrazone linkages which are typically formed by reaction of a hydrazide and an aldehyde; acetal linkages that are typically formed by reaction between an aldehyde and an alcohol; orthoester linkages that are, for example, formed by reaction between a formate and an alcohol; amide linkages formed by an amine group, e.g., at an end of a polymer such as PEG, and a carboxyl group of another PEG chain; urethane linkages formed from reaction of, e.g., a PEG with a terminal isocyanate group and a PEG alcohol; peptide linkages formed by an amine group, e.g., at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by, for example, a phosphoramidite group, e.g., at the end of a polymer, and a 5' hydroxyl group of an oligonucleotide. - 2 1 - [0088] Such optional features of the conjugate, i.e., the introduction of one or more degradable linkages into the polymer chain or to the cholinesterase moiety, may provide for additional control over the final desired pharmacological properties of the conjugate upon administration. For example, a large and relatively inert conjugate (i.e., having one or more high molecular weight PEG chains attached thereto, for example, one or more PEG chains having a molecular weight greater than about 10,000, wherein the conjugate possesses essentially no bioactivity) may be administered, which is hydrolyzed to generate a bioactive conjugate possessing a portion of the original PEG chain. In this way, the properties of the conjugate can be more effectively tailored to balance the bioactivity of the conjugate over time. [0089] The water-soluble polymer associated with the conjugate can also be "cleavable." That is, the water-soluble polymer cleaves (either through hydrolysis, enzymatic processes, or otherwise), thereby resulting in the imconjugated cholinesterase moiety. In some instances, cleavable polymers detach from the cholinesterase moiety in vivo without leaving any fragment of the water-soluble polymer. In other instances, cleavable polymers detach from the cholinesterase moiety in vivo leaving a relatively small fragment (e.g., a succinate tag) from the water-soluble polymer. An exemplary cleavable polymer includes one that attaches to the cholinesterase moiety via a carbonate linkage. [0090] Those of ordinary skill in the art will recognize that the foregoing discussion concerning nonpeptidic and water-soluble polymer is by no means exhaustive and is merely illustrative, and that all polymeric materials having the qualities described above are contemplated. As used herein, the term "polymeric reagent" generally refers to an entire molecule, which can comprise a water-soluble polymer segment and a flmctional group. [0091] As described above, a conjugate of the invention comprises a water-soluble polymer covalently attached to a cholinesterase moiety. Typically, for any given conjugate, there will be one to three water-soluble polymers covalently attached to one or more moieties having cholinesterase activity. In some instances, however, the conjugate may have 1, 2, 3,4, 5, 6, 7, 8 or more water-soluble polymers individually attached to a cholinesterase moiety. Any given water-soluble polymer may be covalently attached to either an amino acid of the cholinesterase moiety, or, when the cholinesterase moiety is (for example) a glycoprotein, to a carbohydrate of the cholinesterase moiety. Attachment to a carbohydrate may be carried out, e.g., using metabolic functionalization employing sialic acid-azide chemistry [Luchansky et al. - 2 2 - (2004) Biochemistry 43(38): 12358-123661 or other suitable approaches such as the use of glycidol to facilitate the introduction of aldehyde groups [Heldt et al. (2007) European Journal of Organic Chemistry 32:5429-5433]. [0092] The particular linkage within the moiety having cholinesterase activity and the polymer depends on a number of factors. Such factors include, for example, the particular linkage chemistry employed, the particular cholinesterase moiety, the available functional groups within the cholinesterase moiety (either for attachment to a polymer or conversion to a suitable attachment site), the presence of additional reactive functional groups within the cholinesterase moiety, and the like. [0093] The conjugates of the invention can be, although not necessarily, prodrugs, meaning that the linkage between the polymer and the cholinesterase moiety is hydrolytically degradable to allow release of the parent moiety. Exemplary degradable linkages include carboxylate ester, phosphate ester, thiolester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides. Such linkages can be readily prepared by appropriate modification of either the cholinesterase moiety (e.g., the carboxyl group C terminus of the protein, or a side chain hydroxyl group of an amino acid such as serine or threonine contained within the protein, or a similar functionality within the carbohydrate) and/or the polymeric reagent using coupling methods commonly employed in the art. Most preferred, however, are hydrolyzable linkages that are readily formed by reaction of a suitably activated polymer with a non-modified fimctional group contained within the moiety having cholinesterase activity. [0094] Alternatively, a hydrolj^cally stable linkage, such as an amide, urethane (also known as carbamate), amine, thioether (also known as sulfide), or urea (also known as carbamide) linkage can also be employed as the linkage for coupling the cholinesterase moiety. Again, a preferred hydrolytically stable linkage is an amide. In one approach, a water-soluble polymer bearing an activated ester can be reacted with an amine group on the cholinesterase moiety to thereby result in an amide linkage. [0095] The conjugates (as opposed to an unconjugated cholinesterase moiety) may or may not possess a measmable degree of cholinesterase activity. That is to say, a polymer-cholinesterase moiety conjugate in accordance with the invention will possesses anywhere from about 0.1% to about 100% of the bioactivity of the unmodified parent - 2 3 - cholinesterase moiety. In some instances, the polymer-cholinesterase moiety conjugates may have greater than 100% bioactivity of the unmodified parent cholinesterase moiety. Preferably, conjugates possessing little or no cholinesterase activity contain a hydrolyzable linkage coimecting the polymer to the moiety, so that regardless of the lack (or relatively lack) of activity in the conjugate, the active parent molecule (or a derivative thereof) is released upon aqueous-induced cleavage of the hydrolyzable linkage. Such activity may be determined using a suitable in-vivo or in-vitro model, depending upon the known activity of the particular moiety having cholinesterase activity employed. [0096] For conjugates possessing a hydrolytically stable linkage that couples the moiety having cholinesterase activity to the polymer, the conjugate will typically possess a measurable degree of bioactivity. For instance, such conjugates are typically characterized as having a bioactivity satisfying one or more of the following percentages relative to that of the unconjugated cholinesterase moiety: at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 100%, and more than 105% (when measured in a suitable model, such as those well known in the art). Preferably, conjugates having a hydrolytically stable linkage (e.g., an amide linkage) will possess at least some degree of the bioactivity of the