CA2653061A1 - Stabilized proteins - Google Patents

Abstract

Methods of stabilizing proteins having at least one oxidizable amino acid thereon, such as recombinant human adenosine deaminase (rhADA) are disclosed. The methods include treating a protein containing an oxidizable amino acid with a sufficient amount of a capping agent such as glutathione under reaction conditions sufficient to cap the oxidizable amino acid without substantially inactivating the protein. Stabilized capped proteins, polymer conjugates of capped proteins, and methods of treatment using the same are also disclosed.

Description

STABILIZED PROTEINS

This patent application claims the benefit of U.S. Patent Application Ser. No.
11/738,012, filed Apri120, 2007, which in turn claims the benefit of U.S.
Provisional Application Ser. No. 60/805,417, filed on June 21, 2006, the contents of each of which are incorporated by reference herein in their entirety_ FIELD OF THE INVENTION
The invention provides proteins, such as adenosine deaminase enzymes, that are stabilized by protecting or capping one or more oxidizable cysteine residues that are exposed to the solution environment of the protein.

BACKGROUND OF THE INVENTION
Adenosine deaminase (ADA) has been used in the treatment of an enzyme deficiency disorder called severe combined immunodeficiency disease (SCTD) or "Bubble boy" disease for some time. For more than 15 years, Enzon Phannaceuticals has made a PEGylated ADA available for patients using a bovine source of enzyme.
Recently, there have been efforts to replace the bovine source enzyme with a recombinant source enzyme (hereinafter "rADA"). Both recombinant human ("rhADA") and recombinant bovine ("rbADA") have been considered as replacements for purified natural bovine ADA_ The rbADA and rhADA enzymes are somewhat less stable than the native purified bovine enzyme that is currently employed. Both rhADA
and rbADA are believed to degrade in a manner consistent with cysteine degradation:
addition of oxygen; formation of dithiols; increasing degradation as pH
increases;
precipitation, especially as the pH is increased and the samples are concentrated. In the reduced state, cysteine contains a reactive -SH group (sulfliydryl) which is the form responsible for the degradation.
We have developed evidence suggesting that a single exposed cysteine may be responsible for the degradation that is seen for both rbADA and rhADA. ADA
purified from a bovine source has a structure very similar to that of rhADA: both bovine ADA
and rhADA have the same number of cysteines in the same positions of the primary sequence. Currently available recombinant bovine ADA contains degradants/impurities (dithiols) that are consistent with cysteine reactivity.
Native bovine ADA differs structurally from recombinant bovine ADA in that it has a single mole of cystein.e bound to each mole of ADA and native bovine ADA is stable to high pH, suggesting that the cysteine bound to the ADA is functioning as a protecting group.
In spite of the foregoing, it would be beneficial to provide rbADA and/or rhADA that are stable, i.e., without significant degradation, at pH levels which are useful for optimum PEGylation of the enzyme when the desired activated PEG's are used. Other proteins and enzymes having free eysteine group(s) that may be involved in instability such as certain interferons, for example, would similarly b enef t from stabilization. The present invention addresses this need.

SUMMARY OF THE INVENTION
Accordingly, the invention provides for a capped protein coinprising at least one amino acid residue that is capped with a capping agent selected to inhibit the oxidation of the amino acid when the protein is in an aqueous medium, so that the capped protein is substantially more stable in aqueous medium than the equivalent uncapped protein.
The amino acid is an oxidizable amino acid, and can be, for example, a cysteine, tryptophan or methionine. The capped protein can be any protein that is of interest, including, for example, an adenosine deaminase protein, whether native or recombinantly expressed, or an interferon, such as, e.g., beta interferon, an alpha interferon and a gamma interferon.. Preferably, the adenosine deaminase is a recombinant human adenosine deaminase (e.g., SEQ ID NO: 2) or a recombinant bovine adenosine deaminase (e.g., SEQ ID NO: 4). Optionally, the recombinant adenosine deaminase lacks the six C-terminal residues indicated by the theoretical translation of the corresponding DNA open reading. frame. In an optional alternative aspect, the N-terminal Met indicated by the theoretical translation of the corresponding DNA open reading frame is present in the capped protein.
The inventive capped protein is capped with a capping agent such as oxidized glutathione, iodoaceta.mide, iodoacetic acid, cystine, other dithiols and mixtures thereof.

The inventive capped protein is optionally conjugated to a substantially non-antigenic polymer to form a polymer conjugated capped recombinantly expressed protein, e.g, a polyalkylene oxide such as polyethylene glycol, wherein the polyalkylene oxide ranges in size from about 2,000 to about 100,000 Daltons.
The polyalkylene oxide is preferably conjugated to the protein via linker chemistry including, e.g., succinimidyl carbonate, thiazolidine thione, urethane, and amide based linkers. The polyalkylene oxide is preferably covalently attached to an epsilon amino group of a Lys on said cysteine-stabilized recombinant human adenosine deaminase, although other sites for covalent attachment are well known to the art. The inventive capped protein preferably comprises at least 5 polyethylene glycol strands attached to epsilon amino groups of Lys on said protein, but alternatively, can comprise about 11-18 PEG strands attached to epsilon amino groups of Lys on said protein.
The invention further provides a method of stabilizing a protein having an oxidizable amino acid thereon, comprising, treating a protein containing an oxidizable amino acid with a sufficient amount of a capping agent, in aqueous solution, under reaction conditions sufficient to cap the reactive amino acid without substantially inactivating the recombinantly expressed protein. The reactive amino acid can include cysteine or methionine and/or any other art-known reactive protein. The protein can be any protein of interest, as set forth above. The capping ageni is, e.g., oxidized glutathione, iodoacetamide, iodoacetic acid, cystin.e, other dithiols and mixtures thereof, and oxidized glutathione is preferred. Preferably, the oxidized glutathione is reacted with the protein at a concentration of from about 20 to about 100 mM
or alternatively, the oxidized glutathione is reacted with the protein at a concentration of from about 22 to about 30 mM.
Preferably, the inventive method provides for the reaction conditions to comprise an aqueous solution at a pH of from about 6.5 to about 8.4. More particularly, the reaction conditions include an aqueous solution at pH of from about 7.2 to about 7.8.
In a preferred aspect, the aqueous solution includes a buffer selected from the group consisting of sodium phosphate, potassium phosphate, Tris, and Hepes at concentrations ranging from 20 to 150 mM.

