the present invention relates to substantially homogenous preparations of chemically modified proteins , and methods therefor . “ substantially homogenous ” as used herein means that the only chemically modified proteins observed are those having one “ modifier ” ( e . g ., dtpa , edta 2 , succinyl ) moiety . the preparation may contain unreacted ( i . e ., lacking modifier moiety ) protein . as ascertained by peptide mapping and n - terminal sequencing , one example below provides for a preparation which is at least 90 % modified protein , and at most 10 % unmodified protein . preferably , the chemically modified material is at least 95 % of the preparation ( as in the working example below ) and most preferably , the chemically modified material is 99 % of the preparation or more . the chemically modified material has biological activity . the present “ substantially homogenous ” monosuccinylated leptin , dtpa - leptin , and edta 2 - leptin preparations provided herein are those which are homogenous enough to display the advantages of a homogenous preparation , e . g ., ease in clinical application in predictability of lot to lot pharmacokinetics . as used herein , biologically active agents refers to recombinant or naturally occurring proteins , whether human or animal , useful for prophylactic , therapeutic or diagnostic application . the biologically active agent can be natural , synthetic , semi - synthetic or derivatives thereof . in addition , biologically active agents of the present invention can be perceptible . a wide range of biologically active agents are contemplated . these include but are not limited to hormones , cytokines , hematopoietic factors , growth factors , antiobesity factors , trophic factors , anti - inflammatory factors , and enzymes ( see also u . s . pat . no . 4 , 695 , 463 for additional examples of useful biologically active agents ). one skilled in the art will readily be able to adapt a desired biologically active agent to the compositions of present invention . such proteins would include but are not limited to interferons ( see , u . s . pat . nos . 5 , 372 , 808 , 5 , 541 , 293 , 4 , 897 , 471 , and 4 , 695 , 623 hereby incorporated by reference including drawings ), interleukins ( see , u . s . pat . no . 5 , 075 , 222 , hereby incorporated by reference including drawings ), erythropoietins ( see , u . s . pat . nos . 4 , 703 , 008 , 5 , 441 , 868 , 5 , 618 , 698 , 5 , 547 , 933 , and 5 , 621 , 080 hereby incorporated by reference including drawings ), granulocyte - colony stimulating factors ( see , u . s . pat . nos . 4 , 810 , 643 , 4 , 999 , 291 , 5 , 581 , 476 , 5 , 582 , 823 , and pct publication no . 94 / 17185 , hereby incorporated by reference including drawings ), stem cell factor ( pct publication nos . 91 / 05795 , 92 / 17505 and 95 / 17206 , hereby incorporated by reference including drawings ), and leptin ( ob protein ) ( see pct publication nos . 96 / 40912 , 96 / 05309 , 97 / 00128 , 97 / 01010 and 97 / 06816 hereby incorporated by reference including figures ). pct publication no . wo 96 / 05309 , published feb . 22 , 1996 , entitled , “ modulators of body weight , corresponding nucleic acids and proteins , and diagnostic and therapeutic uses thereof ” fully sets forth ob protein and related compositions and methods , and is herein incorporated by reference . an amino acid sequence for human ob protein is set forth at wo 96 / 05309 seq . id nos . 4 and 6 ( at pages 172 and 174 of that publication ), and the first amino acid residue of the mature protein is at position 22 and is a valine residue . the mature protein is 146 residues ( or 145 if the glutamine at position 49 is absent , seq . id no . 