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`US 6,875,432 B2
`(10) Patent N0.:
`(12) United States Patent
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`Liu et a1.
`(45) Date of Patent:
`Apr. 5, 2005
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`USOO6875432B2
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`(54) REDUCED-VISCOSITY CONCENTRATED
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`PROTEIN FORNIULATIONS
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`(75)
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`Inventors: Jun Liu, Pacifiea, CA (US); Steven J.
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`Shire, Belmont, CA (US)
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`(73) Assignees: Genentech, Inc., South San Francisco,
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`CA (US); Novartis AG, Basel (CH)
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`( * ) N otice:
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`Subject to any disclaimer, the term of this
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`patent is extended or adjusted under 35
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`U.S.C. 154(b) by 229 days.
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`(21) Appl. No.: 09/971,511
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`Filed:
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`Oct. 4, 2001
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`Prior Publication Data
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`US 2002/0045571 A1 Apr. 18, 2002
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`(22)
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`(65)
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`......... sac/387.3
`5.994.511 A . 11/1999 Lowman et a1.
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`6,252,055 B1 *
`6/2001 Relton ..........
`530/414
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`............ 424/1301
`6,267,958 B1 *
`7/2001 Andya et a1.
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`2003/0092607 A1
`5/2003 Carpenter et a].
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`FOREIGN PATENT DOCUMENTS
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`WO 93/04173
`3/1993
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`WO 97/04801
`/1997
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`97/45140
`* 12/1997
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`WO 99101556
`11999
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`OTHER PUBLICATIONS
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`W0
`W0
`WO
`W0
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`
`a1, Principles of Biochemistry, 3RD Edition,
`White et
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`McGraW—Hill Book Company, p. 540, 1964*
`
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`
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`Presta et al., “Humanization of an Antibody Directed
`
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`
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`Against IgE” J.
`Immzmnl. 1.51
`(5) 262372632 (Sep. 1,
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`1993).
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`Zietkiewicr et al., “In Vivo Studies on the Action on the
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`Tissue of the Osmolality of Parenterally Administered
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`Drugs.” Grzyby Drazdzopodabne.
`(English Translation
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`Attached) 23:869—870 (1971).
`
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`Jones, A., “Analysis of Polypeptides and Proteins.” Adv.
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`Drug Delivery Rev. (10)29—90 (1993).
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`* cited by examiner
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`Primary Examiner—David Saunders
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`(74) Attorney, Agent, or Firm—Craig G. Svoboda
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`ABSTRACT
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`(57)
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`The present application concerns concentrated protein for-
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`mulations With reduced viscosity, which are particularly
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`suitable [or subcutaneous administration. The application
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`further concerns a method for reducing the viscosity of
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`concentrated protein formulations.
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`(60)
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`(51)
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`Related US. Application Data
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`Provisional application No. 60/293,834, filed on May 24,
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`2001, and provisional application No. 60/240,107, filed on
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`Oct. 12, 2000.
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`Int. Cl.7 ...................... A61K 38/16; A61K 39/345;
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`C07K 14/00; C07K 16/00
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`(52) US. Cl.
`................................ 424/130.1; 424/133.1;
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`424/141.1; 424/143.1; 424/174.1; 424/1761;
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`424/1771; 514/2; 530/350; 530/3871;
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`530/3873; 530/388.1; 530/388.22; 530/388.85;
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`530/389.7; 530/390.1, 530/390.5
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`424/1301, 133.1,
`(58) Field of Search .................
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`, 176.1, 177.1; 514/2;
`424/1411, 143.1, 17 .
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`530/350, 387.1, 387.3, 388.1, 388.22, 388.85,
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`389.7, 3901, 390.5
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`(56)
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`References Cited
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`U.S. PATENT DOCUMENTS
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`5,608,038 A *
`3/1997 Eibl et a1.