The inventive process further includes reacting the capped protein in the aqueous solution with an activated polymer under condition sufficient to form capped protein-polymer conjugates, e.g., an activated polyethylene glycol, such as, a succinimidyl carbonate-activated polyethylene glycol, a thiazolidine thione-activated polyethylene glycol, and/or a urethane linkage or amide-linkage forming -activated polyethylene glycol, where the polyethylene glycol preferably has a molecular weight of from about 2,000 to about 100,000 Daltons or more particularly, wherein the polyethylene glycol has a molecular weight of from about 4,000 to about 45,000 Daltons.
The inventive process f rther includes a method of stabilizing a recombinantly expressed protein having an oxidizable cysteine thereon, comprising, recombinantly expressing a protein in a suitable prokaryotic expression system and recovering the recombinantly expressed protein from a cell extract media containing a sufficient .
amount of a capping agent whereby the oxidizable amino acid on the recombinan.tly.
expressed protein is stabilized upon secretion from the prokaryotic cells in said suitable prokaryotic cell expression system. Preferably, the prokaryotic expression system is an E. coli expression system capable of producing recombinant adenosine deaminase, and the recombinantly expressed protein is adenosine deaminase and/or interferon.
The inventive process further includes a method for producing a stabilized derivative of an oxidizable protein of interest, comprising the steps of (a) identifying an oxidizable protein of interest, wherein the protein is oxidizable at. one or more amino acid residues when the protein is in aqueous soluti.on, (b) identifying at least one oxidizable amino acid of the protein of interest, (c) reacting the oxidizable protein of interest with a capping agent selected to prevent the oxidation of the oxidizable amino acid.
The protein is, e.g., an adenosine deaminase or interferon, the oxidizable amino acid is cysteine or methionine and the capping agent is selected from the group consisting of oxidized glutathione, iodoacetamide, iodoacetic acid, cystine, other dithiols and mixtures thereo Still further aspects of the invention include cysteine-stabilized recombinant proteins, enzymes or the like, and methods of making the same. A preferred embodiment in this regard is cysteine stabilized rbADA and/ar rhADA. Polymer conjugates thereof are also contemplated as embodiments of the invention. The polymer is preferably a polyalkylene oxide, e.g, more preferably a polyethylene glcyol.
The conjugates are prepared by reacting the capped protein with an activated polymer, including, e.g., succinimidyl carbonate-activated polyethylene glycol ("SC-PEG"), thiazolidine thione-activated polyethylene glycol ("T-PEG"), and/or a urethane linkage or amide-linkage forrriing -activated polyethylene glycol. SC-PEG is preferred.
A still further aspect of the invention includes methods of treatment using a stabilized enzyme in polymer conjugated or unconjugated form. Specifically, the inventive methods include administering an effective amount of the cysteine-stabilized rADA to a patient in need thereof in order to treat SCID or other enzyme deficiency-related conditions.
For purposes of the present invention "effective amount" shall be understood to mean an amount which achieves a desired clinical result, i.e. reduction, slowing, remission, etc. or reversal of the enzynle deficiency or SCID condition in the patient, i.e., in a mammal or a human.
Further, the use of singular terms for convenience in description is in no way intended to be so limiting. Thus, for example, reference to a composition comprising a cell includes reference to one or more of such cells.
DETAILED DESCRIPTION OF THE INVENTION
A. Overview Broadly, the invention provides capped or protected proteins with enhanced stability. In particular, there are provided proteins expressed with one or more oxidizable or reactive cysteine residues that are exposed to the solution environment of the protein so that the protein exhibits instability in solution, e.g., during storage. The protein is preferably a recombinantly produced ADA enzyme.
The number of oxi.dizable amino acid residues that can be capped according to the invention will depend on the number of such amino acid residues that are present in the protein of interest, and on their location in the tertiary structure of the protein.
Without meaning to be bound by any theory or hypothesis, it is thought that oxidizable amino acids that are exposed to solution when the protein is dissolved or suspended in an aqueous medium, are those most likely to cause oxidation-mediated degradation of the protein. Identifying and capping such amino acids thus inhibits oxidation of such amino acid residues, and thereby provides capped proteins with enhanced stability relative to uncapped proteins. Thus, for example, the residues to be capped can be determined by identifying breakdown products of a protein of interest that are unstable in an aqueous medium, and d.etermining the location of fragment breaks relative to the presence of oxidizable amino acids. The number of residues to be capped can be as few as one, up to as many as necessary to stabilize a protein of interest, e.g., up to as many as 50, or more. More particularly, the number of residues to be capped more typically ranges from 1 to about 20, and even more particularly, from 1 to about 10 amino acid residues.
The invention also provides a method of stabilizing a recombinantly expressed protein having an oxidizable cysteine thereon. The method includes treating a recombinantly expressed protein containing an oxidizable cysteine with a sufficient amount of a capping agent under reaction conditions sufficient to cap the oxidizable cysteine without substantially inactivating the recombinantly expressed protein.
As will be appreciated by those of ordinary skill, "treating" in this aspect, includes allowing the recombinantly expressed protein to react with the capping agent by contacting of the two principal reactants in a suitable buffered solution which will protect the biological activity of the protein while allowing the selective oxidation of the reactive cysteine to occur. The "treating" can occur at various stages of the recornl?inant production after expression but preferably for this aspect of the invention, during one or more of the post expression purification steps. For example, initial purification of ADA proteins from cell extract can be achieved through an anion exchange chromatography in the presence of reducing agent like dithiothreitol (DTT).
Subsequently, upon removal of DTT, the semi-purified ADA can be treated with a sufficient amount of capping agent to block the reactive cysteine. The same procedure can be followed either at any step of purification or with the purified protein at the end of purification and prior to PEGylation. Once capped, no reducing agent is required to maintain stability of the modified ADA.

B. ADA Proteins In certain preferred aspects of the invention, the recombinantly expressed protein is rhADA or rbADA. More preferably, the translated rhADA has the amino acid sequence of SEQ ID NO. 2, prior to capping, and the translated rbADA has the amino acid sequence of SEQ ID NO: 4, prior to capping. It should be noted that SEQ
ID NO:s 2 and 4 are the predicted mature translation sequences, and reflect the post translational cleavage of the N-terminal Met residue. Thus, in the translated respective mature ADA proteins, the Cys residue is at position 74. In addition, it should be noted that the natural bovine ADA as isolated from bovine intestine also has six residues posttranslationally removed from the C-terniinal. It is an optional feature of the present invention that the rbADA is either expressed without the six C-terminal residues (as a mutein) or is posttranslationally modified to remove the same C-terminal residues lacking in the purified natural bovine ADA.
It should fiirther be noted that natural bovine ADA as isolated from bovine intestine has'polymorphisms. Thus, with reference to SEQ ID NO: 4, bovine ADA
polymorphisms include, e.g., glutamine at position 198 in place of lysine, alanine at position 245 in place of threonine; arginine at position 351 instead of glycine. It is therefore contemplated that recombinant position 74 mutein bovine ADA
according to the invention, can also have additional substitutions at one or more of the noted positions or analogs of those positions: Gln in place of Lys198; Ala in place of Thr245;
Arg in place of Gly351.
In addition, the rhADA is expressed by a DNA with codons optimized for E.
coli expression, according to SEQ ID NO: I and the rbADA is expressed by a DNA
with codons optimized for E. coli expression according to SEQ ID NO: 3. A
suitable expression vector can be prepared from genornic or cDNA encoding rhADA or rbADA, respectively, that is optionally under the control of a suitable operably connected inducible promoter.
As exemplified hereinbelow, the vector (pET9d) containing the basic rhADA
nucleotide sequence was obtained from Mike Hershfield of Duke Medical Center, North Carolina (as the HADA1092 clone). The HADA1092 clone was then optimized for bacterial expression, and inserted into a suitable expression vector under the control of an inducible promoter and transformed into the BLR(XDE3) strain. This recombinant clone was designated as EN760.
Similarly, a recombinant rbADA expression clone was constructed by transforming BioCatalytics plasmid (p2TrcCrro-RoADA2-23) into fresh E. col.i chemically competent cells (Novagen, catalog number 69449-4, lot number N60830, Genotype F ompT hsdS,s (rB mB ) gal dcrn). This clone contains the plasmid p2TrcGro-RoADA2-23 that has the bovine ADA gene provided by Roche and the E.
coli GroEL/CrroES chaperone operon (U.S. 6,366,860). This new transformed clone was cultured on a plate containing CFB medium (10 g/L soy peptone, 5 g/L yeast extract, 10 g/L sodium chloride), 2% (w/v) agar, and 25 ~tg/mL kanamycin at 30 C.
The expression of rbADA was induced by IPTG.

