11•1111111111111
`
`US005656730A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,656,730
`Aug. 12, 1997
`
`United States Patent [19]
`Lee
`
`[54] STABILIZED MONOMERIC PROTEIN
`COMPOSITIONS
`
`[75]
`
`Inventor: Lihsyng Stanford Lee, Princeton
`Junction, N.J.
`
`[73] Assignee: Enzon, Inc •• Piscataway, N.J.
`
`[21] Appl. No.: 417,855
`
`Apr. 7, 1995
`
`[22] Filed:
`Int. Cl.6
`................................................... A61K 39/395
`[51]
`[52] U.S. Cl ...................................... 530/357.3; 424/133.1;
`424/135.1
`[58] Field of Search ......................... 514/970; 424/133.1,
`424/135.1, 155.1. 174.1; 435/69.6, 69.7,
`172.1, 172.3, 240.3; 530/387.3, 387.7
`
`[56]
`
`References Cited
`
`U.S. PATENf DOCUMENrS
`
`3,950,513
`4,496,537
`4,597,966
`4,645,830
`4,704,692
`4,777,043
`4,806,524
`4,946,778
`5,096,885
`5,260,203
`5,358,708
`
`4/1976 Jenson ....................................... 424/94
`111985 Kwan ........................................ 424/85
`7 /1986 Zolton et al. .. ........................... 424/85
`2/1987 Yasushi et al .......................... 530/351
`11/1987 Ladner .................................... 364/496
`10/1988 Bennett et al ....................... 424194.64
`2/1989 Kawaguchi et al ......................... 514/8
`8/1990 Ladner et al .
`......................... 435/69.6
`3/1992 Pearlman et al . ......................... 514/12
`11/1993 Ladner et al. .. ..................... 435/172.3
`10/1994 Patel ....................................... 424/85.1
`
`FOREIGN PATENf DOCUMENTS
`
`Japan.
`2/1982
`57-026587
`Japan .
`59-181224 10/1984
`700132 11/1979 U.S.S.R .
`
`OTHER PUBLICATIONS
`
`L. Stanford Lee, ''Protein Folding and Polymerization study
`with a Recombinant Single Chain Antibody", The FASEB
`Jouma~ Experimental Biology 95, Abstracts 1-3621,
`Atlanta Georgia (Apr. 9-13, 1995), Abstract 2377.
`Crowe et al 1987 Biochem. J. vol. 242:1-10.
`Adams et al., Highly Specific in Vivo Tumor Targeting by
`Monovalent and Divalent forms of 741F8 Anti-c-erbB-2
`Single-Chain Fv 1, Cancer Research 53 :4026-4034 (Sep.
`1993).
`Buchner et al., Renaturation, Purification and Characteriza(cid:173)
`tion of Recombinant Fab-Fragments Produced in Escheri(cid:173)
`chia coli, Bio/Technology 9:157-162 (Feb. 1991).
`Buchner et al., Amethod for Increasing the Yield of Properly
`Folded Recombinant Fusion Proteins: Single-Chain
`Immonotoxins from Renaturation of Bacterial Inclusion
`Bodies, Analytical Biochemistry 205:263-270 (1992).
`Colcher, et al. In Vivo Tumor Targeting of a Recombinant
`Single-Chain Antigen-Binding Protein, J. Natl. Cancer
`Inst. 82:1191-1197 (Jul. 1990).
`Colombo et al .. Protein Solvation in Allosteric Regulation:
`A Water Effect on Homoglobin, Science 256:655-659 (May
`.
`1992).
`Hollinger et al., "Diabodies": Small Bivalent and Bispecific
`Antibody Fragments, Proc. Nat'l Acad. Sci. USA
`90:6444-6448 (Jul. 1993).
`
`Levine, Oxidative Modification of Glutamine Synthetase,
`The Journal of Biological Chemistry 258:11828-11833
`(Oct 1983).
`Mezes et al., Third Annual IBC Intem'l Conf. on Antibody
`Engineering, San Diego, CA, one page (1992).