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`................ 530/3871
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`59 Claims, 7 Drawing Sheets
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`Page 1 of 25
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`C SL EXHIBIT 1061
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`Page 1 of 25
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`CSL EXHIBIT 1061
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`US. Patent
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`Apr. 5,2005
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`Sheet 1 0f 7
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`US 6,875,432 132
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`Figure1
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`Page 2 of 25
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`80 rhuMAbE25Concentration
`(mg/ml)
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`Page 2 of 25
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`US. Patent
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`Apr. 5, 2005
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`Sheet 2 0f 7
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`US 6,875,432 B2
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`Figure 4
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`US. Patent
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`Apr. 5, 2005
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`Sheet 5 0f 7
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`US 6,875,432 B2
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`Sheet 6 0f 7
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`US. Patent
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`Apr. 5,2005
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`Sheet 7 of 7
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`US 6,875,432 B2
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`US 6,875,432 B2
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`1
`REDUCED-VISCOSITY CONCENTRATED
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`PROTEIN FORMULATIONS
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`RELATED APPLICATIONS
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`This application is a non-provisional application claiming
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`priority under 35 U.S.C. § 119(e) to provisional application
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`Ser. No. 60/240,107, filed Oct. 12, 2000 and to Ser. No.
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`60/293,834, filed May 24, 2001, the contents of which are
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`incorporated herein by reference.
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`BACKGROUND OF THE INVENTION
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`1. Field of the Invention
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`This invention pertains to concentrated protein formula-
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`tions with reduced viscosity, which are particularly suitable
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`for subcutaneous administration. The invention further con-
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`cerns a method of reducing viscosity of concentrated protein
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`formulations.
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`2. Description of the Related Art
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`In the past ten years, advances in biotechnology have
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`made it possible to produce a variety of proteins for phar-
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`maceutical applications using recombinant DNA techniques.
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`Because proteins are larger and more complex than tradi-
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`tional organic and inorganic drugs (i.e. possessing multiple
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`functional groups in addition to complex three-dimensional
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`structures), the formulation of such proteins poses special
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`problems. One of the problems is the elevated viscosity
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`values of protein formulations, especially at high concen-
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`tration. The delivery of high protein concentration is often
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`required for subcutaneous administration due to the volume
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`limitations (E15 ml) and dose requirements (usually :50
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`mg, preferably 2100 mg). For example, if a protein is to be
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`administered to patients at 2 mg/kg on a weekly basis, the
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`average weekly dose will be 130 mg considering 65 kg as
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`the average weight of patients. Since injection volumes of
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`more than 1.5 ml are not well tolerated for subcutaneous
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`administration, the protein concentration for a weekly sub-
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`cutaneous administration would have to be approximately
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`100 mg/ml (130 mg protein in less than 1.5 ml volume).
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`However, highly concentrated protein formulations pose
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`several problems. One problem is the tendency of proteins
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`to form particulates during processing and/or storage, which
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`makes manipulation during further processing difficult. In
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`the case of reconstituted liquid formulations, this is usually
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`circumvented by adding a suitable surfactant
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`a
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`polysorbate) during lyophilization or after lyophilization
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`while reconstituting the formulation. Although surfactants
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`have been shown to significantly reduce the degree of
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`particulate formation of proteins,
`they do not address
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`another problem associated with manipulating and admin-
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`istering concentrated protein formulations. Proteins tend to
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`form viscous solutions at high concentration because of their
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`macromolecular nature and potential
`intermolecular
`for
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`interactions. Moreover, many proteins are often lyophilized
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`in the presence of large amounts of lyoprotectants, such as
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`sugar to maintain their stability. The sugar can enhance the
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`intermolecular interactions and increase the viscosity.
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`Highly viscous formulations are difficult to manufacture,
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`draw into a syringe and inject subcutaneously. The use of
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`force in manipulating the viscous formulations leads to
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`excessive frothing, and the resultant detergent-like action of
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`froth has the potential to denature and inactivate the thera-
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`peutically active protein. Moreover, viscous solution
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`increases the back-pressure during UF/DF process and
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`makes recovery of protein difficult. This can result in con-
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`siderable loss of protein product. Satisfactory solution of
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`this problem is lacking in the prior art. Therefore, there is a
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`2
`need to develop a method of reducing the viscosity of a
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`formulation containing high concentration of protein.