C. Capping Agents and Reaction Conditions The capping agents included in the methods of the invenfion include, without limitation, oxidized glutathione (preferred), iodoacetamide, iodoacetic acid, cystine, and other dithiols known to those of ordinary skill, and mixtures thereof. The amount and concentration of the capping agent included during the reacting phase of the methods described herein will depend somewhat upon the specific capping agent used and the needs of the artisan but will not be subject to undue experimentation.
Using oxidized glutathione as a prototype, the concentration of used when reacted with the recombinant protein such as rhADA can range from about 25 }M to about 100 mM.
Preferably, the oxidized glutathione is reacted with the recombinant protein at a concentration of from about 5 mM to about 25 mM.
The reaction conditions employed during the reacting of the capping agent and the recombinant protein further include the use of an aqueous solution having a pH of from about 6.5 to about 8.4, preferably from about 7_2 to about 7.8. Jn addition, the aqueous solution preferably includes a suitable buffer such as sodium phosphate, potassium phosphate, Tris, and Hepes and mixtures thereof at concentrations ranging from 10 to 150 mM (Comments: capping can take place out of this buffer range, lower than 10 or high than 150 mM). The reaction conditions further include allowing the reaction to proceed at temperatures which will not contribute to degradation of the protein, i.e. from about 4-37 C. Optionally, capping can take place outside of this temperature range, e.g., at a temperature range lower than 0-4 or higher than 37 C.
The reaction is conducted for a time sufficient to achieve the desired stabilization "of the reactive cysteine_ Simply by way of example, the reaction is conducted for a time ranging from about 5 seconds to about 8 hours (e.g., overnight).
While the invention is being described in terms of a preferred recombinant protein, i.e. rhADA, or rbADA, it will be understood that the processes described herein can be carried out using any number of recombinantly prepared proteins, enzymes, etc. having free cystezne group(s) that may be involved in instability, such as certain interferons. For example, a,, j3, or y interferon can stabilized by the inventive methods. Without lizxa.itation, other suitable proteins, polypeptides and peptides of interest include, but are not limited to, hemoglobin, serum proteins such as blood factors includi-ng Factors VII, VIII, and IX; immunoglobulins, cytokines such as interleukins, i.e. IL=1 through IL-13, etc., colony stimulating factors including granulocyte colony stimulating factors, platelet derived growth factors, etc.
Other proteins of biological or therapeutic interest include insulin, plant proteins such as lectins and ricins, tumor necrosis factors and related proteins, growth factors such as transforming growth factors; such as TGFoc's or (3's and epidermal growth factors, hormones, somatomedins, erythropoietin, pigmentary hormones, hypothalamic releasing factors, antidiuretic hormones, prolactin, chorionic gonadotropin, follicle-stim.ulating hormone, thyroid-stimulating hormone, tissue plasminogen activator, and the like. Immunoglobulins of interest include IgG, IgE, Igm, IgA, IgD and fragments and single chain constructs thereof.
Enzymes of interest include carbohydrate-specific enzymes, proteolytic enzymes, oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.
Without being limited to particular enzymes, examples of enzymes of interest include L-asparaginase, arginase, arginine deiminase, superoxide dismutase, endotoxinases, catalases, chymotrypsin, lipases, uricases, adenosine diphosphatase, tyrosinases and bilirubin oxidase. Carbohydrate-specific enzymes of interest include glucose oxidases, glucodases, galactosidases, glueocerebrosidases, glucouronidases, etc.
The foregoing list is simply illustrative of the proteins which are contemplated to be stabilized using the methods of the invention. It is to be understood that those proteins, enzymes, etc. as defined herein, not specifically mentioned but having reactive cysteines are also intended to be embraced by the inventive method.
As a result of the methods of the present invention, the stabilized protein has a capping group attached thereto at a reactive cysteine. For example, the glutathione or other capping group is conjugated to the rhADA via a disulfide bond as such:
ADA - S -S - glutathione where one cysteine in the primary sequence of ADA is bound to a molecule of the glutathione, shown below with the -SH of the ADA Cys reacting with the -SH of the glutathione shown below:
HS
O O O
H
HO N ",////N OH
H
O NHZ

Such disulfide bonds are stable towards the oxidative degradation pathways and thus render rhADA more stable, especially at a pH above 7.4. In addition, the MW of glutathione-adduct of the stabilized protein such as rhADA will be higher than the pre-stabilized form by an amount about equal to the MW of the capping agent used.
This will allow separation of the desired adduct, if desired. The stabilized adduct will also have different physical and chemical properties which will allow chromatographic separation and isolation as well.

D. Polymer Conjugates In another aspect of the invention, the cysteine capped or stabilized recombinant protein is conjugated to a suitable polymer in order to make polymer conjugates. The polymer conjugation is preferably a PEGylation reaction as such reactions are known to those of ordinary skill. Briefly stated, rhADA or rbADA, for example, is reacted with an activated polymer in an aqueous solution under condition sufficient to form cysteine capped or stabilized polymer conjugates. In this regard, a wide variety of activated polyethylene glycols can be used, including those described, for example i.n commonly assigned US Patent Nos. 5,122,614, 5,324,844, 5,612,460 and 5,808,096 (succinimidyl carbonate-activated polyethylene glycol (SC-PEG) and related activated PEG's), 5,349,001 (cyclic imide thione activated PEG's), 5,650,234, and others known to those of ordinary skill. The disclosure of each of the foregoing is incorporated herein by reference. See also activated polymers available from Nektar /
Shearwater Polymers. The activated PEG's can be linear, branched or U-PEG derivatives such as those described in U.S_ Patents Nos. 5,605,976, 5,643,575, 5,919,455 and 6,113,906 (also incorporated herein by reference) or branched derivatives. It will be further understood that in addition to the PEG-based polymers, a number of other polyalkylene oxides can also be used. For example, the conjugates of the present invention can be made by methods which include converting the multi-arm PEG-OH or "star-PEG"
products such as those described in NOF Corp. Drug Delivery System catalog, Ver. 8, April 2006, the disclosure of which is incorporated herein by reference, into a suitably activated polymer, using the activation techniques described in the aforementioned'614 or'096 patents. Specifically, the PEG can be of the formula:

, (CH2CHZO),; `
~ p CH2CH2_O~
-O-CH2CHg-(OCH2CH2)u'~0 O
, (CH2CH2O),,' -CHZCH2_O_ 'IC
~_O-CH2CH 0 2-OCH CH ~
( Z 2)u Star or k0-CH2CH2~(OCH2CH2)u'-O ro-`-c 0-(CH2CH2O),,=-CH2CHz O1~
1`O-CH2CH2-(OCH2CH2)u" 0 O-- (CH2CH2O),; -CH2CH2'O-4 Multi-arm wherein:
u' is an integer from about 4 to about 455, to preferably provide polymers having a total molecular weight of from about 5,000 to about 40,000; and up to terminal portions of the residue is/are capped with a methyl or other lower al.kyl.
In some preferred embodiments, all 4 of the PEG arms are converted to suitable leaving groups, i.e. SC, etc., for facilitating attachment to the recombinant protein.
Such compounds prior to conversion include:

(CH2CHz0)u 'CH2CH2-OH
H3C-(OCHZCf-2)u~0 0 0, (CH2CH20),; , CH

H3C_ (OCHZCH2)u~

,(CH2CHZ0)1"
CHZCH2~OH

H3C- (OCHZCHz)u~0 O
(CHZCH2O)u'-CH2CH2'O OH

H C_(OCHZCHz),' 0 (CH2CH2O)u'lCH2CH2- OH
H3C`(OCH2CHz)u~0 0, (CH2CH20)"_CH2CH2`
HO, CH CH ~O OH
2 z-(OCH2CHz)õ~

,(CH2CHz0)õ ~CHZCH2~OH
HO- CH2CHz-(OCHZCHz)u~O O 0 , (CHZCH2O),; , CHZCH2, HO,CH CHz-~0 OH
2 (OCH2CHz)õ.

