(12> Umted States Patent
`Hellerbrand et al.
`
`US006238664B1
`(16) Patent N6.=
`US 6,238,664 B1
`(45) Date 0f Patent:
`May 29, 2001
`
`(54) PROCESS FOR STABILIZING PROTEINS
`
`FOREIGN PATENT DOCUMENTS
`
`.
`.
`(75) Inventors. Klaus Hellerbrand, Geltendorf,
`App0ll0n Papadlmltrlou, B1chl,
`
`Gerhard Winter, Dossenheim, all of (DE)
`
`0 018 609
`0 025 275
`0 314 095
`
`0 315 968 0 318 081
`
`11/1980 (EP) .
`3/1981 (EP) '
`5/1989 (EP)
`
`5/1989 (EP) 5/1989 (EP) .
`
`(73) Assignee: Boehringer Mannheim GmbH,
`Mannheim (DE)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U_S_C_ 154(k)) by 0 days
`
`.
`(21) Appl. No.. 09/196,090
`_
`NOV‘ 19’ 1998
`(22) Flled:
`(30)
`Foreign Application Priority Data
`(
`)
`EP ............................................... .. 97120528
`(EP) ............................................... .. 98102846
`
`NOV. 22, 1997
`Feb. 19, 1998
`
`
`
`0670328A1 * 9/1995 0671410111 * 9/1995 (EP) _
`
`OTHER PUBLICATIONS
`
`DerWent AbstractAN 98—555428 Abstract only Yu, R.M. et
`al RU 2109290 Apr. 20, 1998.
`Chemical Abstracts No. 195334 Abstract only Heller MC et
`al 1997 Biotechnology Prog 13:590—596.
`Heller, Biotechnol. Prog. 13 pp. 590—596 (1997).
`HWang, et al, Effect of Phosphate salts on the emulsion
`stability of soy protein isolate, J. Kor. Agric. Chem. Soc.,
`vol. 35, No. 3, pp. 152—156 (1992).
`
`* Cited
`
`examiner
`
`Int. Cl.7 ............................. .. C12Q 1/34; C12N 9/96;
`51
`C12N 5/02; G01N 33/53; A61K 39/395
`(
`)
`(52) US. Cl. ..................... .. 424/130.1; 435/188; 435/963;
`435/390- 530/387 1- 530/350- 530/380
`’
`'
`’
`’424/177 1’
`
`.
`.
`.
`Primary Examuyer—Dav1d Saunders
`A7825?” Exam‘? ’ ‘my DIZQOi‘G
`( .). Howey’
`gem’ Or m” Gorge
`William H. Epstein
`
`_
`W J h t
`'
`O “S on’
`
`(58) Field Of Search ................................... .. 530/350, 380,
`530/387.1, 390.5; 435/3905, 188, 963,
`18; 424/1771, 1301
`
`(56)
`
`References Cited
`
`U-S- PATENT DOCUMENTS
`
`11/1988 Thurow.
`4,783,441
`5,429,928 * 7/1995 Blaustein et al. .
`
`(57)
`
`ABSTRACT
`_
`_
`_
`_
`An aqueous protein solution buffered W1th a potassium
`phosphate buffer, in Which the ratio of potassium ions to
`sodium ions in the solution is at least 10:1, is resistant to the
`formation of protein aggregates and particles under condi
`tions of freezing, thawing, lyophiliZation, and reconstitution.
`
`45 Claims, 4 Drawing Sheets
`
`Ex. 1038 - Page 1 of 10
`
`AMGEN INC.
`Exhibit 1038
`
`

`

`US. Patent
`
`May 29, 2001
`
`Sheet 1 0f 4
`
`US 6,238,664 B1
`
`]
`
`[1050HM(A43U103\Jon RESISTANCEO—‘N
`
`
`
`
`
`
`
`22
`-32"3°-28'25-24'
`-2o'1 46—14-12—10-8 ‘5 -4 '2 -o
`
`—44
`
`56
`
`—4o‘
`
`8 -
`
`—°--10 % KCI
`+70 mM K-P,pH 7.2
`
`TEMPERATURE [°C]
`
`+10 mM K-P, 150 mM
`N°C'
`
`FIG. 1
`
`22 -2o --18 -—16 —14 —12 -1o -—8 -—6 --4 --2 —o
`
`TEMPERATURE [°c]
`
`—o—-—- 50 MM K—Ph, 100 MM
`NoCI,pH 7.2
`—u— 10 mM K-Ph, 20 mM
`NaCI,pH 7.2
`+ 100 mM K—Ph, pH 7.2
`
`FIG. 2
`
`Ex. 1038 - Page 2 of 10
`
`Ex. 1038 - Page 2 of 10
`
`

