(12) United States Patent
`Hellerbrand et al.
`
`USOO6238664B1
`(10) Patent No.:
`US 6,238,664 B1
`(45) Date of Patent:
`May 29, 2001
`
`(54) PROCESS FOR STABILIZING PROTEINS
`(75) Inventors: Klaus Hellerbrand, Geltendorf;
`Appollon Papadimitriou, Bichl;
`Gerhard Winter, Dossenheim, all of
`(DE)
`(73) Assignee: Boehringer Mannheim GmbH,
`Mannheim (DE)
`Subj
`y disclai
`h
`f thi
`ubject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`* Y Not
`Otice:
`
`(21) Appl. No.: 09/196,090
`(22) Filed:
`Nov. 19, 1998
`(30)
`Foreign Application Priority Data
`Nov. 22, 1997
`(EP) ................................................. 97120528
`Feb. 19, 1998
`(EP) ................................................. 98102846
`(51) Int. Cl. ............................... C12O 1/34; C12N 9/96;
`C12N 5/02; G01N 33/53; A61K 39/395
`(52) U.S. Cl. ....................... 424/130.1; 435/188; 435/963;
`435/390; 530/387.1; 530/350, 530/380;
`424/177.1
`(58) Field of Search ..................................... 530/350, 380,
`530/387.1, 390.5; 435/390.5, 188,963,
`18; 424/177.1, 130.1
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`4,783,441
`5,429,928 *
`
`11/1988 Thurow .
`7/1995 Blaustein et al. .
`
`FOREIGN PATENT DOCUMENTS
`0 018 609
`11/1980 (EP).
`0 025 275
`3/1981 (EP).
`O 314 095
`5/1989 (EP).
`O 315968
`5/1989 (EP).
`O 318 081
`5/1989 (EP).
`9/1995 (EP).
`9/1995 (EP).
`OTHER PUBLICATIONS
`
`Derwent Abstract AN 98-555428 Abstract only Yu, R.M. et
`al RU 2109290 Apr. 20, 1998.
`Chemical Abstracts No. 195334 Abstract only Heller MC et
`all 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 by examiner
`
`Primary Examiner David Saunders
`Assistant Examiner Amy DeCloux
`(74) Attorney, Agent, or Firm--George W. Johnston;
`William H. Epstein
`ABSTRACT
`(57)
`An acqueous protein Solution buffered with 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
`
`MAIA Exhibit 1046
`MAIA V. BRACCO
`IPR PETITION
`
`

`

`U.S. Patent
`
`May 29, 2001
`
`Sheet 1 of 4
`
`US 6,238,664 B1
`
`nine
`
`406-320-226-2422-2018-1614-12-10-8-6-4-2-0
`-44 .-56
`22
`8
`-0-10 % KC
`TEMPERATURE (C)
`--70 mM K-P,pH 7.2
`-a- 10 mM K-P, 150 mM
`NOC
`
`FIG. 1
`
`
`
`-22 -20 - 18 - 16 - 14 - 12 - O --8 --6 --4 --2 - 0
`TEMPERATURE (C)
`-- 50 mM K-Ph, 100 mM
`NoClpH 7.2
`-o- 10 mM K-Ph, 20 mM
`NaClipH 7.2
`-- 100 mM K-Ph, pH 7.2
`FIG. 2
`
`

`

`U.S. Patent
`US. Patent
`
`May 29, 2001
`May 29, 2001
`
`Sheet 2 of 4
`Sheet 2 0f 4
`
`US 6,238,664 B1
`US 6,238,664 B1
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`

`

`U.S. Patent
`
`May 29, 2001
`
`Sheet 3 of 4
`
`US 6,238,664 B1
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`

`

`U.S. Patent
`
`May 29, 2001
`
`Sheet 4 of 4
`
`US 6,238,664 B1
`
`
`
`160
`
`100
`
`s 50
`
`-40
`
`0.0
`
`5.0
`
`100 15.200 250 300
`FIG. 5A
`
`36.0
`
`1 13.05
`
`LOLLO
`290 mm
`
`225
`200
`
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`s 100
`
`50
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`5.0
`
`10.0 15,200 25.0
`FIG. 5B
`
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`
`56.0
`
`