immodified parent moiety having cholinesterase activity. [0097] Exemplary conjugates in accordance with the invention will now be described wherein the cholinesterase moiety is a protein. Typically, such a protein is expected to share (at least in part) a similar amino acid sequence as the sequence provided in SEQ ID NO: 1 or SEQ ID NO 2. Thus, while reference will be made to specific locations or atoms within SEQ ID NOS: 1 or 2, such a reference is for convenience only and one having ordinary skill in the art will be able to readily determine the corresponding location or atom in other moieties having cholinesterase activity. In particular, the description provided herein for native hiunan cholinesterase is often applicable to firagments, deletion variants, substitution variants or addition variants of any of the foregoing. [0098] Amino groups on cholinesterase moieties provide a point of attachment between the cholinesterase moiety and the water-soluble polymer. Using the amino acid sequence provided in SEQ ID NOs: 1 through 2, it is evident that there are several lysine residues in each -24- having an e-amino acid that may be available for conjugation. Fiirther, the N-terminai amine of any protein can also serve as a point of attachment. [0099] There are a number of examples of suitable polymeric reagents useful for forming covalent linkages with available amines of a cholinesterase moiety. Specific examples, along with the corresponding conjugate, are provided in Table 1, below. In the table, the variable (n) represents the number of repeating monomeric units and "-NH-(ChE)" represents the residue of the cholinesterase moiety following conjugation to the polymeric reagent. While each polymeric portion [e.g., (OCH2CH2)n or (CH2CH20)„] presented in Table 1 terminates in a "CH3" group, other groups (such as H and benzyl) can be substituted therefor. Table 1 Amine-Selective Polymeric Reagents and the Cholinesterase Moiety Conjugate Formed ^ Therefrom _^^^^^^_^____ Polymeric Reagent Corresponding Conjugate g __ = o « / * * N II H3CO-(CH2CH20)n-C~N J HsCO-CCHzCHzO^-C-NH-CChE) ^^***^ Carbamate Linkage mPEG-Oxycarbonylimidazole Reagents O O 11 / = \ II H3CO-(CH2CH20)n-C-0-4 >-N02 H3CO-(CH2CH20)n-C-NH-(ChE) mPEG Nitrophenyl Reagents CI O H3C0-(CH2CH2O)„4-O-^-Cl H3CO-(CH2CH20)„-ll-NH-(ChE) CI Carbamate Linkage mPEG-Trichlorophenyl Carbonate Reagents _ _ H3C-{OCH2CHz)n-0-CH2-C-0-N J H3C-(OCH2CH2)n-0-CH2-C-N-(ChE) ° Amide Linkage mPEG-Succinunidyl Reagents T3 5 O ^ ? ° r-A. II II / ^ (Q£)—m--c-CHjCi^-(OCJ«>yi,-oo«>trC—»t-{ChE) [ N-0-C-CH2CH2-(OCH2CH2)„-aCH2CH2-&0-N I ^ia.—TZV-!~ Homobifunctional PEG-Succinimidyl Reagents Amide Linkages HN-^NH o o '"iJ'» o ^(CH,),-NH-CH2CH2^0CH,CH2)„-OCH,CH,i!oNf;] Q-(CH,,,-NH^H,CH,^OCH,CH,,„-OCH,CH,CNH-(ChE) Heterobifimctional PEG-Succinimidyl Reagents - 2 5 - Polymeric Reagent Corresponding Conjugate H3C-(OCH2CH2)n-0-CH2CH2-C-ON J H3C-(OCH2CHj)„-0-CH,CH2-C-NH-(ChE) ^^r Amide Linkage O mPEG-SuccLnimidyl Reagents O^ GO o o V-^ " " II II / ^ H3CO-(CH2CH20)n-CH2CH2NH-C-CH2CH2-C-NH-(ChE) H3CO-{CH2CH20)n-CH2CH2NH-C-CH2CH2-CO-N J O Amide Linkage mPEG-Succinimdyl Reagents ^ O o O V II II / ^ H3CO-(CH2CH20)n-CH2CH2SH-CH2CH2-C-NH-(ChE) H3C0-(CH2CH2O)n-CH2CH2SH-CH2CH2-C-O-N J O mPEG Succinimidyl Reagents Amide Linkage H3C-(OCH2CH2)n-0-CH2CH2CH2-C-0-N I || y ^ H3q.1j-(CCHjCH)n-<><>V>V>tCH2-NH HCCH2CH2CH2-(OCH2CH2)n-0-CH2CH2CH2-CH ^^ (^ Homobifimctional PEG Butyraldehyde Reagents Secondary Amine Linkages C5 il H3C-(0CH2CH2)„-CM:H2CH2CH2-CH2 NH (ChE) H3C-(OCH2CH2)n-0-CH2CH2CH2-CH I mPEG Butryaldehyde Reagents Secondary Amine Linkage - 2 8 - Polymeric Reagent Corresponding Conjugate o o o II II H H3C-(0CH2CH2)n-O-CNH-(CH2CH2O)4—CH2CH2CH2CH H3p-(OCH2CH2)„•<>C-^H-(C>l2C^y3)4-a^2C^tCH2CH2-NH (Chq mPEG Butryaldehyde Reagents Secondary Amine Linkage C-(0CH2CH2)„-O-CNH-(CH2CH20),-CH2CH2CH2CH J-f-H^-^H^^-—(CH.CH^U-CH,CH,CH,CH,-NH-v».-av:>viVH,-i*i KjCtOCHiCHjV-OONBOVCttOVCHz^ O O tWXOCH^vocw' 1 o CH-c-^*^-(CH^CH2q)cC^y>^2CH^CH «*E) H3C-(cx>y>y„-oci>« Secondary Amine Linkage Branched mPEG2 Butyraldehyde Reagents H3C-(0CH,CH,),-NH-C-0-CH, O O '«-l°°*'°«--"<-S-°-f' g I II It n HC-OCHjCHiCHa-C-NH-(CH,ClV>>,-CHjCHiCHjCHa-NH-(ChE) HC-OCHjCH2-CH,-C-NH-(CH2CHjO)4—CHjCHjCHzCH n I 0 | v^i" 2 • ! " i - H,C-|OCtV;HJ„-NH-C-0-CH, HjC-CCX^IjCHjJn-NH-C-O-CHj Secondary Amine Linkage Branched mPEG2 Butyraldehyde Reagents —OCH2CH3 H 3 C - ( O C H . C H a } „ - 0 - C H i H - O C H , C H 3 H3C-(OCH2CH2)„-0-CH3CH,-NH-(ChE) mPEG Acetal Reagents Secondary Amine Linkage O II /—v H3C-{OCH2CH2)n-C>CH2CH2-C-N )-NH-(ChE) H3C-(0CH2CH2)n-O-CH2CH2-C-N^^ >=0 V_y mPEG Piperidone Reagents Secondary Amine Linkage (to a secondary carbon) NH (ChE) O I ' ' II H3C-{OCH2CH2)n-0-(CH2)2-5—CH-CH3 H3C-(OCH2CH2)n-0-(CH2)2-5— C - C H3 „_ „ , , ^, „ ^ T5 . secondary amine linkage mPEG Methylketone Reagents ,^ •' , , ". (to a secondary carbon) O II H3CO-(CH2CH20)n-CH2CH2-NH—(ChE) H3CO-(CH2CH20)n-S-CH2-CF3 O mPEG Tresylate Reagents Secondary Amine Linkage - 2 9 - Polymeric Reagent Corresponding Conjugate / | 1 /^">—NI+(ChE) H3C-(0CH2CH2)n-O-CH2CH2-N \\ H3C-(OCH2CH2)n-CM>l2CH2—N I O O mPEG Maleimide Reagents „ j A • T - i , . . . ,. . , , „^ Secondary Amme Linkage (under certain reaction conditions such as pH > 8) j j _ _ ? / ^ 9 AVNH-(ChE) H3C-(0CH2CH2)n-O-CH2CH2-NH-C-CH2CH2-N | H3C-(OCHjCHj)„^CHjCHj-NH-C-CHaCH,-N I mPEG Maleimide Reagents (under certain reaction conditions such as pH > 8) Secondary Amine Linkage H3C-(OCH2CH2)„-0-CH2CH2-C-NH-CH2CH2-NH-C-CH2CH2-N | H3C-(OCHjCH3X,-0-CHjCHj-C-NH-CH3CH2-NlfC-CH,CH,-N^| . y ° mPEG Maleimide Reagents (under certain reaction conditions such as pH > 8) Secondary Amine Linkage NH-CHjCHj-NH-C-CHzCHj-N H NH<:Hpt-'*l-c-aipH,-w J o=c V °=? y O CH, ° If ?^ H3C-(0CH2CH2)„-O-CH2CH2-C-NH-| ^ i^^T?"™ if-nz ^ CH, f Pt, NH-CH2CH2-NH-C-CH2CH2-N I y^ o Secondary Amine Linkages mPEG Forked Maleimide Reagents (under certain reaction conditions such as pH > 8) D o H 3 C - ( O C H 2 C H 2 ) „ - 0 - ( I N H Hj:-l(X»f»,),00*M CH2 ?"= I ^ CHj 9^2 CHj ' O. l u f f /-T-NH-(aiE) CH2 o O 7"-^ O C C-NM-CKjCHj-NH-C-CHjCHj-N O CH—C-NH-CHzCHa-NH-C-CHzCHj-N |l HjC-loCHjCHzJu-OC-NH ^ H3C-(ocH2CH2)„-o-c^H )< Sccondaiy Amine Linkage branched mPEG2 Maleimide Reagents (under certain reaction conditions such as pH > 8) O OH / \ I H3C-(OCH2CH2)n-0-CH2CHCH2 H3C-(OCH2CH2)„-0-CH2CHCH2-NH-(ChE) mPEG Epoxide Reagents Secondary Amine Linkage (under certain reaction conditions such as pH > 8) [0100] Conjugation of a polymeric reagent to an amino group of a cholinesterase moiety can be accomplished by a variety of techniques. In one approach, a cholinesterase moiety can be conjugated to a polymeric reagent functionalized with a succinimidyl derivative (or other activated ester group, wherein approaches similar to those described for these - 3 0 - altemative activated ester group-containing polymeric reagents can be used). In this approach, the polymer bearing a succLoimidyl derivative can be attached to the cholinesterase moiety in an aqueous media at a pH of 7 to 9.0, although using different reaction conditions (e.g., a lower pH such as 6 to 7, or different temperatures and/or less than 15 °C) can result in the attachment of the polymer to a different location on the cholinesterase moiety. La addition, an amide linkage can be formed by reacting an amine-terminated nonpeptidic, water-soluble polymer with a cholinesterase moiety bearing an activating a carboxylic