(12) United States Patent
`Liu et al.
`
`111111111111111111111111111111111111111111111111111111111111111111111111111
`US006875432B2
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 6,875,432 B2
`Apr. 5, 2005
`
`(54) REDUCED-VISCOSITY CONCENTRATED
`PROTEIN FORMULATIONS
`
`(75)
`
`Inventors: Jun Liu, Pacifica, CA (US); Steven J.
`Shire, Belmont, CA (US)
`
`(73)
`
`Assignees: Genentech, Inc., South San Francisco,
`CA (US); Novartis AG, Basel (CH)
`
`( *)
`
`Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 229 days.
`
`5,994,511 A * 11/1999 Lowman eta!. ......... 530/387.3
`6,252,055 B1 * 6/2001 Relton ........................ 530/414
`6,267,958 B1 * 7/2001 Andya et a!. ............ 424/130.1
`2003/0092607 A1 * 5!2003 Carpenter et a!.
`
`FOREIGN PATENT DOCUMENTS
`wo 93/04173
`wo 97/04801
`97/45140
`wo 99/01556
`
`3/1993
`2/1997
`* 12/1997
`1!1999
`
`wo
`wo
`wo
`wo
`
`OTHER PUBLICATIONS
`
`(21) Appl. No.: 09/971,511
`
`(22) Filed:
`
`Oct. 4, 2001
`
`(65)
`
`Prior Publication Data
`
`US 2002/0045571 A1 Apr. 18, 2002
`
`(51)
`
`Related U.S. Application Data
`( 60) Provisional application No. 60/293,834, filed on May 24,
`2001, and provisional application No. 60/240,107, filed on
`Oct. 12, 2000.
`Int. Cl? ...................... A61K 38/16; A61K 39/345;
`C07K 14/00; C07K 16/00
`(52) U.S. Cl. ................................ 424/130.1; 424/133.1;
`424/141.1; 424/143.1; 424/174.1; 424/176.1;
`424/177.1; 514/2; 530/350; 530/387.1;
`530/387.3; 530/388.1; 530/388.22; 530/388.85;
`530/389.7; 530/390.1; 530/390.5
`(58) Field of Search ........................... 424/130.1, 133.1,
`424/141.1, 143.1, 174.1, 176.1, 177.1; 514/2;
`530/350, 387.1, 387.3, 388.1, 388.22, 388.85,
`389.7, 390.1, 390.5
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`5,608,038 A * 3/1997 Eibl et a!. ................ 530/387.1
`
`White et al, Principles of Biochemistry, 3RD Edition,
`McGraw-Hill Book Company, p. 540, 1964.*
`Presta et al., "Humanization of an Antibody Directed
`Against IgE" J. Immunol. 151 (5) :2623-2632 (Sep. 1,
`1993).
`Zietkiewicz et al., "In Vivo Studies on the Action on the
`Tissue of the Osmolality of Parenterally Administered
`Drugs." Grzyby Drozdzopodobne.
`(English Translation
`Attached) 23:869-870 (1971).
`Jones, A, "Analysis of Polypeptides and Proteins." Adv.
`Drug Delivery Rev. (10)29-90 (1993).
`
`* cited by examiner
`
`Primary Examiner-David Saunders
`(74) Attorney, Agent, or Firm-Craig G. Svoboda
`
`(57)
`
`ABSTRACT
`
`The present application concerns concentrated protein for(cid:173)
`mulations with reduced viscosity, which are particularly
`suitable for subcutaneous administration. The application
`further concerns a method for reducing the viscosity of
`concentrated protein formulations.
`
`59 Claims, 7 Drawing Sheets
`
`CSL EXHIBIT 1061
`CSL v. Shire
`
`Page 1 of 25
`
`

`

`U.S. Patent
`US. Patent
`
`Apr. 5,2005
`Apr. 5, 2005
`
`Sheet 1 0f 7
`Sheet 1 of 7
`
`US 6,875,432 132
`US 6,875,432 B2
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`Apr. 5, 2005
`
`Sheet 2 of 7
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`US 6,875,432 B2
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`U.S. Patent
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`Apr. 5, 2005
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`US 6,875,432 B2
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`U.S. Patent
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`Apr. 5, 2005
`
`Sheet 6 of 7
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`US 6,875,432 B2
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`U.S. Patent
`US. Patent
`
`Apr. 5,2005
`Apr. 5, 2005
`
`Sheet 7 0f 7
`Sheet 7 of 7
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`US 6,875,432 132
`US 6,875,432 B2
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`Page 8 of 25
`
`

`

`US 6,875,432 B2
`
`1
`REDUCED-VISCOSITY CONCENTRATED
`PROTEIN FORMULATIONS
`
`RELATED APPLICATIONS
`
`This application is a non-provisional application claiming
`priority under 35 U.S.C. § 119(e) to provisional application
`Ser. No. 60/240,107, filed Oct. 12, 2000 and to Ser. No.