4 ). in addition , biologically active agents can also include but are not limited to insulin , gastrin , prolactin , adrenocorticotropic hormone ( acth ), thyroid stimulating hormone ( tsh ), luteinizing hormone ( lh ), follicle stimulating hormone ( fsh ), human chorionic gonadotropin ( hcg ), motilin , interferons ( alpha , beta , gamma ), interleukins ( il - 1 to il - 12 ), tumor necrosis factor ( tnf ), tumor necrosis factor - binding protein ( tnf - bp ), brain derived neurotrophic factor ( bdnf ), glial derived neurotrophic factor ( gdnf ), neurotrophic factor 3 ( nt3 ), fibroblast growth factors ( fgf ), neurotrophic growth factor ( ngf ), bone growth factors such as osteoprotegerin ( opg ), insulin - like growth factors ( igfs ), macrophage colony stimulating factor ( m - csf ), granulocyte macrophage colony stimulating factor ( gm - csf ), megakaryocyte derived growth factor ( mgdf ), keratinocyte growth factor ( kgf ), thrombopoietin , platelet - derived growth factor ( pgdf ), colony simulating growth factors ( csfs ), bone morphogenetic protein ( bmp ), superoxide dismutase ( sod ), tissue plasminogen activator ( tpa ), urokinase , streptokinase and kallikrein . the term proteins , as used herein , includes peptides , polypeptides , consensus molecules , analogs , derivatives or combinations thereof . in general , comprehended by the invention are pharmaceutical compositions comprising effective amounts of chemically modified protein , or derivative products , together with pharmaceutically acceptable diluents , preservatives , solubilizers , emulsifiers , adjuvants and / or carriers needed for administration . ( see pct 97 / 01331 hereby incorporated by reference .) the optimal pharmaceutical formulation for a desired biologically active agent will be determined by one skilled in the art depending upon the route of administration and desired dosage . exemplary pharmaceutical compositions are disclosed in remington &# 39 ; s pharmaceutical sciences ( mack publishing co ., 18th ed ., easton , pa ., pgs . 1435 - 1712 ( 1990 )). the pharmaceutical compositions of the present invention may be administered by oral and non - oral preparations ( e . g ., intramuscular , subcutaneous , transdermal , visceral , iv ( intravenous ), ip ( intraperitoneal ), intraarticular , placement in the ear , icv ( intracerebralventricular ), ip ( intraperitoneal ), intraarterial , intrathecal , intracapsular , intraorbital , injectable , pulmonary , nasal , rectal , and uterine - transmucosal preparations ). therapeutic uses of the compositions of the present invention depend on the biologically active agent used . one skilled in the art will readily be able to adapt a desired biologically active agent to the present invention for its intended therapeutic uses . therapeutic uses for such agents are set forth in greater detail in the following publications hereby incorporated by reference including drawings . therapeutic uses include but are not limited to uses for proteins like interferons ( see , u . s . pat . nos . 5 , 372 , 808 , 5 , 541 , 293 , hereby incorporated by reference including drawings ), interleukins ( see , u . s . pat . no . 5 , 075 , 222 , hereby incorporated by reference including drawings ), erythropoietins ( see , u . s . pat . nos . 4 , 703 , 008 , 5 , 441 , 868 , 5 , 618 , 698 , 5 , 547 , 933 , and 5 , 621 , 080 hereby incorporated by reference including drawings ), granulocyte - colony stimulating factors ( see , u . s . pat . nos . 4 , 999 , 291 , 5 , 581 , 476 , 5 , 582 , 823 , 4 , 810 , 643 and pct publication no . 94 / 17185 , hereby incorporated by reference including drawings ), stem cell factor ( pct publication nos . 91 / 05795 , 92 / 17505 and 95 / 17206 , hereby incorporated by reference including drawings ), and the ob protein ( see pct publication nos . 96 / 40912 , 96 / 05309 , 97 / 00128 , 97 / 01010 and 97 / 06816 hereby incorporated by reference including figures ). in addition , the present compositions may also be used for manufacture of one or more medicaments for treatment or amelioration of the conditions the biologically active agent is intended to treat . the principal embodiment of the method for making the substantially homogenous preparation of monosuccinylated protein comprises : ( a ) reacting a protein with 3 - 7 fold molar excess of succinic anhydride ; ( b ) stirring the reaction mixture 2 - 16 hours at 4 ° c . ; ( c ) dialyzing said mixture against 20 mm tris - hcl , ph 7 . 2 ; and ( d ) isolating said monosuccinylated protein . optionally , the method can comprise , just after step ( b ), the steps of : adding solid hydroxylamine to said mixture while maintaining the ph above 6 . 