acid group. [0101] An exemplary conjugate comprises the following structure O II H3CO-(CH2CH20)„-X-CH-C-NH-(ChE) R1 wherein: (n) is an integer having a value of from 2 to 4000; X is a spacer moiety; R' is an organic radical; and ChE is a residue of a cholinesterzise moiety. [0102] Another exemplary conjugate of the present invention comprises the following structure: O II H3CO-(CH2CH20)n-CH2-CH-C-NH-(ChE) CH3 wherein (n) an integer having a value of from 2 to 4000 and ChE is a residue of a cholinesterase moiety. [0103] Typical of another approach useful for conjugating the cholinesterase moiety to a polymeric reagent is use of reductive amination to conjugate a primary amine of a cholinesterase moiety with a polymeric reagent functionalized with a ketone, aldehyde or a hydrated form thereof (e.g., ketone hydrate, aldehyde hydrate). In this approach, the primary amine fit)m the cholinesterase moiety reacts with the carbonyl group of the aldehyde or ketone (or the corresponding hydroxyl-containing group of a hydrated aldehyde or ketone), thereby forming a Schiffbase. The Schiff base, in timi, can then be reductively converted to a stable conjugate through use of a reducing agent such as sodium borohydride. Selective reactions (e.g., at the N-tenninus) are possible, particularly with a polymer functionalized with a ketone - 3 1 - or an alpha-methyl branched aldehyde and/or under specific reaction conditions (e.g., reduced PH). [0104] Exemplary conjugates of the invention wherein the water-soluble polymer is in a branched form include those wherein the water-soluble polymer comprises the following structure: O H3CO-(CH2CH20)n—CH2CH2-NH-C-Oo -o- H3CO-{CH2CH20)n-CH2CH2-NH-C-0- wherein each (n) is independently an integer having a value of from 2 to 4000. [0105] Exemplary conjugates of the invention comprise the following structure: O H3CO-(CH2CH20)n-CH2CH2-NH-C-0-i FRJ I Q -0-X-(CH2CH20)b-C--NH-(ChE) H3C0-(CH2CH20)n-CH2CH2-NH-C-O-' [H J j, wherein: each (n) is independently an integer having a value of from 2 to 4000; X is spacer moiety; (b) is an integer having a value 2 through 6; (c) is an integer having a value 2 through 6; R'^, in each occurrence, is independently H or lower alkyl; and ChE is a residue of a cholinesterase moiety. [0106] An exemplary conjugate of the invention comprises the following structure: o H3CO-{CH2CH20)„—CHaCHj-NH-C-O-i Q •• Q k-OCH2CH2CH2-C-NH-(CH2CH20)4-CH2CH2CH2CH2-(ChE) H3CO-(CH2CH20)n—CH2CH2-NH-C-O-J wherein: each (n) is independently an integer having a value of from 2 to 4000; and ChE is a residue of a cholinesterase moiety. [0107] Another exemplary conjugate of the invention comprises the following structure: - 3 2 - O H3CO-(CH2CH20)n-CH2CH2-NH-C-On FR^I O I 11 Q -0-(X)a-(CH2CH20)b-- -C- -C-NH-(ChE) H3CO-(CH2CH20)n-CH2CH2-NH-C-0-' LR^JC wherein: each (n) is independently an integer having a value of from 2 to 4000; (a) is either zero or one; X, when present, is a spacer moiety comprised of one or more atoms; (b') is zero or an integer having a value of one through ten; (c) is an integer having a value of one through ten; R'^, in each occurrence, is independently H or an organic radical; R^, in each occurrence, is independently H or an organic radical; and ChE is a residue of a cholinesterase moiety. [0108] An exemplary conjugates of the invention comprises the following structure: O H3CO-(CH2CH20)n-CH2CH2~NH-C-0-i O Q -0-CH2CH2CH2C-NH-(ChE) H3CO-(CH2CH20)n-CH2CH2-NH-C-0- wherein: each (n) is independently an integer having a value of from 2 to 4000; and ChE is a residue of cholinesterase moiety. [0109] Carboxyl groups represent another functional group that can serve as a point of attachment on the cholinesterase moiety. Structurally, the conjugate will comprise the following: O II (ChE)-C-X-POLY where (ChE) and the adjacent carbonyl group corresponds to the carboxyl-containing cholinesterase moiety, X is a Unkage, preferably a heteroatom selected from O, N(H), and S, and POLY is a water-soluble polymer such as PEG, optionally terminating in an end-capping moiety. [0110] The C(0)-X linkage results from the reaction between a polymeric derivative bearing a terminal fimctional group and a carboxyl-containing cholinesterase moiety. As - 3 3 - discussed above, the specific linkage will depend on the type of functional group utilized. If the polymer is end-functionalized or "activated" with a hydroxyl group, the resulting linkage will be a carboxylic acid ester and X will be O. If the polymer backbone is fiinctionalized with a thiol group, the resulting linkage will be a thioester and X will be S. When certain multi-arm, branched or forked polymers are employed, the C(0)X moiety, and in particular the X moiety, may be relatively more complex and may include a longer linkage structure. [0111] Water-soluble derivatives containing a hydrazide moiety are also useful for conjugation at a carbonyl and carboxylic acid. To the extent that the cholinesterase moiety does not contain a carbonyl moiety or a carboxylic acid, one can be added using techniques known to one of ordinary skill in the art. For example, a carbonyl moiety can be introduced by reducing a carboxylic acid (e.g., the C-terminal carboxylic acid) and/or by providing glycosylated or glycated (wherein the added sugars have a carbonyl moiety) versions of the cholinesterase moiety. With respect to cholinesterase moieties containing a carboxylic acid, a PEG-hydrazine reagent can, in the presence of a coupling agent (e.g., DCC), covalently attach to the cholinesterase moiety [e.g., mPEG-OCH2C(0)NHNH2 + HOC(0)-(ChE) results in niPEG-0CH2C(0)NHNHC(0)-ChE]. Specific examples of water-soluble derivatives containing a hydrazide moiety, along wifli the corresponding conjugates, are provided in Table 2, below. In addition, any water-soluble derivative containing an activated ester (e.g., a succinimidyl group) can be converted to contain a hydrazide moiety by reacting flie water-soluble polymer derivative containing the activated ester with hydrazine (NH2-NH2) or tert-butyl carbazate [NH2NHC02C(CH3)3]. In the table, the variable (n) represents the number of repeating monomeric units and "=C-(ChE)" represents the residue of the cholinesterase moiety following conjugation to the polymeric reagent. Optionally, the hydrazone linkage can be reduced using a suitable reducing agent. While each polymeric portion [e.g., (OCH2CH2)n or (CH2CH20)n] presented in Table 2 terminates in a "CH3" group, other groups (such as H and benzyl) can be substituted therefor. - 3 4 - Table 2 Carboxyl-Specific Polymeric Reagents and the Cholinesterase Moiety Conjugate Formed Therefrom Polymeric Reagent Corresponding Conjugate ^^ O O 11 II HaCCKObCHzO^CHzO^-C-NH-NHz H3CCKCH2CH20)nCH2CH2-C-NH-N=C-(ChE) mPEG-Hydrazine Reagents Hydrazone Linkage O O II II H3CCHCH2CH20)„CH2CH2-0-CH2-C-NH-NH2 H3CXHCH2CH20)nCH2CH2-0-CH2-C-NH-N=C-(ChE) mPEG-Hydrazine Reagents Hydrazone Lmkagc O II O H3C(>(CH2CH20)nCH2CH2-NH-C-NH-NH2 || H3CCKCH2CH20)nCH2CH2-NH-C-NH-N=C-(ChE) mPEG-Hydrazine Reagents Hydrazone Linkage O O II II htCCKCH2CH20)nCH2CH2-NH-NH-C-NH-NH2 H3CCKCH2CH20)nCH2CH2-NH-NH-C-NH-N=G{CH2CHP)„CH2CH2-NH-NH-C-NH-NH2 H3CC>{CH2CH20)nCH2CH2-NH-NH-C-NH-N=GtO)„CH2CH2-NH-C-NH-NH-C-NH-|«>{GC) mPEG-Hydrazine Reagents Hydrazone Linkage O O II 11 H3CCKCH^H20)nCH2CH2-0—C-NH-NHj H3a>(CH2CH20)hCH2CH2-0-C-NH-N=0(aiE) mPEG-Hydrazine Reagents Hydrazone Linkage - 3 5 - Polymeric Reagent Corresponding Conjugate