`

`US. Patent
`
`May 29, 2001
`
`Sheet 2 0f 4
`
`US 6,238,664 B1
`
`0‘3
`
`9.
`E1.
`
`
`
`zc.SmE._,w_o5:528;
`
`
`
`SEE—Ema20:536—..
`
`zQSmEm520:529.
`
`Ex. 1038 - Page 3 of 10
`
`Ex. 1038 - Page 3 of 10
`
`
`
`

`

`US. Patent
`
`May 29, 2001
`
`Sheet 3 0f 4
`
`US 6,238,664 B1
`
`08
`
`
`
`20.5925...202.5320;
`
`8m
`
`00.on0N
`
`
`
`20.59255202.520;
`
`w.03
`
`8m
`
`00.onon
`
`
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`E:is“.
`
`0—0
`
`20.50.25.20:50.51
`
`2959256
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`
`
`
`20.592552025.50;
`
`202.5809.
`
`8N 20.59255
`20:580.”.
`
`SN
`
`8.3
`
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`
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`
`20.5925520:50.50
`
`Ex. 1038 - Page 4 of 10
`
`Ex. 1038 - Page 4 of 10
`
`
`

`

`U.S. Patent
`
`May 29, 2001
`
`Sheet 4 0f 4
`
`US 6,238,664 B1
`
`2 13.17
`
`LOLLO
`290 mm
`
`160
`
`100
`
`MAU
`
`1 10.88
`
`__40
`
`0.0
`
`.
`
`5.0
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`10.0
`
`15.0M|N20.0
`25.0
`FIG. 5A
`
`30.0
`
`36.0
`
`1 13.05
`
`LOLLO
`290 mm
`
`1
`
`225
`200
`
`160
`
`I
`
`MAU 100
`
`I
`
`0.
`
`J
`
`0.0
`
`5.0
`
`10.0
`
`15.0M|N20.0
`25.0
`FIG. 5B
`
`30.0
`
`36.0
`
`Ex. 1038 - Page 5 of 10
`
`