`Milenic et al., Construction, Binding Properties, Metabo(cid:173)
`lism, and Tumor Targeting of a Single-Chain Fv Derived
`from the Pancarcinoma Monoclonal Antibody CC49, Can(cid:173)
`cer Res. 51:6363-6371 (Dec. 1991).
`Muller et al., Ligand Binding to Anti-FluorescylAntibodies:
`Stability of the Antigen Binding Site, Biochemistry
`33:6221-6227 (May 1994).
`Newton et al,, Expression and Characterization of Recom(cid:173)
`binant Human Eosinophil-derived Neurotoxin and Eosino(cid:173)
`phil-derived Neurotoxin-Anti-transferrin Receptor sFv,
`The Journal of Biological Chemistry 269:26739-26745
`(Oct 1994).
`Pantoliano et al., Conformational Stability, Folding, and
`Ligand-Binding Affinity of Single-Chain Fv Immunoglo(cid:173)
`bulin Fragments Expressed in Escherichia Coli, Biochem(cid:173)
`istry 30:10117-10125 (1991).
`Rand, Raising Water to New Heights, Science 256:618 (May
`1992).
`Sawyer et al., The Effects of Induction Conditions on
`Production of a Soluble Anti-Tumor sFv in Escherichia coli,
`Protein Engineering 7:1401-1406 (Aug. 1994).
`Wang et al., Parenteral Formulations of Proteins and Pep(cid:173)
`tides: Stability and Stabilizers, Journal of Parenteral Sci(cid:173)
`ence & Technology 42:S12-S13, Supplement (1988).
`Weidner et al., Molecular Stabilization Effects of Interac(cid:173)
`tions between Anti-Metatype Antibodies and Liganded
`Antibody, The
`Journal of Biological Chemistry
`267:10281-10288 (May 1992).
`Whitlow et al., Single-Chain Fv Proteins and Their Fusion
`Proteins, Methods: A Companion to Methods in Enzymology
`2:97-105 (Apr. 1991).
`Whitlow et al., An Improved Linker for Single-Chain Fv
`with Reduced Aggregation and Enhanced Proteolytic Sta(cid:173)
`bility, Protein Engng. 6:989-995 (1993).
`Whitlow et al., Multivalent Fvs: Chaterization of Single(cid:173)
`Chain Fv Oligomers and Preparation of a Bispecific Fv,
`Protien Engineering 7:1017-1026 (Aug. 1994).
`
`Primary Examiner-Lila Feisee
`Assistant Examiner-Julie E. Reeves
`Attorney, Agent, or Finn-Sterne, Kessler, Goldstein & Fox,
`P.L.L.C.
`
`[57]
`
`ABSTRACT
`
`The present invention relates to a stabilized protein compo(cid:173)
`sition comprising a monomeric protein and a storage(cid:173)
`stabilizing amount of sucrose, histidine or glycine, which is
`sufficient to inhibit aggregation of the protein molecules
`during freeze/thaw cycles, and methods therefor. More
`particularly, this invention relates to pharmaceutically(cid:173)
`acceptable, single-chain antigen-binding protein composi(cid:173)
`tions having increased frozen-storage stability, especially to
`freeze/thaw cycles.
`
`10 Claims, No Drawings
`
`CFAD Exhibit 1089
`CFAD v. NPS
`IPR2015-00990
`
`1
`
`

`
`5,656,730
`
`1
`STABILIZED MONOMERIC PROTEIN
`COMPOSITIONS
`
`FIELD OF THE INVENTION
`
`The present invention relates to a stabilized protein com(cid:173)
`position comprising a monomeric protein and a storage(cid:173)
`stabilizing mount of sucrose, histidine or glycine, which is
`sufficient to inhibit aggregation of the protein molecules
`during freeze/thaw cycles, and methods therefor. More
`particularly, this invention relates to pharmaceutically
`acceptable, monomeric single-chain antigen-binding protein
`compositions having increased frozen-storage stability,
`especially to freeze/thaw cycles.
`
`2
`more particularly in U.S. Pat. No. 4,704,692. A description
`of the theory and production of single-chain antigen-binding
`proteins is found in U.S. Pat Nos. 4,946,778 and 5,260,203.
