EP2038424A2 - Stabilisierte proteine - Google Patents

Stabilisierte proteine

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Publication number
EP2038424A2
EP2038424A2 EP07797875A EP07797875A EP2038424A2 EP 2038424 A2 EP2038424 A2 EP 2038424A2 EP 07797875 A EP07797875 A EP 07797875A EP 07797875 A EP07797875 A EP 07797875A EP 2038424 A2 EP2038424 A2 EP 2038424A2
Authority
EP
European Patent Office
Prior art keywords
protein
capped
adenosine deaminase
amino acid
oxidizable
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07797875A
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English (en)
French (fr)
Inventor
Stephen K. Youngster
Amartya Basu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Enzon Pharmaceuticals Inc
Original Assignee
Enzon Pharmaceuticals Inc
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Filing date
Publication date
Application filed by Enzon Pharmaceuticals Inc filed Critical Enzon Pharmaceuticals Inc
Publication of EP2038424A2 publication Critical patent/EP2038424A2/de
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • 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.
  • AD A Adenosine deaminase
  • SCID severe combined immunodeficiency disease
  • rADA recombinant source enzyme
  • rhADA rhADA
  • rbADA recombinant bovine
  • 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 (sulfhydryl) which is the form responsible for the degradation.
  • 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.
  • 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 cysteine 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.
  • the invention provides for a capped protein comprising 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.
  • 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).
  • the recombinant adenosine deaminase lacks the six C-terminal residues indicated by the theoretical translation of the corresponding DNA open reading frame.
  • 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, iodoacetamide, 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.
  • 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 agent is, e.g., oxidized glutathione, iodoacetamide, iodoacetic acid, cystine, other dithiols and mixtures thereof, and oxidized glutathione is preferred.
  • 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.
  • 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 atpH of from about 7.2 to about 7.8.
  • 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.
  • an activated polyethylene glycol such as, a succinimidyl carbonate-activated polyethylene glycol,
  • the inventive process further 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 recombinantly expressed protein is stabilized upon secretion from the prokaryotic cells in said suitable prokaryotic cell expression system.
  • 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
  • the protein is, e.g., an adenosine deaminase or interferon
  • the oxidizable amino acid is cysteine or methionine
  • the capping agent is selected from the group consisting of oxidized glutathione, iodoacetamide, iodoacetic acid, cystine, other dithiols and mixtures thereof.
  • cysteine-stabilized recombinant proteins 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/or 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 forming -activated polyethylene glycol.
  • SC-PEG succinimidyl carbonate- activated polyethylene glycol
  • T-PEG thiazolidine thione-activated polyethylene glycol
  • a urethane linkage or amide-linkage forming -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.
  • 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.
  • 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 enzyme deficiency or SCID condition in the patient, i.e., in a mammal or a human.
  • composition comprising a cell includes reference to one or more of such cells.
  • the invention provides capped or protected proteins with enhanced stability.
  • 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 oxidizable 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.
  • 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.
  • 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 determining 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.
  • 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 recombinant production after expression but preferably for this aspect of the invention, during one or more of the post expression purification steps.
  • 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).
  • DTT dithiothreitol
  • 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.
  • the recombinant ⁇ expressed protein is rhADA or rbAD A. 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.
  • SEQ ID NO: s 2 and 4 are the predicted mature translation sequences, and reflect the post translational cleavage of the N-termmal Met residue. Thus, in the translated respective mature ADA proteins, the Cys residue is at position 74.
  • the natural bovine ADA as isolated from bovine intestine also has six residues posttranslationally removed from the C-terminal.
  • the rbADA is either expressed without the six C-term ⁇ ial residues (as a mutein) or is posttranslationally modified to remove the same C-terminal residues lacking in the purified natural bovine ADA.
  • bovine ADA as isolated from bovine intestine has polymorphisms.
  • 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: GIn in place of Lysi 98 ; Ala in place of Thr245; Arg in place of GIy 351 .
  • the rhADA is expressed by a DNA with codons optimized for E. coli expression, according to SEQ ID NO: 1 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 genomic or cDNA encoding rhADA or rbADA, respectively, that is optionally under the control of a suitable operably connected inducible promoter.
  • 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( ⁇ DE3) strain. This recombinant clone was designated as EN760.
  • rbADA expression clone was constructed by transforming BioCatalytics plasmid (p2TrcGro-RoADA2-23) into fresh E. coli BL21 chemically competent cells (Novagen, catalog number 69449-4, lot number N60830, Genotype F " ompT ⁇ sdS ⁇ (I B me ) gal dcm).