5 until said hydroxylamine is completely dissolved , followed by elevating the ph to 8 . 5 using 5 n naoh , followed by stirring said mixture another 1 - 2 hours at 4 ° c . the general process is shown schematically in example 1 . the principal embodiment of the method for making the substantially homogenous preparation of dtpa - protein comprises : ( a ) reacting a protein with 1 - 5 fold molar excess of dtpa ; ( b ) stirring the reaction mixture 2 - 16 hours at 4 ° c . ; ( c ) dialyzing said mixture against 20 mm tris - hcl , ph 7 . 2 ; and ( d ) isolating said dtpa - protein . the general process is shown schematically in example 1 . the principal embodiment of the method for making the substantially homogenous preparation of edta 2 - protein comprises : ( a ) reacting a protein with 0 . 5 - 5 fold molar excess of edta 2 ; ( b ) stirring the reaction mixture 2 - 16 hours at 4 ° c . ; ( c ) filtering said reaction mixture ; ( d ) concentrating said reaction mixture ; and ( e ) isolating said edta 2 - protein . the general process is shown schematically in example 1 . the following examples are offered to more fully illustrate the invention , but are not to be construed as limiting the scope thereof . example 1 describes the preparation of monosuccinylated leptin , monosubstituted dtpa - leptin monomers and dimers , and edta 2 - leptin monomers and dimers . example 2 describes the physiochemical characterization of the modified leptin species prepared in example 1 . example 3 describes the receptor binding studies performed on the modified leptin species prepared in example 1 . example 4 describes the solubility testing performed on the modified leptin species prepared in example 1 . example 5 describes the in vivo bioactivity studies performed on the modified leptin species prepared in example 1 . example 6 describes the injection site evaluation performed on the modified leptin species prepared in example 1 . this example describes the preparation of monosuccinylated leptin , monosubstituted dtpa - leptin monomers and dimers , and edta 2 - leptin monomers and dimers . the protein succinylation method of the present invention can be generally depicted as follows : recombinant human - methionyl - leptin ( rhu - met - leptin ) protein ( prepared as described in materials and methods , infra ) at 2 - 3 mg / ml in 20 mm nahpo 4 , ph 7 . 0 , was reacted with 3 - 7 fold molar excess of solid succinic anhydride ( sigma chemical , st . louis , mo . ), with a 5 fold molar excess preferred , and the reaction stirred 2 - 16 hours at 4 ° c . solid hydroxylamine ( sigma chemical , st . louis , mo .) is then added to the reaction while maintaining the ph above 6 . 5 . after the hydroxylamine has dissolved completely the ph is elevated to 8 . 5 using 5 n naoh and the reaction allowed to stir another 1 - 2 hours at 4 ° c . ( the hydroxylamine step may be omitted with a small decrease in yield ). finally , the reaction is dialyzed against 20 mm tris - hcl , ph 7 . 2 . the monosuccinylated rhu - met - leptin is isolated by anion exchange chromatography with a high performance sepharose q column ( pharmacia , piscataway , n . j .) in 20 mm tris , ph 7 . 2 , with a 0 - 0 . 5 m nacl gradient ( see fig1 ). the product is recognized in the eluant by an isoelectric shift of − 0 . 7 pi units observed with isoelectric focusing ( ief ) page using a 5 % polyacrylamide , ph 3 - 7 gel ( novex , inc ., san diego , calif .) ( fig2 ). final recovery of monosuccinylated rhu - met - leptin is typically 45 - 47 %. the dtpa modification method of the present invention can be generally depicted as follows : recombinant human - methionyl - leptin ( rhu - met - leptin ) protein ( prepared as described in materials and methods , infra ) at 2 - 3 mg / ml in 20 mm nahpo 4 , ph 7 . 0 , was reacted with a 1 - 5 fold molar excess of solid dtpa ( sigma chemical , st . louis , mo . ), with 2 - 3 fold molar excess preferred , and the reaction stirred 2 - 16 hours at 4 ° c . finally , the reaction is dialyzed against 20 mm tris - hcl , ph 7 . 2 . the dtpa modified rhu - met - leptin is isolated by anion exchange chromatography with a high performance sepharose q column ( pharmacia , piscataway , n . j .) in 20 mm tris , ph 7 . 