`60/293,834, filed May 24, 2001, the contents of which are
`incorporated herein by reference.
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`This invention pertains to concentrated protein formula(cid:173)
`tions with reduced viscosity, which are particularly suitable 15
`for subcutaneous administration. The invention further con-
`cerns a method of reducing viscosity of concentrated protein
`formulations.
`2. Description of the Related Art
`In the past ten years, advances in biotechnology have
`made it possible to produce a variety of proteins for phar(cid:173)
`maceutical applications using recombinant DNA techniques.
`Because proteins are larger and more complex than tradi(cid:173)
`tional organic and inorganic drugs (i.e. possessing multiple
`functional groups in addition to complex three-dimensional
`structures), the formulation of such proteins poses special
`problems. One of the problems is the elevated viscosity
`values of protein formulations, especially at high concen(cid:173)
`tration. The delivery of high protein concentration is often
`required for subcutaneous administration due to the volume
`limitations ( ~ 1.5 ml) and dose requirements (usually ~50
`mg, preferably ~100 mg). For example, if a protein is to be
`administered to patients at 2 mg!kg on a weekly basis, the
`average weekly dose will be 130 mg considering 65 kg as
`the average weight of patients. Since injection volumes of 35
`more than 1.5 ml are not well tolerated for subcutaneous
`administration, the protein concentration for a weekly sub(cid:173)
`cutaneous administration would have to be approximately
`100 mg/ml (130 mg protein in less than 1.5 ml volume).
`However, highly concentrated protein formulations pose
`several problems. One problem is the tendency of proteins
`to form particulates during processing and/or storage, which
`makes manipulation during further processing difficult. In
`the case of reconstituted liquid formulations, this is usually
`circumvented by adding a suitable surfactant (e.g. a 45
`polysorbate) during lyophilization or after lyophilization
`while reconstituting the formulation. Although surfactants
`have been shown to significantly reduce the degree of
`particulate formation of proteins, they do not address
`another problem associated with manipulating and admin- 50
`istering concentrated protein formulations. Proteins tend to
`form viscous solutions at high concentration because of their
`macromolecular nature and potential for intermolecular
`interactions. Moreover, many proteins are often lyophilized
`in the presence of large amounts of lyoprotectants, such as 55
`sugar to maintain their stability. The sugar can enhance the
`intermolecular interactions and increase the viscosity.
`Highly viscous formulations are difficult to manufacture,
`draw into a syringe and inject subcutaneously. The use of
`force in manipulating the viscous formulations leads to 60
`excessive frothing, and the resultant detergent-like action of
`froth has the potential to denature and inactivate the thera(cid:173)
`peutically active protein. Moreover, viscous solution
`increases the back-pressure during UF/DF process and
`makes recovery of protein difficult. This can result in con- 65
`siderable loss of protein product. Satisfactory solution of
`this problem is lacking in the prior art. Therefore, there is a
`
`2
`need to develop a method of reducing the viscosity of a
`formulation containing high concentration of protein.
`Stable isotonic lyophilized protein formulations are dis(cid:173)
`closed in PCT publication WO 97/04801, published on Feb.
`5 13, 1997, the entire disclosure of which is hereby expressly
`incorporated by reference. The disclosed lyophilized formu(cid:173)
`lations can be reconstituted to generate high protein(cid:173)
`concentration liquid formulations without apparent loss of
`stability. However, the potential issues associated with the
`10 high viscosity of the reconstituted formulations are not
`addressed.
`Applicants have discovered that the preparation of
`proteinaceous, lyophilized formulation with 100 mM NaCl
`diluent can result in a slightly hypertonic solution. It had
`been previously believed that pharmaceutical formulations
`must be maintained at physiological pH and be isotonic.