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`Stable isotonic lyophilized protein formulations are dis-
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`closed in PCT publication WO 97/04801, published on Feb.
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`13, 1997, the entire disclosure of which is hereby expressly
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`incorporated by reference. The disclosed lyophilized formu-
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`lations can be reconstituted to generate high protein-
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`concentration liquid formulations without apparent loss of
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`stability. However, the potential issues associated with the
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`high viscosity of the reconstituted formulations are not
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`addressed.
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`the preparation of
`Applicants have discovered that
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`proteinaceous, lyophilized formulation with 100 mM NaCl
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`diluent can result in a slightly hypertonic solution. It had
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`been previously believed that pharmaceutical formulations
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`must be maintained at physiological pH and be isotonic.
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`This belief was based at least in part on the perception that
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`the administration of hypertonic formulation could lead to
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`dehydration and therefore could damage the tissue at the site
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`of injection. However,
`the belief of the requirement for
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`absolute isotonicity of a pharmaceutical formulation may
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`not be well-founded. For example, Zietkiewicz et al., Grzyby
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`Drozdzopodobne 23: 869—870 (1971) have shown that abso-
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`lute isotonicity of the drugs is not necessary. It was found to
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`be sufficient to avoid the drug solutions that exceed the
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`critical limits of hypertonicity. For example, tissue damage
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`was observed only when hypertonic solution of 1300
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`mOsmol/Kg (~650 mM NaCl) or higher was administered
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`subcutaneously or intramuscularly to experimental animals.
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`As a result, formulations which are slightly hypertonic, or
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`outside of the physiological pH range do not appear to
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`present a risk of tissue damage at the site of administration.
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`Applicants have further found that proteinaceous solu-
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`tions having a lowered (4.0—5.3) or elevated (6.5—12.0) pH
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`was also effective at reducing the viscosity of high concen-
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`tration protein formulations.
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`The present invention is directed to providing a high
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`concentration protein formulation with reduced viscosity,
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`which is easy to handle and is suitable for subcutaneous
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`administration. The present invention is further directed to
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`providing a method of reducing viscosity of concentrated
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`protein formulations.
`
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`SUMMARY OF THE INVENTION
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`The present invention concerns a method of lowering the
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`viscosity of concentrated protein composition by:
`(1)
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`increasing the total ionic strength of the formulation through
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`the addition of salts or buffer components; or (2) altering the
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`pH of the formulation to be lower (z4.0 to z5 .3) or elevated
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`(z6.5 to z12.0), without significantly compromising stability
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`or biological activity. Accordingly, the invention concerns
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`methods and means for reducing the viscosity of concen-
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`trated protein formulations, primarily to ensure easy
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`manipulation before and during administration to a patient.
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`In one aspect,
`the present invention provides a stable
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`formulation of reduced viscosity comprising a protein in an
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`amount of at least about 80 mg/ml and a salt or a buffer in
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`an amount of at least about 50 mM, and having a kinematic
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`viscosity of about 50 cs or less. The salts and/or buffers are
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`pharmaceutically acceptable and are derived from various
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`known acids (inorganic and organic) or base forming metals
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`and amines. Alternatively, the salts and/or buffers may be
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`derived from amino acids. In a specific aspect, the salts are
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`chosen from the group consisting of sodium chloride, argi-
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`nine hydrochloride, sodium thiocyanate, ammonium
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`thiocyanate, ammonium sulfate, ammonium chloride, cal-
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`10
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`Page 9 0f 25
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`Page 9 of 25
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`

`

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`US 6,875,432 B2
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`3
`cium chloride, Zinc chloride and sodium acetate. In another
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`aspect, the salts or buffers are monovalent. In yet another
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`the formulation contains the above salt or buffer
`aspect,
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`components in an amount of about 50—200 mM, and has a
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`viscosity of about 2 to 30 cs. In a particular embodiment, the
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`protein in the formulation has a molecular weight of at least
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`about 15—20 kD. In another particular embodiment,
`the
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`formulation is hypertonic. In yet another particular aspect,
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`the formulation may further comprise a surfactant such as
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`polysorbate. The invention also contemplates a reconstituted
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`formulation that further comprises a lyoprotectant such as
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`sugars. In yet another particular aspect, the lyoprotectant
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`sugar can be, for example, sucrose or trehalose, and may be
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`present in an amount of about 60—300 mM. In another
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`specific aspect, the protein concentration in the reconstituted
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`formulation is about 2—40 times greater than the protein
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`concentration in the mixture before lyophilization.