2 , with a 0 - 0 . 5 m nacl gradient . alternatively , monomeric and dimeric forms of monosubstituted dtpa - rhu - met - leptin or rhu - met - leptin are separated by size exclusion chromatography on a sephacryl 100 column ( pharmacia , piscataway , n . j .) in pbs ( life technologies , grand island , n . y . )( see fig3 ). the products are recognized in the eluant by an isoelectric shift observed with the monomeric dtpa - leptin by isoelectric focusing ( ief ) page using a 5 % polyacrylamide , ph 3 - 7 gel ( novex , inc ., san diego , calif . )( fig2 ) or the mass increase of a crosslinked dimer observed with sds - page using a 4 - 20 % polyacrylamide gel ( novex , inc ., san diego , calif . )( see fig4 ). final recovery of dtpa - rhu - met - leptin dimer is approximately 30 %. the edta 2 modification method of the present invention can be generally depicted as follows : recombinant human - methionyl - leptin ( rhu - met - leptin ) protein ( prepared as described in materials and methods , infra ) at 2 - 3 mg / ml in 20 mm nahpo 4 , ph 7 . 0 , was reacted with a 0 . 5 - 5 fold molar excess of edta 2 ( aldrich chemical co ., milwaukee , wis .) either as a solid or dissolved in dmso , with 0 . 75 fold molar excess edta 2 in dmso preferred , and the reaction stirred 2 - 16 hours at 4 ° c . the reaction is then filtered through a 0 . 45 micron filter ( nalgene ), concentrated by stirred cell over 10 kda molecular weight cutoff membrane to ˜ 20 mg / ml and the monomeric and dimeric forms of monosubstituted edta 2 - rhu - met - leptin then separated by size exclusion chromatography on a sephacryl 100 column ( pharmacia , piscataway , n . j .) equilibrated in pbs ( see fig5 ). alternatively , the reaction may be purified by hydrophobic interaction chromatography using a high performance phenyl - sepharose column ( pharmacia , piscataway , n . j .) eluted with a 0 . 8 - 0 m ammonium sulfate gradient in 20 mm nahpo 4 , ph 7 . 0 . the products are recognized in the eluant by an isoelectric shift observed with the monomeric edta 2 - rhu - met - leptin by isoelectric focusing ( ief ) page using a 5 % polyacrylamide , ph 3 - 7 gel ( novex , inc ., san diego , calif .) ( fig2 ) or the mass increase of a crosslinked dimer observed with sds - page using a 4 - 20 % polyacrylamide gel ( novex , inc ., san diego , calif . )( fig4 ). final recovery of edta 2 - rhu - met - leptin dimer exceeds 50 %. this example describes the physiochemical characterization of the leptin conjugates prepared in example 1 . modification of succinyl - leptin , dtpa - leptin monomers and dimers , and edta 2 - leptin monomers and dimers was evaluated by a combination of peptide mapping of lys - c digests on reverse phase hplc , maldi - tof mass spectrometry and peptide sequencing . lys - c digests of unmodified leptin and the various modified leptins were performed by reaction of 100 μg of protein with 4 μg of endoproteinase lys - c ( boehringer mannheim ) in 50 mm tris - hcl , ph 8 . 5 ( 200 μl ) for four hours at room temperature . peptide maps of the various samples were generated by reverse phase hplc on a 4 . 6 × 250 mm , 5 μ c4 column ( vydak , hesperia , calif .) equilibrated in 0 . 1 % triflouroacetic acid ( tfa ) with elution over a 0 - 90 % acetonitrile gradient ( see fig6 - 8 ). as evidenced by the plots depicted in fig6 - 8 , only the n - terminal peptide ( m1 - k6 ) shows any change in retention time as a result of chemical modification . this result indicates that lysine at position 6 is unmodified and accessible to lys - c digestion and suggests that the chemical modification occurs at the α - amine of the n - terminus . n - terminal modification is further supported by efforts at n - terminal sequencing which indicate that the n - terminus is blocked ( data not shown ). mass determinations for succinyl - leptin and dtpa - and edta 2 - leptin dimers were made on a kompact maldi iv ( kratos , ramsey , n . j .) using a 12 pmol sample in a sinapinic acid matrix . each conjugate indicates a single chemical modification per molecule . in addition to the analysis above , the effects on the secondary structure of the succinyl - leptin was evaluated using circular dichroism spectroscopy . far - uv circular dichroism spectra of unmodified and succinylated leptin in phosphate buffered saline were collected using a 0 . 