`

`US 6,238,664 B1
`
`1
`PROCESS FOR STABILIZING PROTEINS
`
`FIELD OF THE INVENTION
`This invention is in the field of proteins and protein
`Solutions.
`
`15
`
`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
`25
`Stabilization of antibodies by adding block polymers com
`posed of polyoxy-propylene and polyoxy-ethylene and by
`phospholipids is described in EP-A 0318081. 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 0018 609), glycerin,
`albumin and dextran sulfate (U.S. Pat. No. 4,808,705),
`detergents such as Tween(R20 (DE 2652 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 0
`C. or when a Solution is reconstituted after lyophilization.
`In EP-A 0314095 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).
`Alyophilisate of a plasma protein Such as factor VIII is
`described in EP-A O 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
`
`35
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`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(R80, 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(E) 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
`0° 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 specific 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 4 C. to 30
`C. In a more specific 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
`
`

`

`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 specific 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 specific 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 Specific 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.
`A pharmaceutical 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 e.g. 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
`
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`US 6,238,664 B1
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`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 e.g. 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 +0.5 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 m0sm after reconstitution (+20 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 e.g.
`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 um but can also be in the range above 10 lim. The
`particles can be determined by Suitable particle counting
`methods using commercial particle counting instruments
`Such as e.g. 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 um/ml is <3000
`or the number of particles between 10 and 400 um/ml is
`2000 or less. According to the USP (US-Pharmacopoeia) a
`
`

`

`US 6,238,664 B1
`
`S
`maximum of 6000 particles in the range above 10 um and a
`maximum of 600 particles in the range above 25 um 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 Specific 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 modified
`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(E) 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 amorphousand 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 first addition of salt or/and buffer, or to
`rebuffer at a later time in the isolation and purification
`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
`purification 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.
`97120528.1 is incorporated herein by reference.
`The invention will be better understood by reference to
`the following examples. These Examples are illustrative,
`
`6
`and do not limit the invention which is defined 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.
`
`15
`
`EXAMPLE 2
`
`25
`
`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
`
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`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(E80,
`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(R80, 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(E80,
`pH 7.2
`a) centrifuged (starting material)
`b) after shear stress (30 sec. vortexing)
`
`

`

`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
`O° 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
`
`5
`
`15
`
`25
`
`The antibodies MAB L-selectin, MAB, HBV; MAB
`PDGF-R and MABLNGF-R in various buffers were analy
`Sed for particle content before and after freeze/thaw stress
`(6xfreezing/thawing) (Accu Sizer, Particle Sizing Systems)
`(results cf. table 1, C. protein concentration). Particles
`with a size of 2-400 um per ml are stated. It is clear that the
`particle formation is Suppressed according to the invention
`by using Sodium-free potassium phosphate buffers (KP).
`This effect can be increased by adding a nonionic detergent.
`
`35
`
`40
`
`TABLE 1.
`
`particles/ml
`without stress
`Comg/ml) 2-400 um
`
`Particles/ml
`6 x freezing?
`thawing
`2-400 um
`
`45
`
`21.40
`
`18.50
`
`17.85
`
`18.30
`
`1.70
`
`1.70
`
`875
`
`276
`
`544
`
`740
`
`130
`
`691
`
`6245
`
`332
`
`19085
`
`695
`
`33.795
`
`677
`
`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
`in buffer
`
`10 mM KP, 30 mM NaCl,
`pH 6.6
`100 mM KP, 0.01% by
`weight
`Tween80, pH 7.2
`MABPDGF-R
`in buffer
`
`10 mM KP, 150 mM
`NaCl, pH 7.2
`50 mM KP, 0.01% by
`weight
`Tween80, pH 7.2
`MABLNGF-R
`
`US 6,238,664 B1
`
`8
`
`TABLE 1-continued
`
`particles/ml
`without stress
`Comg/ml) 2-400 um
`
`Particles/ml
`6 x freezing?
`thawing
`2-400 um
`
`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
`4° 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 acqueous 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 4°C. to 30° C.
`15. The process of claim 10 wherein the osmolarity of the
`liquid aqueous solution is from 100 to 500 mC)sm.
`
`

`

`US 6,238,664 B1
`
`9
`16. The process of claim 15 wherein the osmolarity of the
`liquid aqueous solution is from 280 mC)sm to 320 mC)sm.
`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 mC)sm.
`25. The process of claim 24 wherein the osmolarity of the
`liquid aqueous solution is from 280 mC)sm to 320 mC)sm.
`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;
`is buffered with potassium phosphate buffer in a con
`centration 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) lyophilizing the aqueous buffered Solution.
`
`1O
`
`15
`
`25
`
`35
`
`40
`
`45
`
`50
`
`55
`
`10
`29. The process of claim 28 wherein the protein is an
`antibody.
`30. The process of claim 28 wherein the dissolved protein
`is present in the aqeuous Solution in a concentration from 1
`mg/