as used herein , factor ix includes both plasma derived and recombinantly or synthetically produced . factor ix concentration is conveniently expressed as mg / ml or as u / ml , with 1 mg usually representing & gt ; 150 u ± 100 u or more . one unit of activity is defined as the amount of factor ix clotting activity in one milliliter of normal human plasma . the specific activity is the ratio of clotting activity concentration to protein concentration , expressed as u / mg of protein . amounts shown are understood to be ± about 10 %, e . g ., about 50 mm includes 50 mm ± 5 mm ; e . g ., 4 % includes 4 % ± 0 . 4 %, etc . as used herein , the term &# 34 ; tonicity modifier &# 34 ; includes agents which contribute to the osmolality of the solution . examples of tonicity modifiers include , but are not limited to , amino acids such as arginine , histidine , and glycine , salts such as sodium chloride , potassium chloride , and sodium citrate , and saccharides such as sucrose , glucose , and mannitol , and the like . the term &# 34 ; cryoprotectant &# 34 ; generally includes agents which provide stability to the protein from freezing - induced stresses ; however , cryoprotectants may also provide general stability , for example for bulk drug formulations during storage from non - freezing - induced stresses . exemplary cryoprotectants include polyols , and saccharides such as mannitol and sucrose , as well as surfactants such as polysorbate , or polyethyleneglycol , and the like . while preferred concentrations of cryoprotectant range from about 0 . 2 to 4 % ( weight / volume ), relatively high concentrations , for example greater than 5 %, are also suitable ; the levels used are limited only by those customarily used in clinical practice . the upper concentration limits for bulk drug may be higher than for finished dosage , e . g ., greater than 5 %. &# 34 ; surfactants &# 34 ; generally include those agents which protect the protein from air / solution interface induced stresses and solution / surface induced stresses ( e . g ., resulting in protein aggregation ), and may include detergents such as polysorbate - 80 ( tween ), for example , about 0 . 005 to 1 % ( volume / volume ), or polyethyleneglycol ( peg ), such as peg8000 , for example . optionally , relatively high concentrations , e . g ., up to 0 . 5 %, are suitable for maintaining protein stability ; however , the levels used in actual practice are customarily limited by clinical practice . the term &# 34 ; buffering agent &# 34 ; encompasses those agents which maintain the solution ph in an acceptable range and may include histidine , phosphate ( sodium or potassium ), citrate ( sodium or potassium ), maleic acid , ammonium acetate , tris ( tris ( hydroxymethyl ) aminomethane ), diethanolamine , and the like . the upper concentration limits may be higher for bulk protein than for finished dosage protein forms as is readily appreciated by one skilled in the art . for example , while buffer concentrations can range from several millimolar up to the upper limit of their solubility , e . g ., citrate , could be as high as 200 mm , one skilled in the art would also take into consideration both achieving and maintaining a physiologically appropriate concentration . percentages are weight / volume when referring to solids dissolved in solution and volume / volume when referring to liquids mixed into solutions . for example , for sucrose , it is weight dry sucrose / volume of solution and for tween , it is the volume of 100 % stock / volume of solution . the term &# 34 ; isotonic with serum ,&# 34 ; 300 ± 50 milliosmolal , is meant to be a measure of osmolality of the solution prior to administration . maintaining physiological osmolality is important for the dosage formulations to be injectable without prior dilution . however , for bulk formulations , much higher osmolalities can be effectively utilized as long as the solution is made isotonic prior to use . the term &# 34 ; excipients &# 34 ; includes pharmaceutically acceptable reagents to provide appropriate tonicity , cryoprotection of the protein , maintenance of ph , and proper conformation of the protein during storage so that substantial retention of biological activity