O H300-{CH2CH2O)nCH2-C-NH-NH2 jj |f mPEG-Hydrazine Reagents H3C0KCH,CH,0)„CHa-C-NH NH-c-(ChE) ^ ^ I C(0)NHNHC(0) Linkage [0112] Thiol groups contained within the cholinesterase moiety can serve as effective sites of attachment for the water-soluble polymer. In particular, cysteine residues provide thiol groups when the cholinesterase moiety is a protein. The thiol groups in such cysteine residues can then be reacted with an activated PEG that is specific for reaction with thiol groups, e.g., an N-maleimidyl polymer or other derivative as described in U.S. Patent No. 5,739,208 and in WO 01/62827. In addition, a protected thiol may be incorporated into an oligosaccharide side chain of an activated glycoprotein, followed by deprotection with a thiol-reactive water-soluble polymer. [0113] Specific examples of reagents, along with the corresponding conjugate, are provided in Table 3, below. In the table, the variable (n) represents the number of repeating monomeric units and "-S-(ChE)" represents the cholinesterase moiety residue following conjugation to the water-soluble polymer. While each polymeric portion [e.g., (OCH2CH2)n or (CHiCiijOyn] presented in Table 3 terminates in a "CH3" group, other groups (such as H and benzyl) can be substituted therefor. [0114] With respect to SEQ ED NOs: 1 ihrough 2 corresponding to exemplary cholinesterase moieties, it can be seen that there are many thiol-containing cysteine residues. Thus, preferred thiol attachment sites are associated vwth one of these seven cysteine residues. Although it is preferred not to disrupt any disulfide bonds, it may be possible to attach a polymer within the side chain of one or more of these cysteine residues and retain a degree of activity. A preferred location to attach a water-soluble polymer is at the thiol-containing cysteine residue corresponding to Cys66 of SEQ ID NO: 2. In addition, it is possible to add a cysteioe residue to the cholinesterase moiety using conventional synthetic techniques. See, for example, the procedure described in WO 90/12874 for adding cysteine residues, wherein such procedure can be adapted for a cholinesterase moiety. In addition, conventional genetic engineering processes can also be used to introduce a cysteine residue into the cholinesterase - 3 6 - moiety. In some embodiments, however, it is preferred not to introduce an additional cysteine residue and/or thiol group. Table 3 Thiol-S elective Polymeric Reagents and the Cholinesterase Moiety Conjugate Formed Therefrom Polymeric Reagent Corresponding Conjugate HsC-COCHzCHaJn-O-CHzCHa—N J H3C-(OCH2CH2)n-0-CH2CH2—N mPEG Maleimide Reagent Thioether Linkage H3CO-(CH2CH20)„-CH2CH2CHj-N || H3CO-(CH2CH20)n-CH2CH2CH2-N J mPEG. Maleimide Reagent Thioether Linkage H,CO-(CH^H^).-C-NH-CH^(y3CH^HpCHjCHjNH-C-CH^H,CH,-N H H,CO-{CHjCMjO),-C-Nrt-CH2CH,OCHjCH20CHjCHjNHCCH,CH:^H,-N J mPEG Maleimide Reagent ^, . , ^ . , Thioether Lmkage ^ ^ (ChE)-S-r^ 7^S-(ChE) 1 N—{CHjCHjOJn-CHzCHj—N J I N—(CHjCH20)„-CH2CHj—N I Homobiflmctionjd mPEG Maleimide Thioether Linkages Reagent S 7^ O /-l-S-(ChE) H3C-(OCH2CH2)n-0-CH2CH2-NH-C-CH2CH2-N | H3C-{0CH2CH2)„-O-CH2CH2-NH-C-CH2CH2-N I mPEG Maleimide Reagent Thioether Lmkage mPEG Maleimide Reagent " Thioether Linkage . o - ^ o f . - - " - - o — p - . h i . o ^ o f N ^ ° ^ o — ; r — N ^ ^ ^ ^ ^ ' o o mPEG Maleimide Reagent | Thioether Linkage -37- Polymeric Reagent Corresponding Conjugate o=c V 0=9 V NH-CH2CH2-NH-C-CH2CH2-N J t*l-CH2CHi-NH-C-CH2CHj-r^ mPEG Forked Maleimide Reagent Thioether Linkage H]C-(0CH2CH2l,-O-C-NH H5C-(0CHjCH2l,-O-C-NH ?"» CHj ?"» CH2 ?"' O 9"^ O CH-C-NH-CH2CH2-KH-C-CH2CH2-N 1] ^ i-S-NH-CH^CH^^JH-C-CKbCH^-N''^ J ^ " ' " "^ branched mPEG2 Maleimide Reagent Thioether Linkage o o HjC-(OCH,CHjl,-NH-C-0-CHj O O °^^ H5C-(0CH^H,)„-NH-C-O-CH, O O °V HC-OCHJCHJ-CHJ-E-NH-CHJCHJ-NH-CCIVCHJ-NI HC-OCHjCHyCHj-C-NHCHjCHjNH-CCHjCHj-M 1 H,C-(OCH,CHjli,-NH-C-0-CH, O HJC-(0CH,CH2),-NH4-O-CH3 O branched mPEG2 Maleimide Reagent Thioether Linkage jj ^ NH-CH2CH2-NH-S-CH.CH.-NQ ^ N H - C H . C H 2 - N H - L H 2 C H 2 - N M ~ ^ " " ^' O CH-=-«H-^ I y CHj II / cK, o c—c-m^ H,C-(0CH2CH2),-OO«< l' O^ fl /" CH ° ^ ? / ^ K,C-(OCH2CH2)„-CW>NH | ^ O NH-CH2CH2-NH-C-CH2CH2-N J 0=C O / ^ S - ( C l £) i r NH-CHjCHj-NH-C-CiyiKz-N J Branched mPEG2 Forked Maleunide o^ Reagent Thioether Linkages tw^a^f^y[m. H3C0-(CH2CH2O)„-C-NH-CH2CHjOCH,CHj0CHjCHjNH-C-CH2CH:CHi-N | 30kDa PEG Bearing a Maleimide Group (n defined to provide a ~30kDa PEG) - 5 5 - ° 9 7^S-(ChE) H,CO-(CHjCH^)„-C-NH-CH,CH20CH2CH20CH3CHjNHCCHjCHjCHj-N J Example IB Conjugate (n defined to provide a ~301cDa PEG) [0176] The starting concentration of the butylcholinesterase protein stock solution was ±90 mg/mL and the protein was dissolved in a buffer containing 10 mM NaP04 (pH 7.4), 1 mM EDTA and 35 mM NaCl. One gram of protein (11 mL of the above stock solution) was diluted with 8.1 mL of dilution buffer (2 mM NaP04, 1 mM EDTA (pH 7.4)) such that the final protein concentration was 52-53 mg/mL. [0177] While stirring this protein solution, 0.74 mL of Tris buffer (IM Tris, pH 8.2) wEis added. This was followed by addition of 0.238 mL of Tris base (1 M Tris base). The resulting pH of the solution was pH 8.30. [0178] hi a separate container an amount of the PEG reagent, equal to 6 mol equivalents of the protein quantity, was dissolved in PEG dilution buffer (2 mM NaP04, 1 mM EDTA, pH 6.1) to a 16.7% (w/v) solution. While stirring the protein solution the PEG reagent solution was added to the diluted protein solution. This mixture is hereafter referred to as the PEGylation reaction. The PEGylation reaction was allowed to stir for six hours at room temperature (22 °C). [0179] After this time a second PEG reagent solution was made by dissolving an amoimt of PEG reagent equal to 4 mol equivalents of the protein quantity in PEG dilution buffer. While continuing to stir the PEGylation reaction this additional PEG reagent solution was added to the PEGylation reaction. The PEGylation reaction was allowed to stir for an additional 6-18 hours at room temperature (22 °C). The PEGylation reaction was then stored at 4 °C until the PEGylation products were purified, but usually within 48 hours. - 5 6 - Example IC PEGylation of rBChE with a Branched 40kDa PEG Bearing a Maleimide Group o HaC-COCHjCHjj^-NH-C-O-CHa O O °V_ HC-OCH^HjCHa-C-NH-CHjCHj-NH-CCHz-CHz-N j ) H3C-(OCH2CH2)n-NH-C-0-CH2 O Branched 40kDa PEG Bearing a Maleimide Group (each n defined to provide a ~20kDa PEG) o H3C-(0CHjCHa)„-NH-C-0-CHj O O °V I II n 7^S-(ChE) HC-OCHiCHj-CHj-C-NH-CHzCHz-NH-C-CHz-CHj—N J II I '^^ HjC-COCHjCHjJn-NH-C-O-CHj O Example IC Conjugate (each n defined to provide a ~20kDa PEG) [0180] The starting concentration of the butylcholinesterase protein stock solution was ±90 mg/mL and the protein was dissolved in a buffer containing 10 mM NaP04 (pH 7.4), 1 niM EDTA and 35 mM NaCl. One gram of protein (11 mL of the above solution) was diluted with 8.1 mL of dilution buffer (2 mM NaP04,1 mM EDTA (pH 7.4)) such that the final protein concentration was 52-53 mg/mL. [0181] While stirring this protein solution, 0.74 mL of Tris buffer (IM Tris, pH 8.2) was added. This was followed by addition of 0.238 mL of Tris base (1 M Tris base). The resulting pH of the solution was pH 8.30. [0182] In a separate container an amount of the PEG reagent, equal to 6 mol equivalents of the protein quantity, was dissolved in PEG dilution buffer (2 mM NaP04,1 mM EDTA, pH 6.1) to