H3C (OCH2CHz)u -O 0-(CHZCH20)õ=-CH2CHz-OH
rO~
H3C-(OCH2CH2)1'- O 0 '(CH2CH20)u'-CH3 H3C-(OCH2CH2)L.-0 O-"-C 0---(CH2CH20)Ll'-CH3 r H3C-(OCH2CH2)u'- 0 O, (CH2CH20)u.-CH2CH2-OH
H3C-(OCH2CH2),'-O ro---C 0-(CH2CH20),'-CH2CH2-OH
H3C-(OCH2CH2)1'- 0 O-(CH2CH20),; -CH2CHZ---OH

HO-CH2CH2-(OCH2CH2)õ'-OrO O-(CH2CH20)U'-CHZCH2-OH
~
H3C-(OCH2CH2)u '__ O O-- (CH2CH2O)õ'-CH3 H3C-(OCH2CH2),,=-OrO~0-(CH2CH20)u,-CH2CH2-OH
HO-CH2CH2_(OCH2CH2)õ '_ O O-- (CH2CHZO),,=-CH3 H3C-(OCH2CH2),'-O 0-(CH2CH20)õ'-CH2CH2 OH
HO--CH2CH2-(OCH2CH2)u'_ 0 O_~ (CH2CH2O)U.---CH2CHZ OH
HO-CH2CH2-(OCH2CHZ),Y-O CO_(CH2cH20)u H3C-(OCHZCH2),,'_ O O, (CH2CH2O)õ,-CH2CH2-OH
and HO-CH2CH2-(OCH2CH2)u'--O ro-`c O-(CH2CH2O},;-CH2CH2-OH
HO---CH2CH2-(OCH2CH2),, '_ O O, (CH2CH2O),'_CH2CH2-OH

In most preferred aspects of the invention, the activated polyethylene glycol is one which provides a urethane linkage or amide-linkage with the protein.
In yet alternative aspects, the activated polymers can employ a hindered ester-based linker. See US Provisional Application No. 60/844,942 entitled "Polyalkylene Oxides Having Hindered Ester-Based Biodegradable Linkers", the contents of which are incorporated by reference. For exarnple, a non-limiting list of such compounds include:

O
CH3-O(CH2CH2O)0',- ~ NH
C
II , O
O
H
CH3-O(CH2CH2O)u H2C`C---~N
I , O

CH3-0(CH2CH20)u H2C `C NI~~O
fl O

O
CH3-O(CH2CH20)u- H2C `
C I
O
O

CH3-O(CH2CH2O)9-HZC `
C NH
II >
5. 0 and CH3-O(CH2CH2O)u HzC ` C ~ NH wherein "u" is an integer ranging from 10 to about 455.

Suitable polymers will. vary substantially by weight. Polymers having molecular number average weights ranging from about 2,000 to about 100,000 are usually selected for the purposes of the present invention. Molecular weights of from about 4,000 to about 45,000 are preferred and 5,000 to about 12,000 are particularly preferred.
Methods of preparing polymers having terminal carboxylic acids in high purity are described in US Patent Application No. 11/328,662, the contents of which are incorporated hereizi by reference. The methods include first preparing a tertiary alkyl ester of a polyalkylene oxide followed by conversion to the carboxylic acid derivative thereof. The first step of the preparation of the PAO carboxylic acids of the process includes forming an intermediate such as t-butyl ester of polyalkylene oxide carboxylic acid. This intermediate is formed by reacting a PAO with a t-butyl haloacetate in the presence of a base such as potassium t-butoxide. Once the t-butyl ester intermediate has been formed, the carboxyl.ic acid derivative of the polyalkylene oxide can be readily provided in purities exceeding 92%, preferably exceeding 97%, more preferably exceeding 99% and most preferably exceeding 99.5% purity.
In yet alternative aspects, polymers having terminal amine groups can be employed to make the ADA conjugates. The methods of preparing polymers containing terminal amines in high purity are described in US Patent Application Nos.
11/508,507 and 11/537,172, the contents of each of which are incorporated by reference. For example, polymers having azides react with phosphine-based reducing agent such as triphenylphosphine or an alkali metal borohydride reducing agent such as NaBH4. Alterraatively, polym.ers including leaving groups react with protected amine salts such as potassium salt of methyl-tert-butyl imidodicarbonate (KNMeBoc) or the potassium salt of di-tert-butyl imidodicarbonate (KNBoc2) followed by deprotecting the protected amine group. The purity of the polymers containing the terminal amines fozTned by these processes is greater than about 95% and preferably greater than 99%.
The branching afforded by the polymers of the 5,643,575 patent, cited above, allows secondary or tertiary branching as a way of increasiza.g polymer loading on a biologically active molecule from a single point of attachment_ It will be understood that the water-soluble polymer can be functionalized for attachrnent to the bifunctional linkage groups if required without undue experimentation.
The polymeric substances included herein are preferably water-soluble at room temperature. A non-limiting list of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, ~. _ polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained. In addition to mPEG, C1-4 alkyl-terrninated polymers are also useful.

As an altemative to PAO-based polymers, effectively non-antigenic materials such as dextran, polyvinyl pyrrolidones, polyacrylamides such as HPMA's (hydroxypropylmethacrylamides), polyvinyl alcohols, carbohydrate-based polymers, copolymers of the foregoing, and the like can be used. Those of ordinary skill in the art will realize that the foregoing list is merely illustrative and that all polymer materials having the qualities described herein are contemplated. For purposes of the present itivention, "substantially or effectively non-antigenic" means all materials understood in the art as being nontoxic and not eliciting an appreciable immunogenic response in inazxamals.

E. Cell Extract Media Containing Capping Agent In an alternative aspect of the invention, there is provided another method of stabilizing a recombinantly expressed protein having a reactive cysteine thereori. In this process, the steps include a recoznbinantly expressed protein in a suitable prokaryotic expression system and recovering the recombinantly expressed protein from a cell extract media containing a sufficient amount of a capping agent whereby the reactive cysteine on the recombinantly expressed protein is stabilized substantially upon secretion from the prokaryotic cells in the suitabie prokaryotic cell expression system. As was the case in the first aspect of the invention.; the preferred protein being expressed is rhADA or rbADA. A suitable prokaryotic expression system is an E.
co.lz expression system capable of producing the rhADA or rbADA.
The rhADA eDNA containing expression vector (pET9d) was introduced into an E.coli expression strain, BL21(kDE3) or an equivalent expression vector and bacterial strain combination and the recombinant protein was allowed to synthesize in the bacteria by IPTG induction. Cells were then lysed in Tris buffer at pH 7.4 supplemented with EDTA and desired concentration of capping agent, 5- 50 mM
herein, and an extract was prepared that contained expressed rhADA.
EXAMPLES
The following examples serve to provide further appreciation of the invention but are not meant in any way to restrict the effective scope of the invention.

GENERATION OF RECOMBINANT BACTERIAL
CLONE CARRY]NG SYNTHETIC rhADA cDNA

The vector (pET9d-HCADA 1092) containing the basic human adenosine deaminase nucleotide (rhADA) sequence was obtained from Mike Hershfield of Duke Medical Center, North Carolina (as the HADA1092 clone). The cDNA was codon optimized for bacterial expression by following the standard bacterial codon usage for Eschexichia colr K12, using the codon data described by Grantham R. et al.;
1981;
"Codon catalogue usage in genome strategy modulated for gene expressivity,"
Nucleic Acid Res. 9:r43-r47, and Lathe, R. 1985; "Synthetic oligonucleotide probes deduced from amino acid sequence data, Theoretical and Practical considerations." J.
Mol Biol;
183:1-12, both of which are incorporated by reference herein in their entireties. The corresponding RNA sequence was then analyzed for the formation of hairpin structure or loop formation and was subjected to minimum free energy calculations, and NcoI
and HindIH flanking sites were synthetically inserted to allow for cloning into expression vector pET28a (commercially available from Novagen). The resulting recombinant plasmid containing human ADA cDNA, was designated as pIIADApET28a, and was transformed into the BLR(XDE3) strain of E.co1r (Novagen) to create a viable expression clone for rhADA. This recombinant clone, with the rhADA gene under control of an isopropyl-beta-D-thiogalactopyranoside (IPTG) IPTG-inducible promoter, was designated as the EN760. The codon optimized DNA
molecule encoding the rhADA protein is shown as SEQ ID NO: 1.