`This belief was based at least in part on the perception that
`the administration of hypertonic formulation could lead to
`dehydration and therefore could damage the tissue at the site
`20 of injection. However, the belief of the requirement for
`absolute isotonicity of a pharmaceutical formulation may
`not be well-founded. For example, Zietkiewicz et al., Grzyby
`Drozdzopodobne 23: 869-870 (1971) have shown that abso(cid:173)
`lute isotonicity of the drugs is not necessary. It was found to
`25 be sufficient to avoid the drug solutions that exceed the
`critical limits of hypertonicity. For example, tissue damage
`was observed only when hypertonic solution of 1300
`mOsmol/Kg (-650 mM NaCl) or higher was administered
`subcutaneously or intramuscularly to experimental animals.
`30 As a result, formulations which are slightly hypertonic, or
`outside of the physiological pH range do not appear to
`present a risk of tissue damage at the site of administration.
`Applicants have further found that proteinaceous solu(cid:173)
`tions having a lowered (4.0-5.3) or elevated (6.5-12.0) pH
`was also effective at reducing the viscosity of high concen(cid:173)
`tration protein formulations.
`The present invention is directed to providing a high
`concentration protein formulation with reduced viscosity,
`40 which is easy to handle and is suitable for subcutaneous
`administration. The present invention is further directed to
`providing a method of reducing viscosity of concentrated
`protein formulations.
`
`SUMMARY OF THE INVENTION
`
`The present invention concerns a method of lowering the
`viscosity of concentrated protein composition by: (1)
`increasing the total ionic strength of the formulation through
`the addition of salts or buffer components; or (2) altering the
`pH of the formulation to be lower (""4.0 to ""5.3) or elevated
`(""6.5 to ""12.0), without significantly compromising stability
`or biological activity. Accordingly, the invention concerns
`methods and means for reducing the viscosity of concen(cid:173)
`trated protein formulations, primarily to ensure easy
`manipulation before and during administration to a patient.
`In one aspect, the present invention provides a stable
`formulation of reduced viscosity comprising a protein in an
`amount of at least about 80 mg/ml and a salt or a buffer in
`an amount of at least about 50 mM, and having a kinematic
`viscosity of about 50 cs or less. The salts and/or buffers are
`pharmaceutically acceptable and are derived from various
`known acids (inorganic and organic) or base forming metals
`and amines. Alternatively, the salts and/or buffers may be
`derived from amino acids. In a specific aspect, the salts are
`chosen from the group consisting of sodium chloride, argi(cid:173)
`nine hydrochloride, sodium thiocyanate, ammonium
`thiocyanate, ammonium sulfate, ammonium chloride, cal-
`
`Page 9 of 25
`
`

`

`US 6,875,432 B2
`
`3
`cium chloride, zinc chloride and sodium acetate. In another
`aspect, the salts or buffers are monovalent. In yet another
`aspect, the formulation contains the above salt or buffer
`components in an amount of about 50--200 mM, and has a
`viscosity of about 2 to 30 cs. In a particular embodiment, the
`protein in the formulation has a molecular weight of at least
`about 15-20 kD. In another particular embodiment, the
`formulation is hypertonic. In yet another particular aspect,
`the formulation may further comprise a surfactant such as
`polysorbate. The invention also contemplates a reconstituted
`formulation that further comprises a lyoprotectant such as
`sugars. In yet another particular aspect, the lyoprotectant
`sugar can be, for example, sucrose or trehalose, and may be
`present in an amount of about 60-300 mM. In another
`specific aspect, the protein concentration in the reconstituted
`formulation is about 2-40 times greater than the protein
`concentration in the mixture before lyophilization.