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`In another embodiment, the invention provides a stable
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`formulation of reduced viscosity comprising a protein in an
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`amount of at least about 80 mg/ml by having a pH lower
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`(z4.0 to z5.3) or elevated (z6.5 to z12.0), wherein the
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`kinematic viscosity is reduced to 50 cs or less. In a specific
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`aspect, the viscosity is reduced to about 2 to 30 cs. In another
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`specific aspect, the pH is altered through the addition of a
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`pharmaceutically acceptable acid, base or buffer, and is
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`added in an amount of at least about 10 mM, preferably
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`about 50—200 mM, more preferably about 100—200 mM,
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`most preferably about 150 mM. In a specific aspect, the acid,
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`base and/or buffers are monovalent.
`In another specific
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`aspect, the acid, base and/or buffers are selected from the
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`group consisting of acetic acid, hydrochloric acid, and
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`arginine. In another particular aspect, the formulation may
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`further comprise a surfactant such as polysorbate. The
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`invention also contemplates a reconstituted formulation that
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`further comprises a lyoprotectant such as sugars.
`In a
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`particular aspect,
`the lyoprotectant sugar can be,
`for
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`example, sucrose or trehalose, and may be present in an
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`amount of about 60—300 mM. In another preferred aspect,
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`the protein concentration in the reconstituted formulation is
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`about 2—40 times greater than the protein concentration in
`
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`the mixture before lyophilization. In a particular aspect, the
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`pH is any tenth pH value within those enumerated above; for
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`example, for the lower pH value, example values are pH 4.0,
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`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.
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`At the higher pH range, example values are 6.5, 6.6, 6.7, 6.8,
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`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,
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`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,
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`9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7,
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`10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8,
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`11.9 and 12.0.
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`the invention provides a
`In a particular embodiment,
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`formulation containing high concentrations of large molecu-
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`lar weight proteins, such as immunoglobulins. The immu-
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`noglobulins may, for example, be antibodies directed against
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`a particular predetermined antigen. In a specific aspect, the
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`antigen is IgE (e.g.,
`rhuMAbE-25,
`rhuMAbE-26 and
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`rhuMAbE-27 described in WO 99/0155 6). Alternatively, the
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`antigen may include:
`the CD proteins CD3, CD4, CD8,
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`CD19, CD20 and CD34; members of the HER receptor
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`family such as EGF receptor, HER2, HER3 or HER4
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`receptor; cell adhesion molecules such as LFA-1, Mol,
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`p150,95, VLA-4,
`ICAM-1, VCAM and (xv/[33 integrin
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`including the (X- and B-subunits thereof (e.g., anti-CD11a,
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`anti-CD18 or anti-CD11b antibodies); growth factors such
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`as VEGF; blood group antigens; flk2/fit3 receptor; obesity
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`(OB) receptor; and protein C.
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`The formulations of the present invention may be phar-
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`maceutical formulations, in particular, formulations for sub-
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`cutaneous administration.
