NZ751543B2 - Formulations with reduced oxidation - Google Patents

Abstract

The invention provides formulations comprising a protein in combination with a compound that prevents oxidation of the protein. The invention also provides methods for making such formulations and methods of using such formulations. The invention further provides methods of screening for compounds that prevent oxidation of a protein in a protein composition and methods of preventing oxidation of a protein in a formulation. Compounds which prevent said oxidation of protein include N-acetyl-L-tryptophan, N-acetyl-L-tryptophanamide, 5-amino-DL-tryptophan, 5-hydroxyindole-3-acetic acid and melatonin. hat prevent oxidation of a protein in a protein composition and methods of preventing oxidation of a protein in a formulation. Compounds which prevent said oxidation of protein include N-acetyl-L-tryptophan, N-acetyl-L-tryptophanamide, 5-amino-DL-tryptophan, 5-hydroxyindole-3-acetic acid and melatonin.

Description

FORMULATIONS WITH REDUCED OXIDATION CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisional of New Zealand patent application 711566, which is the national phase entry in New Zealand of PCT international application (published as WO2014/160495). This application claims the benefit of U.S. Provisional Application No. 61/780,845, filed March 13, 2013, and U.S.

Provisional ation No. 61/909,813, filed November 27, 2013, all of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION This ion relates to liquid formulations comprising a protein and further comprising a compound that prevents oxidation of said protein, s for producing and using the liquid formulations as well as methods of screening for compounds that prevent protein ion in protein compositions.

BACKGROUND OF THE INVENTION Oxidative degradation of amino acid residues is a commonly observed phenomenon in protein pharmaceuticals. A number of amino acid residues are susceptible to oxidation, particularly methionine (Met), cysteine (Cys), histidine (His), tryptophan (Trp), and tyrosine (Tyr) (Li et al., Biotechnology and Bioengineering 48:490-500 (1995)). ion is typically observed when the protein is exposed to hydrogen peroxide, light, metal ions or a combination of these during various processing steps (Li et al., Biotechnology and Bioengineering 48:490-500 (1995)). In particular, proteins exposed to light (Wei, et al., Analytical Chemistry 79(7):2797-2805 (2007)), AAPH or Fenton reagents (Ji et al., J Pharm Sci 98(12):4485-500 (2009)) have shown increased levels of oxidation on phan residues, whereas those exposed to hydrogen peroxide have typically shown only methionine ion (Ji et al., J Pharm Sci :4485-500 (2009)). Light exposure can result in protein oxidation through the formation of reactive oxygen species (ROS) including singlet oxygen, en peroxide and superoxide (Li et al., Biotechnology and Bioengineering 48:490-500 (1995); Wei, et al., Analytical Chemistry 79(7):2797-2805 (2007); Ji et al., J Pharm Sci :4485-500 (2009); Frokjaer et al., Nat Rev Drug Discov 4(4):298-306 (2005)), whereas protein ion typically occurs via yl radicals in the Fenton ed reaction (Prousek et al., Pure and Applied Chemistry 79(12):2325-2338 (2007)) and via alkoxyl peroxides in the AAPH ed reaction (Werber et al., J Pharm Sci 100(8):3307-15 (2011)). Oxidation of tryptophan leads to a myriad of ion products, including hydroxytryptophan, kynurenine, and N-formylkynurenine, and has the potential to impact safety and efficacy (Li et al., Biotechnology and Bioengineering 48:490-500 (1995); Ji et al., J Pharm Sci 98(12):4485-500 (2009); Frokjaer et al., Nat Rev Drug Discov 4(4):298-306 (2005)).

Oxidation of a particular tryptophan residue in the heavy chain mentarity determining region (CDR) of a onal antibody that correlated to loss of biological function has been reported (Wei, et al., Analytical Chemistry 79(7):2797-2805 (2007)).

Trp oxidation mediated by a histidine coordinated metal ion has ly been reported for a Fab molecule (Lam et al., Pharm Res 28(10):2543-55 (2011)). Autoxidation of polysorbate 20 in the Fab ation, leading to the generation of s peroxides, has also been d in the same report. Autoxidation-induced generation of these peroxides can also lead to methionine oxidation in the protein during long-term storage of the drug product since Met residues in ns have been suggested to act as internal antioxidants (Levine et al., Proceedings of the National Academy of Sciences of the United States of America 93(26):15036-15040 (1996)) and are easily oxidized by peroxides. Oxidation of amino acid es has the potential to impact the biological activity of the protein. This may be ally true for monoclonal dies (mAbs). Methionine oxidation at Met254 and Met430 in an IgG1 mAb potentially impacts serum half-life in transgenic mice (Wang et al., Molecular Immunology 48(6-7):860-866 (2011)) and also impacts binding of human IgG1 to FcRn and Fc-gamma receptors (Bertolotti-Ciarlet et al., Molecular Immunology 46(8-9)1878-82 (2009)).

The stability of proteins, especially in liquid state, needs to be evaluated during drug product manufacturing and storage. The development of pharmaceutical formulations sometimes includes addition of idants to prevent oxidation of the active ingredient.

Addition of L-methionine to formulations has ed in reduction of methionine residue oxidation in proteins and peptides (Ji et al., J Pharm Sci 98(12):4485-500 (2009); Lam et al., Journal of Pharmaceutical Sciences 86(11):1250-1255 (1997)). Likewise, addition of L-tryptophan has been shown to reduce ion of tryptophan residues (Ji et al., J Pharm Sci 98(12):4485-500 (2009); Lam et al., Pharm Res 28(10):2543-55 (2011)). L- Trp, however, possesses strong absorbance in the UV region (260-290nm) making it a y target during photo-oxidation (Creed, D., Photochemistry and Photobiology 39(4):537-562 (1984)). Trp has been hypothesized as an endogenous photosensitizer enhancing the oxygen dependent photo-oxidation of tyrosine (Babu et al., Indian J Biochem Biophys 29(3):296-8 (1992)) and other amino acids (Bent et al., Journal of the American Chemical Society 97(10):2612-2619 (1975)). It has been demonstrated that L- Trp can generate hydrogen de when exposed to light and that L-Trp under UV light produces en peroxide via the superoxide anion (McCormick et al., Science 191(4226):468-9 (1976); Wentworth et al., Science 293(5536):1806-11 (2001); McCormick et al., Journal of the American Chemical Society 100:312-313 (1978)). onally, tryptophan is known to produce singlet oxygen upon exposure to light (Davies, M.J., Biochem Biophys Res Commun 305(3):761-70 (2003)). Similar to the protein oxidation induced by autoxidation of polysorbate 20, it is possible that protein ion can occur upon ROS generation by other excipients in the protein formulation (e.g. L-Trp) under normal handling conditions.