a 16.7% (w/v) solution. While stirring the protein solution the PEG reagent solution was added to the diluted protein solution. This mixture is hereafter referred to as the PEGylation reaction. The PEGylation reaction was allowed to stir for 6 hours at room temperature (22 °C). [0183] After this time a second PEG reagent solution was made by dissolving an amoxint of PEG reagent equal to 4 mol equivalents of the protein quantity in PEG dilution buffer. While continuing to stir the PEGylation reaction this additional PEG reagent solution was added to the PEGylation reaction. The PEGylation reaction was allowed to stir for an - 5 7 - additional 6-18 hours at room temperature (22 °C). The PEGylation reaction was then stored at 4 °C until the PEGylation products were purified, but usually within 48 hours. Example ID PEGylation of rBChE with a Branched 60kDa PEG Bearing a Maleimide Group o HaC-tOCHzCHzJn-NH-C-O-CHa n O °'!Sw I II II 7~Ti HC-OCHiCHz-CHz-C-NH-CHzCHz-NH-CCHz-CHa-N |j H3C-(OCH2CH2)„-NH-C-0-CH2 O Branched 60kDa PEG Bearing a Maleimide Group (each n defined to provide a ~30kDa PEG) o H3C-(OCHaCH2)„-NH-C-0-CH2 O O °V I H II A-T-S-(ChE) HC-OCHiCHz-CHj-C-NH-CHaCHj-NH-C-CHj-CHa-N J H3C-(OCH2CH2)„-NH-C-0-CH2 O Example ID Conjugate (each n defined to provide a ~30kDa PEG) [0184] The starting concentration of the butylcholinesterase protein stock solution was ±90 mg/mL and the protein w£is dissolved in a buflFer containing 10 mM NaP04 (pH 7.4), 1 mM EDTA and 35 mM NaCl. One gram of protein (11 mL of the above stock solution) was diluted with 8.1 mL of dilution buffer (2 mM NaP04, 1 mM EDTA (pH 7.4)) such that the final protein concentration was 52-53 mg/mL. [01851 While stirring this protein solution, 0.74 mL of Tris buffer (IM Tris, pH 8.2) was added. This was followed by addition of 0.2J8 mL of Tris base (1 M Tris base). The resulting pH of the solution was pH 8.30. [0186] In a separate container an amoimt of the PEG reagent, equal to 6 mol equivalents of the protein quantity, was dissolved in PEG dilution buffer (2 mM NaP04,1 mM EDTA, pH 6.1) to a 16.7% (w/v) solution. While stirring the protein solution the PEG reagent solution was added to the diluted protein solution. This mixture is hereafter referred to as the PEGylation reaction. The PEGylation reaction was allowed to stir for six hours at room temperature (22 °C). - 5 8 - [0187] After this time a second PEG reagent solution was made by dissolving an amount of PEG reagent equal to 4 mol equivalents of the protein quantity in PEG dilution buffer. While continuing to stir the PEGylation reaction this additional PEG reagent solution was added to the PEGylation reaction. The PEGylation reaction was allowed to stir for an additional 6-18 hours at room temperature (22 °C). The PEGylation reaction was then stored at 4 "C until the PEGylation products were purified, but usually within 48 hours. Example 2 Alternative PEGylation Conditions Achieving 65-70% PEGylation Yield Using mPEG-40k-Maleimide [0188] The reaction time for this PEGylation reaction was six days at 10 °C with stirring using a magnetic stir bar and plate. [0189] PEG reagent was added to a stock solution in batch mode with stirring, adding 1 mol equivalent of dry PEG reagent on each day as shown in the Table 4, below. [0190] To achieve a high level of PEGylation (i.e., > 65% PEGylation with respect to the monomer), the concentration of the protein was maintained at the highest possible level. Under these conditions, the reaction mixture was '"miDcy/cloudy" due to the formation of a reversible protein aggregate. However, if more than one mol equivalent of PEG was added without minimal dilution of the PEGylation reaction, aggregation was too great (determined empirically) and the PEGylation efficiency was reduced. Therefore, before the addition of dry PEG, the PEGylation reaction was diluted with buffer as shown in the Table 4, below. [0191] For the PEG reagent additions where reaction dilution buffer is also added, the PEG reagent could also be dissolved in the buffer before adding the mixture to the PEGylation reaction. [0192] The protein was dissolved in buffer containing 10 mM NaP04 (pH 7.3), 1 mM EDTA and 35 mM NaCl (reaction dilution buffer) at a starting concentration of 83 mg/mL and the reaction quantities below describe PEGylation of 20 grams of rhBChE protein in a starting volume of 241 mL. - 5 9 - [0193] Buffer and PEG reagent additions were made at room temperature according to the specifications set forth in Table 4. Table 4 Specificarion of Additions Day Reaction dilution PEG reagent buffer added (mL) added (grams) 0 0 10.35 1 160 10.35 2 160 10.35 3 160 10.35 4 160 10.35 5 0 10.35 6 Reaction stopped by storing at 4 °C [0194] After this reaction the PEGylated protein was purified as described in Example 3. Example 3 Purification of Di-PEGvlated Dimer [0195] The PEGylation method described in Examples 1A-ID and 2 generates a protein solution where 65 to 70% of the protein is PEGylated with respect to the monomer form of the protein. The PEGylation reaction was analyzed by RP-HPLC after the protein was reduced to monomer. However, the biological form desired from this reaction was the di-PEGylated dimer and the PEGylation reaction did not produce fully PEGylated dimer. The level of PEGylation could be marginally increased by further additions of PEG reagent, but ultimately the moderate increase in PEGylation would be offset by the increased cost of the PEG reagent. Analysis of reaction mixtures showed that approximately 50% of the reaction mixture was in the form of mono-PEGylated dimer and 45% in the form of di-PEGylated dimer. Furthermore, after pxorification, only 30 to 35% of di-PEGylated dimer could be recovered. The level of recovery would be too low for an economically viable process. [0196] A method is therefore described where substantially all the mono-PEGylated monomer form of the protein which was present in the mono-PEGylated dimer JBraction could - 6 0 - be recovered and converted to di-PEGylated dimer. This method enabled a total process yield ofatleast55 to60%. [0197] For purification of the 1 gram PEGylation reaction, a 22 mm diameter anion exchange column (Q-sepharose fast flow) was packed (using methods known to those skilled in the art) such that the bed volume would result in a 5 mg/mL protein loading. The colimm was equilibrated in chromatography buffer A (10 mM NaP04 (pH 7.8), 1 mM EDTA, 5 mM cysteine). [0198] The protein in the PEGylation reaction was reduced to the monomer form by addition of 1 PEGylation reaction volume of reducing buffer (10 mM NaP04 (pH 7.8), 1 mM EDTA, 20 mM cysteine) and incubating at room temperature for one hour. [0199] Thereafter, seven PEGylation reaction voliunes of chromatography buffer A and one PEGylation reaction volume of water were added resulting in a 10-fold dilution of the original PEGylation reaction. [0200] An appropriate chromatography instrament was programmed so that the entire diluted PEGylation reaction was loaded onto the column and any unbound proteins washed out with two column bed volumes of chromatography buffer A. Thereafter, several gradient steps were used to elute the PEGylated monomer of BChE. [0201] Gradient step 1 was a continuous gradient from 0 to 25 % chromatography buffer B (equivalent to buffer A but also containing 0.5 M NaCl) over 2.5 bed volumes. Fractions were collected. [0202] Gradient step 2 was a hold