`The single-chain antigen-binding proteins produced under
`5 the process recited in U.S. Pat. Nos. 4,946,778 and 5,260,
`203 have binding specificity and affinity substantially simi(cid:173)
`lar to that of the corresponding Fab fragment. Several
`different single-chain molecules that can successfully bind
`antigen have now been constructed using a variety of
`10 peptide linkers. For a review. see, Whitlow and Filpula,
`Methods: Compan. Methods Enzymol. 2:97-105 (1991).
`Many of the early single-chain F v products were insoluble
`aggregates, requiring solubilization under strong denaturing
`solutions, followed by renaturing and proper refolding
`15 before they could manifest siingle-chain antigen binding
`ability. See, e.g., Buchner et al., Bio/l'echnol. 9:157 (1991)
`The useful lifetime of proteins, particularly those devel(cid:173)
`and Buchner et al., Analyt. Biochem. 205:263 (1992). Thus,
`oped for pharmaceutical administration, is extended by
`it has been found to be particularly advantageous to provide
`storage as frozen, lyophilized or refrigerated aqueous com(cid:173)
`for the expression of a single-chain antigen-binding mol-
`positions. However, upon thawing, frozen non-lyophilized
`20 ecule as a soluble product, which can be purified directly
`monomeric proteins have a tendency to form polymeric
`from the periplasrnic fraction without the need for in vitro
`aggregates. Although the formation of a multivalent com(cid:173)
`manipulation and refolding. See, Sawyer et al., Protein
`pound may be desirable in specific situations. unwanted
`Engineering 7:1401 (1994).
`aggregation is disadvantageous in a monomeric protein
`However, if the soluble, monomeric protein composition
`composition.
`25 forms aggregates during frozen storage of the non(cid:173)
`Antibodies represent a specific class of proteins generated
`lyophilized product, or as a result of the freeze/thaw process,
`by the immune system to provide a molecule capable of
`a heterogeneous, unstable composition may result in which
`complexhag with an invading molecule, termed an antigen.
`immunoreactivity may be diminished. At the very least,
`Natural antibodies have two identical antigen-binding bind(cid:173)
`disadvantageous aggregation of the protein monomers as a
`30 result of storage instabilities may require additional purifi(cid:173)
`ing sites, both of which are specific to a particular antigen.
`The antibody molecule "recognizes" the antigen by com(cid:173)
`cation steps (including denaturation and refolding) to restore
`plexing its antigen-binding sites with areas termed epitopes.
`the protein to a composition of homogeneous monomers.
`The epitopes fit into the conformational architecture of the
`Biological consistency and stability are essential for most
`antigen-binding sites of the antibody, enabling the antibody
`clinical applications of a monomeric, single-chain antigen-
`to bind to the antigen.
`35 binding protein composition.
`Studies have been made. at both the academic and clinical
`The antibody molecule is composed of two identical
`heavy and two identical light chains, held together by
`levels, of the decomposition mechanisms of proteins. As a
`result, the art has recognized that different ixoteins exhibit
`disulfide bonds. Covalent interchain and intrachain bonding
`serves to stabilize the various chains of antibody molecules.
`highly variable inactivation responses. For example, R. L.
`Levine has reported in J. Biol. Chem. 258:11828 (1983) an
`The light chain comprises one variable region (termed VJ
`analysis of the effects of 24 amino acids and sulfhydryl
`and one constant region (CJ, while the heavy chain com-
`compounds as stabilizers to inhibit glutamine synthetase
`prises one variable region (termed V H) and three constant
`regions (CHl, CH2 and CH2). Pairs of regions associate to
`degradation in a system containing oxygen, ascorbate and
`form discrete structures. The light-chain variable region, V L• 45 trace metal. Levine disclosed that only cysteine and histidine
`showed significant activity in ixeventing loss of activity for
`and the heavy-chain variable region, V H• of a particular
`antibody molecule have specific amino acid sequences that
`the enzyme, whereas some of the compounds tested actually
`allow the antigen-binding site to assume a conformation that
`stimulated the inactivation reaction. For instance, the inac-
`binds to the antigen epitope recognized by that particular
`tivation of the enzyme creatine kinase by ascorbate has been
`antibody. More specifically, the light and heavy chain vari- 50 shown to be stimulated by histidine.