and protein stability is maintained . the following examples illustrate practice of the invention . these examples are for illustrative purposes only and are not intended in any way to limit the scope of the invention claimed . example 1 describes the effect of calcium addition and the effect of ph on clotting activity . example 2 describes the effects of specific buffering agents on the formation of high molecular weight aggregates ( hmw ). example 3 illustrates the use of the invention for higher concentrations of factor ix . example 4 illustrates the complexity of excipient interactions in stabilizing factor ix . example 5 describes factor ix in various formulations relating to freeze / thaw stability . the preparation of recombinant factor ix has been described in u . s . pat . no . 4 , 770 , 999 , kaufman , et al . one suitable purification method is that described in hrinda , et al ., preclinical studies of a monoclonal antibody -- purified factor ix , mononine ™ seminars in hematology , 28 ( 3 ): 6 ( july 1991 ). other methods of preparation include the use of conformation - specific monoclonal antibodies as described by tharakan , et al ., &# 34 ; physical and biochemical properties of five commercial resins for immunoaffinity purification of factor ix .&# 34 ; journal of chromatography 595 : 103 - 111 ( 1992 ); and by liebman , et al ., &# 34 ; immunoaffinity purification of factor ix ( christmas factor ) by using conformation - specific antibodies directed against the factor ix - metal complex .&# 34 ; proc . nat . acad . sci ., usa 82 : 3879 - 3883 ( 1985 ); as well as conventional chromatographic procedures , for example , as described by hashimoto , et al ., &# 34 ; a method for systematic purification from bovine plasma of six vitamin k - dependent coagulation factors : prothrombin , factor x , factor ix , protein c , and protein z .&# 34 ; j . biochem . 97 : 1347 - 1355 ( 1985 ), and bajaj , p . et al . prep . biochem . 11 : 397 ( 1981 ). &# 34 ; large - scale preparation and biochemical characterization of a new high purity factor ix concentrate prepared by metal chelate affinity chromatography &# 34 ;, p . a . feldman et . al ., blood coagulation and fibrinolysis 5 : 939 - 948 ( 1994 ). yet another method of purification is described in u . s . ser . no . 08 / 472 , 823 , filed jun . 7 , 1995 ; and incorporated herein by reference . a well characterized property of factor ix is its ability to bind ca 2 + ions . structural studies indicate that ca 2 + binding may confer a more stable structure , reducing the probability of molecular motion (&# 34 ; structure of the metal - free γ - carboxyglutamic acid - rich membrane binding region of factor ix by two - dimensional nmr spectroscopy &# 34 ;, s . j . freedman , b . c . furie , b . furie , and j . d . baleja , j . biol . chem . 270 ( 14 ): 7980 - 7987 ( 1995 ); &# 34 ; structure of the calcium ion - bound γ - carboxyglutamic acid - rich domain of factor ix ,&# 34 ; s . j . freedman , b . c . furie , b . furie , and j . d . baleja , biochemistry 34 : 12126 - 12137 ( 1995 ); &# 34 ; the structure of a ca 2 + - binding epidermal growth factor - like domain : its role in protein - protein interactions &# 34 ;, s . rao , p . handford , m . mayhew , v . knott , g . brownlee , and d . stuart , cell 82 : 131 - 141 ( 1995 ); &# 34 ; structure of ca 2 + prothrombin fragment 1 including the conformation of the gla domain &# 34 ;, m . soriano - garcia , c . h . park , a . tulinsky , k . g . ravichandran , and e . skrzypczak - jankun , biochem . 28 : 6805 - 6810 ( 1989 )). presumably , less mobility accords a lower probability of molecular interaction , thereby reducing the probability of degrading processes . surprisingly , this turns out not to be the case . samples are prepared in the formulations set forth in table i below , at a recombinant factor ix protein concentration of ˜ 0 . 5 mg / mil ( 100 u / ml ) and an osmolality of 300 ± 50 milliosmolal . all samples contain a recombinant form of factor ix . to examine the potential utility of ca 2 + as a stabilizing agent , a set of samples was prepared in the formulations listed in table 1 . the formulation of sample a is the formulation used for commercially available plasma - derived lyophilized factor ix ( mononine ™). all samples contain a recombinant form of factor ix . table 1______________________________________sample formulations saltsample ph buffer ( 10 mm ) ( tonicity modifier ) other excipient______________________________________a 7 . 