step at 25 % buffer B for 2.5 bed volumes. Fractions were collected. [0203] Gradient step 3 was a direct step to 100% B followed by a hold step at 100% B (gradient step 4) for 2 bed volxmies. Fractions were collected. [0204] The mono-PEGylated monomer eluted over a broad peak during gradient steps 1 and 2. The non-PEGylated monomer eluted during gradient step 4. [0205] The column was regenerated using standard methods. - 6 1 - 10206] Appropriate fiactions were pooled and buffer exchanged / concentrated using Tangential Flow Filtration (TFF) and standard methods as described by the manufacturer of the filtration units. [0207] The mono-PEGylated monomer solution was concentrated by TFF to a protein concentration of 25 mg/mL and seven buffer volume changes of TFF buffer (10 mM NaP04 (pH 7.5), 1 mM EDTA, 35 mM NaCl) were applied. The solution WEIS filter sterilized using a 0.22 [im filtration unit. [0208] At this point, the cysteine used to reduce the protein to monomer had been removed (by the TFF process) from the protein solution and incubation at room temperature for 48 hours followed by storage at 4 °C allowed the dimer form of the protein to regenerate. The final product was therefore the di-PEGylated dimer form of the protein. Example 4 PEGvlation of rChE with Branched mPEG-N-Hydroxysuccinimide Derivative, 40kDa H3C-(-OCH2CH2)-NH-C-0—I O ^ " O -OCH2CH2CH2-C-O-N J ] H3C-{-OCH2CH2)-NH-C-0—' )f n O [0209] PEGylation reactions are designed such that after addition of all the reaction components and buffers, the final rChE concentration is 2.5 mg/ml. PEG2-NHS, 40kDa, stored at -20 °C under argon, is warmed to ambient temperature. A quantity of the PEG reagent equal to 10-50 mol equivalents of the rChE to be PEGylated is weighed out and dissolved in 20mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12% reagent solution. The 12% PEG reagent solution is quickly added to the aliquot of stock rChE solution and stirred for 3 - 18 hours at room temperature to allow for coupling of the mPEG2-NHS to rChE via an amide linkage, resulting in a conjugate solution. The conjugate solution is quenched with a lysine solution (pH 7.5) such that the final lysine molar concentration is 10 — 100 times the PEG reagent molar concentration. [0210] mPEG2-NHS is found to provide a relatively large molecular volume of active N-hydroxysuccinimide ("NHS") ester, which selectively reacts with lysine and terminal amines. - 6 2 - [0211] Using this same approach, other conjugates are prepared using mPEG2-NHS having other weight average molecular weights. [0212] Conjugates using PEG2-NHS, 40kDa, were prepared substantially in accordance with the procedm-e set forth in this Example wherein PEG2-NHS, 40kDa, at a mol equivalent of 10, 25 and 50 was used in three separate attempts. The SDS-PAGE analysis of the resulting conjugate solutions is provided in FIG. 1. Example 5 PEGylation of rChE with Linear mPEG-Butyraldehyde Derivative. 30kDa p CHaO-AcHaCHzO^C-NH-^-CHaCHzO^CHaCHjCHzCHO Linear mPEG-Butyraldehyde Derivative, 30kDa ("mPEG-ButyrALD") [0213] PEGylation reactions are designed such that after addition of all the reaction components and buffers, the final rChE concentration is 2.5 mg/ml. mPEG-ButyrALD, 30kDa, stored at -20 °C imder argon, is warmed to ambient temperature. A quantity of the PEG reagent equal to 10 — 50 mol equivalents of the rChE to be PEGylated is weighed out and dissolved in 20mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12% reagent solution. The 12% PEG reagent solution is added to the aliquot of stock rChE solution and stirred for 15 - 30 minutes. A reducing agent, sodium cyanoborohydride (NaCNBHs), is then added at 10 - 100 molar excess relative to the PEG reagent anid the reaction stirred for 5 — 18 hours at room temperature to ensure coupling via a secondary amine linkage to thereby form a conjugate solution. [0214] The aldehyde group of mPEG-ButyrALD is found to react with the primary amines associated with rChE and covalently bond to them via secondary amine upon reduction by a reducing reagent such as sodium cyanoborohydride. [0215] Using this same approach, other conjugates are prepared using mPEG-BuryrALD having other weight average molecular weights. [0216] Conjugates using mPEG-ButyrALD, 30kDa,were prepared substantially in accordance with the procedure set forth in this Example wherein mPEG-ButyrALD, 30kDa, at - 6 3 - a mol equivalent of 10, 25 and 50 was used in three separate attempts. The SDS-PAGE analysis of the resulting conjugate solutions is provided in FIG. 1. Example 6 PEGylation of rChE with Branched mPEG-Butyraldehyde Derivative, 40kDa O H3C-(-OCH2CH2)-NH-C-0—I O " O —OCH2CH2CH2-C-NH-(-CH2CH20|-CH2CH2CH2CHO HaC-foCHzCHzJ-NH-C-O-J '^ Branched mPEG-Butyraldehyde Derivative, 40kDa ("mPEG2-ButyrALD") [0217] PEGylation reactions are designed such that after addition of all the reaction components and buffers, the final rChE concentration is 2.5 mg/ml. mPEG2-ButyrALD, 40kDa, stored at -20 °C under argon, is warmed to ambient temperature. A quantity of the PEG reagent equal to 10 - 50 mol equivalents of the rChE to be PEGylated is weighed out and dissolved in 20mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12% reagent solution. The 12% PEG reagent solution is added to the aliquot of stock rChE solution and stirred for 15 — 30 minutes. A reducing agent, sodium cyanoborohydiide (NaCNBHs), is then added at 10 - 100 molar excess relative to the PEG reagent and the reaction stirred for 5 — 18 hours at room temperature to ensure coupling via a secondary amine linkage to thereby form a conjugate solution. [0218] The aldehyde group of niPEG2-ButyrALD is found to react with the primary amines associated with rChE and covalently bond to them via secondary amine upon reduction by a reducing reagent such as sodium cyanoborohydride. [0219] Using this same approach, other conjugates are prepared using mPEG2-BuryrALD having other weight average molecular weights. [0220] Conjugates using mPEG2-ButyrALD, 40kDa, were prepared substantially in accordance with the procedure set forth in this Example wherein mPEG2-ButyrALD, 40kDa, at a mol equivalent of 10, 25 and 50 was used in three separate attempts. The SDS-PAGE analysis of the resulting conjugate solutions is provided in FIG. 1. - 6 4 - Example 7 PEGvIation of rChE with Linear mPEG-Succinimidyl g-Methylbutanoate Derivative, 30kPa o 1 CH304CH2CH20V-CH2CH2CH-C-0-N 7] o Linear mPEG-Succinimidyl a-Methylbutanoate Derivative, 30kDa ("mPEG-SMB") [0221] PEGylation reactions are designed such that after addition of all the reaction components and buffers, the final rChE concentration is 2.5 mg/ml. mPEG-SMB, 30kDa, stored at -20 °C under argon, is warmed to ambient temperature. A quantity of the PEG reagent equal to 10-50 mol equivalents of the rChE to be PEGylated is weighed out and dissolved in 20mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12% reagent solution. The 12% PEG reagent solution is added to the aliquot of stock rChE solution and stirred for 5 - 18 hoirrs at room temperature thereby resulting in a conjugate solution. The conjugate solution is quenched with a lysine solution (pH 7.5) such that the final lysine molar concentration is 10 - 100 times the PEG reagent molar concentration. [0222] The mPEG-SMB derivative is found to provide a sterically hindered active NHS ester, which selectively reacts with lysine and terminal amines. [0223] Using this