`able regions, V L and V H associate to form an "Fv'' area
`Several authors have described methods for stabilizing a
`which contains the antigen-binding site.
`lyophilized protein composition. For example, in U.S. Pat.
`No. 4,496,537, Kwan describes the enhanced storage sta-
`Cleavage of the naturally-occurring antibody molecule
`with a proteolytic enzyme generates fragments which retain
`bility of lyophilized alpha-type inteferon formulations, by
`their antigen binding site. Fragments of this type. commonly 55 incorporating glycine or alanine prior to lyophilization. The
`resulting lyophilized formulations can be stored without loss
`known as Fab (for Fragment, antigen binding) are composed
`oftheCL,VL.CHlandVHregionsoftheantibody.wherein
`of activity for more than six months at 20° C before
`the light chain and the fragment of the heavy chain are
`reconstitution with water.
`covalently linked by a disulfide linkage.
`An abstract of Japanese Patent Application 59-181224
`Recent advances in immunobiology, recombinant DNA 60 discloses an enhanced stability for interferons having added
`an amino acid and, optionally, human serum albumin before
`technology, and computer science have allowed the creation
`of single polypeptide chain molecules that bind antigen.
`freeze drying. Yasushi et al. disclose in U.S. Pat. No.
`These single-chain antigen-binding molecules contain only
`4,645,830, that interleukin-2 is stabilized against loss of
`the variable domains of the antibody, incorporating a linker
`activity during freezing, lyophilization or storage, by for-
`polypeptide to bridge the variable regions, V L and V H, into 65 mulating the composition to include human serum albumin,
`a single, monomeric polypeptide chain, the "sFv." A
`a reducing compound or both, and by adjusting the pH to
`computer-assisted method for linker design is described
`between 3 and 6. According to Yasushi et al., the
`
`BACKGROUND OF THE INVENTION
`
`40
`
`2
`
`

`
`5,656,730
`
`4
`Therefore, the invention of the presently formulated, stabi(cid:173)
`lized protein composition, its method of preparation, and
`pharmaceutically-acceptable compositions prepared thereby
`will significantly advance the art by ensuring a stable supply
`5 of such proteins as consistent, essentially homogeneous
`monomers.
`
`SUMMARY OF THE INVENTION
`
`20
`
`30
`
`35
`
`40
`
`3
`interleukin-2 formulation may also contain an amino acid,
`particularly glycine, a monosaccharide, and/or a sugar alco(cid:173)
`hol.
`To avoid the inconvenience of reconstituting a lyophilized
`product and because of the potential for introducing an error
`during such procedures, other authors have analyzed meth(cid:173)
`ods for stabilizing a refrigerated, aqueous protein composi(cid:173)
`tion. An abstract of Japanese Patent Application 57-26587
`describes the stabilization of ascotbit acid oxidase by adding
`one or more of the following: arginine, lysine, histidine and 10
`borates. In addition, in U.S. Pat. No. 4,806,524, Kawaguchi
`et al. describe the stabilization of either freeze-chied or
`aqueous erythropoietin formulations against decomposition,
`by adding one or more of the following: polyethylene glycol,
`proteins, sugars, amino acids, inorganic salts, organic salts 15
`and sulfur-containing reducing agents. Furthermore, in U.S.
`Pat. No. 4,777,043, Bennett et al. report that an increased
`solubility and stability is obtained when human tissue plas(cid:173)
`minogen activator is formulated to contain arginine, as the
`protonated cation "argininium ion."
`More recently, Patel, in U.S. Pat. No. 5,358,708, has
`disclosed a method for increasing the storage stability of an
`aqueous formulation containing a protein component
`selected from among the group consisting of interferons,
`granulocyte-macrophage colony-stimulating factors and 25
`interleukins, by the addition of a stabilizing amount of
`methionine, histidine or mixtures thereof. Patel reports that
`without such treatment, aqueous protein formulations typi(cid:173)
`cally have short useful storage lives after reconstitution,
`even when stored at low temperatures (e.g., 5° C.).