`In another embodiment, the invention provides a stable
`formulation of reduced viscosity comprising a protein in an
`amount of at least about 80 mg/ml by having a pH lower
`(>4.0 to ""5.3) or elevated (""6.5 to ""12.0), wherein the
`kinematic viscosity is reduced to 50 cs or less. In a specific
`aspect, the viscosity is reduced to about 2 to 30 cs. In another
`specific aspect, the pH is altered through the addition of a
`pharmaceutically acceptable acid, base or buffer, and is
`added in an amount of at least about 10 mM, preferably
`about 50-200 mM, more preferably about 100--200 mM,
`most preferably about 150 mM. In a specific aspect, the acid,
`base and/or buffers are monovalent. In another specific
`aspect, the acid, base and/or buffers are selected from the
`group consisting of acetic acid, hydrochloric acid, and
`arginine. In another particular aspect, the formulation may
`further comprise a surfactant such as polysorbate. The
`invention also contemplates a reconstituted formulation that
`further comprises a lyoprotectant such as sugars. In a
`particular aspect, the lyoprotectant sugar can be, for
`example, sucrose or trehalose, and may be present in an
`amount of about 60-300 mM. In another preferred aspect,
`the protein concentration in the reconstituted formulation is
`about 2-40 times greater than the protein concentration in
`the mixture before lyophilization. In a particular aspect, the
`pH is any tenth pH value within those enumerated above; for
`example, for the lower pH value, example values are pH 4.0,
`4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2 and 5.3.
`At the higher pH range, example values are 6.5, 6.6, 6.7, 6.8,
`6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,
`8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,
`9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7,
`10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8,
`11.9 and 12.0.
`In a particular embodiment, the invention provides a
`formulation containing high concentrations of large molecu(cid:173)
`lar weight proteins, such as immunoglobulins. The immu(cid:173)
`noglobulins may, for example, be antibodies directed against
`a particular predetermined antigen. In a specific aspect, the
`antigen is IgE (e.g., rhuMAbE-25, rhuMAbE-26 and
`rhuMAbE-27 described in WO 99/01556). Alternatively, the
`antigen may include: the CD proteins CD3, CD4, CDS,
`CD19, CD20 and CD34; members of the HER receptor
`family such as EGF receptor, HER2, HER3 or HER4
`receptor; cell adhesion molecules such as LFA-1, Mol,
`p150,95, VLA-4, ICAM-1, VCAM and av/~3 integrin
`including the a- and ~-subunits thereof (e.g., anti-CD11a,
`anti-CD18 or anti-CD11b antibodies); growth factors such
`as VEGF; blood group antigens; fik2/fit3 receptor; obesity
`(OB) receptor; and protein C.
`The formulations of the present invention may be phar(cid:173)
`maceutical formulations, in particular, formulations for sub(cid:173)
`cutaneous administration.
`
`10
`
`30
`
`4
`In another aspect, the invention provides a method of
`reducing the viscosity of a formulation containing a protein
`in an amount of at least about 80 mg/ml by the addition of
`a salt or buffer component in an amount of at least about 50
`5 mM, wherein the kinematic viscosity is reduced to 50 cs or
`less. In a specific aspect, the viscosity is reduced to about 2
`to 30 cs. In another specific aspect, the salts or buffer
`components may be added in an amount of at least about 100
`mM, preferably about 50--200 mM, more preferably about
`100-200 mM, most preferably about 150 mM. The salts
`and/or buffers are pharmaceutically acceptable and are
`derived from various known acids (inorganic and organic)
`with "base forming" metals or amines. Alternatively, the
`salts and/or buffers may be derived from amino acids. In yet
`another specific aspect, the salts and/or buffers are monova-
`15 lent. In yet another specific aspect, the salts are selected from
`the group consisting of sodium chloride, arginine
`hydrochloride, sodium thiocyanate, ammonium thiocyanate,
`ammonium sulfate, ammonium chloride, calcium chloride,
`zinc chloride and sodium acetate. In yet another aspect, the
`20 formulation contains the above salt or buffer components in
`an amount of about 50-200 mM, and has a viscosity of about
`2 to 30 cs. In yet another aspect, the protein in the formu(cid:173)
`lation has a molecular weight of at least about 15-20 kD. In
`another particular embodiment, the formulation may further
`25 comprise a surfactant such as polysorbate. The invention
`also contemplates a reconstituted formulation that further
`comprises a lyoprotectant such a sugar. In a particular
`aspect, the lyoprotectant sugar can be, for example, sucrose
`or trehalose, and may be present in an amount of about
`60--300 mM. In a specific aspect, the formulation can be
`reconstituted with a diluent comprising the buffer or salt. In
`a preferred embodiment, the protein concentration in the
`reconstituted formulation is about 2-40 times greater than
`the protein concentration in the mixture before lyophiliza(cid:173)
`tion.