`
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`10
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`15
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`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
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`50
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`55
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`60
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`65
`
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`4
`
`the invention provides a method of
`In another aspect,
`
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`
`
`
`
`
`
`
`reducing the viscosity of a formulation containing a protein
`
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`
`
`in an amount of at least about 80 mg/ml by the addition of
`
`
`
`
`
`
`
`
`
`
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`
`
`a salt or buffer component in an amount of at least about 50
`
`
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`
`
`
`mM, wherein the kinematic viscosity is reduced to 50 cs or
`
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`
`less. In a specific aspect, the viscosity is reduced to about 2
`
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`
`
`to 30 cs. In another specific aspect,
`the salts or buffer
`
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`
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`
`
`
`components may be added in an amount of at least about 100
`
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`mM, preferably about 50—200 mM, more preferably about
`
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`
`
`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-
`
`
`
`
`
`
`
`
`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
`
`
`
`
`
`
`
`
`
`
`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-
`
`
`
`
`
`
`
`
`
`
`
`
`lation has a molecular weight of at least about 15—20 kD. In
`
`
`
`
`
`
`
`
`
`
`
`
`another particular embodiment, the formulation may further
`
`
`
`
`
`
`
`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-
`
`
`
`
`
`
`
`tion.
`
`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
`
`
`
`
`
`
`
`
`
`
`
`
`
`(z4.0 to z5 .3) or elevated (z6.5 to z12.0), 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 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
`
`
`
`
`
`
`
`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
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`example, sucrose or trehalose, and may be present in an
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`amount of about 60—300 mM. In a preferred embodiment,
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`the protein concentration in the reconstituted formulation is
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`about 2—40 times greater than the protein concentration in
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`the mixture before lyophilization. In a particular aspect, the
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`pH is any tenth pH value within those enumerated above; for
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`example, for the lower pH value, example values are pH 4.0,
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`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.
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`At the higher pH range, example values are 6.5, 6.6, 6.7, 6.8,
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`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,
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`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,
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`9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7,
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`10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8,
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`11.9 and 12.0.
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`Page 10 0f 25
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`Page 10 of 25
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`

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`US 6,875,432 B2
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`5
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`In yet another aspect, the invention provides a method of
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`reducing the viscosity of a formulation of a protein having
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`a molecular weight of at least about 15—20 kD, including
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`immunoglobulins, specifically antibodies which specifically
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`bind to a particular antigen. In a specific aspect, the method
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`is used to prepare a reconstitutable formulation, especially
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`those that are concentrated to a much greater concentration
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`of therapeutic protein (e.g., 2—40 times) after the concen-
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`tration step (e.g., lyophilization) compared to before.
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`In yet another embodiment,
`the invention provides a
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`method for the treatment, prophylactic or therapeutic, of a
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`disorder treatable by the protein (e.g., antibody) formulated,
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`using the formulations disclosed herein. Such formulations
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`are particularly useful for subcutaneous administration.
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`Also provided is an article of manufacture comprising a
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`container enclosing a formulation disclosed herein.
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`In yet another embodiment,
`the present invention dis-
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`closes a method of preventing self-association of proteins in
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`concentrated liquid formulations by (1) adding a salt or a
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`buffer component in an amount of at least about 50 mM; or
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`(2) altering the pH by lowering to (z4.0 to z5.3) or elevating
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`to (z6.5 to z12.0). In a specific aspect, the self-association
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`to be prevented is that
`induced or exacerbated by the
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`presence of sugars (e.g., sucrose or trehalose) that are
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`commonly used as lyoprotectants. Accordingly, this method
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`is particularly useful for preventing self-association of
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`reconstituted lyophilized formulations.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`FIG. 1 shows the effects of protein concentration on
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`viscosity of reconstituted formulation containing the anti-
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`IgE antibody rhuMAb E25, 16 mM histidine. 266 mM
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`sucrose and 0.03% Polysorbate 20 at 25° C.
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`FIG. 2 depicts the effects of NaCl concentration on
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`viscosity of reconstituted formulation containing 125 mg/ml
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`of the antibody IgE antibody rhuMAb E25, 16 mM histidine,
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`266 mM sucrose, 0.03% Polysorbate 20 and various
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`amounts of NaCl at 25° C.
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`FIG. 3 shows the effects of various salts on viscosity of
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`reconstituted formulation containing 40 mg/ml of the anti-
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`IgE antibody rhuMAb E25, 10 mM histidine, 250 mM
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`sucrose, 0.01% Polysorbate 20 and various amounts of salts
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`at 25° C.