`Finally, certain authors have described particular methods
`for the stabilization of protein compositions designed spe(cid:173)
`cifically for pharmaceutical applications. According to
`Jensen in U.S. Pat No. 3,950,513, the solubility and stability
`of plasmin solutions for parenteral administration are
`enhanced by the addition of physiologically non-toxic amino
`acids. While, in Technical Report No. 10, entitled
`"Parenteral Formulations of Proteins and Peptides: Stability
`and Stabilizers," J. Parenteral Sci. & Technol. 42:S12-Sl3,
`Supplement 1988, Y-C. J. Wang and M.A. Hanson review
`the use of amino acids to stabilize parenteral formulations of
`proteins and peptides.
`Recent publications have reported that besides the protein
`structure itself, the surrounding solvent shell is of crucial 45
`importance for the stability and dynamic properties of the
`protein composition. See Muller et al. Biochemistry 33:6221
`(1994). Moreover, even the role of water in protein aggre(cid:173)
`gation and protein reactions has received considerable atten(cid:173)
`tion (Rand, Science 256:618 (1992); Colombo et at, Science 50
`256:655 (1992).
`A number of investigators have reported the spontaneous
`aggregation of sFyS. Weidner et al., J. Biol. Chem.
`267:10281 (1992) observed aggregates of the 4-4-20/212
`sFv; Mezes et al., Third Annual IBC Intern'l Conf. on
`Antibody Engineering, San Diego, Calif. (1992) reported
`aggregates of the CC49/205c sFv; Adams et al., Cancer Res.
`53:4026 (1993) and Hollinger et al., Proc. Nat'l Acad. Sci.
`USA 90:6444 (1993) described divalent sFvof anti-c-erbB-2
`sFv; and Whitlow et al, Protein Eng. 7:1017 (1994) inves(cid:173)
`tigated heterodimers of the CC49 and 4-4-20 sFyS.
`However, until the discovery of the present invention,
`there remained a long-felt need in the art for a method of
`stabilizing frozen, non-lyophilized, monomeric protein com(cid:173)
`positions to inhibit or prevent unwanted aggregation of the 65
`soluble polypeptide molecules, particularly when the com(cid:173)
`position is exposed to repeated freeze/thaw cycles.
`
`In view of the importance of maintaining a consistent,
`essentially homogeneous supply of storage-stable protein
`compositions, the present invention relates to a stabilized
`protein composition comprising a monomeric single-chain
`antigen-binding protein and a storage-stabilizing amount of
`sucrose, histidine or glycine, which is sufficient to inhibit
`aggregation of the protein monomers as a result of frozen
`storage. Moreover, the stabilized monomeric single-chain
`antigen-binding protein composition comprising a storage(cid:173)
`stabilizing amount of sucrose, histidine or glycine will
`withstand repeated freeze/thaw cycles without significant
`aggregation of the polypeptide molecules.
`Accordingly, the invention is directed to a sucrose, histi(cid:173)
`dine or glycine storage-stabilized monomeric single-chain
`antigen-binding protein formulation , its method of
`preparation, and pharmaceutically-acceptable compositions
`prepared thereby.
`Also provided is a method of inhibiting aggregation of a
`monomeric single-chain antigen-binding protein composi(cid:173)
`tion in frozen storage comprising adding a storage(cid:173)
`stabilizing amount of sucrose, histidine or glycine, wherein
`the resulting protein composition will withstand repeated
`freeze/thaw cycles without significant aggregation of the
`peptide molecules.
`Preferably, the invention provides a method of inhibiting
`aggregation of a monomeric single-chain antigen-binding
`protein composition comprising adding a frozen storage(cid:173)
`stabilizing amount of histidine. The thus provided stabilized
`monomeric protein composition will withstand repeated
`freeze/thaw cycles without significant aggregation of the
`peptide molecules.
`Another aspect of the invention includes the storage(cid:173)
`stabilized monomeric protein product of the above-disclosed
`method of inhibiting aggregation.
`A further aspect of the invention is a pharmaceutically(cid:173)
`acceptable, storage-stabilized, protein composition compris(cid:173)
`ing a monomeric single-chain antigen-binding protein and
`an mount of sucrose, histidine or glycine which is sufficient
`to inhibit aggregation of said monomers during frozen
`storage and a pharmaceutically acceptable buffer therefor.