`

`US 6,238,664 B1
`
`1
`PROCESS FOR STABILIZING PROTEINS
`
`FIELD OF THE INVENTION
`This invention is in the ?eld of proteins and protein
`solutions.
`
`BACKGROUND OF THE INVENTION
`
`Proteins such as enZymes or antibodies as Well as frag
`ments thereof are unstable and susceptible to loss of activity
`and/or to formation of soluble or insoluble aggregates in
`aqueous solutions and When stored at loW temperatures
`(beloW 0° C.) and in particular in repeated freezing and
`thaWing processes and these aggregates become apparent by
`forming particles and thus as turbidities. HoWever, such
`aggregate and/or particle formation cannot be tolerated or at
`least only in traces for pharmaceutical compositions of
`proteins. A pharmaceutical composition should be a clear
`solution and if it is present as a lyophilisate it should also
`lead to a clear particle-free solution When reconstituted
`Which is also free of soluble protein aggregates.
`Numerous processes and additives are knoWn for the
`stabiliZation of proteins in solutions. For example the sta
`biliZation of proteins by adding heat-shock proteins such as
`HSP25 is for example described in EP-A 0 599 344. The
`stabiliZation of antibodies by adding block polymers com
`posed of polyoxy-propylene and polyoxy-ethylene and by
`phospholipids is described in EP-A 0 318 081. EP-A 0 025
`275 describes the stabiliZation of immunoglobulin by adding
`a salt of a basic substance containing nitrogen such as
`arginine, guanidine or imidaZole. Other suitable additives
`for stabiliZation are polyethers (EP-A 0 018 609), glycerin,
`albumin and dextran sulfate (US. Pat. No. 4,808,705),
`detergents such as TWeen®20 (DE 26 52 636, GB 8514349),
`chaperones such as GroEL (Mendoza, J. A. Biotechnol.
`Tech. 10 (1991) 535—540), citrate buffer (WO 93/22335) or
`chelating agents (WO 91/15509). Although these additives
`enable proteins to be stabiliZed to a certain extent in aqueous
`solutions. It has, hoWever, turned out that none of the
`processes knoWn in the prior art is suitable for stabiliZing
`proteins during repeated freeZing and thaWing processes in
`such a Way that no soluble or insoluble aggregates or only
`negligible amounts for therapeutic purposes are formed
`during rethaWing, during storage at temperatures below 00
`C. or When a solution is reconstituted after lyophiliZation.
`In EP-A0 314 095 a lyophilisate of a plasma protein such
`as factor VIII is described Which contains histidine buffer as
`a buffer substance and calcium chloride as an additive and
`is present in a high ionic strength (0.35 to 1.2 mol/l NaCl).
`A lyophilisate of a plasma protein such as factor VIII is
`described in EP-A 0 315 968 Which contains 0.5 to 15
`mmol/l sodium chloride or potassium chloride, 0.01 to 10
`mmol/l lysine hydrochloride and 0.2 to 5 mmol/l histidine as
`a buffer ion. HoWever, histidine buffer is not suitable for
`stabiliZing proteins and for preventing aggregate and particle
`formation When lyophilisates of proteins are reconstituted.
`
`SUMMARY OF THE INVENTION
`This invention provides a composition comprising an
`aqueous buffered solution having a protein dissolved
`therein, Wherein the solution contains potassium ions and
`either contains no sodium ions or contains sodium ions such
`that the ratio of potassium ions to sodium ions in the solution
`is at least 10:1; the solution being buffered With a potassium
`phosphate buffer.
`This invention further provides a process for forming an
`aqueous buffered solution having a protein dissolved therein
`
`10
`
`15
`
`20
`
`25
`
`30
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`comprising: dissolving the protein in an aqueous solution;
`and adjusting the aqueous solution With a potassium phos
`phate buffer so that the aqueous solution having the protein
`dissolved therein contains potassium ions and either con
`tains no sodium ions or contains sodium ions such that the
`ratio of potassium ions to sodium ions in the solution is at
`least 10:1.
`The aqueous buffered protein solution of this invention is
`resistant to the formation of protein aggregation and particle
`formation under conditions of freeZing, thaWing,
`lyophiliZation, and reconstitution. Reduction of protein
`aggregation and particle formation is desirable during the
`manipulation of protein solutions generally, to reduce loss of
`protein and protein activity. It is also desirable in pharma
`ceutical uses, in Which limiting the number of aggregates is
`particularly important.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`FIG. 1 shoWs the determination of the eutectic points of
`various buffers and salt solutions.
`FIG. 2 shoWs the shift of the pH value during freeZing of
`phosphate buffers.
`FIGS. 3A to 3C shoW the particle formation of solutions
`of an antibody (against L-selectin) in various buffer solu
`tions (A, B, C) after shear or freeZe/thaW stress. A: AB in 10
`mmol/l KP, 150 mmol/l NaCl, pH 7; B: AB in 100 mmol/l
`KP, pH 7.2; C: AB in 100 mmol/l KP 0.01% by Weight
`TWeen®80, pH 7.2; a: centrifuged (starting material); b:
`after shearing stress (30 sec vortexing); c: after six freeZe/
`thaW cycles (—20° C.).
`FIGS. 4A to 4C shoW the particle formation of solutions
`of an antibody against HBV in various buffer solutions (A,
`B, C) after shear or freeZe/thaW stress. A: AB in 10 mmol/l
`KP, 30 mmol/l NaCl, pH 6.5; B: AB in 100 mmol/l KP, pH
`7.2; C: AB in 100 mmol/l KP, 0.01% by Weight Tween@ 80,
`pH 7.2;
`FIGS. 5A to 5B shoW the siZe exclusion HPLC analysis
`of soluble aggregates in protein solutions (humaniZed IgG
`according to example 3) after storage at temperatures below
`00 C. A: AB in 10 mmol/l KP, 150 mmol/l NaCl, pH 7.0; B:
`AB in 100 mmol/l KP, pH 7.2.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`This invention provides a composition comprising an
`aqueous buffered solution having a protein dissolved
`therein, Wherein the solution contains potassium ions and
`either contains no sodium ions or contains sodium ions such
`that the ratio of potassium ions to sodium ions in the solution
`is at least 10:1. In an embodiment of this invention the
`solution is buffered With a potassium phosphate buffer in a
`concentration of from 10 to 300 mmol/liter in the solution.
`In a more speci?c embodiment, the concentration of the
`potassium phosphate buffer in the solution is from 50 to 250
`mmol/liter. In an embodiment the solution is at a pH of from
`6 to 8 When measured at a temperature of from 40 C. to 30°
`C. In a more speci?c embodiment the pH of the solution is
`from 6.5 to 7.5 When measured at a temperature of from 4°
`C. to 30° C. In an embodiment of this invention the ratio of
`potassium ions to sodium ions is at least 50: 1. Preferably the
`buffer is substantially free of sodium ions.
`This invention also provides a process for forming an
`aqueous buffered solution having a protein dissolved therein
`comprising: a) dissolving the protein in an aqueous solution;
`and b) adjusting the aqueous solution With a potassium
`
`Ex. 1038 - Page 6 of 10
`
`