0 histidine 0 . 066 m nacl 165 mm mannitol ( 0 . 385 %) b 7 . 0 histidine 260 mm glycine 29 mm sucrosec 7 . 0 histidine 250 mm glycine , 29 mm sucrose 5 mm ca . sup . 2 + d 7 . 5 tris 260 mm glycine 29 mm sucrosee 7 . 5 tris 250 mm glycine , 29 mm sucrose 5 mm ca . sup . 2 + f 7 . 5 diethanolamine 260 mm glycine 29 mm sucroseg 7 . 5 diethanolamine 250 mm glycine , 29 mm sucrose 5 mm ca . sup . 2 + ______________________________________ samples of factor ix in each formulation were stored at 4 ° c . for 2 . 5 months . samples were assayed for protein concentration and clotting activity . factor ix activity is determined according to the method of pittman , d ., et al ., blood 79 : 389 - 397 ( 1992 ) utilizing factor ix - deficient blood . the ratio of clotting activity to protein concentration , the specific activity , expressed as units / mg of protein , is given in table 2 . an acceptable specific activity would be no more than 20 % greater than the starting specific activity . table 2______________________________________factor ix specific activitysample time zero 2 . 5 months______________________________________a 219 . 9 161 . 3b 191 . 8 153 . 2c 239 . 4 964 . 1d 209 . 3 135 . 8e 212 . 1 1956 . 9f 190 . 1 123 . 5g 217 . 3 2570 . 8______________________________________ the samples containing calcium , i . e ., samples c , e , and g , have higher specific activities after 2 . 5 months of storage . this is due to the inclusion of ca 2 + and indicates that the factor ix has undergone a conversion to an activated - like molecule . activated factor ix is factor ix that has been cleaved at residues r 145 - a 146 and r 180 - v 181 and is then able to catalyze clotting . normally , factor ix circulates as intact protein and is not converted to its activated form unless there is initiation of the clotting cascade . injecting someone with activated rhfix could have thrombotic implications . therefore inclusion of ca 2 + at a concentration of 5 mm is destabilizing and is to be avoided . the average specific activity after eight months of 4 ° c . storage of samples formulated in buffer / excipient combinations similar to and including those in table 1 , but without calcium , at ph 7 . 0 is 112 . 5 ± 10 . 5 u / mg , but at ph 7 . 5 is only 84 . 0 ± 22 . 1 u / mg , indicating subtle shifts in ph are significant for maintaining long - term factor ix stability . factor ix is prepared in a set of isotonic experimental formulations as summarized in table 3 , including several different excipient combinations for each buffering agent and some including less than 5 mm edta . factor ix concentrations are approximately 1 mg / ml ( average 161 u / ml ). samples are assayed for the amount of high molecular weight material ( hmw ) present and for clotting activity . the formation of significant (& gt ; 3 %) amounts of hmw is undesirable and as indicative of physical degradation of factor ix with possible impact on product safety and efficacy . table 3______________________________________sample formulationsbuffering agent ( 10 - 15 mm ) excipients______________________________________phosphate arginine - hcl , sodium ( either sodium or potassium chloride , glycine , sucrose , phosphate , ph 7 . 0 ) mannitol , glucosecitrate sorbitol , glucose , glycine ,( sodium , ph 6 . 0 - 6 . 5 ) sucrose , arginine - hclammonium acetate mannose , mannitol , sodium ( ph 6 . 5 - 7 . 0 ) chloride , arginine - hclmaleic acid glycine , mannose ( ph 6 . 5 ) ______________________________________ table 4 shows the effects of the different buffering agents on hmw generation as measured by size exclusion chromatography ( sec - hplc ). samples were stored at 30 ° c . for six weeks . table 4 gives the average increase expressed as ( hmw / total protein × 100 %) at six weeks minus that at time zero . table 4______________________________________percent increase hmw generationbuffering agent avg . increase (% of total ) ______________________________________phosphate : 4 . 