FERMENTATION Of EN760 Clone Expressing rhADA

A single colony from a BLR (XDE3) plate was inoculated in a 50m1 tube in 5m1 LB media containing kariaanycin 10 g/ml and tetracycline 12.5~tg/ml. The tube was incubated in a 37 C/250 rpm shaker for 7 hours and then, 250 1 of the culture was used to inoculate a 1 L flask containing 250m1 of fresh complete LB media. This flask was incubated in a 37 C/250 rpm shaker overnight. The overnight culture (OD600 value 4.401) was used to inoculate a BioFlo 3000 fermenter (5L; North Brunswick Scientific Company, Edison, NJ) containing 4.5L complete SuperBroth (complete: kanamycin 10~Lg/inl, tetracycline 12.5~tg/ml, 5m1/L glycerol, and 1.68m1/L MgSO4.7H20) resulting in an initial OD600 of 0.2445. The culture was grown to an OD600 value of 22.5 (EFT 6 hours) and induced with 5 mM IPTG to begin the production phase. The culture was harvested 2 h post induction. (OD600 value 44) using a Beckman Coulter Centrifuge (7000rpm/4 C/20min) yielding a wet cell weight of 282 g.
Medium composition Composition of SuperBroth (DifcoTMSelectAPSTM SuperBroth from BD
BioSciences, Inc.) Autoclave components:
Soy Hydrolysate .12.0g/L
Yeast Extract 24.0g/L
Di-potassium phosphate 11.4g/L
Mono-potassium phosphate 1.7g/L
Filtered components (supplements):
Kanamycin 10~tg/m.l Tetracyclin 12.5 ~tg/ml Fermentation Parameters:
Agitation: 1000 rpm Tenaperature: 37 C
pH: 7.0 Air: 1.5/2.0 (induction) vvm Kanamycin: 10gg/znl Tetracycline: 12.5 ~tg/ml MgSO4.7H2.0: 1.68m1/L

GENERATION OF RECOMBINANT BACTERIAL
CLONE CARRYING SYNTHETIC rbADA cDNA

The purified mature ADA protein derived from bovine intestinal preparations is a 356 amino acid protein lacking the N-terminal methionine and final six C-terminal residues predicted from the eDNA sequence (GenBank NP_776312, incorporated by reference herein). In brief, the defined polypeptide sequence was provided to BioCatalytics Inc. for whole gene synthesis of a new gene having codons optimized for expression in E. colr, using their methods that include chemical synthesis of overlapping oligonucleotide segments. The BioCatalytics methods are described in greater detail by U.S. Patent No. 6,366,860, the contents of which are incorporated by reference herein in their entirety.
Bovine ADA expression was investigated in several expression systems. For example, the flanking restriction sites, NdeTand BamHlwere included at the termini of the gene. Following digestion of the synthetic DNA with the restriction enzymes Ndel and BamHI, the 1.1 kilobase gene was ligated via T4 DNA ligase into the plasmid vector pET-9d (Novagen Corporation), which had also been digested with these two enzymes. The recombinant plasmid was introduced into E. coli strain BLR (DE3) or HMS174 (DE3) by electroporation using a BTX Electro Cell Manipulator 600 according to the manufacturer's instructions. The transformation mixture was plated on LB agar plates containing kanamycin (15 }ig/ml). This allowed for selection of colonies containing the plasmid pET-9d/bADA (designated bADAJpET9d: BLR(DE3) or bADA/pET9d:HMS174(DE3)). The ADA variant gene nucleotide sequence was verified by DNA sequence analysis with ABI Prism 310 Genetic Analyzer using Big Dye Tenninators. Isolated colonies were fu-ther purified by plating and analyzed for IPTG inducible gene expression in LB medium by standard methods such as those described in Novagen pET System Manual. Ninth Edition. Several induction parameters were examined including time, temperature and inducer concentration_ A
preferred condition was induction with 0.3 % lactose for 12 hrs at 37 C, which allowed high level production of ADA within the cytoplasm of the host bacteria at about 20% of total cell protein. The ADA product was confirmed on SDS PAGE analysis to exhibit PURIFICATION Of RECOMBINANT ADA PROTEIN PRODUCED BY THE
CLONE EXPRESSING rbADA OF EXAMPLE 3 The purification of rbADA was carried out in a 3 chromatographic protocol developed by Enzon. Briefly, thawed cell paste (obtained from Biocatalytics, respectively) of 200 g which was stored at -80 C was re-suspended in 1800 ml buffer of 20 mM Bis-Tris, 1mM EDTA, pH 7.4, and homogenized at 1200 RPM for 10 seconds with Tempest Virtis (SentryTM, Microprocessor, Boston, MA). This suspension was passed through a stainless steel mesh (Opening micrometer 250 , No.60, W.S
Tyler) to removed big particles. The homogenous cell suspension was microfluidized for 3 cycles at 15,000 psi (unit was ice-bathed) (Micro Fluidizer, Microfluidics Corp., Model# 110Y, Boston, MA). At the end of micro fluidization, 200 ml of the same buffer as above was used to rinse the unit and this solution was combined with the above suspension. The soluble protein from cell lysates was extracted by centrifugation at 7100 rpm (12000 x g) for 60 minutes at 4 C (Avanti J-201, Beckman Coulter; Rotor# JLA8.1000). The supematant was collected carefully to avoid unwanted mixing_ The resulting volume of supernatant was 2100 ml, total protein concentration by BCA method was 13.2 mg/ml.
To remove nucleotides in this cell extract, 31 ml of 10% PEI solution (final PEI
0.15%) was added to the above supernatant and mixed thoroughly by stirring for min. The cell extract was stored at 4 C over night. The precipitant from this over night sample was removed by a centrifugation at 7100 rpm (12000 x g), for 60 minutes at 4 C (Avanti J-201, Beckman Coulter; Rotor# JLA8.1000). Similarly, the supematant vcras collected carefully to avoid any unwanted mixing. Now the volume of supematant was 1940 ml, total protein concentration by BCA method was 3.53 mg/ml.
Protection of rbADA or rhADA from oxidation of cysteine was carried out by adding 5-50 mM oxidized glutathione at pH 2-10. To help ADA bind to the first column, 10%
PEG4600 was added to this cell extract slowly and the pH of this cell extract was adjusted to 6.5 slowly with 1 N NaOH and 1N HCI. This supernatant was centrifuged again at 7100 rpm (12000 x g), for 60 minutes at 4 C(Avanti J-201, Beckman Coulter;
Rotor# JLA8.1000) before loading to the next column.
The cell extract was loaded to a pre-equilibrated Capto Q column (Cat# 17-5316-01, GE Healthcare, Piscataway, NJ. Bed volume 350 ml pre packed in a XK-column) with a buffer of 20 mM Tris-Bis, 1 mM EDTA, pH 6.5. Before ADA was eluted off from the column at 80 mM NaCl in the equilibration buffer, elutions at 60 mM and 70 mM NaC1 were first performed to remove impurities. The elution profile was analysed by ADA activity, SDS-PAGE analysis, Western Blots, and RP-HPLC.
After Capto Q column, 2 hydrophobic interaction chromatographic purifications were used one by one to further polish the purity of the protein. The first HIC was Octyl Sepharose 4FF (Cat# 17-0946-02, GE Healthcare, Piscataway, NJ). The pool of ADA fractions from Capto Q column was adjusted to 1.5 M(NH4)2SO4 with animonium sulfate powder directly and the pH was adjusted to 6.5. The filtered sample (Nalgene Nunc, CAT #540887, MEMB 0.2 PES, Rochester, NY) was loaded to the 1St HIC column which was pre-equilibrated with 1.5 M (NH4)2SO4,20 MM potassium phosphate, 1 mM EDTA, pH 6.5 (Bed volume 150 ml, in XK-50, GE Healthcare, Piscataway, NJ). The ADA protein was eluted with an ammonium sulfate gradient and the purity profile of this elution was determined by SDS-PAGE and RP-HPLC. The ADA protein in the fractions of first HIC column was pooled and adjusted to 1 M
(NH4)2SO4 and loaded directly to the second HIC column (Bed volume 150m1, XK-50, HIC Phenyl HP, Cat# 17-1082-01, Piscataway, NJ) which was pre-equilibrated with 1 M (NH4)2S04,20 mM KH2PO4-KZHPO4, 1 mM EDTA, pH 6.5. ADA was eluted with an ammonium sulfate gradient from 1 M to 300 mM in the 20 mM KH2P04-KZHPO4, I
mM EDTA, pH 6.5. ADA purity of these fractions was analyzed by SDS-PAGE and RP-HPLC. The purified rbADA or rhADA was further desalted and concentrated in a LabScaleTM TFF systems (Membrane BioMax 5, Bedford, MA).