`

`US 6,238,664 B1
`
`3
`phosphate buffer so that the aqueous solution having the
`protein dissolved therein contains potassium ions and either
`contains no sodium ions or contains sodium ions such that
`the ratio of potassium ions to sodium ions in the solution is
`at least 10:1. After the buffered solution is formed, it can be
`froZen or lyophiliZed if desired, in accordance With conven
`tional techniques. In a further embodiments, the froZen
`solution is thaWed and the lyophiliZed solution is reconsti
`tuted.
`In an embodiment of the composition and process of this
`invention the solution is buffered With a potassium phos
`phate buffer in a concentration of from 10 to 300 mmol/liter
`in the solution. In a more speci?c embodiment, the concen
`tration of the potassium phosphate buffer in the solution is
`from 50 to 250 mmol/liter. In an embodiment the solution is
`at a pH of from 6 to 8 When measured at a temperature of
`from 4° C. to 30° C. In a more speci?c embodiment the pH
`of the solution is from 6.5 to 7.5 When measured at a
`temperature of from 4° C. to 30° C. In an embodiment of this
`invention the ratio of potassium ions to sodium ions is at
`least 50:1. Preferably the buffer is substantially free of
`sodium ions.
`In accordance With this invention, any protein can be
`utiliZed. The speci?c identity of the protein is not critical,
`provided that an aqueous solution of the protein can be
`formed. In an embodiment of this invention the protein is an
`antibody. Furthermore, the concentration of the protein in
`the solution is not critical. Any amount of protein Which can
`be dissolved in the aqueous buffered solution can be utiliZed.
`Typically, the concentration of protein Which is dissolved in
`solution Will be from 1 mg/ml to 50 mg/ml.
`This invention provides a process for preventing the
`formation of protein aggregates in a solution of a pharma
`ceutical composition of a protein, preferably of an antibody,
`that is reconstituted from a lyophilisate Wherein an aqueous
`buffered solution of the protein is froZen, thaWed, divided
`into compartments of injectable amounts and these compart
`ments are lyophiliZed Which is characteriZed in that the
`aqueous buffered solution of the protein contains potassium
`phosphate buffer as a buffer substance and the ratio of
`potassium to sodium ions in the solution is 10:1 or larger.
`The aqueous buffer solution preferably contains essentially
`no sodium ions.
`The invention enables pharmaceutical compositions of
`proteins, in particular proteins Which have a tendency to
`dimeriZe or multimeriZe such as antibodies, to be formulated
`into. a stable pharmaceutical composition in a neutral pH
`range (pH 6—8, preferably pH 6.5—7.5). Proteins such as
`antibodies tend to aggregate in the neutral pH range espe
`cially if the solutions are froZen (optionally lyophiliZed)
`once or several times and thaWed again.
`Apharmaceutical composition is especially advantageous
`in potassium phosphate buffer in the pH range betWeen 6 and
`8, at a buffer concentration betWeen 10 and 300 mmol/l,
`preferably betWeen 50 and 250 mmol/l in Which the loWest
`possible number of sodium ions are present in the pharma
`ceutical composition. A suitable ratio of potassium to
`sodium ions in the solution is 10:1 or more. It is particularly
`preferable that potassium phosphate buffer is used alone as
`the buffer substance in the pharmaceutical composition and
`no sodium salt (such as eg sodium chloride) is added. In
`such a case almost no sodium ions are present in the
`pharmaceutical composition or it only contains them in such
`loW amounts that they do not cause formation of aggregates
`of proteins during repeated freeZing or thaWing.
`It has turned out that lyophilisates of protein solutions
`Which have been froZen at least once during the production
`
`10
`
`15
`
`25
`
`35
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`45
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`55