21citrate : 0 . 80ammonium acetate : 3 . 42maleic acid : 1 . 67______________________________________ the citrate buffered samples had , on average , the smallest amount of hmw generated , regardless of the other excipients included . an appropriate buffer does not allow greater than a 2 % increase . all samples are stored further for six months at 4 ° c . and assayed for clotting activity . the average amount of activity remaining for samples containing the various saccharides varied greatly ; sucrose - containing samples maintained an average 71 % of the starting activity , mannitol 53 %, glucose 52 %, and mannose only 27 %. surprisingly , not all saccharides are equally effective at maintaining factor ix activity , despite the addition of other excipients . another set of formulations is prepared comprising higher concentrations of factor ix ; samples are prepared in the formulations listed in table 5 at a concentration of 8 mg / ml ( 2000 u / ml ). all contain 15 mm sodium citrate and are buffered at ph 6 . 8 , without surfactant . bg4 is slightly hypertonic , the rest are isotonic . table 5______________________________________sample formulations______________________________________bg1 : 2 % sucrose , 2 % arginine - hcl , 1 mm edtabg2 : 4 % sucrose , 1 % glycine , 1 mm edtabg3 : 15 % arginine - hcl , 1 % glycinebg4 : 5 % arginine - hcl , 1 mm edtabg5 : 4 % sucrose , 1 % glycine______________________________________ samples are stored in both glass vials and glass prefillable syringes for eight months at 4 ° c . to determine whether the amount of air / solution interface or siliconized stopper / solution interface would impact the stability of the product . no significant differences were seen by any stability indicating analytical methods between the vials and syringes . the results of several analytical methods are shown in table 6 . &# 34 ; specific activity &# 34 ; and &# 34 ; hmw &# 34 ; have been described previously . &# 34 ; sds - page &# 34 ; is polyacrylamide gel electrophoresis ; gels were scanned and bands quantified . reversed phase hplc is used to evaluate product heterogeneity and changes in peak ratios may indicate changes in the product , for example , oxidation of oligosaccharides . table 6______________________________________specific hmw , as % full - length reversed phasesam - activity , determined by fix , by sds - hplc ratiople as % of control sec - hplc page assay______________________________________bg - 1 91 % 0 . 31 98 . 2 0 . 33bg - 2 82 % 0 . 33 98 . 0 0 . 32bg - 3 91 % 0 . 35 98 . 2 0 . 34bg - 4 88 % 0 . 25 98 . 6 0 . 33bg - 5 79 % 0 . 40 98 . 3 0 . 31con - 100 % 0 . 21 98 . 6 0 . 31trol______________________________________ even at the higher concentration of factor ix these formulations demonstrate feasibility of the invention . another set of factor ix formulations , all containing citrate , is prepared as summarized in table 7 . all formulations are isotonic , contain factor ix at concentrations of 1 to 2 mg / ml ( average 208 to 481 u / ml ), use sodium citrate as the ph buffering agent , and are adjusted to ph 6 . 8 . table 7______________________________________sample formulationsmajor excipient ( range of concentration , wt / vol %) used in combination with : ______________________________________mannitol arginine - hcl , edta , glycine , tween - 80 ,( 55 - 275 mm , 1 - 5 %) sucrose , nacl , kclarginine - hcl mannitol , edta , sucrose , glycine , tween - 80 ,( 47 - 237 mm , 1 - 5 %) glucoseglycine mannitol , arginine - hcl , glucose , tween - 80 ,( 66 - 306 mm , 0 . 5 - 2 . 3 %) edtasucrose mannitol , arginine - hcl , glycine , nacl , edta ,( 29 - 234 mm , 1 - 8 %) tween - 80glucose arginine - hcl , glycine , nacl , kcl , edta ( 55 - 278 mm , 1 - 5 %) nacl sucrose , glucose , mannitol , edta ( 100 mm , 0 . 58 %) kcl glucose , mannitol ( 100 mm , 0 . 75 %) ______________________________________ samples are stored at 4 ° c . and assayed at several points in time . after eight months of 4 ° c . storage , nine samples maintain ˜ 100 % of the clotting activity of the starting material . the formulations of these nine are shown in table 8 ( all include 15 mm sodium citrate , are ph 6 . 