`35 In yet another embodiment, the invention provides a method
`for reducing the viscosity comprising a protein in an amount
`of at least about 80 mg/ml by altering the pH to be lower
`(""4.0 to ""5.3) or elevated (""6.5 to ""12.0), wherein the
`kinematic viscosity is reduced to 50 cs or less. In a specific
`40 aspect, the viscosity is reduced to about 2 to 30 cs. In another
`specific aspect, the pH is altered through the addition of a
`pharmaceutically acceptable acid, base or buffer, and is
`added in an amount of at least about 10 mM, preferably
`about 50-200 mM, more preferably about 100-200 mM,
`45 most preferably about 150 mM. In a specific aspect, the acid,
`base and/or buffers are monovalent. In an another specific
`aspect, the acid, base and/or buffers are selected from the
`group consisting of acetic acid, hydrochloric acid, and
`arginine. In another particular embodiment, the formulation
`50 may further comprise a surfactant such as polysorbate. The
`invention also contemplates a reconstituted formulation that
`further comprises a lyoprotectant such as sugars. In a
`particular aspect, the lyoprotectant sugar can be, for
`example, sucrose or trehalose, and may be present in an
`55 amount of about 60-300 mM. In a preferred embodiment,
`the protein concentration in the reconstituted formulation is
`about 2-40 times greater than the protein concentration in
`the mixture before lyophilization. In a particular aspect, the
`pH is any tenth pH value within those enumerated above; for
`60 example, for the lower pH value, example values are pH 4.0,
`4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2 and 5.3.
`At the higher pH range, example values are 6.5, 6.6, 6.7, 6.8,
`6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,
`8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,
`65 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7,
`10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8,
`11.9 and 12.0.
`
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`US 6,875,432 B2
`
`5
`In yet another aspect, the invention provides a method of
`reducing the viscosity of a formulation of a protein having
`a molecular weight of at least about 15-20 kD, including
`immunoglobulins, specifically antibodies which specifically
`bind to a particular antigen. In a specific aspect, the method 5
`is used to prepare a reconstitutable formulation, especially
`those that are concentrated to a much greater concentration
`of therapeutic protein (e.g., 2-40 times) after the concen(cid:173)
`tration step (e.g., lyophilization) compared to before.
`In yet another embodiment, the invention provides a 10
`method for the treatment, prophylactic or therapeutic, of a
`disorder treatable by the protein (e.g., antibody) formulated,
`using the formulations disclosed herein. Such formulations
`are particularly useful for subcutaneous administration.
`Also provided is an article of manufacture comprising a 15
`container enclosing a formulation disclosed herein.
`In yet another embodiment, the present invention dis(cid:173)
`closes a method of preventing self-association of proteins in
`concentrated liquid formulations by (1) adding a salt or a
`buffer component in an amount of at least about 50 mM; or 20
`(2) altering the pH by lowering to (""4.0 to ""5.3) or elevating
`to (""6.5 to ""12.0). In a specific aspect, the self-association
`to be prevented is that induced or exacerbated by the
`presence of sugars (e.g., sucrose or trehalose) that are
`commonly used as lyoprotectants. Accordingly, this method 25
`is particularly useful for preventing self-association of
`reconstituted lyophilized formulations.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 shows the effects of protein concentration on
`viscosity of reconstituted formulation containing the anti(cid:173)
`IgE antibody rhuMAb E25, 16 mM histidine. 266 mM
`sucrose and 0.03% Polysorbate 20 at 25° C.
`FIG. 2 depicts the effects of NaCl concentration on
`viscosity of reconstituted formulation containing 125 mg/ml
`of the antibody IgE antibody rhuMAb E25, 16 mM histidine,
`266 mM sucrose, 0.03% Polysorbate 20 and various
`amounts of NaCl at 25° C.
`FIG. 3 shows the effects of various salts on viscosity of
`reconstituted formulation containing 40 mg/ml of the anti(cid:173)
`IgE antibody rhuMAb E25, 10 mM histidine, 250 mM
`sucrose, 0.01% Polysorbate 20 and various amounts of salts
`at 25° C.
`FIG. 4 shows the effects of buffer concentration on
`viscosity of a liquid formulation containing SO mg/ml of the
`anti-IgE antibody rhuMAb E25, 50 mM histidine, 150 mM
`trehalose, 0.05% Polysorbate 20 and various amounts of
`histidine, acetate, or succinate components at 25° C.