`Preferably, the invention provides a pharmaceutically-
`acceptable, frozen storage-stabilized monomeric protein
`composition comprising the single-chain antigen-binding
`protein and a storage-stabilizing mount of histidine, so that
`the resulting protein composition will withstand repeated
`freeze/thaw cycles without significant aggregation of the
`polypeptide molecules.
`Other aspects, objects, features and characteristics of the
`present invention will become more apparent upon consid-
`60 eration of the following description and appended claims.
`
`55
`
`DEfAJLED DESCRIPTION OF THE
`PREFERRED EMBODIMENfS
`
`This invention relates to the discovery that the inclusion
`of sucrose, histidine or glycine, or their equivalents, in a
`monomeric protein composition significantly increases the
`stability and solubility of the protein composition by inhib-
`
`3
`
`

`
`5,656,730
`
`5
`iting the fonnation of polypeptide aggregates during frozen
`storage and repeated freeze/thaw manipulations. Thus, the
`present invention makes possible the stable frozen storage of
`monomeric protein compositions, and the methods of pre(cid:173)
`paring such stable compositions.
`In general, the monomeric protein compositions of the
`present invention are single-chain antigen-binding proteins.
`The compositions may contain other components in mounts
`which will preferably not detract from the preparation of the
`frozen storage-stable forms thereof. Moreover, the preferred
`monomeric single-chain antigen-binding protein composi(cid:173)
`tions will be prepared using components of the type and in
`mounts acceptable for safe, effective pharmaceutical admin(cid:173)
`istration of the storage-stabilized product.
`For the purposes of this application, "monomer" or
`"monomeric"refers to the a molecule having only a single
`peptide chain, regardless of the number of antigen-binding
`sites contained therein. Thus, the storage-stabilized mono(cid:173)
`meric single-chain antigen-binding protein of the present
`invention may be exemplified by a single chain molecule
`having one binding site, or by a multivalent antigen-binding
`molecule having more than one antigen-binding site, per(cid:173)
`mitting hi- and multi-specific binding, so long as such
`multivalent antigen-binding molecules are monomeric pro(cid:173)
`teins. However. it is the unwanted formation of polypeptide
`aggregates in a stored monomeric protein composition that
`is inhibited by the frozen storage-stabilizing methods of the
`present invention.
`The terms "single-chain molecule" or "single-chain pro(cid:173)
`tein" are used interchangeably here. They are structurally
`defined as a first polypeptide, comprising the binding por(cid:173)
`tion of the variable region of an antibody heavy or light
`chain, associated with a second polypeptide, comprising the
`binding portion of the variable region of an antibody heavy
`or light chain, the two polypeptides being joined by a
`peptide linker linking the first polypeptide and second
`polypeptide into a single polypeptide chain. The single
`polypeptide chain thus comprises a pair of variable regions
`connected by a polypeptide linker. The regions may asso-
`ciate to form a functional antigen-binding site, as in the case
`wherein the regions comprise a light-chain and a heavy(cid:173)
`chain variable region pair with appropriately paired comple(cid:173)
`mentarity determining regions (CDRs). In such a case. the
`single-chain protein is referred to as a "single-chain antigen
`binding protein" or "single-chain antigen-binding mol(cid:173)
`ecule." A similar single-chain antigen-binding protein com(cid:173)
`prising multiple pairs of heavy and light chain variable
`regions is also a part of this invention.
`The variable regions may have unnaturally paired CDRs 50
`or may both be derived from the same kind of antibody
`chain, either heavy or light, in which case the resulting
`single-chain molecule may not display a functional antigen(cid:173)
`binding site. 1\vo or more such single-chain molecules may
`associate to form a multivalent antigen-binding protein. The 55
`single-chain antigen-binding protein molecule is more fully
`described in U.S. Pat. Nos. 4,946,778 and 5,260,203 both of
`which are incorporated herein by reference in their entirety.