`
`65
`
`4
`process can then be reconstituted substantially Without for
`mation of turbidities if potassium phosphate buffer is used as
`the buffer substance. The usual buffers such as sodium
`phosphate buffer, histidine buffer or citrate buffer lead to the
`formation of aggregates in such a process Which are mainly
`composed of the protein and thus also lead to turbidities to
`a considerable degree. The froZen protein solutions are
`already completely froZen through beloW ca. —15° C., have
`eutectic points above ca. —15° C. and can thus already be
`stored at this temperature or at loWer temperatures prefer
`ably eg at —20° C. Since a solution is only completely
`froZen through beloW the eutectic temperature, this means
`that a protein in a phosphate buffer containing sodium ions
`is subjected to a higher stress during the froZen storage
`(usually at —20° C.) and during the freeZing/thaWing process
`than in a buffer free of sodium ions or in a buffer in Which
`the sodium ion concentration is very loW. According to the
`invention this stress is avoided in the above-mentioned
`formulations resulting in a suppression of aggregate and
`particle formation. This formulation enables a stable storage
`of the protein solution at —20° C. Which can save costs.
`Potassium phosphate buffers in contrast to sodium phos
`phate buffers have only a slight pH shift (preferably at most
`:1 pH unit, particularly preferably at most 10.5 pH units)
`during the freeZing process.
`It has turned out that the concentration of the phosphate
`buffer should be at least 10 mmol/l, preferably about 50
`mmol/l or higher in order to effectively prevent particle
`formation. Since the osmolarity should not be too high (it
`should advantageously be in the physiological range, pref
`erably ca. 300 mOsm after reconstitution (120 mOsm, a
`range of 100 to 500 mOsm is also suitable)) in pharmaceu
`tical compositions (i.e. preferably in the reconstituted
`solution), the concentration of the buffer substance or
`optionally the sum of buffer substance and salt should be not
`more than 250—300 mmol/l. The buffer concentration is
`preferably betWeen 50 and 250 mmol/l in the compartment.
`HoWever, higher concentrations of buffer substance and/or
`salt can be tolerated in the production of the solutions
`(bulkWare) used to produce the compartments.
`If a salt additive is desired in the pharmaceutical compo
`sition especially to adjust the ionic strength, it is advanta
`geous according to the invention to also not use sodium salts
`or to select a concentration of the sodium ions Which is
`substantially loWer than the concentration of the potassium
`ions. It is therefore eXpedient to add a potassium salt such as
`potassium chloride instead of the otherWise usual sodium
`chloride. HoWever, it has turned out that loW amounts of
`sodium salts (e.g. ca. 10 mmol/l or less) do not interfere
`provided the ratio of potassium ions to sodium ions is 10:1
`or higher. It is not possible to add calcium salts such as eg
`calcium chloride since calcium phosphate is precipitated by
`such an addition and hence, apart from the formation of
`undesired turbidity, the buffer effect of the potassium phos
`phate according to the invention is abolished.
`Non-soluble aggregates Whose formation should be pre
`vented in the process according to the invention are essen
`tially understood as protein aggregates Whose siZe is usually
`at least 1 pm but can also be in the range above 10 pm. The
`particles can be determined by suitable particle counting
`methods using commercial particle counting instruments
`such as eg the particle counting instrument AccuSiZer 700
`from PSS (Particle SiZing Systems, USA). According to the
`invention an improvement of the process is achieved When
`the number of particles betWeen 2 and 400 pm/ml is <3000
`or the number of particles betWeen 10 and 400 pm/ml is
`2000 or less. According to the USP (US-Pharmacopoeia) a
`
`Ex. 1038 - Page 7 of 10
`
`