`FIG. 5 shows the effects of NaCl concentration on vis(cid:173)
`cosity of a reconstituted formulation containing 21 mg/ml
`rhuMAb E26, 5 mM histidine, 275 mM sucrose at 6° C.
`FIG. 6 shows the effects of pH on viscosity of liquid
`formulations containing 130 mg/ml rhuMAb E25, 2-17.5
`mM of acetate or arginine with and without 150 mM NaCl
`at 25° C.
`FIG. 7 shows the effects of pH on viscosity of reconsti(cid:173)
`tuted lyophilized formulations containing 94 mg/ml
`rhuMAb E25, 250 mM trehalose, 20 mM histidine, at 25° C.
`
`DETAILED DESCRIPTION OF THE
`PREFERRED EMBODIMENT
`
`I. Definitions
`By "protein" is meant a sequence of amino acids for
`which the chain length is sufficient to produce the higher
`levels of tertiary and/or quaternary structure. Thus, proteins
`
`6
`are distinguished from "peptides" which are also amino
`acid-based molecules that do not have such structure.
`Typically, a protein for use herein will have a molecular
`weight of at least about 15-20 kD, preferably at least about
`20 kD.
`Examples of proteins encompassed within the definition
`herein include mammalian proteins, such as, e.g., growth
`hormone, including human growth hormone and bovine
`growth hormone; growth hormone releasing factor; parathy(cid:173)
`roid hormone; thyroid stimulating hormone; lipoproteins;
`a-1-antitrypsin; insulin A-chain; insulin B-chain; proinsu-
`lin; follicle stimulating hormone; calcitonin; luteinizing hor(cid:173)
`mone; glucagon; clotting factors such as factor VIIIC, factor
`IX, tissue factor, and von Willebrands factor; anti-clotting
`factors such as Protein C; atrial natriuretic factor; lung
`surfactant; a plasminogen activator, such as urokinase or
`tissue-type plasminogen activator (t-PA, e.g., Activase®,
`TNKase®, Retevase®); bombazine; thrombin; tumor necro(cid:173)
`sis factor-a and -~; enkephalinase; RANTES (regulated on
`activation normally T-cell expressed and secreted); human
`macrophage inflammatory protein (MIP-1-a); serum albu-
`min such as human serum albumin; mullerian-inhibiting
`substance; relaxin A-chain; relaxin B-chain; prorelaxin;
`mouse gonadotropin-associated peptide; DNase; inhibin;
`activin; vascular endothelial growth factor (VEGF); recep(cid:173)
`tors for hormones or growth factors; an integrin; protein A
`or D; rheumatoid factors; a neurotrophic factor such as
`bone-derived neurotrophic factor (BDNF), neurotrophin-3,
`-4, -5, or -6 (NT-3, NT-4, NT5, or NT-6), or a nerve growth
`30 factor such as NGF-~; platelet-derived growth factor
`(PDGF); fibroblast growth factor such as aFGF and bFGF;
`epidermal growth factor (EGF); transforming growth factor
`(TGF) such as TGF-a and TGF-~, including TGF-~1, TGF(cid:173)
`~2, TGF-~3, TGF-~4, or TGF-~5; insulin-like growth
`35 factor-! and -II (IGF-1 and IGF-11); des(l-3)-IGF-1 (brain
`IGF-1); insulin-like growth factor binding proteins; CD
`proteins such as CD3, CD4, CDS, CD19 and CD20; eryth(cid:173)
`ropoietin (EPO); thrombopoietin (TPO); osteoinductive fac(cid:173)
`tors; immunotoxins; a bone morphogenetic protein (BMP);
`40 an interferon such as interferon-a, -~,and-y; colony stimu(cid:173)
`lating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF;
`interleukins (ILs), e.g., IL-l to IL-10; superoxide dismutase;
`T-cell receptors; surface membrane proteins; decay acceler(cid:173)
`ating factor (DAF); a viral antigen such as, for example, a
`45 portion of the AIDS envelope; transport proteins; homing
`receptors; addressins; regulatory proteins; immunoadhesins;
`antibodies; and biologically active fragments or variants of
`any of the above-listed polypeptides.
`The protein which is formulated is preferably essentially
`50 pure and desirably essentially homogeneous (i.e. free from
`contaminating proteins). "Essentially pure" protein means a
`composition comprising at least about 90% by weight of the
`protein, based on total weight of the composition, preferably
`at least about 95% by weight. "Essentially homogeneous"
`55 protein means a composition comprising at least about 99%
`by weight of protein, based on total weight of the compo(cid:173)
`sition.