`The monomeric protein compositions stabilized by the
`present invention may be actually produced for the purpose 60
`of creating a frozen stock or they may be procured from
`whatever commercial or institutional source makes them
`available. The "single-chain antigen-binding proteins" may
`be produced by any process. including the process set forth
`in U.S. Pat. No. 4,946.778 (Lactner et al.).
`The single-chain antigen-binding protein used to exem(cid:173)
`plify the stabilized monomeric protein compositions of the
`
`6
`present invention include, for example, single-chain proteins
`comprising the CC49 V L region connected through the 218
`linker polypeptide to the CC49 V H region (CC49/218).
`Many such single-chain antigen-binding proteins. including
`5 a variety of linker and domain combinations, have been
`described previously (Colcher et al.. Natl Cancer Inst.
`82:1191 (1990); Milehie et al., Cancer Res. 51:6363 (1991);
`Pantoliano et al., Biochemistry 30:10117 (1991); and Whit(cid:173)
`low et at., Protein Engng. 6:989 (1993)), each of which is
`herein incorporated by reference if the description of such
`10 single-chain antigen-binding proteins is deemed relevant.
`Without being bound by any particular theory, the inven(cid:173)
`tors speculate on several models which can equally explain
`the phenomenon of multivalent aggregation observed as a
`result of frozen storage of monomeric protein compositions,
`15 and which becomes particularly evident following repeated
`freeze/thaw manipulations. Stabilization of a protein mono(cid:173)
`mer appears to be enhanced by creating a suitable rnicroen(cid:173)
`vironrnent surrounding the molecule. However, the inven(cid:173)
`tion is useful and operable to inhibit such aggregation,
`20 regardless of the precise mechanism behind the fonnation of
`polymeric aggregates or behind the effective method of
`monomeric protein stabilization.
`"Frozen storage" refers to freezing and maintaining a
`previously aqueous monomeric protein sample at a tempera(cid:173)
`ture below 0° C, preferably -20° C or lower.
`"Freeze/thaw cycles" or "freeze/thaw manipulations"
`refer to known techniques for using a protein sample in
`frozen storage, wherein the temperature of the sample is
`30 raised to a level which will restore its aqueous state for a
`sufficient period of time to permit use of the Rampie,
`followed by freezing to a temperature below 0° C and return
`to frozen storage. preferably at a temperature of -20° C or
`lower.
`In a preferred embodiment, the monomeric protein com(cid:173)
`position is storage-stabilized by the addition of sucrose,
`histidine or glycine. The sucrose, histidine or glycine addi(cid:173)
`tive concentration is between 0.1 and 20 rnM, preferably
`formulated in a pharmaceutically acceptable buffer. More
`preferably the sucrose, histidine or glycine additive concen(cid:173)
`tration is between 0.4 and 15 rnM, most preferably between
`0.4 and 10 mM, and often at approximately 10 rnM. The
`invention further may include combinations of sucrose,
`histidine and/or glycine at the above concentration.
`In the most preferred embodiment of the present
`invention, the monomeric protein composition is storage(cid:173)
`stabilized by the addition of histidine at the above concen(cid:173)
`tration.
`Preferably the concentration of monomeric protein in the
`compositions of the present invention will range from about
`0.1 mg/ml to about 10 mg/ml of stabilized solution. The ratio
`of sucrose, glycine or histidine to the monomeric protein in
`the composition of the present invention will be about 5
`moles to about 400 moles for each 1 mole of monomer,
`preferably about 50 moles to about 350 moles for each 1
`mole of monomer, more preferably about 100 moles to about
`300 moles for each 1 mole of monomer. and most preferably
`about 150 moles to about 250 moles for each 1 mole of
`monomer. Often the ratio of sucrose, glycine or histidine to
`the monomeric protein in the composition of the present
`invention will be about 200 moles for each 1 mole of
`monomer. However. the exact ratio required for a particular
`combination of protein, formulation components, amino
`acid or mixture and prescribed storage conditions can be
`65 determined by simple experimentation, using the usual
`analytical techniques for protein activity or aggregation over
`the desired storage lifetime.