PREPARATION OF rhADA CELL EXTRACT
TREATED WITH OXIDIZED GLUTATHIONE

Frozen cells prepared according to Example 2, were thawed and resuspended in resuspension buffer contqining 20 mM Bis-Tris 1mM EDTA at pH 7.4. The thawed, resuspended cells were homogenized, filtered through a 250~t stainless steel mesh and microfluidized at 15000 psi for 3 cycles to ensure complete lysis. The resultant cell extract was clarified by centrifugation and half of the supemant was added to 25 mM
oxidized glutathione ("GSSG") (final concentration: GSSG was made in 250 mM
stock in 200 mM Bis-Tris, pH 7.4). The other half of the extract was treated with an additiona125 mM GSSG (therefore, total concentration of GSSG was 50 mM) before further processing. Both samples were then treated overnight with 0.1%
polyethyleneimine ("PEI", pH 7.4) at 4 C and centrifuged to remove nucleic acid and protein precipitates and subsequently filtered. The clarified sample was then processed further through anion exchange chromatography. The details of the cell extract preparation are compiled in Table 1, as follows.

Rr ~ O ~r1. =

c3 N O OO E-~ y ~ `ti N a) L:
=~~õ' ~ ,--~ 00 bA - C) p 2 o p Z ~~.~ yi ,, p o > p o a) o v~ o;
E cn O N z~~ d U c~ ~a u, a =~ U ~ OA ~ =~ N N.Y.+ G9 0 0 51..~
Q m -0 4.-P-M Vl O p a _ to =n y. "d 4] O
N
~ Vi .~" +-+ =~ ~ CC i-+ V p ` ~

~S oa n LC , =

P'I Q tl o0 M M
f~ U R+ V ~
p E'~ ',~% .~-p o 0 o a 6\
>~ Q N N N (~i d Q. n n n n C/]

v~ O Uj k/'Y
~
co CD
~Q ~rC7~vC
14 N kn N
r--LF) CC a~ ~
~
,~-\w 15; T-ct~
V ~ [C r+ N L, .~ ~ ~ =++ "'' -~ ~
h0 .=-y~. 'a O cG ia. = ~ ~ ~ ~ E
~
~ ¾' = ~ r~-i , ~ Z4 ~ N ~ "tl ¾' tl ~ ~ '"' bj) 0 'C7 W ~ _"G P-' y m ~ ,-r"+ ~ ~ =y C ~1 ~ y ~ p ~ tl 'tl cd ; ~ ID rZ4 C/] 0 O.-.+
r..} V] Q w~¾a A. n C..) The clarification of cell extracts by PEI treatment was confirmed by SDS-PAGE.
The protection of ADA protein by GSSG was confirmed by reverse-phase HPLC.

EXA.MPLE 6 PURIFICATION OF rhADA USING
ANION EXCHANGE CHROMATOGRAPHY
IN THE PRESENCE OF OXIDIZED GLUTATIHIONE

The cell extract (100 m l) obtained above was subjected to an anion ion-exchange chromatogaphy column (can be Q Sepharose fast flow, DEAE Sepharose fast flow, Capto Q Sepharose fast flow, etc, GE Healthcare, Piscataway, NJ). Two independent chromatographies were conducted to purify the rhADA from 25 mM- and 50 mM GSSG-treated cell extracts. Briefly, the pH and conductivity of the cell extract were adjusted to 6.5 and 1.5 mS/cm, respectively with addition of 1N NaOH, followed by dilution with distilled water. Approximately 100 ml of adjusted-cell extract (treated with 25 mM GSSG) was passed through a 27ml DEAE column pre-equilibrated with equilibration buffer containing 20 mM Bis-Tris and 1 mM EDTA at pH 6.5. Non-specifically bound proteins were removed from the column with 2 cv (ie., column volumes) of equilibration buffer, and human ADA was eluted from the column with a 20 cv linear gradient of 20 mM
KCI
prepared in the above buffer. Fractions corresponding to the peak were collected, analyzed by 4-20% reducing gel and, fractions containing human ADA were pooled.
Subsequent to protein elution, column was sanitized with NaOH and acetic acid and was equilibrated wIth equlllbratlon buffer prior to process and puriy hu~i~an ADA from 50 mM
GSSG-treated cell extract following an identical protocol. In order to retain integrity of the glutathione-modified species, no reducing agent was used during purification steps.
The separation of glutathione capped rhADA (GS-rhADA) from other iunpurities by this chromatographic method was confirmed by running peak fractions on standard SDS-PAGE. The ADA activity of GS-rhADA was confirmed by standard enzyrnatic assay.

PURIFICATION OF rbADA USING
CHROMATOGRAPHIC METHOD
IN THE PRESENCE OF OXIDIZED GLUTATHIONE

The purification of rbADA was carried out in a 3 chromatographic protocol.
Briefly, thawed cell paste of 200 g (as obtained from Example 4, supra) which was stored at -80 C, was re-suspended in 1800 ml buffer of 20 mM Bis-Tris, 1mM EDTA, pH
7.4, and homogenized at 1200 RPM for 10 seconds with Tempest Virtis (SentryTM, Microprocessor, Boston, MA). This suspension was passed through a stainless steel mesh (Opening micrometer 250 , No.60, W.S Tyler) to removed big particles. The homogenous cell suspension was microfluidized for 3 cycles at 15,000 psi (unit was ice-bathed) (Micro Fluidizer, Microfluidics Corp., Model# 110Y, Boston, MA). At the end of micro fluidization, 200 ml of the same buffer as above was used to rinse the unit and this solution was combined with the above suspension. The soluble protein from cell lysates was extracted by centrifugation at 7100 rpm (12000 x g) for 60 minutes at 4 C
(Avanti J-201, Beckman Cou.lter; Rotor# JLA8.1000). The supernatant was collected carefully to avoid unwanted mixing. Now the volume of supematant was 2100 ml, total protein concentration by BCA method was 13.2 mg/ml.
To remove nucleotides in this cell extract, a 31 ml of 10% PET solution (final PEI
0.15%) was added to the above supernatant and mixed thoroughly by stirring for 10 min_ This cell extract was then stored overnight at 4 C. The precipitant from this overnight sample was removed by a centrifugation at 7100 rpm (12000Xg), for 60 minutes at 4 C
(Avanti J-201, Beckrnan Coulter; Rotor# JLA8.1000). Similarly, the supernatant was collected carefully to avoid any unwanted mixing. Now the volume of supernatant was 1940 ml, total protein concentration by BCA method was 3.53 mg/ml.
Protection of cysteine 74 (ie., Cys75 if the N-terminal Met is present) was performed by capping this amino acid with 25 mM oxidized glutathione at pH
6.5. To help ADA bind to the first column, 10% PEG4600 was added to this cell extract slowly and the pH of this cell extract was adjusted to 6.5 slowly with I N NaOH. This supernatant was centrifuged again at 7100 rpm (12000 x g), for 60 minutes at 4 C (Avanti 7-201, Beckman Coulter; Rotor# JLA8.1000) before loaded to the next column.
The cell extract was loaded to a pre-equilibrated Capto Q column (Cat# 17-5316-01, GE Healthcare, Piscataway, NJ. Bed volume 350 ml pre packed in a XK-50 column) with a buffer of 20 mM Tris-Bis, 1 mM EDTA, pH 6.5. Before ADA was eluted off from the column at 80 mM NaCI in the equilibration buffer, elutions at 60 mM and 70 mM NaCl were first performed to remove impurities. The elution profile was analysed by ADA
activity, SDS-PAGE analysis, Western Blots, and RP-HPLC.
After the Capto Q column, two hydrophobic interaction chromatographic purifications were used one by one to further polish the purity of the protein. The first HIC
was Octyl Sepharose 4FF (Cat# 17-0946-02, GE Healthcare, Piscataway, NJ). The pool of ADA fractions from Capto Q column was adjusted to 1.5 M(NH4)2SO4 with ammonium sulfate powder directly and the pH was adjusted to 6.5. The filtered sample (Nalgene Nunc, CAT #540887, MEMB 0.2 PES, Rochester, NY) was loaded to the lst HIC
column which was pre-equilibrated with 1.5 M(NHa)2SO4, 20 mM potassium phosphate, 1 mM
EDTA, pH 6.5 (Bed volume 150 ml, in XK-50, GE Healtlicare, Piscataway, NJ).
The ADA protein was eluted with an ammonium sulfate gradient and the purity profile of this elution was determined by SDS-PAGE and RP-HPLC. The ADA protein in the fractions of first HIC column was pooled and adjusted to 1 M(NHa)z.SO4 and loaded directly to the second HIC column (Bed volume 150m1, XK-50, HIC Phenyl HP, Cat# 17-1082-01, Piscataway, NJ) which was pre-equilibrated with 1 M(NI-Ia2SO4, 20 MM KHZPO4-KZHPO4, 1 mM EDTA, pH 6.5. ADA was eluted off with an ammonium sulfate gradient from 1 M to 300 mM in the 20 mM KH2PO4-K2HPO4, 1 mM EDTA, pH 6.5. ADA purity of these fractions was analyzed by SDS-PAGE and RP-HPLC. The purified rbADA
was further desalted and concentrated in a LabScaleTM TFF systems (Membrane BioMax 5, Bedford, MA).