`

`US 6,238,664 B1
`
`5
`maximum of 6000 particles in the range above 10 pm and a
`maximum of 600 particles in the range above 25 pm are
`allowed per injected dose of a pharmaceutical preparation.
`This can be achieved according to the invention in a simple
`manner for therapeutic compositions of proteins.
`In accordance With this invention any protein can be
`utiliZed. The invention is based on the use of the aqueous
`buffered solution in accordance With this invention, and is
`not limited as to the speci?c protein dissolved therein.
`Proteins (polypeptides) are understood Within the sense of
`the invention as naturally occurring and recombinant pro
`teins or protein fragments as Well as chemically modi?ed
`proteins and proteins containing amino acid substitutions
`and additions. Proteins Which are desirably stabiliZed for
`pharmaceutical compositions are preferably antibodies, anti
`body fusion proteins such as immunotoxins, enZymes and
`protein hormones such as erythropoietin, somatostatin,
`insulin, cytokines, interferons or plasminogen activators.
`Compartments Within the sense of the invention are
`understood as aliquots of the protein solution Which, option
`ally after further processing (addition of further pharmaceu
`tically acceptable substances), are suitable as pharmaceuti
`cal compositions preferably for injection in the patients.
`The pH range in Which the pharmaceutical composition is
`stabiliZed by the potassium phosphate buffer is preferably a
`slightly acidic, neutral or slightly alkaline range (ca. pH 6—8,
`preferably about pH 7).
`According to the invention it is preferable to add a
`nonionic detergent such as polysorbate (e.g. Tween@ 80),
`preferably at a concentration of at most 0.1% by Weight and
`at least 0.01% by Weight.
`In addition it is preferable to add cryoprotectors or glass
`formers such as a non-reducing sugar (preferably sucrose or
`trehalose), advantageously at a concentration of at least 10
`mg/ml, preferably of ca. 30—100 mg/ml.
`Consequently a further subject matter of the invention is
`a loW aggregate, meltable solid storage form of a protein
`Which is essentially amorphous and is composed of a froZen
`solution of the protein and potassium phosphate buffer as the
`main buffer substance in Which the ratio of potassium ions
`to sodium ions in the solution is at least 10:1.
`Independent of the concentration of potassium ions and
`the residual content of sodium ions, the ratio of potassium to
`sodium ions should be at least 10:1, preferably at least 50:1.
`It is particularly preferable to use essentially sodium-ion
`free potassium buffer.
`In a further preferred embodiment of the invention the
`pharmaceutical composition contains a protein Which has
`been produced by an in vitro cell culture (for example
`recombinant production or culture of a hybridoma cell line
`to produce monoclonal antibodies). In this case it is expe
`dient to either add potassium salt and/or potassium phos
`phate buffer With the ?rst addition of salt or/and buffer, or to
`rebuffer at a later time in the isolation and puri?cation
`process. This enables the interim stable storage of the
`polypeptide preparation beloW 0° C. Rebuffering is under
`stood as an exchange of ions for example by dialysis. In the
`puri?cation and isolation process of the protein the buffer or
`salt concentration can indeed be higher than 50—100 mmol/l
`before compartmentation since these compositions are not
`used therapeutically. HoWever, it is essential that an osmo
`larity that is suitable for an injectable composition is
`adjusted before the compartmentation.
`The disclosure of European Patent Application No.
`971205281 is incorporated herein by reference.
`The invention Will be better understood by reference to
`the folloWing examples. These Examples are illustrative,
`
`10
`
`15
`
`20
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`25
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`30
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`35
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`40
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`45
`
`50
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`
`6
`and do not limit the invention Which is de?ned in the claims
`Which folloW thereafter.
`
`EXAMPLE 1
`
`Eutectic temperatures of various buffer and salt
`solutions
`
`From FIG. 1 it is clear that the eutectic temperature of
`NaCl containing buffers is ca. 10° C. loWer than that of
`NaCl-free buffers or solutions Which contain KCl instead of
`NaCl. Since a solution is only completely froZen through
`beloW the eutectic temperature, this means that a protein in
`an NaCl-containing phosphate buffer is subjected to a higher
`stress than in NaCl-free buffer during froZen storage (usually
`at —20° C.) and during the freeZe/thaW process. According to
`the invention this stress is avoided in the above-mentioned
`formulations Which suppresses the formation of aggregates
`and particles. This formulation enables a stable storage of
`the protein solution at —20° C. by Which means cost savings
`can be achieved.
`
`EXAMPLE 2
`
`Shift of the pH value during freeZing of phosphate
`buffers
`
`It is clear from FIG. 2 that in NaCl-containing phosphate
`buffers the pH value greatly decreases during the freeZing
`process due to precipitated disodium hydrogen phosphate.
`The pH value remains largely constant in NaCl-free potas
`sium phosphate buffer.
`
`EXAMPLE 3
`
`Particle formation in protein solutions after shear or
`freeZe/thaW stress
`Solutions of a humaniZed IgG (antibody against
`L-selectin) in various buffers (A, B, C) Were analysed for
`particle content (Accu SiZer, Particle SiZing Systems, USA):
`A) AB in 10 mmol/l KP, 150 mmol/l NaCl, pH 7
`B) AB in 100 mmol/l KP, pH 7.2
`C) AB in 100 mmol/l KP, 0.01% by Weight TWeen®80,
`pH 7.2
`a) centrifuged (starting material)
`b) after shear stress (30 sec. vortexing)
`c) after six freeZe/thaW cycles (—20° C.)
`The data in FIG. 3 each refer to 0.7 ml sample.
`It can be seen from FIG. 3 that particle formation is
`suppressed according to the invention by using sodium-free
`potassium phosphate buffers. This effect can be increased by
`the 5 addition of a nonionic detergent (TWeen®80, 0.01% by
`Weight).
`
`EXAMPLE 4
`
`Particle formation in protein solutions after shear or
`freeZe/thaW stress
`
`Solutions of an antibody against HBV in various buffers
`(A, B, C) Were analysed for particle content (Accu SiZer,
`Particle SiZing Systems):
`A) AB in 10 mmol/l KP, 30 mmol/l NaCl, pH 6.5
`B) AB in 100 mmol/l KP, pH 7.2
`C) AB in 100 mmol/l KP, 0.01% by Weight TWeen®80,
`pH 7.2
`a) centrifuged (starting material)
`b) after shear stress (30 sec. vortexing)
`
`Ex. 1038 - Page 8 of 10
`
`