same approach, other conjugates are prepared using mPEG-SMB having other weight average molecular weights. - 6 5 - Example 8 PEGvlation of rChE with mPEG-PIP, ZOkPa [0224] The basic structure of the polymeric reagent is provided below: O , . CH30-(CH2CH20)„CH2CH2-C-N Vo CH30-(CH2CH20)„CH2CH2-C-N }<°^ \ r °^ (hydrated foim) [0225] PEGylation reactions are designed such that after addition of all the reaction components and buffers, the final rChE concentration is 2.5 mg/ml. mPEG-PIP, 20kDa, stored at -20 °C under argon, is warmed to ambient temperature. A quantity of the PEG reagent equal to 10 — 50 mol equivalents of the iChE to be PEGylated is weighed out and dissolved in 20mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12% reagent solution. The 12% PEG reagent solution is added to the aliquot of stock rChE solution and stirred for 15 - 30 minutes. A reducing agent, sodium cyanoborohydride (NaCNBHs), is then added at 10 - 100 molar excess relative to the PEG reagent and the reaction stirred for 5 - 1 8 hours at room temperature to ensure coupling via a secondary amine linkage (to a secondary carbon) to thereby form a conjugate solution. The conjugate solution is quenched with a lysine solution (pH 7.5) such that the final lysine molar concentration is 10 - 100 times the PEG reagent molar concentration. [0226] The ketone group of mPEG-PIP is found to react with the primary imaines associated with rChE and covalently bond to them via a secondary amine upon reduction by a reducing reagent such as sodium cyanoborohydride. [0227] Using this same approach, other conjugates are prepared using mPEG-PIP having other weight average molecular weights. - 6 6 - Example 9 Activity of Exemplary frChE)-PEG Conjueates [0228] The activities of the (rChE)-PEG conjugates described in the preceding Examples are determined. All of the rChE conjugates are believed to be pharmacologically active. -67- <110> NEKTAR THERAPEUTICS AL, CORPORATION Bossard, Mary J Zappe, Harold Lee, Seoju Fernando, Lai A.R. <120> CONJUGATES OF A CHOLINESTERASE MOIETY AND A POLYMER <130> SHE0169.PCT <140> Not yet assigned <141> 2009-05-15 <150> US 61/127,928 <151> 2008-05-16 <160> 2 <170> Patentin version 3.4 <210> 1 <211> 602 <212> PRT <213> Unknown <220> <223> Unknown origin <400> 1 Met His Ser Lys Val Thr lie lie Cys lie Arg Phe Leu Phe Trp Phe 1 5 10 15 Leu Leu Leu Cys Met Leu lie Gly Lys Ser His Thr Glu Asp Asp lie 20 25 30 lie lie Ala Thr Lys Asn Gly Lys Val Arg Gly Met Asn Leu Thr Val 35 40 45 Phe Gly Gly Thr Val Thr Ala Phe Leu Gly H e Pro Tyr Ala Gin Pro 50 55 60 Pro Leu Gly Arg Leu Arg Phe Lys Lys Pro Gin Ser Leu Thr Lys Trp 65 70 75 80 Ser Asp H e Trp Asn Ala Thr Lys Tyr Ala Asn Ser Cys Cys Gin Asn 85 90 95 H e Asp Gin Ser Phe Pro Gly Phe His Gly Ser Glu Met Trp Asn Pro 100 105 110 -68- Asn Thr Asp Leu Ser Glu Asp Cys Leu Tyr Leu Asn Val Trp lie Pro 115 120 125 Ala Pro Lys Pro Lys Asn Ala Thr Val Leu lie Trp lie Tyr Gly Gly 130 135 140 Gly Phe Gin Thr Gly Thr Ser Ser Leu His Val Tyr Asp Gly Lys Phe 145 150 155 160 Leu Ala Arg Val Glu Arg Val H e Val Val Ser Met Asn Tyr Arg Val 165 170 175 Gly Ala Leu Gly Phe Leu Ala Leu Pro Gly Asn Pro Glu Ala Pro Gly 180 185 190 Asn Met Gly Leu Phe Asp Gin Gin Leu Ala Leu Gin Trp Val Gin Lys 195 200 205 Asn H e Ala Ala Phe Gly Gly Asn Pro Lys Ser Val Thr Leu Phe Gly 210 215 220 Glu Ser Ala Gly Ala Ala Ser Val Ser Leu His Leu Leu Ser Pro Gly 225 230 235 240 Ser His Ser Leu Phe Thr Arg Ala H e Leu Gin Ser Gly Ser Phe Asn 245 250 255 Ala Pro Trp Ala Val Thr Ser Leu Tyr Glu Ala Arg Asn Arg Thr Leu 260 265 270 Asn Leu Ala Lys Leu Thr Gly Cys Ser Arg Glu Asn Glu Thr Glu H e 275 280 285 H e Lys Cys Leu Arg Asn Lys Asp Pro Gin Glu H e Leu Leu Asn Glu 290 295 300 Ala Phe Val Val Pro Tyr Gly Thr Pro Leu Ser Val Asn Phe Gly Pro 305 310 315 320 Thr Val Asp Gly Asp Phe Leu Thr Asp Met Pro Asp H e Leu Leu Glu 325 330 335 -69- Leu Gly Gin Phe Lys Lys Thr Gin lie Leu Val Gly Val Asn Lys Asp 340 345 350 Glu Gly Thr Ala Phe Leu Val Tyr Gly Ala Pro Gly Phe Ser Lys Asp 355 360 365 Asn Asn Ser lie lie Thr Arg Lys Glu Phe Gin Glu Gly Leu Lys lie 370 375 380 Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser lie Leu Phe His 385 390 395 400 Tyr Thr Asp Trp Val Asp Asp Gin Arg Pro Glu Asn Tyr Arg Glu Ala 405 410 415 Leu Gly Asp Val Val Gly Asp Tyr Asn Phe lie Cys Pro Ala Leu Glu 420 425 430 Phe Thr Lys Lys Phe Ser Glu Trp Gly Asn Asn Ala Phe Phe Tyr Tyr 435 440 445 Phe Glu His Arg Ser Ser Lys Leu Pro Trp Pro Glu Trp Met Gly Val 450 455 460 Met His Gly Tyr Glu lie Glu Phe Val Phe Gly Leu Pro Leu Glu Arg 465 470 475 480 Arg Asp Asn Tyr Thr Lys Ala Glu Glu lie Leu Ser Arg Ser lie Val 485 490 495 Lys Arg Trp Ala Asn Phe Ala Lys Tyr Gly Asn Pro Asn Glu Thr Gin 500 505 510 Asn Asn Ser Thr Ser Trp Pro Val Phe Lys Ser Thr Glu Gin Lys Tyr 515 520 525 Leu Thr Leu Asn Thr Glu Ser Thr Arg lie Met Thr Lys Leu Arg Ala 530 535 540 Gin Gin Cys Arg Phe Trp Thr Ser Phe Phe Pro Lys Val Leu Glu Met 545 550 555 560 -70- Thr Gly Asn lie Asp Glu Ala Glu Trp Glu Trp Lys Ala Gly Phe His 565 570 575 Arg Trp Asn Asn Tyr Met Met Asp Trp Lys Asn Gin Phe Asn Asp Tyr 580 585 590 Thr Ser Lys Lys Glu Ser Cys Val Gly Leu 595 600 <210> 2 <211> 574 <212> PRT <213> Unknown <220> <223> Unknovm origin <400> 2 Glu Asp Asp lie lie lie Ala Thr Lys Asn Gly Lys Val Arg Gly Met 1 5 10 15 Asn Leu Thr Val Phe Gly Gly Thr Val Thr Ala Phe Leu Gly lie Pro 20 25 30 Tyr Ala Gin Pro Pro Leu Gly Arg Leu Arg Phe Lys Lys Pro Gin Ser 35 40 45 Leu Thr Lys Trp Ser Asp lie Trp Asn Ala Thr Lys Tyr Ala Asn Ser 50 55 •60 Cys Cys Gin Asn lie Asp Gin Ser Phe Pro Gly Phe His Gly Ser Glu 65 70 75 80 Met Trp Asn Pro Asn Thr Asp Leu Ser Glu Asp Cys Leu Tyr Leu Asn 85 90 95 Val Trp lie Pro Ala Pro Lys Pro Lys Asn Ala Thr Val Leu lie Trp 100 105 110 lie Tyr Gly Gly Gly Phe Gin Thr Gly Thr Ser Ser Leu His Val Tyr 115 120 125 Asp Gly Lys Phe Leu Ala Arg Val Glu Arg Val lie Val Val Ser Met 130 135 140 -71- Asn Tyr Arg Val Gly Ala Leu Gly Phe Leu Ala Leu Pro Gly Asn, Pro 145 150 155 160 Glu Ala Pro Gly Asn Met Gly Leu Phe Asp Gin Gin Leu Ala Leu Gin 165 170 175 Trp Val Gin Lys Asn lie Ala Ala Phe Gly Gly Asn Pro Lys Ser Val 180 185 190 Thr Leu Phe Gly Glu Ser Ala Gly Ala Ala Ser Val Ser Leu His Leu 195 200 205 Leu Ser Pro Gly Ser His Ser Leu Phe Thr Arg Ala lie Leu Gin Ser 210 215 220 Gly Ser Phe Asn Ala Pro Trp Ala Val Thr Ser Leu Tyr Glu Ala Arg 225 230 235 240 Asn Arg Thr Leu Asn Leu Ala Lys Leu Thr Gly Cys Ser Arg Glu Asn 245 250 255 Glu Thr Glu lie lie Lys Cys Leu Arg Asn Lys Asp Pro Gin Glu lie 260 265 270 Leu Leu Asn Glu Ala Phe Val Val Pro Tyr Gly Thr Pro Leu Ser Val 275 280 285 Asn Phe Gly Pro Thr Val Asp Gly Asp Phe Leu Thr Asp Met Pro Asp 290 295 300 lie Leu Leu Glu Leu Gly Gin Phe Lys Lys Thr Gin lie Leu Val Gly 305 310 315 320 Val Asn Lys Asp Glu Gly Thr Ala Phe Leu Val Tyr Gly Ala Pro Gly 325 330 335 Phe Ser Lys Asp Asn Asn Ser lie H e Thr Arg Lys Glu Phe Gin Glu 340 345 350 Gly Leu Lys H e Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser 355 360 365 -72- Ile Leu Phe His Tyr Thr Asp Trp Val Asp Asp Gin Arg Pro Glu Asn 370 375 380 Tyr Arg Glu Ala Leu Gly Asp Val Val Gly Asp Tyr Asn Phe lie Cys 385 390 • 395 400 Pro Ala Leu Glu Phe Thr Lys Lys Phe Ser Glu Trp Gly Asn Asn Ala 405 410 415 Phe Phe Tyr Tyr Phe Glu His Arg Ser Ser Lys Leu Pro Trp Pro Glu 420 425 430 Trp Met Gly Val Met His Gly Tyr Glu lie Glu Phe Val Phe Gly Leu 435 440 445 Pro Leu Glu Arg Arg Asp Asn Tyr Thr Lys Ala Glu Glu lie Leu Ser 450 455 460 Arg Ser lie Val Lys Arg Trp Ala Asn Phe Ala Lys Tyr Gly Asn Pro 465 470 475 480 Asn Glu Thr Gin Asn Asn Ser Thr Ser Trp Pro Val Phe Lys Ser Thr 485 490 495 Glu Gin Lys Tyr Leu Thr Leu Asn Thr Glu Ser Thr Arg lie Met Thr 500 505 510 Lys Leu Arg Ala Gin Gin Cys Arg Phe Trp Thr Ser Phe Phe Pro Lys 515 520 525 Val Leu Glu Met Thr Gly Asn H e Asp Glu Ala Glu Trp Glu Trp Lys 530 535 540 Ala Gly Phe His Arg Trp Asn Asn Tyr Met Met Asp Trp Lys Asn Gin 545 550 555 560 Phe Asn Asp Tyr Thr Ser Lys Lys Glu Ser Cys Val Gly Leu 565 570 - 7 3 - What is claimed is: 1. A conjugate comprising a residue of a cholinesterase moiety covalently attached to a water-soluble polymer, wherein the residue of the cholinesterase moiety is covalently attached to the water-soluble polymer through a cysteine residue within the residue of the cholinesterase moiety. 