CHARACTERIZATION OF PARTIALLY PURIFIED rhADA BY MASS
SPECTROMETRY AND REVERSE PHASE HPLC (RP-IIPLC) A glutathione-modified recombinant human ADA cell extract was partially purified by anion exchange chromatography (Capto Q column, GE Healthcare, Piscataway, NJ) using 80 mM NaCI in 20 mM Tris-Bis; 1 mM EDTA (pH 6.5) for elution. The major product-containing fraction was subjected to liquid chromatography / mass spectrometry ("LCIMS") and analyzed for the presence of the glutathione adduct of ADA. The analysis indicated that the major peak, comprising 85% of the total area of the chromatogram, had a mass of 40,940 Da, which corresponds to the theoretical mass of glutathione-modified rhADA. Several other impurities were present, including a minor component of non-modified rhADA, and accounted for the remaining 15% of the chromatogram.
In addition, an experiment was perforrned to determine the effect of glutathione concentration on the degree of rhADA modification. The crude cell extract was divided into two portions which were subsequently treated with 25 mM and 50 mM
glutathione, respectively. Each preparation was then partially purified by anion exchange chromatography and analyzed by LC/MS. Results of LC/MS analysis indicated that the major peak from each preparation had a mass corresponding to glutathione-modif ed rhADA, with peak area comprising 83-85% of the total area of the chromatogram.
Several other iznpurities were present, including minor amounts of non-modified rhADA, accounting for the remaining 15-17% of the chromatograms. The results indicate that both mM and 50 mM glutathione treatments efficiently convert rhADA to glutathione-modified rhADA.

CONFIRMATION OF PROTECTIVE EFFECT FOR rhADA OF CAPPED
CYSTEINE WITH IAA

The following experiment was done to confirm the protective effect of capping rhADA with IAA. rhADA, at a concentration of approximately 0.6 mg/mL, was reacted with 125 mM iodoacetamide (IAA) in sodium phosphate buffer at pH 7.4 for 16 hours at 37 C. Within several minutes of beginning the reaction, analysis of the sample by RP-HPLC with UV and mass spectrometric detection showed that approximately 70.9%
of the rhADA was monoderivatized with IAA and 17.2% was derivatized at two sites.
After 2 and 16 hours incubation at 37 C, the chromatographic profile was not significantly changed, with no indication that oxidized species were formed, indicating that the IAA
derivative was stable towards the oxidative degradation pathways typical of rhADA.
When. a sample of rhADA that was not derivatized with IAA was incubated under the same conditions (16 hours at 37 C and pH 7.4) and analyzed similarly, the rhADA
protein degraded to oxidized species to an extent of 30%.
Thus, capping with IAA protected rhADA from the oxidative degradation that occurs at 37 C and pH 7.4.

CONFIRMATION OF PROTECTIVE
EFFECT FOR rhADA OF CAPPED CYSTEINE WITH GLUTATHIONE
As an alternative to the IAA, it was decided to attempt derivatization of the reactive cysteine with oxidized glutathione, a normal biological constituent. rhADA, at a concentration of 0.6 mg/mL, was reacted with 25 mM oxidized glutathione in sodium phosphate buffer at pH 7.4. After incubation for 4, 8, and 12 hours, the sample was analyzed by RP-HPLC with UV and mass spectrometric detection_ The monoderivatized form of GS-rhADA was formed exclusively and the derivative was stable after 12 hours incubation at 25 C. Additionally, the activity of the mono-glutathionylated rhADA
compound was not found to be significantly different from the activity of the non-derivatized rhADA. Thus, capping the reactive cysteine in rhADA with oxidized glutathione yields a predominantly mono-derivatized species that is stable under conditions where the non-derivatized rhADA is prone to oxidative degradation and the derivative has activity comparable to that of the non-derivatized form of rhADA.

CONFIRMATION OF PROTECTIVE EFFECT FOR GS-rbADA
Samples of mature rbADA, capped at cys74 with glutathione (GS-rbADA) at concentrations of approximately 0.5 mg/mL in sodium phosphate buffer (pH 7.8) were used for the stability study. Stability was monitored by reversed-phase HPLC
(RP-HPLC) using both UV detection at 220 nm and mass spectrometric detection (Micromass Q-TOF
electrospray mass spectrometer). The HPLC conditions were as follows:
Column: Zorbax 300 SB-C8 (Agilent, 250 x 4.6 mm, 300 angstrom pore size, 5 micron particle size Mobile Phase A: 0.1 % trifluoroacetic acid in water Mobile Phase B: 0.1% trifluoroacetic acid in acetonitrile/water (80/20; v/v) Gradient: Time % Mobile Phase B

Column temperature: 40 C
.p'low rate: 1.0 mL/min Injection volume: 50 L

Purity of the compounds was determined by RP-HPLC analysis at the initial time the stability study was started and at various timepoints, including 4, 8, and 17 days, after initiation of the study. It should be noted that the rbADA and GS-rbADA
samples were approximately two months old at the start of this study and had already suffered some degradation. However, for the purpose of the present study, the difference in purity between the initial tim.epoint and after 17 days incubation at 25 C is the relevant parameter to examine. As shown in Table 1, the purity of rbADA was 83.7% at the initial timepoint and decreased to 66.1% after 17 days, indicating that 17.6% of rbADA has degraded over this time period. Mass spectrometric analysis of the peaks separated chromatographically indicated that the major degradant eluting at 31.851 min, accounting for 30.5%
of the area of the chromatogram, had a mass 32 Da higher than that of rbADA. This mass change is consistent with the addition of 2 oxygens to rbADA to form the sulfinic acid degradant of the free cysteine at position 74 of rbADA. The smaller degradant peak, eluting at 32.538 min, had a mass consistent with the addition of I oxygen to rbADA to form the sulfenic acid degradant of the free cysteine at position 74 of rbADA. In contrast to the extensive degradation found for rbADA, GS-rbADA did not exhibit significant degradation when stored under the same conditions, with the purity at 81.9% at the initial timepoint and purity at 87.4% after 17 days at 25 C. The apparent increase in purity is likely due to chronnatographic variations occurring as is more evident when the % purities at cach of the 4 successive timepoints are examined. There is no evidence of the extensive oxidative degradation seen in rbADA demonstrating that capping the reactive cysteine in rbADA
protects the enzyme from this degradation pathway. This proves that cysteine74 is indeed the source of the oxidative degradation that occurs in ADA_ Table 2. Stability of rbADA, GS-rbADA, in sodium phosphate buffer (pH 7.8) at C

Timepoint % Purity as Determined by RP-HPLC
rbADA GS-rbADA
Initial 83.7 81.9 4 Days 83.6 86.1 8 Days 76.3 82.5 17 Days 66.1 87.4

Claims (48)

1. A capped protein comprising at least one amino acid residue that is linked to a capping agent, wherein the capped protein is substantially more stable in aqueous medium than the equivalent uncapped protein, wherein the linked amino acid residue is an amino acid that is oxidizable, and wherein the capping agent is selected to inhibit the oxidation of the amino acid.