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`FIG. 4 shows the effects of buffer concentration on
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`viscosity of a liquid formulation containing 80 mg/ml of the
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`anti-IgE antibody rhuMAb E25, 50 mM histidine, 150 mM
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`trehalose, 0.05% Polysorbate 20 and various amounts of
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`histidine, acetate, or succinate components at 25° C.
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`FIG. 5 shows the effects of NaCl concentration on vis-
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`cosity of a reconstituted formulation containing 21 mg/ml
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`rhuMAb E26, 5 mM histidine, 275 mM sucrose at 6° C.
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`FIG. 6 shows the effects of pH on viscosity of liquid
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`formulations containing 130 mg/ml rhuMAb E25, 2—17.5
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`mM of acetate or arginine with and without 150 mM NaCl
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`at 25° C.
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`FIG. 7 shows the effects of pH on viscosity of reconsti-
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`tuted lyophilized formulations containing 94 mg/ml
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`rhuMAb E25, 250 mM trehalose, 20 mM histidine, at 25° C.
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`DETAILED DESCRIPTION OF THE
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`PREFERRED EMBODIMENT
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`I. Definitions
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`By “protein” is meant a sequence of amino acids for
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`which the chain length is sufficient to produce the higher
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`levels of tertiary and/or quaternary structure. Thus, proteins
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`Page 11 0f 25
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`6
`are distinguished from “peptides” which are also amino
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`acid-based molecules that do not have such structure.
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`Typically, a protein for use herein will have a molecular
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`weight of at least about 15—20 kD, preferably at least about
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`20 kD.
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`Examples of proteins encompassed within the definition
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`herein include mammalian proteins, such as, e.g., growth
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`hormone,
`including human growth hormone and bovine
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`growth hormone; growth hormone releasing factor; parathy-
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`roid hormone;
`thyroid stimulating hormone;
`lipoproteins;
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`ot-1-antitrypsin; insulin A-chain; insulin B-chain; proinsu-
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`lin; follicle stimulating hormone; calcitonin; luteinizing hor-
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`mone; glucagon; clotting factors such as factor VIIIC, factor
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`IX, tissue factor, and von Willebrands factor; anti-clotting
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`lung
`factors such as Protein C; atrial natriuretic factor;
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`surfactant; a plasminogen activator, such as urokinase or
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`tissue-type plasminogen activator (t-PA, e.g., Activase®,
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`TNKase®, Retevase®); bombazine; thrombin; tumor necro-
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`sis factor-ot and -[3; enkephalinase; RANTES (regulated on
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`activation normally T-cell expressed and secreted); human
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`macrophage inflammatory protein (MIP-1-ot); serum albu-
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`min such as human serum albumin; mullerian-inhibiting
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`substance;
`relaxin A-chain;
`relaxin B-chain; prorelaxin;
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`inhibin;
`mouse gonadotropin-associated peptide; DNase;
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`activin; vascular endothelial growth factor (VEGF); recep-
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`tors for hormones or growth factors; an integrin; protein A
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`or D; rheumatoid factors; a neurotrophic factor such as
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`bone-derived neurotrophic factor (BDNF), neurotrophin-3,
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`-4, -5, or -6 (NT-3, NT-4, NT5, or NT-6), or a nerve growth
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`factor such as NGF-B; platelet-derived growth factor
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`(PDGF); fibroblast growth factor such as aFGF and bFGF;
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`epidermal growth factor (EGF); transforming growth factor
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`(TGF) such as TGF-ot and TGF-B, including TGF-B1, TGF-
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`B2, TGF-B3, TGF-B4, or TGF-BS;
`insulin-like growth
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`factor-I and -II (IGF-I and IGF-II); des(1—3)-IGF-I (brain
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`insulin-like growth factor binding proteins; CD
`IGF-I);
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`proteins such as CD3, CD4, CD8, CD19 and CD20; eryth-
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`ropoietin (EPO); thrombopoietin (TPO); osteoinductive fac-
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`tors; immunotoxins; a bone morphogenetic protein (BMP);
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`an interferon such as interferon