It is apparent from recent studies that the addition of rd ents, such as L- Trp and rbates, to protein compositions that are meant to stabilize the protein can result in unexpected and undesired consequences such as ROS-induced oxidation of the protein. Therefore, there remains a need for the identification of ative excipients for use in protein compositions and the development of such compositions. It is an object of the invention to go at least some way to meeting this need; and/ or to at least provide the public with a useful .

BRIEF SUMMARY OF THE INVENTION ] In a first aspect, the ion relates to a liquid formulation comprising a protein and a nd which prevents oxidation of the n in the liquid formulation, wherein the compound is selected from the group consisting of N-acetyl-L-trytophan, N-acetyl-L- tryptophanamide, 5-amino-DL-tryptophan, and 5-hydroxyindoleacetic acid, or a pharmaceutically acceptable salt thereof.

] In a second aspect, the invention relates to a method of making a protein formulation comprising adding a compound that prevents oxidation of a protein to the protein formulation, wherein the compound is selected from the group consisting of N-acetyl-L- tryptophan, N-acetyl-L-tryptophanamide, 5-amino-DL-tryptophan, and oxyindole acetic acid, or a pharmaceutically acceptable salt f. [0005C] In a third aspect, the invention relates to a method of preventing oxidation of a protein in a protein formulation comprising adding a compound that prevents oxidation of the protein to the formulation, wherein the compound is selected from the group consisting of N- acetyl-L-tryptophan, N-acetyl-L-tryptophanamide, 5-amino-DL-tryptophan, 5- hydroxyindoleacetic acid and melatonin, or a ceutically able salt thereof.

BRIEF DESCRIPTION OF THE INVENTION Described herein are formulations comprising a protein and a compound that prevents ion of the protein in the formulation, methods of making the formulations, and methods of screening compounds that prevent oxidation of a protein in a n composition.

In one embodiment, described herein is a liquid formulation sing a protein and a compound which prevents oxidation of the protein in the liquid formulation, wherein the compound is selected from the group consisting of 5-hydroxy-tryptophan, 5-hydroxy indole, 7-hydroxy , and serotonin.

In some embodiments, the liquid formulation is a pharmaceutical formulation suitable for administration to a t. In some embodiments, the formulation is aqueous.

In some embodiments, the compound that prevents oxidation of the protein in the formulation is from about 0.3 mM to about 10 mM, or up to the highest concentration that the compound is soluble to in the formulation. In some embodiments, the compound that prevents oxidation of the protein in the formulation is from about 0.3 mM to about 9 mM, from about 0.3 mM to about 8 mM, from about 0.3 mM to about 7 mM, from about 0.3 mM to about 6 mM, from about 0.3 mM to about 5 mM, from about 0.3 mM to about 4 mM, from about 0.3 mM to about 3 mM, from about 0.3 mM to about 2 mM, from about 0.5 mM to about 2 mM, from about 0.6 mM to about 1.5 mM, or from about 0.8 mM to about 1.25 mM. In some embodiments, the nd that prevents oxidation of the protein in the formulation is about 0.3 mM to about 1 mM. In some embodiments, the compound that prevents oxidation of the protein in the formulation is from about 0.3 mM to about 5 mM. In some embodiments, the compound that prevents oxidation of the n in the formulation is about 1 mM. In some embodiments, the compound prevents oxidation of tryptophan and/or methionine in the protein. In some embodiments, the compound prevents ion of the protein by a reactive oxygen species. In some embodiments, the reactive oxygen species is selected from the group consisting of singlet oxygen, a superoxide (O2-), hydrogen peroxide, a hydroxyl radical, and an alkyl peroxide.

In some embodiments, the protein in the ation is susceptible to oxidation. In some embodiments, tryptophan in the protein is susceptible to ion. In some embodiments, the protein is an antibody (e.g., a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, or antibody fragment). In some embodiments, the protein concentration in the formulation is about 1 mg/mL to about 250 mg/mL.

In some embodiments, the ation further comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, a tant, and a tonicity agent. In some embodiments, the formulation has a pH of about 4.5 to about 7.0.

In another ment, described herein is a method of making a liquid formulation comprising adding an amount of a compound that prevents oxidation of a n to the liquid formulation, wherein the compound is selected from the group ting of 5- y-tryptophan, 5-hydroxy indole, 7-hydroxy indole, and serotonin. In another embodiment, described herein is a method of preventing oxidation of a protein in a liquid formulation comprising adding an amount of a compound that prevents oxidation of the protein to the liquid ation, wherein the compound is ed from the group consisting of 5-hydroxy-tryptophan, 5-hydroxy indole, 7-hydroxy indole, and serotonin.

In some embodiments of the methods described herein, the formulation is a pharmaceutical formulation suitable for administration to a subject. In some ments, the formulation is aqueous.

In some embodiments of the methods described , the compound in the formulation is from about 0.3 mM to about 10 mM, or up to the highest concentration that the compound is soluble to in the formulation. In some embodiments, the compound in the ation is at a concentration from about 0.3 mM to about 9 mM, from about 0.3 mM to about 8 mM, from about 0.3 mM to about 7 mM, from about 0.3 mM to about 6 mM, from about 0.3 mM to about 5 mM, from about 0.3 mM to about 4 mM, from about 0.3 mM to about 3 mM, from about 0.3 mM to about 2 mM, from about 0.5 mM to about 2 mM, from about 0.6 mM to about 1.5 mM, or from about 0.8 mM to about 1.25 mM. In some embodiments, the compound in the formulation is about 0.3 mM to about 1 mM. In some embodiments, the compound in the formulation is from about 0.3 mM to about 5 mM. In some embodiments, the compound in the formulation is from about 1 mM. In some embodiments, the compound prevents oxidation of tryptophan and/or nine in the protein. In some embodiments, the compound prevents oxidation of the protein by a reactive oxygen species. In some ments, the reactive oxygen species is selected from the group consisting of singlet oxygen, a superoxide (O2-), hydrogen peroxide, a hydroxyl radical, and an alkyl peroxide.

In some embodiments, the protein is tible to oxidation. In some embodiments, tryptophan in the protein is susceptible to oxidation. In some embodiments, the protein is an antibody (e.g., a polyclonal antibody, a onal antibody, a humanized antibody, a human antibody, a chimeric antibody, or antibody fragment). In some embodiments, the protein concentration in the formulation is about 1 mg/mL to about 250 mg/mL.