05 cm cell in a jasco j - 710 circular dichroism spectrophotometer ( jasco , tokyo , japan ). the spectra are depicted in fig9 and demonstrate that the secondary structure of succinylated - leptin is preserved . in sum , the example 2 data confirms the modification of succinyl - leptin , dtpa - leptin monomers and dimers , and edta 2 - leptin monomers and dimers at the n - terminus , as well as preservation of secondary structure with succinyl - leptin . this example describes the receptor binding studies performed on each of the leptin conjugates prepared in example 1 . each of the leptin conjugates prepared in example 1 was evaluated using an in vitro receptor binding assay which measures the relative affinity of leptin conjugates based on their ability to displace radiolabeled human leptin from a human leptin receptor expressed in immobilized cell membranes . as evidenced by the fig1 data , the chemically modified isoforms , succinyl -, dtpa -, and edta 2 - leptin each showed relative affinities for human leptin receptor equal to the unmodified leptin over the entire range of ligand binding (˜ 1 - 100 ng / ml ), with ed 50 &# 39 ; s of approximately 10 ng / ml . the example 3 data thus show that the monosubstituted succinyl - leptin , monosubstituted dtpa - leptin dimer , and edta 2 - leptin dimer demonstrate preservation of in vitro receptor binding activity as compared to unmodified leptin . this example describes the solubility testing performed on each of the leptin conjugates prepared in example 1 . the leptin conjugates were dialyzed into pbs then concentrated with centriprep concentrators , 10 kda molecular weight cutoff ( amicon ) to the point that precipitates were observed . the sample was clarified by centrifugation and the conjugate protein concentration in the supernatant determined . the samples were then kept at room temperature (≈ 22 ° c .) for 48 hours and at regular time points centrifuged and the conjugate protein concentration in the supernatant redetermined . the solubility of the conjugate protein in pbs is thus defined as the steady state protein concentration at room temperature observed in the supernatant after centrifugation ( see table 2 ). this example describes the in vivo bioactivity studies performed on the leptin conjugates prepared in example 1 . the described leptin conjugates were tested in both mouse and dog animal models to determine bioefficacy relative to the unmodified leptin . mice were injected daily for 5 - 7 days with monosubstituted succinyl - leptin , dtpa - leptin dimer , dtpa - leptin monomer and edta 2 - leptin dimer at dosages of 1 , 10 and 50 mg / kg body weight . bioefficacy was measured as a percentage weight loss from day 0 , normalized to the vehicle alone control and compared to the weight loss observed with the unmodified protein . all samples for dosages of 1 and 10 mg / kg were formulated in pbs at 0 . 2 and 2 . 0 mg / ml respectively . higher dosages were formulated in pbs at 20 - 50 mg / ml for the chemically modified forms , however the solubility limits of the unmodified leptin necessitated its formulation at high concentrations in a ph 4 acetate buffer . in addition dogs were injected with 0 . 05 , 0 . 15 and 0 . 5 mg / kg daily dosages of succinyl - leptin at 5 mg / ml over 28 days while monitoring weight loss followed by a recovery period . bioactivity , as judged by drug induced weight loss in animal models , for succinyl leptin was equivalent to the unmodified leptin in both dogs and mice ( fig1 ). similarly , both dtpa - leptin monomers and dimers and edta 2 - leptin dimers caused equivalent weight loss in mice as compared to the unmodified leptin ( fig1 & amp ; 12 ). the fig1 & amp ; 12 data show that the monosubstituted succinyl - leptin , monosubstituted dtpa - leptin monomers and dimers , and edta 2 - leptin dimer demonstrate preservation of in vivo bioefficacy as compared to unmodified leptin . this example describes the injection site evaluation performed on the leptin conjugates