2. The capped protein of claim 1 wherein the oxidizable amino acid is selected from the group consisting of a cysteine residue, a methionine residue, a tryptophan residue, and combinations thereof.

3. The capped protein of claim 1 that is an adenosine deaminase protein or an interferon.

4. The capped protein of claim 3 wherein the adenosine deaminase is selected from the group consisting of recombinant human adenosine deaminase and recombinant bovine adenosine deaminase.

5. The capped protein of claim 4 wherein the recombinant human adenosine deaminase comprises SEQ ID NO: 2, prior to capping; and the recombinant bovine adenosine deaminase comprises SEQ ID NO: 4, prior to capping.

6. The capped protein of claim 5 wherein the six C-terminal residues of the bovine adenosine deaminase are not present.

7. The capped protein of claim 1 wherein the capping agent is selected from the group consisting of oxidized glutathione, iodoacetamide, iodoacetic acid, cystine, other dithiols and mixtures thereof.

8. The capped protein of claim 4 that is conjugated to a substantially non-antigenic polymer to form a polymer conjugated capped protein.

9. The polymer conjugated capped protein of claim 8 wherein the polymer is a polyalkylene oxide.

10. The polymer conjugated capped protein of claim 9 wherein the polyalkylene oxide ranges in size from about 2,000 to about 100,000 Daltons.

11. The polymer conjugated capped protein of claim 9 wherein the polyalkylene oxide is a polyethylene glycol.

12. The polymer conjugated capped protein of claim 11 wherein the polyethylene glycol is conjugated to the protein via a linker chemistry selected from the group consisting of succinimidyl carbonate, thiazolidine thione, urethane, and amide based linkers.

13. The polymer conjugated capped protein of claim 11 wherein the polyethylene glycol is covalently attached to an epsilon amino group of a Lys on said capped protein.

14. The polymer conjugated capped recombinantly expressed protein of claim 11, wherein protein has at least 5 polyethylene glycol strands attached to epsilon amino groups of Lys on said capped protein.

15. The polymer conjugated capped recombinantly expressed protein of claim 11, wherein the protein has about 11-18 PEG strands attached to epsilon amino groups of Lys on said protein.

16. A method of capping a protein having at least one oxidizable amino acid thereon, comprising, treating the protein containing an oxidizable amino acid with a sufficient amount of a capping agent under reaction conditions sufficient to cap the oxidizable amino acid without substantially inactivating the protein, to provide a stabilized protein.

17. The method of claim 16, wherein the protein is a recombinant adenosine deaminase selected from the group consisting of recombinant human adenosine deaminase and recombinant bovine adenosine deaminase.

18. The method of claim 17, wherein the recombinant human adenosine deaminase comprises SEQ ID NO: 2, prior to capping; and the recombinant bovine adenosine deaminase comprises SEQ ID NO: 4, prior to capping.

19. The method of claim 16, wherein the capping agent is selected from the group consisting of oxidized glutathione, iodoacetamide, iodoacetic acid, cystine, other dithiols and mixtures thereof.

20. The method of claim 19, wherein the capping agent is oxidized glutathione.

21. The method of claim 20, wherein the oxidized glutathione is reacted with the protein at a concentration of from about 20 to about 100 mM.

22. The method of claim 20, wherein the oxidized glutathione is reacted with the protein at a concentration of from about 22 to about 30 mM.

23. The method of claim 16, wherein the reaction conditions include an aqueous solution at pH of from about 6.5 to about 8.4.

24. The method of claim 23, wherein the reaction conditions include an aqueous solution at pH of from about 7.2 to about 7.8.

25. The method of claim 23, wherein the aqueous solution includes a buffer selected from the group consisting of sodium phosphate, potassium phosphate, Tris, and Hepes at concentrations ranging from 20 to 150 mM.

26. The method of claim 16, further comprising reacting the capped protein in said aqueous solution with an activated polymer under condition sufficient to form capped protein-polymer conjugates.

27. The method of claim 26, wherein the activated polymer is an activated polyethylene glycol.

28. The method of claim 27, wherein the activated polyethylene glycol is selected from the group consisting of a succinimidyl carbonate-activated polyethylene glycol, a thiazolidine thione-activated polyethylene glycol, and a urethane linkage or amide-linkage forming -activated polyethylene glycol.

29. The method of claim 27, wherein the polyethylene glycol has a molecular weight of from about 2,000 to about 100,000 Daltons.

30. The method of claim 29, wherein the polyethylene glycol has a molecular weight of from about 4,000 to about 45,000 Daltons.

31. The method of claim 16, wherein the protein is an interferon.

32. The method of claim 31, wherein the interferon is selected from the group consisting of a beta interferon, an alpha interferon and a gamma interferon.

33. A method of stabilizing a recombinantly expressed protein having a reactive cysteine thereon, comprising, recombinantly expressing a protein in a suitable prokaryotic expression system and recovering the recombinantly expressed protein from a cell extract media containing a sufficient amount of a capping agent whereby the reactive cysteine on the recombinantly expressed protein is stabilized upon secretion from the prokaryotic cells in said suitable prokaryotic cell expression system.

34. The method of claim 35, wherein the prokaryotic expression system is an E.
coli expression system capable of producing recombinant adenosine deaminase.

35. The method of claim 33, wherein the recombinantly expressed protein is selected from the group consisting of adenosine deaminase and interferon.

36. The method of claim 35, wherein the recombinantly expressed adenosine deaminase has an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, prior to capping.

37. The method of claim 33, wherein the capping agent is selected from the group consisting of oxidized glutathione, iodoacetamide, iodoacetic acid, cystine, other dithiols and mixtures thereof.

38. The method of claim 37, wherein the capping agent is oxidized glutathione.

39. The method of claim 38, wherein the glutathione in said cell extract media is present in a concentration of from about 20 to about 100 mM.

40. The method of claim 39, wherein the glutathione in said cell extract media is present in a concentration of from about 20 to about 50 mM.

41. The method of claim 32, wherein the cell extract media includes 20 mM Bis-Tris and 1.0 mM EDTA at pH 7.4.

42. A method of treating an adenosine deaminase -mediated condition in mammals, comprising administering an effective amount of a polymer conjugated capped recombinantly expressed adenosine deaminase of claim 15.

43. The method of claim 42, wherein the adenosine deaminase-mediated condition is severe combined immune deficiency.

44. A method for producing a stabilized derivative of an oxidizable protein of interest, comprising the steps of (a) identifying an oxidizable protein of interest, wherein the protein is oxidizable at one or more amino acid residues when the protein is in aqueous solution, (b) identifying at least one oxidizable amino acid of the protein of interest, (c) reacting the oxidizable protein of interest with a capping agent selected to prevent the oxidation of the oxidizable amino acid.

45. The method of claim 44 wherein the protein is adenosine deaminase or interferon.

46. The method of claim 44 wherein the oxidizable amino acid is cysteine.

47. The method of claim 44 wherein the capping agent is selected from the group consisting of oxidized glutathione, iodoacetamide, iodoacetic acid, cystine, other dithiols and mixtures thereof.

48. The method of claim 23, wherein the reaction conditions include an aqueous solution at pH of about 6.5.