In some ments, one or more ents selected from the group consisting of a stabilizer, a , a tant, and a tonicity agent are added into the formulation. In some embodiments, the formulation has a pH of about 4.5 to about 7.0.

In another embodiment, described herein is a method of ing a compound that ts oxidation of a protein in a n composition, comprising selecting a compound that has lower oxidation potential and less photosensitivity as compared to L- tryptophan, and testing the effect of the selected nd on preventing oxidation of the protein.

In some embodiments of the methods, the photosensitivity is measured based on the amount of H2O2 produced by the compound upon light exposure. In some embodiments, the compound that produces less than about 20% of the amount of H2O2 produced by L- phan is selected. In some embodiments, the oxidation potential is measured by cyclic voltammetry. In some ments, the selected compound is tested for the effect on preventing oxidation of the protein by reactive oxygen species generated by 2,2’- azobis(2-amidinopropane) dihydrochloride (AAPH), light, and/or a Fenton reagent.

It is to be understood that one, some, or all of the properties of the various embodiments bed herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. These and other embodiments of the invention are further described by the detailed description that follows. [0019A] Certain statements that appear herein are broader than what appears in the statements of the invention. These statements are provided in the interests of providing the reader with a better understanding of the invention and its practice. The reader is directed to the accompanying claim set which defines the scope of the invention. [0019B] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be ued as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common l knowledge in the art.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a series of graphs demonstrating the oxidation of A) Fab in mAb1, and B) Fc in mAb1 after eight hours of light exposure at 250 W/m2. mAb1 was present at 5 mg/mL in 20mM histidine acetate, 250mM trehalose, 0.02% polysorbate 20. All vials were placed in the lightbox except the mAb1 Ref Mat. Foil CTRL vials were covered in foil before placement in the lightbox. Three separate experimental vials were averaged for each sample, except “10mM Met, 1mM Trp” (*) was the average of two mental vials, and mAb1 Ref Mat was one mental vial with three independent injections on the HPLC. Error bars represent one standard deviation.

Figure 2 is a graph showing dose dependent H2O2 tion by L-Trp. Diamonds indicate L-Trp alone; Triangles te L-Trp + SOD; Circles and Squares indicate LTrp + NaN3 + SOD. All studies were performed in 20mM L-His HCl, pH 5.5.

Figure 3 is a series of graphs demonstrating A) Hydrogen peroxide (H2O2) production in 50mg/mL mAb1 formulations containing 3.2mM L-Trp when exposed to ambient light conditions for 1, 3 and 7 days and B) Percent (%) Fab oxidation in mAb1 formulations containing 3.2mM L-Trp after 10 days of exposure to ambient light conditions.

Figure 4 is a series of graphs showing en peroxide tion by tryptophan derivatives and indole derivatives under light stress for 4 hours at 250 W/m2. A) Screening of tryptophan derivatives (1 mM) for hydrogen peroxide (µM) generation in a mM HisAc pH5.5 formulation. B) Screening of indole derivatives (1 mM) for hydrogen peroxide (µM) generation in a 20 mM HisAc pH5.5 formulation.

Figure 5 is a graph showing the effect of NaN3 on H2O2 production by various Trp derivatives upon light exposure. Data is shown as a ratio with t to peroxide generated by L-Trp.

Figure 6 is a graph showing the correlation n oxidation potential and light- induced peroxide formation. The boxed region shows candidate antioxidant compounds.

Figure 7 is a series of graphs showing the oxidation of A) Fab in mAb1, and B) Fc in mAb1 after AAPH incubation. All samples were incubated with AAPH except mAb1 Ref Mat and No AAPH. All samples were incubated at 40°C except mAb1 Ref Mat. Data shown are the average of three experimental samples ± 1SD, except mAb1 Ref Mat which is the e of six HPLC injections without error bars.

Figure 8 is series of graphs showing the oxidation of A) Fab in mAb1, and B) Fc in mAb1 after sixteen hours of light exposure at 250 W/m2. All vials were placed in the lightbox except the mAb1 Ref Mat. Foil CTRL vials were covered in foil before placement in the lightbox. Three te mental vials were averaged for each sample, except L-tryptophanamide (*)was the average of two experimental vials and mAb1 Ref Mat was one vial with three independent injections on the HPLC. Error bars represent one standard ion.

Figure 9 is a series of graphs showing the oxidation of A) Fab in 3 mg/mL mAb1, and B) Fc in 3 mg/mL mAb1 following the Fenton reaction using 10 ppm of H2O2 and 0.2 mM of Fe(III). The reaction was incubated at 40oC for 3 hours, quenched with 100 mM L-Met and analyzed using RP-HPLC after papain digest. All samples are the average of three separate vials, and mAb1 control (Ref Mat) was one vial with five independent injections on the HPLC. Error bars represent one standard ion.

Figure 10 is a series of diagrams showing the putative ism of A) L-Trp and B) -hydroxy-L-tryptophan excitation and in generating and quenching 1O 2. k 25C represents the second order rate constant for quenching of 1O2 (Dad et al., J Photochem Photobiol B, 78(3):245-51 (2005)) while Eox is the oxidation potential of the molecule versus Ag/AgCl.

DETAILED DESCRIPTION I. Definitions.

Before describing the invention in , it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of the active ient to be ive, and which contains no additional components which are unacceptably toxic to a t to which the formulation would be administered. Such formulations are sterile.

A “sterile” formulation is aseptic or free or essentially free from all living microorganisms and their spores.

A “stable” formulation is one in which the protein therein ially retains its physical stability and/or chemical stability and/or biological activity upon storage.

Preferably, the formulation essentially retains its physical and chemical stability, as well as its biological activity upon storage. The storage period is generally selected based on the intended life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example. Stability can be measured at a selected amount of light exposure and/or temperature for a selected time period. ity can be evaluated qualitatively and/or quantitatively in a variety of different ways, including evaluation of aggregate formation (for example using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); evaluation of ROS formation (for e by using a light stress assay or a 2,2’- (2-Amidinopropane) Dihydrochloride (AAPH) stress assay); oxidation of specific amino acid es of the protein (for example a Trp residue and/or a Met residue of a monoclonal dy); by assessing charge geneity using cation ge chromatography, image capillary isoelectric focusing (icIEF) or ary zone electrophoresis; amino-terminal or carboxy-terminal sequence is; mass spectrometric analysis; SDS-PAGE analysis to compare d and intact antibody; peptide map (for example tryptic or LYS-C) analysis; evaluating biological activity or target binding function of the protein (e.g., antigen binding function of an antibody); etc.