`In certain embodiments, the protein is an antibody. The
`antibody may bind to any of the above-mentioned
`60 molecules, for example. Exemplary molecular targets for
`antibodies encompassed by the present invention include
`CD proteins such as CD3, CD4, CDS, CD19, CD20 and
`CD34; members of the HER receptor family such as the
`EGF receptor, HER2, HER3 or HER4 receptor; cell adhe-
`65 sian molecules such as LFA-1, Mol, p150,95, VLA-4,
`ICAM-1, VCAM and av/~3 integrin including either a or~
`subunits thereof (e.g. anti-CDlla, anti-CDlS or anti-CDllb
`
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`US 6,875,432 B2
`
`10
`
`7
`antibodies); growth factors such as VEGF; IgE; blood group
`antigens; fik2/fit3 receptor; obesity (OB) receptor; protein C
`etc.
`The term "antibody" is used in the broadest sense and
`specifically covers monoclonal antibodies (including full 5
`length antibodies which have an immunoglobulin Fe
`region), antibody compositions with polyepitopic
`specificity, bispecific antibodies, diabodies, and single-chain
`molecules, as well as antibody fragments (e.g., Fab, F(ab')2 ,
`and Fv).
`The basic 4-chain antibody unit is a heterotetrameric
`glycoprotein composed of two identical light (L) chains and
`two identical heavy (H) chains. An IgM antibody consists of
`5 of the basic heterotetramer unit along with an additional
`polypeptide called a 1 chain, and contains 10 antigen binding 15
`sites, while IgA antibodies comprise from 2-5 of the basic
`4-chain units which can polymerize to form polyvalent
`assemblages in combination with the 1 chain. In the case of
`IgGs, the 4-chain unit is generally about 150,000 daltons.
`Each L chain is linked to an H chain by one covalent 20
`disulfide bond, while the two H chains are linked to each
`other by one or more disulfide bonds depending on the H
`chain isotype. Each H and L chain also has regularly spaced
`intrachain disulfide bridges. Each H chain has at the
`N-terminus, a variable domain (V H) followed by three 25
`constant domains (CH) for each of the a and y chains and
`four CH domains for fA and E isotypes. Each L chain has at
`the N-terminus, a variable domain (VL) followed by a
`constant domain at its other end. The VL is aligned with the
`V H and the CL is aligned with the first constant domain of 30
`the heavy chain (CH1). Particular amino acid residues are
`believed to form an interface between the light chain and
`heavy chain variable domains. The pairing of a V Hand VL
`together forms a single antigen-binding site. For the struc(cid:173)
`ture and properties of the different classes of antibodies, see 35
`e.g., Basic and Clinical Immunology, 8th Edition, Daniel P.
`Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton
`& Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.
`The L chain from any vertebrate species can be assigned
`to one of two clearly distinct types, called kappa and lambda, 40
`based on the amino acid sequences of their constant
`domains. Depending on the amino acid sequence of the
`constant domain of their heavy chains (CH), immunoglo(cid:173)
`bulins can be assigned to different classes or isotypes. There
`are five classes of immunoglobulins: IgA, IgD, IgE, IgG and 45
`IgM, having heavy chains designated a, ll, E, y and fA,
`respectively. The y and fA classes are further divided into
`subclasses on the basis of relatively minor differences in the
`CH sequence and function, e.g., humans express the follow(cid:173)
`ing subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
`The term "variable" refers to the fact that certain seg(cid:173)
`ments of the variable domains differ extensively in sequence
`among antibodies. The V domain mediates antigen binding
`and defines the specificity of a particular antibody for its
`particular antigen. However, the variability is not evenly 55
`distributed across the entire span of the variable domains.
`Instead, the V regions consist of relatively invariant
`stretches called framework regions (FRs) of about 15-30
`amino acid residues separated by shorter regions of extreme
`variability called "hypervariable regions" or sometimes 60
`"complementarity determining regions" (CDRs) that are
`each approximately 9-12 amino acid residues in length. The
`variable domains of native heavy and light chains each
`comprise four FRs, largely adopting a ~-sheet configuration,
`connected by three hypervariable regions, which form loops 65
`connecting, and in some cases forming part of, the ~-sheet
`structure. The hyperva