  • This clone contains the plasmid p2TrcGro-RoADA2-23 that has the bovine ADA gene provided by Roche and the E. coli GroEL/GroES 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 ⁇ g/mL kanamycin at 3O 0 C.
  • CFB medium 10 g/L soy peptone, 5 g/L yeast extract, 10 g/L sodium chloride
  • 2% (w/v) agar 2% (w/v) agar
  • 25 ⁇ g/mL kanamycin at 3O 0 C.
  • the expression of rbADA was induced by IPTG.
  • the capping agents included in the methods of the invention 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.
  • the concentration of used when reacted with the recombinant protein such as rhADA can range from about 25 ⁇ M to about 100 mM.
  • 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. to.
  • 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.
  • 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 cysteine group(s) that may be involved in instability, such as certain interferons.
  • ⁇ , ⁇ , or ⁇ interferon can stabilized by the inventive methods.
  • suitable proteins, polypeptides and peptides of interest include, but are not limited to, hemoglobin, serum proteins such as blood factors including Factors VII, VIII, and IX; immunoglobulins, cytokines such as interleukins, i.e. IL-I through IL-13, etc., colony stimulating factors including granulocyte colony stimulating factors, platelet derived growth factors, etc.
  • 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 TGFGC'S or ⁇ 's and epidermal growth factors, hormones, somatomedins, erythropoietin, pigmentary hormones, hypothalamic releasing factors, antidiuretic hormones, prolactin, chorionic gonadotropin, follicle- stimulating 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.
  • examples of enzymes of interest include L-asparaginase, arginase, arginine deiminase, superoxide dismutase, endotoxinases, catalases, chymotrypsin, lipases, uncases, adenosine diphosphatase, tyrosinases and bilirubin oxidase.
  • Carbohydrate-specific enzymes of interest include glucose oxidases, glucodases, galactosidases, glucocerebrosidases, glucuronidases, 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.
  • the stabilized protein has a capping group attached thereto at a reactive cysteine.
  • 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:
  • 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.
  • 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.
  • 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.
  • activated polyethylene glycols can be used, including those described, for example in commonly assigned US Patent Nos.
  • 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 O96 patents.
  • the PEG can be of the formula:
  • 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 3 terminal portions of the residue is/are capped with a methyl or other lower alkyl.
  • all 4 of the PEG arms are converted to suitable leaving groups, i.e. SC, etc., for facilitating attachment to the recombinant protein.
  • suitable leaving groups i.e. SC, etc.
  • the activated polyethylene glycol is one which provides a urethane linkage or amide-linkage with the protein.
  • the activated polymers can employ a hindered ester- based linker.
  • 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.
  • a non-limiting list of such compounds - include: and
  • 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.
  • 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 /-butyl ester of polyalkylene oxide carboxylic acid. This intermediate is formed by reacting a PAO with a /-butyl haloacetate in the presence of a base such as potassium /-butoxide.
  • the carboxylic 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.
  • 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.
  • polymers having azides react with phosphine-based reducing agent such as triphenylphosphine or an alkali metal borohydride reducing agent such as NaBH 4 .
  • polymers including leaving groups react with protected amine salts such as potassium salt of methyl-tert-butyl iniidodicarbonate (KNMeBoc) or the potassium salt of di-tert-butyl imidodicarbonate (KNBoC 2 ) followed by deprotecting the protected amine group.
  • protected amine salts such as potassium salt of methyl-tert-butyl iniidodicarbonate (KNMeBoc) or the potassium salt of di-tert-butyl imidodicarbonate (KNBoC 2 ) followed by deprotecting the protected amine group.
  • KNMeBoc methyl-tert-butyl iniidodicarbonate
  • KNBoC 2 di-tert-butyl imidodicarbonate
  • 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.
  • PEG polyethylene glycol
  • Cj_4 alkyl-terminated polymers are also useful.
  • PAO-based polymers As an alternative 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.
  • HPMA's hydroxypropylmethacrylamides
  • polyvinyl alcohols polyvinyl alcohols
  • carbohydrate-based polymers carbohydrate-based polymers
  • copolymers of the foregoing, and the like can be used.
  • substantially or effectively non-antigenic means all materials understood in the art as being nontoxic and not eliciting an appreciable immunogenic response in mammals.
  • the steps include a recombinantly 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 suitable prokaryotic cell expression system.
  • the preferred protein being expressed is rhAD A or rbAD A.