Instability may involve any one or more of: aggregation, deamidation (e.g. Asn deamidation), ion (e.g. Met oxidation and/or Trp ion), isomerization (e.g.

Asp isomerization), clipping/hydrolysis/fragmentation (e.g. hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc.

A protein “retains its physical stability” in a pharmaceutical formulation if it shows no signs or very little of aggregation, precipitation and/or denaturation upon visual examination of color and/or clarity, or as measured by UV light ring or by size exclusion chromatography.

A protein “retains its chemical stability” in a ceutical formulation, if the chemical stability at a given time is such that the protein is considered to still retain its biological activity as defined below. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Chemical tion may involve protein oxidation which can be evaluated using tryptic e mapping, reverse-phase high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry ), for example. Other types of chemical alteration include charge alteration of the protein which can be evaluated by ion-exchange chromatography or icIEF, for example.

A n “retains its biological activity” in a pharmaceutical formulation, if the ical ty of the protein at a given time is within about 10% (within the errors of the assay) of the ical activity exhibited at the time the pharmaceutical formulation was prepared as determined for example in an antigen binding assay for a monoclonal antibody.

As used herein, “biological activity” of a protein refers to the ability of the protein to bind its target, for example the ability of a monoclonal antibody to bind to an n. It can further include a biological response which can be measured in vitro or in vivo. Such activity may be antagonistic or agonistic.

A protein which is “susceptible to oxidation” is one comprising one or more residue(s) that has been found to be prone to ion such as, but not limited to, methionine (Met), cysteine (Cys), histidine (His), tryptophan (Trp), and tyrosine (Tyr).

For example, a tryptophan amino acid in the Fab portion of a onal antibody or a methionine amino acid in the Fc portion of a monoclonal antibody may be susceptible to oxidation.

By “isotonic” is meant that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or ice-freezing type ter, for example.

As used herein, “buffer” refers to a buffered solution that resists s in pH by the action of its acid-base conjugate components. The buffer described preferably has a pH in the range from about 4.5 to about 8.0. For example, histidine acetate is an example of a buffer that will l the pH in this range.

A “preservative” is a compound which can be optionally included in the formulation to essentially reduce bacterial action therein, thus facilitating the production of a multiuse formulation, for example. Examples of potential vatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl l, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

In one ment, the preservative herein is benzyl l.

As used herein, a “surfactant” refers to a e-active agent, preferably a nonionic surfactant. Examples of surfactants herein include polysorbate (for example, polysorbate and, rbate 80); poloxamer (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or l-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, dopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or aramidopropyl-betaine (e.g. lauroamidopropyl); amidopropyl-, palmidopropyl-, or isostearamidopropyldimethylamine ; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQUATTM series (Mona Industries, Inc., Paterson, N.J.); polyethyl glycol, polypropyl , and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc.); etc. In one embodiment, the surfactant herein is polysorbate 20.

“Pharmaceutically able” excipients or carriers as used herein include pharmaceutically acceptable rs, stabilizers, buffers, acids, bases, sugars, preservatives, surfactants, tonicity agents, and the like, which are well known in the art gton: The Science and Practice of Pharmacy, 22nd Ed., Pharmaceutical Press, 2012). Examples of pharmaceutically acceptable excipients include buffers such as phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid, L-tryptophan and nine; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; metal xes such as Zn-protein complexes; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming rions such as sodium; and/or nonionic surfactants such as polysorbate, poloxamer, polyethylene glycol (PEG), and PLURONICS™. “Pharmaceutically acceptable” excipients or carriers are those which can reasonably be administered to a subject to provide an effective dose of the active ingredient employed and that are nontoxic to the subject being exposed thereto at the dosages and concentrations employed.

The protein which is formulated is preferably essentially pure and desirably essentially neous (e.g., free from contaminating proteins etc.). “Essentially pure” protein means a composition comprising at least about 90% by weight of the protein (e.g., monoclonal antibody), based on total weight of the composition, preferably at least about 95% by weight. “Essentially homogeneous” protein means a ition sing at least about 99% by weight of the protein (e.g., monoclonal antibody), based on total weight of the ition.

The terms “protein” “polypeptide” and “peptide” are used interchangeably herein to refer to rs of amino acids of any . The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid r that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, ation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, ns containing one or more s of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Examples of proteins encompassed within the definition herein include mammalian proteins, such as, e.g., renin; a growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing ; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alphaantitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; leptin; 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 human urine or tissue-type nogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; a tumor necrosis factor receptor such as death receptor 5 and CD120; TNF-related apoptosis-inducing ligand (TRAIL); B-cell maturation antigen (BCMA); hocyte stimulator (BLyS); a proliferation-inducing ligand (APRIL); enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human hage inflammatory n (MIPalpha); a serum albumin such as human serum albumin; Muellerian-inhibiting substance; relaxin n; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial n, such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin; activin; platelet-derived endothelial cell growth factor (PD-ECGF); a vascular endothelial growth factor family protein (e.g., VEGF-A, VEGF-B, VEGF-C, , and P1GF); a platelet-derived growth factor (PDGF) family protein (e.g., PDGF-A, PDGF-B, PDGF-C, PDGF-D, and dimers thereof); fibroblast growth factor (FGF) family such as aFGF, bFGF, FGF4, and FGF9; epidermal growth factor (EGF); ors for hormones or growth factors such as a VEGF receptor(s) (e.g., VEGFR1, VEGFR2, and VEGFR3), epidermal growth factor (EGF) or(s) (e.g., ErbB1, ErbB2, ErbB3, and ErbB4 receptor), platelet-derived growth factor (PDGF) receptor(s) (e.g., PDGFR-α and PDGFR-β), and fibroblast growth factor receptor(s); TIE ligands (Angiopoietins, ANGPT1, ); Angiopoietin receptor such as TIE1 and TIE2; protein A or D; rheumatoid s; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-b; transforming growth factor (TGF) such as TGF-alpha and TGF- beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or ; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins (IGFBPs); CD proteins such as CD3, CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); a ine such as CXCL12 and CXCR4; an interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; a cytokine such as interleukins (ILs), e.g., IL-1 to IL-10; midkine; superoxide ase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for example, a portion of the AIDS pe; transport proteins; homing receptors; addressins; regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and VCAM; ephrins; Bv8; Delta-like ligand 4 (DLL4); Del-1; BMP9; BMP10; Follistatin; Hepatocyte growth factor (HGF)/scatter factor (SF); Alk1; Robo4; ESM1; Perlecan; EGF-like domain, multiple 7 (EGFL7); CTGF and members of its family; thrombospondins such as thrombospondin1 and thrombospondin2; collagens such as en IV and en XVIII; neuropilins such as NRP1 and NRP2; Pleiotrophin (PTN); Progranulin; Proliferin; Notch proteins such as Notch1 and ; semaphorins such as Sema3A, Sema3C, and Sema3F; a tumor associated antigen such as CA125 (ovarian cancer antigen); immunoadhesins; and fragments and/or variants of any of the above-listed proteins as well as antibodies, including antibody fragments, binding to one or more protein, including, for example, any of the above-listed proteins.