8 , and isotonic ). table 8______________________________________1 4 % sucrose , 1 . 4 % glycine , 0 . 005 % tween - 802 1 % mannitol , 2 % arginine - hcl , 0 . 5 % glycine3 2 . 2 % arginine - hcl , 0 . 75 % glycine4 3 % mannitol , 1 % glycine5 3 % mannitol , 1 % glycine , 1 mm edta6 3 % mannitol , 1 . 5 % arginine , 0 . 005 % tween - 807 3 . 3 % arginine - hcl8 2 % mannitol , 2 % sucrose , 1 . 4 % arginine9 4 % sucrose , 1 . 4 % glycine , 1 mm edta______________________________________ several formulations containing similar excipients in similar ratios nevertheless , surprisingly , do not maintain clotting activity nearly as well . shown for these nine formulations are the results of other stability indicating assays . specific activity is expressed as u / mg and an acceptable range is 250 to 275 u / mg . sec - hmw is a measure of high molecular weight aggregates as determined by size - exclusion chromatography and the limit of acceptability is less than 1 %. c - terminal clips is a measure of degradation species as determined by reversed phase chromatography and the limit of acceptability is less than 1 %. table 9______________________________________ recovery of c - terminalsample activity specific activity sec hmw clips______________________________________1 ≧ 100 % 262 0 . 24 % 0 . 31 % 2 ≧ 100 % 256 0 . 25 % 0 . 28 % 3 ≧ 100 % 255 0 . 27 % 0 . 28 % 4 ≧ 100 % 262 0 . 26 % 0 . 33 % 5 ≧ 100 % 272 0 . 23 % 0 . 38 % 6 ≧ 100 % 263 0 . 22 % 0 . 28 % 7 ≧ 100 % 258 0 . 24 % 0 . 19 % 8 ≧ 100 % 251 0 . 20 % 0 . 33 % 9 ≧ 100 % 251 0 . 20 % 0 . 31 % ______________________________________ based on the preferred formulations set forth in tables 8 and 9 , more preferred formulations include as follows : ( all are buffered at ph 6 . 8 with citrate and are isotonic ) ideally , a similar formulation is utilized for bulk protein as is used for the finished dosage form . this demands that the same formulation that stabilizes factor ix from long - term storage stresses also be appropriate for stabilizing factor ix from the stresses normally encountered by bulk protein , such as freezing and thawing . samples are prepared in the formulations set forth in table 10 below , at a protein concentration of ˜ 2 mg / ml ( 500 u / ml ) and an osmolality of 300 ± 50 milliosmolal . all include 10 mm sodium citrate , ph 6 . 8 , and all are prepared both with and without 0 . 005 % tween - 80 ( polysorbate ). table 10______________________________________sample formulations______________________________________a . 2 . 5 % arginine - hcl , 2 . 2 % sucroseb . 1 . 8 % glycine , 2 % sucrosec . 1 . 8 % arginine - hcl , 2 . 4 % mannitold . 2 . 2 % glycine , 0 . 2 % mannitole . 2 . 7 % arginine - hcl , 0 . 8 % mannitolf . 2 % arginine - hcl , 2 % sucrose , 0 . 9 % mannitolg . 1 . 8 % arginine - hcl , 2 % mannitol , 0 . 8 % sucrose______________________________________ samples of factor ix in each formulation were subjected to five freeze - thaw cycles to determine susceptibility to freezing - induced denaturation , which can result in formation of protein aggregates . a series of freeze - thaw cycles is a useful indication of a protein &# 39 ; s susceptibility to increased aggregate formation as may be observed during freezing and long - term storage . samples are assayed for the amount of hmw present . samples with and without tween - 80 ( 0 . 005 %) have minimal aggregation ( less than 0 . 15 % hmw increase ). while the present invention has been described in terms of specific methods , formulations , and compositions , it is understood that variations and modifications will occur to those skilled in the art upon consideration of the present invention . numerous modifications and variations in the invention as described in the above illustrative examples are expected to occur to those skilled in the art and , consequently , only such limitations as appear in the appended claims should be placed thereon . accordingly , it is intended in the appended claims to cover all such equivalent variations which come within the scope of the invention as claimed .
provided by the present invention are novel compositions and methods for obtaining concentrated preparations of factor ix and liquid formulations of factor ix suitable for storage and administration .