`
`25
`
`35
`
`40
`
`45
`
`4
`
`

`
`5,656,730
`
`20
`
`25
`
`30
`
`35
`
`40
`
`7
`Suitable pH ranges for the preparation of the frozen(cid:173)
`storage stabilized monomeric protein compositions are
`about 4 to about 9, preferably in the range of about 6 to about
`8, more preferably in the range of about 6.7 to about 7.5.
`When the stabilized monomeric protein composition is 5
`intended for pharmaceutical use, the most preferred pH is in
`the physiologically acceptable range, i.e., about neutral.
`In addition to sucrose, histidine or glycine, the stabilized
`monomeric protein compositions of the present invention
`also contain a compatible buffer system to maintain the 10
`acceptable pH levels. A preferred buffer system is a com(cid:173)
`bination of sodium dibasic phosphate and sodium monoba-
`sic phosphate, for example, phosphate buffered saline
`(PBS).
`Additionally, the improved, storage-stable, monomeric 15
`single-chain antigen-binding protein composition may
`optionally include one or more nonionic detergents, such as
`Tween 80 (polyoxythylenesorbitan) polysorbate 20, polysor(cid:173)
`bate 80, and the like, in amounts of from 0.001 to about 1 %,
`to enhance the stability of the protein composition to freeze/
`thaw manipulations. Moreover, other pharmaceutically
`acceptable excipients, well known to those skilled in the art,
`may also form a part of such compositions. These may
`include, for example, various bulking agents, additional
`buffering agents , chelating agents, antioxidants ,
`preservatives, cosolvents, and the like. Specific examples of
`such pharmaceutically acceptable excipients could include
`mannitol, trimethamine salts ("Tris buffer"), gelatin, human
`serum albumin or other polypeptides, various small peptides
`such as glycylglycine, and the like.
`Aggregation of the monomers in a frozen-stored, mono(cid:173)
`meric protein composition can be measured by any method
`known in the art. Such methods include gel filtration chro(cid:173)
`matography to separate proteins on the basis of theft
`molecular weight. A "get" is a matrix of water and a
`polymer, such as agarose or polymerized acrylamide. The
`present invention encompasses the use of gel filtration
`HPLC (high performance liquid chromatography), as will be
`appreciated by one of ordinary skill in the art. Elution
`procedures are well known in the chemical and biochemical
`arts, as are methods of manual and automated fraction
`collection.
`Other recognized methods of measuring aggregation
`include cation exchange chromatography, which is the gen- 45
`eral liquid chromatographic technique of ion-exchange
`chromatography utilizing anion columns well-known to
`those of ordinary skill in the art. The cations exchanged in
`the present invention are from the protein molecules. Since
`multivalent protein aggregates will have some multiple of 50
`the net charge of the single-chain antigen-binding protein,
`the aggregates are retained more strongly, and are thus
`separated from the single-chain molecules. A preferred cat(cid:173)
`ionic exchanger is a polyaspartic acid column.
`Thus, a monomeric single-chain antigen-binding protein
`can be readily distinguished from an aggregate. However,
`those of ordinary skill in the art will realize that aggregation
`assays of the invention are not limited to any particular type
`of chromatography column, so long as it is capable of
`separating the two forms of protein molecules.
`The use of the term "to inhibit aggregation" or "aggre(cid:173)
`gation inhibited" when used to describe a composition of
`single-chain antigen-binding protein molecules, means the
`lack of a significant peak corresponding to an aggregated
`polymeric protein molecule when the composition is ana(cid:173)
`lyzed by, e.g., gel filtration chromatography. Gel filtration
`HPLC chromatography (TSK G2000SW column from Toyo
`
`55
`
`60
`
`65
`
`5
`
`8
`Soka, Tokyo, Japan) has been used to identify and separate
`monomeric single-chain and multivalent antigen-binding
`proteins. This procedure has been described by Fukano, et
`al .. J. Chromatography 166:47 (1978).
`A treated composition of the present invention in which
`the monomeric form is retained by more than 85%, and
`preferably by more than 90%, and most preferably by more
`than 94-95% of the protein composition (that is, having less
`than 5-6% aggregation) following frozen storage, would be
`considered to be "aggregation inhibited," or "storage(cid:173)
`stabilized." In the same context, a composition retaining less
`than 70% of its o