The term ody” herein is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal dies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired ical activity.

An “isolated” n (e.g., an isolated antibody) is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic or therapeutic uses for the protein, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. Isolated protein includes the protein in situ within recombinant cells since at least one component of the protein’s natural environment will not be t. Ordinarily, however, isolated protein will be prepared by at least one cation step.

“Native dies” are usually tetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains.

Each light chain is linked to a heavy chain by one covalent ide bond, while the number of disulfide es varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is d with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the n binding site. The nt domain contains the CH1, CH2 and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.

The “variable region” or “variable domain” of an antibody refers to the amino- terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as “VH.” The variable domain of the light chain may be referred to as “VL.” These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

The term “variable” refers to the fact that n portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular dy for its particular n. However, the variability is not evenly distributed hout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet uration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the n-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of an antibody to an antigen, but t various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“κ”) and lambda (“λ”), based on the amino acid sequences of their nt domains.

The term IgG “isotype” or “subclass” as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. Depending on the amino acid ces of the constant domains of their heavy chains, antibodies (immunoglobulins) can be ed to different classes. There are five major classes of globulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that pond to the ent classes of immunoglobulins are called α, γ, ɛ, γ, and µ, respectively. The subunit structures and three-dimensional configurations of ent classes of immunoglobulins are well known and described generally in, for example, Abbas et al. ar and Mol. Immunology, 4th ed., W.B. Saunders, Co., 2000. An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or es.

The terms “full length antibody,” “intact antibody” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an dy with heavy chains that contain an Fc region.

“Antibody fragments” comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from dy fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name ts its ability to crystallize readily. Pepsin treatment yields an F(ab’)2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen. The Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain ing one or more cysteines from the antibody hinge region. Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab’)2 antibody fragments originally were produced as pairs of Fab’ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

“Fv” is the minimum antibody fragment which contains a complete antigen-binding site. In one ment, a two-chain Fv s consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be ntly linked by a flexible e linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three HVRs of each le domain interact to define an antigen-binding site on the surface of the VH-VL dimer. tively, the six HVRs confer antigen-binding icity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs ic for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

“Single-chain Fv” or “scFv” antibody fragments se the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL s which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315, 1994.

The term “diabodies” refers to antibody nts with two n-binding sites, which nts comprise a chain le domain (VH) connected to a lightchain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two s on the same chain, the domains are forced to pair with the complementary domains of another chain and create two n-binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; ; Hudson et al., Nat. Med. 9:129- 134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor s. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody sing a polypeptide sequence that binds a target, wherein the targetbinding polypeptide sequence was obtained by a process that es the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be tood that a ed target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its tion in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody bed herein. In contrast to polyclonal antibody preparations, which typically include ent antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is ed against a single determinant on an antigen. In addition to their specificity, onal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., dies: A tory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display logies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 7 (1992); Sidhu et al., J. Mol. Biol. 338(2): 0 ; Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl.

Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. s 284(1-2): 119-132 (2004), and technologies for producing human or human-like antibodies in s that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., ; WO 1996/34096; ; WO 0741; vits et al., Proc. Natl. Acad. Sci.

USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; ,625,126; 5,633,425; and 5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically e “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a ular antibody class or subclass, while the remainder of the chain(s) is identical with or gous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include PRIMATTZED® antibodies n the antigen-binding region of the antibody is derived from an antibody ed by, e.g., immunizing macaque monkeys with the antigen of interest.

“Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain l sequence derived from non-human immunoglobulin. In one embodiment, a humanized dy is a human immunoglobulin (recipient antibody) in which residues from a HVR of the recipient are replaced by residues from a HVR of a non-human species (donor antibody) such as mouse, rat, , or nonhuman primate having the d icity, affinity, and/or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues.

Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These cations may be made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, le domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), lly that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. . Biol. 2:593- 596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. ctions 23:1035-1038 ; Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising man antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol.

Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the ation of human monoclonal antibodies are s described in Cole et al., Monoclonal Antibodies and Cancer y, Alan R. Liss, p. 77 (1985); Boerner et al., J.

Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin.

Pharmacol., 5: 368-74 (2001). Human antibodies can be ed by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 584 regarding XENOMOUSETM technology). See also, for example, Li et al., Proc. Natl. Acad. Sci.

USA, 103:3557-3562 (2006) regarding human antibodies ted via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refers to the s of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine icity to antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular y 25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e.g., Hamers- man et al., Nature 363:446-448 ; f et al., Nature Struct. Biol. 3:733- 736 (1996).

A number of HVR delineations are in use and are encompassed herein. The Kabat Complementarity Determining s (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, al Institutes of Health, Bethesda, Md. (1991)). a refers instead to the location of the structural loops (Chothia and Lesk J. Mol.

Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling re. The “contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101 HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56 or 50- 56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93- 102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

“Framework” or “FR” residues are those variable domain residues other than the HVR residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or ion into, a FR or HVR of the variable domain. For e, a heavy chain le domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the dy with a “standard” Kabat numbered sequence The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately es 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody.

The expression “linear dies” refers to the antibodies bed in Zapata et al. (1995 Protein Eng, 8(10):1057-1062). Briefly, these dies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

The term “about” as used herein refers to an acceptable error range for the respective value as determined by one of ordinary skill in the art, which will depend in part how the value is measured or determined, i.e., the limitations of the ement system. For example, ” can mean within 1 or more than 1 standard deviations, per the practice in the art. A reference to “about” a value or parameter herein es and bes embodiments that are directed to that value or parameter per se. For example, a description referring to “about X” includes description of “X”.

As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a compound” ally includes a combination of two or more such compounds, and the like.