`

`US 6,238,664 B1
`
`7
`c) after siX freeZe/thaW cycles (—20° C.)
`The data in FIG. 3 each refer to 0.7 ml sample.
`It can be seen from FIG. 4 that particle formation is
`suppressed according to the invention by using sodium-free
`potassium phosphate buffers. This effect can be increased by
`the addition of a nonionic detergent.
`
`EXAMPLE 5
`
`Prevention of the formation of soluble aggregates
`during the storage of protein solutions (humaniZed
`IgG according to eXample 3) at temperatures beloW
`0° C.
`
`Protein solutions Were stored for several Weeks at —20° C.
`in A) 10 mM potassium phosphate, 150 mM NaCl, pH 7.0,
`and B) in 100 mM potassium phosphate, pH 7.2. Analysis of
`the soluble aggregates and the native protein Was carried out
`by siZe exclusion HPLC (FIG. 5). According to the invention
`considerably feWer protein aggregates occur in the NaCl
`free buffer than in the NaCl-containing buffer. This is above
`all due to the fact that a shift of the pH value is substantially
`prevented in the NaCl-free buffer and the storage tempera
`ture is considerably beloW the eutectic temperature. (see also
`eXamples 1 and 2).
`
`EXAMPLE 6
`
`Particle formation in protein solutions after freeZe/
`thaW stress
`
`25
`
`30
`
`The antibodies MAB L-selectin, MAB HBV; MAB
`PDGF-R and MAB LNGF-R in various buffers Were analy
`sed for particle content before and after freeZe/thaW stress
`(6><freeZing/thaWing) (Accu SiZer, Particle SiZing Systems)
`(results cf. table 1, Cpm: protein concentration). Particles
`With a siZe of 2—400 pm per ml are stated. It is clear that the
`particle formation is suppressed according to the invention
`
`by using sodium-free potassium phosphate buffers This effect can be increased by adding a nonionic detergent.
`
`35
`
`TABLE 1
`
`particles/ml
`Without stress
`2-400 ,um
`
`Particles/ml
`6 x freezing/
`thawing
`2400 ,um
`
`cpm [mg/ml]
`
`21.40
`
`18.50
`
`17.85
`
`18.30
`
`1.70
`
`1.70
`
`875
`
`276
`
`544
`
`740
`
`130
`
`691
`
`6245
`
`332
`
`19085
`
`695
`
`33795
`
`677
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`MAB L-selectin in
`buffer
`
`10 mM KP, 150 mM
`NaCl, pH 7.2
`100 mM KP, 0.01% by
`Weight
`TWeen80, pH 7.2
`MAB HBV
`%
`10 mM KP, 30 mM NaCl,
`pH 6.6
`100 mM KP, 0.01% by
`Weight
`TWeen80, pH 7.2
`MAB PDGF-R
`in buffer
`
`10 mM KP, 150 mM
`NaCl, pH 7.2
`50 mM KP, 0.01% by
`Weight
`TWeen80, pH 7.2
`MAB LNGF-R
`
`8
`
`TABLE 1-continued
`
`particles/ml
`Without stress
`2-400 [urn
`
`Particles/ml
`6 x freezing/
`thawing
`2-400 ,um
`
`Cpm [mg/ml]
`
`1.70
`
`1.70
`
`690
`
`1164
`
`28915
`
`1257
`
`in buffer
`
`10 mM KP, 150 mM
`NaCl, pH 7.2
`50 mM KP, 0.01% by
`Weight
`TWeen80, pH 7.2
`
`What is claimed is:
`1. In a process for reconstituting a lyophiliZate formed
`from an aqueous solution having a protein dissolved therein,
`the improvement comprising:
`Wherein the solution contains potassium ions and either
`contains no sodium ions or contains sodium ions such
`that the ratio of potassium ions to sodium ions in the
`solution is at least 10:1; and
`the solution being buffered at a pH from 6 to 8 With a
`potassium phosphate buffer in a concentration of from
`10 to 300 mmol/liter in the solution.
`2. The process of claim 1 Wherein the protein is an
`antibody.
`3. The process of claim 1 Wherein the dissolved protein is
`present in the solution in a concentration from 1 mg/ml to 50
`mg/ml.
`4. The process of claim 1 Wherein the concentration of the
`potassium phosphate buffer in the solution is from 50 to 250
`mmol/liter.
`5. The process of claim 1 Wherein the pH of the solution
`is from 6.5 to 7.5 When measured at a temperature of from
`40 C. to 30° C.
`6. The process of claim 1 Wherein the osmolarity of the
`solution is from 100 to 500 mOsm.
`7. The process of claim 6 Wherein the osmolarity of the
`solution is from 280 mOsm to 320 mOsm.
`8. The process of claim 1 Wherein the ratio of potassium
`ions to sodium ions in the solution is at least 50:1.
`9. The process of claim 1 Wherein the buffer is substan
`tially free of sodium ions.
`10. In a process for thaWing a froZen aqueous solution
`formed from a liquid aqueous solution having a protein
`dissolved therein, the improvement comprising:
`Wherein the liquid aqueous solution contains potassium
`ions and either contains no sodium ions or contains
`sodium ions such that the ratio of potassium ions to
`sodium ions in the solution is at least 10:1; and
`the liquid aqueous solution being buffered at a pH from 6
`to 8 With a potassium phosphate buffer in a concentra
`tion of from 10 to 300 mmol/liter in the liquid aqueous
`solution.
`11. The process of claim 10 Wherein the protein is an
`antibody.
`12. The process of claim 10 Wherein the dissolved protein
`is present in the liquid aqueous solution in a concentration
`from 1 mg/ml to 50 mg/ml.
`13. The process of claim 10 Wherein the concentration of
`the potassium phosphate buffer in the liquid aqueous solu
`tion is from 50 to 250 mmol/liter.
`14. The process of claim 10 Wherein the pH of the liquid
`aqueous solution is from 6.5 to 7.5 When measured at a
`temperature of from 40 C. to 30° C.
`15. The process of claim 10 Wherein the osmolarity of the
`liquid aqueous solution is from 100 to 500 mOsm.
`
`Ex. 1038 - Page 9 of 10
`
`