2. A conjugate comprising a residue of a cholinesterase moiety covalently attached to a water-soluble polymer, wherein the water-soluble polymer, prior to being covalently attached, is a polymeric reagent bearing a maleimide group. 3. A conjugate comprising a residue of a cholinesterase moiety covalently attached to a water-soluble polymer, wherein the water-soluble polymer is a branched water-soluble polymer. 4. The conjugate of any one of claims 1, 2 and 3, wherein the cholinesterase moiety is acetylcholinesterase. 5. The conjugate of any one of claims 1, 2 and 3, wherein the cholinesterase moiety is butyrylcholinesterase. 6. The conjugate of any one of claims 1, 2 and 3, wherein the cholinesterase moiety is recombinantly prepared. 7. The conjugate of any one of claims 1, 2, 3,4, 5, 6 and 7, wherein the water-soluble polymer is a polymer selected jfrom the group consisting of poly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, and poly(acryloylmorpholine). 8. The conjugate of claim 7, wherein the water-soluble polymer is a poly(alkyIene oxide). - 74 - 9. The conjugate of claim 8, wherein the poly{alkylene oxide) is a poly(ethylene glycol). 10. The conjugate of claim 9, wherein the poly{ethylene glycol) is terminally capped with an end-capping moiety selected from the group consisting of hydroxy, alkoxy, substituted alkoxy, alkenoxy, substituted edkenoxy, alkynoxy, substituted alkynoxy, aryloxy and substituted aryloxy. 11. The conjugate of any one of cMms 1, 2, 3, 4, 5, 6 and 7, wherein the water-soluble polymer the poly(ethylene glycol) has a weight-average molecular weight in a range of from about 500 Daltons to about 100,000 Daltons. 12. The conjugate of claim 1, wherein the cysteine residue within the residue of the cholinesterase moiety corresponds to Cys66 of butyrylcholinesterase. 13. The conjugate of claim 2, wherein the polymeric reagent bearing a maleimide group has the following structure: O H3CO-(CH2CH20)n-CH2CH2-NH-C-0-i O o -O-X-NM) H3CO-(CH2CH20)n-CH2CH2-NH-C-0-' O wherein: X is a spacer moiety comprised of one or more atoms; and each (n) is independently an integer having a value of from about 2 to about 4000. 14. The conjugate of claim 13, wherein the polymeric reagent bearing a maleimide group has the following structure: o H3C-(0CH2CHj)„-NH-C-0-CH2 O O "v,, HC-OCHiCHzCHz-C-NH-CHzCHz-NH-CCHa-CHa-N || ? I y^ H3C-(0CH2CH2)„-NH-C-0-CH2 O wherein each (n) is independently an integer having a value of from about 225 to about 1930. - 7 5 - 15. The conjugate of claim 14, wherein each (n) is defined so as to provide -(OCH2CH2)- as having a molecular weight of about 20kDa. 16. The conjugate of claim 3, wherein the branched water-soluble polymer includes the following structure: O H3CO-(CH2CH20)n—CH2CH2-NH-C-OH3CO-( CH2CH20)n-CH2CH2-NH-C-0- wherein each (n) is independently an integer having a value of from 2 to 4000. 17. The conjugate of any one of claims 1, 2 or 3, having the following structure: O H3CO-(CH2CH20)n-CH2CH2-NH-C-0-i O;^. / ^ S - ( C h E ) H3CO-(CH2CH20)n-CH2CH2-NH-C-0-' O wherein: each (n) is independently an integer having a value of from 2 to 4000; X is a spacer moiety comprised of one or more atoms; and ChE is a residue of a cholinesterase moiety. 18. The conjugate of claim 17, having the following structure: o H3C-(OCH2CH2)n-NH-C-0-CH2 O O '"'•V I II II /^S-(ChE) HC-OCH2CH2CH2-C-NH-CH2CH2-NH-CCH2CH2-N I H3C-(OCH2CH2)n-NH-C-0-CH2 O wherein each (n) is independently an integer having a value of from 2 to 4000. 19. The conjugate of any one of claims 1, 2, 3, 4, 5, 6 and 7, wherein the conjugate has from one to two water-soluble polymers attached to the residue of the choliaesterase moiety. - 7 6 - 20. The conjugate of claim 19, wherein the conjugate has two water-soluble polymers attached to the residue of the cholinesterase moiety. 21. The conjugate of any one of claims 1, 2, 3, 4, 5, 6 and 7, wherein the residue of the cholinesterase moiety is in the form of a dimer derived from two separate cholinesterase moieties. 22. The conjugate of claim 21, wherein the conjugate has two water-soluble polymers attached to the residue of the cholinesterase moiety, one water-soluble polymer attached to each of cholinesterase forming the dimer. 23. The conjugate of claim 2, wherein the polymeric reagent bearing a maleimide group has a single maleimide group. 24. The conjugate of any one of claims 1, 2, 3,4, 5, 6 and 7, wherein the cholinesterase moiety is glycosylated. 25. A conjugate comprising a residue of a cholinesterase moiety covalently attached to a water-soluble polymer, wherein the conjugate is in an isolated and monoPEGylated form. 26. A conjugate comprising a residue of a cholinesterase moiety covalently attached to a water-soluble polymer, wherein the water-soluble polymer, prior to being covalently attached, is a polymeric reagent bearing a maleimide group. 27. A conjugate comprising a residue of a cholinesterase moiety covalently attached to a water-soluble polymer, wherein the cholinesterase moiety is a precursor cholinesterase moiety. 28. A pharmaceutical composition comprising a conjugate of any one of claims 1 through 27 and a pharmaceutically acceptable excipient. -77- 29. A method for making a conjugate comprising contactiag, under conjugation conditions, a cholinesterase moiety witii a polymeric reagent bearing a thiol-reactive fimctional group. 30. The method of claim 29, wherein the contact step is carried out at a pH of greater than 8.0. 31. A method for making a conjugate comprising: (a) combining, under conjugation conditions, a reagent composition comprising a plurality of thiol-selective polymeric reagent molecules with a cholinesterase moiety composition comprising a plurality of cholinesterase moiety molecules, each molecule in the form of a dimer to form a conjugate mixture comprising monoconjugated dimers and diconjugated dimers; (b) subjecting the conjugate mixture to reducing conditions to form a reduced mixture comprising reduced unconjugated monomers and reduced monoconjugated monomers; (c) separating the reduced monoconjugated monomers from the reduced mixture to form a composition comprising reduced monoconjugated monomers; and (d) removing the reducing conditions from the composition comprising reduced monoconjugated monomers to thereby form a composition of diconjugated dimers. 32. The method of claim 31, wherein the composition comprising reduced monoconjugated monomers is substantially free of reduced imconjugated monomers. Dated this 29/10/2010 V/j 1 //// / OFREMFRYJKSAGAR ATTORNEY/OR THE AP^ilCANTS.