It is tood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of” aspects and embodiments. [0074A] The term “comprising” as used in this specification and claims means “consisting at least in part of”. When interpreting statements in this specification, and claims which include the term “comprising”, it is to be understood that other features that are additional to the es ed by this term in each statement or claim may also be present. Related terms such as “comprise” and “comprised” are to be interpreted in r manner.

II. Protein Formulations and Preparation The t description s to liquid formulations comprising a protein and a compound which prevents oxidation of the n in the liquid formulation, wherein the compound is ed from the group consisting of 5-hydroxy-tryptophan, 5-hydroxy indole, 7-hydroxy , and serotonin. In some embodiments, the compound in the formulation is from about 0.3 mM to about 10 mM, or up to the highest concentration that the compound is soluble in the formulation. In some embodiments, the compound in the formulation is about 1 mM. In some embodiments, the compound prevents oxidation of one or more amino acids in the n ed from group consisting of tryptophan and methionine. In some embodiments, the compound prevents oxidation of the protein by a reactive oxygen species (ROS). In a further embodiment, the reactive oxygen species is selected from the group consisting of a singlet oxygen, a superoxide (O2-), an alkoxyl radical, a peroxyl l, a hydrogen peroxide (H2O2), a dihydrogen trioxide (H2O3), a hydrotrioxy radical (HO3•), ozone (O3), a hydroxyl radical, and an alkyl peroxide. In some embodiments, a protein described herein is susceptible to oxidation. In some embodiments, methionine, cysteine, histidine, tryptophan, and/or tyrosine in the protein is susceptible to oxidation. In some embodiments, tryptophan and/or nine in the protein is susceptible to oxidation. For e, a tryptophan amino acid in the Fab portion of a monoclonal antibody and/or a methionine amino acid in the Fc portion of a monoclonal antibody can be susceptible to oxidation. In some embodiments, the protein is a therapeutic protein. In some of the embodiments herein, the protein is an antibody.

In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, or an dy fragment. In a r embodiment, the compound prevents oxidation of one or more amino acids in the Fab portion of an antibody. In another r embodiment, the compound prevents oxidation of one or more amino acids in the Fc n of an antibody. In some embodiments, the formulation described herein is a pharmaceutical formulation suitable for stration to a subject. As used herein a “subject” or an “individual” for purposes of treatment or administration refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human. In some embodiments, the formulation is aqueous. In some embodiments herein, the protein (e.g., the antibody) concentration in the ation is about 1 mg/mL to about 250 mg/mL. In some ments, the formulation further one or more excipients selected from the group consisting of a izer, a buffer, a tant, and a tonicity agent. For example, a formulation of the description can comprise a monoclonal antibody, a compound as described herein which prevents oxidation of the n (e.g., 5-hydroxy ), and a buffer that maintains the pH of the formulation to a desirable level. In some embodiments, a formulation described herein has a pH of about 4.5 to about 7.0. ns and antibodies in the formulation may be prepared using methods known in the art. The antibody (e.g., full length antibodies, antibody fragments and multispecific antibodies) in the liquid formulation is prepared using techniques available in the art, nonlimiting exemplary methods of which are described in more detail in the following sections. The methods herein can be adapted by one of skill in the art for the preparation of formulations comprising other proteins such as peptide-based inhibitors. See Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012); Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., 2003); Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 2002); Current Protocols in Protein Science, (Horswill et al., 2006); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (R.I. Freshney, 6th ed., J. Wiley and Sons, 2010) for generally well understood and commonly employed ques and procedures for the production of therapeutic proteins, which are all incorporated herein by nce in their entirety.

A. dy Preparation The dy in the liquid formulations described herein is ed against an antigen of interest. ably, the antigen is a biologically important polypeptide and administration of the antibody to a mammal suffering from a disorder can result in a therapeutic benefit in that mammal. However, antibodies directed against nonpolypeptide antigens are also contemplated.

Where the antigen is a polypeptide, it may be a transmembrane molecule (e.g. receptor) or ligand such as a growth factor. Exemplary antigens include molecules such as vascular endothelial growth factor (VEGF); CD20; ox-LDL; ox-ApoB100; renin; a growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating e; lipoproteins; alphaantitrypsin; insulin n; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing e; 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 human urine or tissue-type nogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; a tumor necrosis factor receptor such as death receptor 5 and CD120; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated on tion normally T-cell expressed and ed); human macrophage inflammatory protein (MIPalpha); a serum albumin such as human serum albumin; Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated n (CTLA), such as CTLA-4; inhibin; activin; receptors for hormones or growth factors; protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor , neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or a nerve growth factor such as NGF-β; plateletderived growth factor ; fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); orming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or ; insulin-like growth factor-I and -II (IGF-I and IGF-II); des (1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins such as CD3, CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as eron-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; xide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrns such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 or HER4 receptor; and fragments of any of the above-listed ptides. (i) n Preparation Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens for generating dies. For transmembrane molecules, such as receptors, fragments of these (e.g. the ellular domain of a receptor) can be used as the immunogen. Alternatively, cells expressing the transmembrane molecule can be used as the immunogen. Such cells can be derived from a natural source (e.g. cancer cell lines) or may be cells which have been transformed by inant techniques to express the transmembrane molecule. Other antigens and forms thereof useful for preparing antibodies will be apparent to those in the art. (ii) Certain Antibody-Based Methods Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant n to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a tional or derivatizing agent, for e, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N- hydroxysuccinimide (through lysine es), glutaraldehyde, succinic anhydride, SOCl2, or R1N=C=NR, where R and R1 are different alkyl groups.

Animals are zed against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 µg or 5 µg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of ’s complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund’s te adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are d until the titer plateaus.

Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different n and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.

Monoclonal antibodies of st can be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), and further described, e.g., in Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: onal dies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) ing human-human hybridomas. onal methods e those bed, for example, in U.S. Pat. No. 7,189,826 regarding production of monoclonal human l IgM antibodies from hybridoma cell lines. Human hybridoma technology (Trioma technology) is described in Vollmers and ein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

For s other hybridoma techniques, see, e.g., US 2006/258841; US 2006/183887 (fully human dies), US 2006/059575; US 2005/287149; US 2005/100546; US 2005/026229; and U.S. Pat. Nos. 7,078,492 and 7,153,507. An exemplary protocol for producing monoclonal dies using the hybridoma method is described as follows. In one embodiment, a mouse or other appropriate host animal, such as a hamster, is zed to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Antibodies are raised in s by multiple subcutaneous (sc) or intraperitoneal (ip) injections of a polypeptide of interest or a fragment thereof, and an adjuvant, such as monophosphoryl lipid A (MPL)/trehalose dicrynomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, Mont.). A polypeptide of interest (e.g., antigen) or a fragment thereof may be prepared using methods well known in the art, such as recombinant methods, some of which are further described herein. Serum from immunized animals is assayed for anti-antigen antibodies, and booster immunizations are optionally administered. Lymphocytes from animals producing anti-antigen dies are isolated. Alternatively, lymphocytes may be immunized in vitro.

Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. See, e.g., Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986). Myeloma cells may be used that fuse ently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.

Exemplary myeloma cells include, but are not limited to, murine myeloma lines, such as those d from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture tion, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies r, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, e.g., a medium that contains one or more nces that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. Preferably, serum-free hybridoma cell culture s are used to reduce use of animal-derived serum such as fetal bovine serum, as described, for e, in Even et al., Trends in Biotechnology, 24(3), 105-108 (2006).

Oligopeptides as tools for improving productivity of hybridoma cell cultures are described in Franek, Trends in Monoclonal Antibody Research, 111-122 .

Specifically, standard culture media are enriched with certain amino acids (alanine, serine, asparagine, proline), or with protein hydrolyzate fractions, and apoptosis may be significantly ssed by synthetic oligopeptides, tuted of three to six amino acid residues. The peptides are present at millimolar or higher concentrations.

Culture medium in which hybridoma cells are growing may be d for production of onal antibodies that bind to an antibody described herein. The binding specificity of monoclonal antibodies produced by hybridoma cells may be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbent assay (ELISA). The binding affinity of the monoclonal antibody can be determined, for example, by ard analysis. See, e.g., Munson et al., Anal. Biochem., 107:220 (1980).

After hybridoma cells are fied that produce antibodies of the d specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods. See, e.g., Goding, supra. Suitable culture media for this purpose include, for example, D-MEM or 640 medium. In addition, hybridoma cells may be grown in vivo as ascites tumors in an . Monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification ures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or ty chromatography. One procedure for isolation of proteins from hybridoma cells is described in US 2005/176122 and U.S. Pat. No. 6,919,436. The method includes using minimal salts, such as lyotropic salts, in the binding process and preferably also using small amounts of organic solvents in the elution process. (iii) Certain Library Screening Methods Antibodies in the formulations and itions described herein can be made by using combinatorial libraries to screen for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and ing such libraries for antibodies possessing the desired binding characteristics. Such methods are described generally in Hoogenboom et al. in s in lar Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, N.J., 2001). For example, one method of generating antibodies of st is through the use of a phage antibody library as described in Lee et al., J. Mol. Biol. (2004), :1073-93.

In ple, synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments e of binding to the desired n are adsorbed to the antigen and thus separated from the non-binding clones in the library. The g clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption/elution. Any of the antibodies can be ed by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length antibody clone using the Fv sequences from the phage clone of interest and suitable nt region (Fc) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH ation 91-3242, Bethesda Md. (1991), vols. 1-3.

In certain embodiments, the antigen-binding domain of an antibody is formed from two variable (V) regions of about 110 amino acids, one each from the light (VL) and heavy (VH) chains, that both present three hypervariable loops (HVRs) or complementarity-determining regions (CDRs). Variable s can be displayed onally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein, scFv encoding phage clones and Fab encoding phage clones are collectively referred to as “Fv phage clones” or “Fv clones.” Repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al., Ann. Rev. l., 12: 433-455 (1994). Libraries from zed sources provide high-affinity antibodies to the immunogen t the requirement of constructing hybridomas. atively, the naive repertoire can be cloned to provide a single source of human dies to a wide range of non-self and also self antigens t any immunization as bed by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described by Hoogenboom and Winter, J. Mol.

Biol., 227: 381-388 (1992).

In certain embodiments, filamentous phage is used to display antibody fragments by fusion to the minor coat protein pIII. The antibody fragments can be displayed as single chain Fv fragments, in which VH and VL domains are connected on the same polypeptide chain by a flexible polypeptide spacer, e.g. as described by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fab fragments, in which one chain is fused to pIII and the other is secreted into the ial host cell periplasm where assembly of a Fab-coat n structure which becomes yed on the phage surface by displacing some of the wild type coat proteins, e.g. as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).

In general, nucleic acids ng antibody gene fragments are obtained from immune cells harvested from humans or animals. If a library biased in favor of antiantigen clones is desired, the subject is immunized with n to generate an antibody response, and spleen cells and/or ating B cells other peripheral blood lymphocytes (PBLs) are recovered for y uction. In one embodiment, a human antibody gene fragment library biased in favor of anti-antigen clones is obtained by generating an anti-antigen antibody response in transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system) such that antigen immunization gives rise to B cells producing human antibodies against antigen. The generation of human antibody-producing transgenic mice is described below.

Additional enrichment for anti-antigen ve cell populations can be obtained by using a suitable screening procedure to isolate B cells expressing antigen-specific ne bound antibody, e.g., by cell separation using antigen affinity chromatography or adsorption of cells to fluorochrome-labeled n followed by flow-activated cell sorting (FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs from an unimmunized donor provides a better representation of the possible antibody repertoire, and also permits the construction of an antibody y using any animal (human or nonhuman ) species in which n is not antigenic. For libraries incorporating in vitro antibody gene construction, stem cells are ted from the subject to provide nucleic acids encoding unrearranged antibody gene segments. The immune cells of interest can be obtained from a y of animal species, such as human, mouse, rat, lagomorpha, luprine, canine, feline, porcine, bovine, equine, and avian species, etc.

Nucleic acid encoding antibody le gene ts (including VH and VL segments) are recovered from the cells of interest and amplified. In the case of rearranged VH and VL gene libraries, the desired DNA can be ed by isolating genomic DNA or mRNA from lymphocytes followed by polymerase chain reaction (PCR) with primers matching the 5’ and 3’ ends of rearranged VH and VL genes as bed in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse V gene oires for expression. The V genes can be amplified from cDNA and genomic DNA, with back primers at the 5’ end of the exon encoding the mature V-domain and forward primers based within the J-segment as described in Orlandi et al. (1989) and in Ward et al., Nature, 341: 544-546 (1989). However, for amplifying from cDNA, back primers can also be based in the leader exon as described in Jones et al., Biotechnol., 9: 88-89 (1991), and forward primers within the nt region as described in Sastry et al., Proc. Natl.

Acad. Sci. (USA), 86: 5728-5732 (1989). To maximize complementarity, degeneracy can be incorporated in the primers as described in i et al. (1989) or Sastry et al. .