prepared in example 1 . tissue sections from the injection sites of three mice from each dosing group were examined histochemically . injection site pathology &# 39 ; s which were identified and scored were necrosis , suppurative ( mixed cell infiltrate composed of eosinophils and neutrophils ), mononuclear cells ( macrophages ), leptin precipitates ( characterized as either fine ppt . or large deposits / clumps ) and giant cells . each reaction was scored using the following grading system : the averaged sum of the scores for each animal were used to define an overall biocompatibility score using the following scoring key : although high concentrations of succinyl - leptin were marginally soluble in pbs at ph 7 . 0 , for the purposes of injection site testing , samples of succinyl - leptin at 20 mg / ml remained soluble in pbs at ph 7 . 2 and at 50 mg / ml in pbs at ph 7 . 5 . table 3 shows the injection site evaluation comparing unmodified leptin at 50 mg / ml delivered in ph 4 . 0 , acetate buffer vs . monosubstituted succinyl - leptin at 50 mg / ml in ph 7 . 5 , pbs , after 7 days . as depicted in table 3 , monosubstituted succinyl - leptin , at high concentration dosages , showed improvement in every category of injection site pathology relative to the unmodified leptin , with the most dramatic improvement seen with the almost complete elimination of leptin precipitates and giant cells in the injection sites . table 4 shows the injection site evaluation comparing unmodified leptin at 43 mg / ml delivered in ph 4 . 0 , acetate buffer vs . monosubstituted succinyl - leptin at 43 mg / ml in ph 4 . 0 , acetate buffer , after 7 days . the table 4 data shows that , surprisingly , it was also observed that high concentrations of monosubstituted succinyl - leptin could be delivered in ph 4 , acetate buffer and still demonstrate the dramatic improvements in injection site reactions observed when monosubstituted succinyl - leptin was delivered in pbs . table 5 shows the injection site evaluation comparing unmodified leptin at 20 mg / ml delivered in ph 4 . 0 , acetate buffer vs . monosubstituted dtpa - leptin dimer at 20 mg / ml in pbs , after 7 days . as depicted in tables 5 & amp ; 6 , dtpa - leptin dimers ( table 5 ) or edta 2 - leptin dimers ( table 6 ) can be administered to mice at high concentration in pbs demonstrating the same improvement in injection site pathology as observed with succinyl - leptin . these conjugates however , are substantially more soluble in ph 7 , pbs and thus provide for a more rugged formulation in this buffer . in sum , the example 6 data shows that the monosubstituted succinyl - leptin , monosubstituted dtpa - leptin monomers and dimers , and edta 2 - leptin monomers and dimers do not precipitate at the injection site when dosed at high concentrations , and importantly , demonstrate substantial improvement in the adverse injection site reactions observed with the unmodified leptin . the present recombinant human methionyl - leptin ( rhu - met - leptin ) may be prepared according to the above incorporated - by - reference pct publication , wo 96 / 05309 at pages 151 - 159 . for the present working examples , a rhu - met - leptin was used which has ( as compared to the amino acid sequence at page 158 ) a lysine at position 35 instead of an arginine , and an isoleucine at position 74 instead of an isoleucine . other recombinant human leptin proteins may be prepared according to methods known generally in the art of expression of proteins using recombinant dna technology . while the present invention has been described in terms of certain preferred embodiments , it is understood that variations and modifications will occur to those skilled in the art . therefore , it is intended that the appended claims cover all such equivalent variations which come within the scope of the invention as claimed .
the present invention broadly relates to chemical modification of biologically active proteins or analogs thereof . more specifically , the present invention describes novel methods for site - specific chemical modification of various proteins , and resultant compositions having improved biocompatibility and bioactivity .