  • a suitable prokaryotic expression system is an E. coli expression system capable of producing the rhAD A or rbAD A.
  • the rhADA cDNA containing expression vector (pET9d) was introduced into an E.coli expression strain, BL21( ⁇ DE3) 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.
  • 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 HADAl 092 clone).
  • the cDNA was codon optimized for bacterial expression by following the standard bacterial codon usage for Escherichia coli 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.
  • RNA sequence was then analyzed for the formation of hairpin structure or loop formation and was subjected to minimum free energy calculations, and Ncol and HindIII 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 pHADApET28a, and was transformed into the BLR( ⁇ DE3) strain of E.coli (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.
  • a single colony from a BLR ( ⁇ DE3) plate was inoculated in a 50ml tube in 5ml LB media containing kanamycin lO ⁇ g/ml and tetracycline 12.5 ⁇ g/ml.
  • the tube was incubated in a 37°C/250 rpm shaker for 7 hours and then, 250 ⁇ l of the culture was used to inoculate a IL flask containing 250ml of fresh complete LB media. This flask was incubated in a 37°C/250 rpm shaker overnight.
  • the overnight culture (OD 6O0 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 lO ⁇ g/ml, tetracycline 12.5 ⁇ g/ml, 5ml/L glycerol, and 1.68ml/L MgSO 4 JH 2 O) resulting in an initial OD 60O of 0.2445.
  • the culture was grown to an OD 6 O 0 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 (ODeoo value 44) using a Beckman Coulter Centrifuge (7000rpm/4°C/20min) yielding a wet cell weight of 282 g.
  • Medium composition ODeoo value 44
  • 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 cDNA sequence (GenBankNP_776312, incorporated by reference herein), hi 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. coli, 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.
  • the flanking restriction sites, Ndel and BamHIwe ⁇ Q included at the termini of the gene.
  • 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. coH strain BLR (DE3) or HMS 174 (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 ⁇ g/ml). This allowed for selection of colonies containing the plasmid pET-9d/bADA (designated bADA/pET9d: 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 Terminators. Isolated colonies were further 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 0 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
  • rbADA 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, ImM 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# 11OY, 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 supernatant 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)
  • 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 NaCl 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.
  • 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 (NHt) 2 SO 4 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 1 st HIC column which was pre-equilibrated with 1.5 M (NH 4 ) 2 SO 4 , 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 eh ⁇ tion 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 (NHU) 2 SO 4 and loaded directly to the second HIC column (Bed volume 150ml, XK-50, HIC Phenyl HP, Cat# 17-1082-01, Piscataway, NJ) which was pre-equilibrated with 1 M (NH 4 ) 2 SO 4 , 20 mM KH 2 PO 4 -K 2 HPO 4 , 1 mM EDTA, pH 6.5.
  • ADA was eluted with an ammonium sulfate gradient from 1 M to 300 mM in the 20 mM KH 2 PO ⁇ K 2 HPO 45 1 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).
  • 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 additional 25 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 0 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.
  • PEI polyethyleneimine
  • the cell extract (100 ml) obtained above was subjected to an anion ion-exchange chromatography 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 IN NaOH, followed by dilution with distilled water.
  • rbADA 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 -8O 0 C, was re-suspended in 1800 ml buffer of 20 roM Bis-Tris, ImM EDTA, pH 7.4, and homogenized at 1200 RPM for 10 seconds with Tempest Virtis (SentryTM,
  • 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.
  • 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. 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 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 niM Tris-B ⁇ s, 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 NaCl were first performed to remove impurities. The elution profile was analysed by ADA activity, SDS-PAGE analysis, Western Blots, and RP-HPLC.
  • the Capto Q column 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 (NHi) 2 SO 4 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 1 st HIC column which was pre-equilibrated with 1.5 M (NHi) 2 SO 4 , 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 (NHi) 2 SO 4 and loaded directly to the second HIC column (Bed volume 150ml, XK-50, HIC Phenyl HP, Cat# 17-1082-01, Piscataway, NJ) which was pre-equilibrated with 1 M (NH ⁇ SO 4 , 20 mM KH 2 PO 4 - K 2 HPO 4 , 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 KH 2 PO 4 -K 2 HPO 4 , 1 mM EDTA, pH 6.5.
  • 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.
  • IAA iodoacetamide
  • analysis of the sample by RP- HPLC with UV and mass spectrometric detection showed that approximately 70.9% of the rhADA was monoder ⁇ vatized with IAA and 17.2% was derivatized at two sites.
  • 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.
  • rhADA 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 0 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.

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