`

`US 6,238,664 B1
`
`9
`16. The process of claim 15 wherein the osmolarity of the
`liquid aqueous solution is from 280 mOsm to 320 mOsm.
`17. The process of claim 10 Wherein the ratio of potassium
`ions to sodium ions in the liquid aqueous solution is at least
`50:1.
`18. The process of claim 10 Wherein the buffer is sub
`stantially free of sodium ions.
`19. A process for forming a froZen aqueous buffered
`solution having a protein dissolved therein comprising:
`a) dissolving the protein in a liquid aqueous solution; and
`b) adjusting the liquid aqueous solution With a potassium
`phosphate buffer so that the liquid aqueous solution
`having the protein dissolved therein contains potassium
`ions and either contains no sodium ions or contains
`sodium ions such that the ratio of potassium ions to
`sodium ions in the liquid solution is at least 10:1;
`is buffered With the potassium phosphate buffer in a
`concentration of from 10 to 300 mmol/liter; and
`is at a pH of from 6 to 8 When measured at a tempera
`ture of from 4° C. to 30° C.; and
`c) freezing the liquid aqueous buffered solution.
`20. The process of claim 19 Wherein the protein is an
`antibody.
`21. The process of claim 19 Wherein the dissolved protein
`is present in the liquid aqueous solution in a concentration
`from 1 mg/ml to 50 mg/ml.
`22. The process of claim 19 Wherein the concentration of
`the potassium phosphate buffer in the liquid aqueous solu
`tion is from 50 to 250 mmol/liter.
`23. The process of claim 19 Wherein the pH of the liquid
`aqueous solution is from 6.5 to 7.5 When measured at a
`temperature of from 4° C. to 30° C.
`24. The process of claim 19 Wherein the osmolarity of the
`liquid aqueous solution is from 100 to 500 mOsm.
`25. The process of claim 24 Wherein the osmolarity of the
`liquid aqueous solution is from 280 mOsm to 320 mOsm.
`26. The process of claim 19 Wherein the ratio of potassium
`ions to sodium ions in the liquid aqueous solution is at least
`50:1.
`27. The process of claim 19 Whereein the buffer is
`substantially free of sodium ions.
`28. A process for forming a lyophiliZate of a protein,
`comprising:
`a) dissolving the protein in an aqueous solution; and
`b) adjusting the aqueous solution With a potassium phos
`phate buffer so that the aqueous solution having the
`protein dissolved therein
`contains potassium ions and either contains no sodium
`ions or contains sodium ions such that the ratio of
`potassium ions to sodium ions in the solution is at
`least 10:1;
`i