In certain embodiments, library diversity is maximized by using PCR primers targeted to each V-gene family in order to amplify all available VH and VL arrangements present in the immune cell nucleic acid sample, e.g. as described in the method of Marks et al., J.

Mol. Biol., 222: 581-597 (1991) or as described in the method of Orum et al., Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of the amplified DNA into expression vectors, rare restriction sites can be introduced within the PCR primer as a tag at one end as described in Orlandi et al. (1989), or by further PCR ication with a tagged primer as described in Clackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically nged V genes can be derived in vitro from V gene segments. Most of the human VH-gene segments have been cloned and sequenced ted in Tomlinson et al., J. Mol. Biol., 227: 776-798 (1992)), and mapped ted in Matsuda et al., Nature Genet., 3: 88-94 (1993); these cloned segments (including all the major conformations of the H1 and H2 loop) can be used to generate diverse VH gene repertoires with PCR primers encoding H3 loops of diverse sequence and length as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). VH repertoires can also be made with all the sequence diversity focused in a long H3 loop of a single length as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992).

Human Vκ and Vλ segments have been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol., 23: 461 (1993)) and can be used to make synthetic light chain repertoires. tic V gene repertoires, based on a range of VH and VL folds, and L3 and H3 lengths, will encode antibodies of considerable structural ity. ing amplification of V-gene encoding DNAs, germline V-gene segments can be rearranged in vitro according to the methods of Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH and VL gene oires together in several ways. Each repertoire can be created in different vectors, and the vectors recombined in vitro, e.g., as described in Hogrefe et al., Gene, 128: 119- 126 (1993), or in vivo by combinatorial infection, e.g., the loxP system described in Waterhouse et al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivo recombination approach ts the two-chain nature of Fab fragments to overcome the limit on y size imposed by E. coli transformation efficiency. Naive VH and VL repertoires are cloned separately, one into a phagemid and the other into a phage vector. The two libraries are then combined by phage infection of phagemid-containing bacteria so that each cell contains a different ation and the library size is limited only by the number of cells present (about 1012 clones). Both vectors contain in vivo recombination signals so that the VH and VL genes are recombined onto a single on and are copackaged into phage virions. These huge libraries provide large numbers of diverse antibodies of good affinity (Kd-1 of about 10-8 M).

Alternatively, the repertoires may be cloned sequentially into the same vector, e.g. as bed in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g. as bed in Clackson et al., Nature, 352: 624-628 (1991). PCR assembly can also be used to join VH and VL DNAs with DNA encoding a flexible peptide spacer to form single chain Fv (scFv) repertoires. In yet another technique, “in cell PCR assembly” is used to combine VH and VL genes within lymphocytes by PCR and then clone repertoires of linked genes as described in Embleton et al., Nucl. Acids Res., : 3831-3837 (1992).

The antibodies produced by naive libraries (either natural or synthetic) can be of te affinity (Kd-1 of about 106 to 107 M-1), but affinity tion can also be mimicked in vitro by constructing and reselecting from secondary libraries as described in Winter et al. (1994), supra. For example, mutation can be introduced at random in vitro by using errorprone polymerase (reported in Leung et al., Technique 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol. Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc.

Natl. Acad. Sci USA, 89: 3576-3580 (1992). Additionally, affinity maturation can be performed by randomly mutating one or more CDRs, e.g. using PCR with primers carrying random sequence ng the CDR of interest, in selected individual Fv clones and screening for higher affinity clones. WO 9607754 (published 14 Mar. 1996) described a method for inducing mutagenesis in a complementarity determining region of an globulin light chain to create a library of light chain genes. Another ive approach is to recombine the VH or VL domains selected by phage display with repertoires of naturally occurring V domain variants obtained from unimmunized donors and screen for higher affinity in several rounds of chain reshuffling as described in Marks et al., Biotechnol., : 779-783 (1992). This technique allows the production of antibodies and antibody nts with affinities of about 10-9 M or less.

Screening of the libraries can be accomplished by various techniques known in the art. For example, antigen can be used to coat the wells of adsorption plates, expressed on host cells d to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning phage display libraries.

The phage library samples are contacted with lized antigen under conditions suitable for binding at least a portion of the phage les with the ent. Normally, the ions, ing pH, ionic strength, temperature and the like are selected to mimic physiological conditions. The phages bound to the solid phase are washed and then eluted by acid, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci USA, 88: 7978-7982 (1991), or by alkali, e.g. as described in Marks et al., J. Mol. Biol., 222: 581-597 (1991), or by n ition, e.g. in a procedure similar to the antigen competition method of Clackson et al., , 352: 624-628 (1991). Phages can be enriched 20-1,000-fold in a single round of selection. Moreover, the ed phages can be grown in bacterial culture and subjected to further rounds of selection.

The efficiency of selection depends on many factors, including the kinetics of dissociation during washing, and whether multiple antibody fragments on a single phage can simultaneously engage with antigen. Antibodies with fast dissociation kinetics (and weak binding affinities) can be retained by use of short washes, multivalent phage display and high coating density of antigen in solid phase. The high density not only stabilizes the phage through multivalent interactions, but favors ing of phage that has iated. The ion of dies with slow dissociation kinetics (and good binding affinities) can be promoted by use of long washes and monovalent phage display as described in Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and a low coating density of antigen as described in Marks et al., Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of different affinities, even with affinities that differ slightly, for antigen. However, random on of a selected antibody (e.g. as performed in some affinity maturation ques) is likely to give rise to many mutants, most binding to n, and a few with higher affinity. With limiting antigen, rare high affinity phage could be competed out. To retain all higher affinity s, phages can be incubated with excess biotinylated antigen, but with the biotinylated antigen at a tration of lower molarity than the target molar affinity nt for antigen. The high affinity-binding phages can then be captured by streptavidin-coated paramagnetic beads.

Such “equilibrium capture” allows the antibodies to be selected according to their affinities of binding, with sensitivity that permits isolation of mutant clones with as little as two-fold higher affinity from a great excess of phages with lower affinity. ions used in washing phages bound to a solid phase can also be manipulated to discriminate on the basis of dissociation kinetics.

Anti-antigen clones may be selected based on activity. In certain embodiments, the description includes anti-antigen antibodies that bind to living cells that naturally express antigen or bind to free floating antigen or antigen attached to other cellular structures. Fv clones corresponding to such anti-antigen antibodies can be selected by (1) isolating antiantigen clones from a phage library as described above, and optionally amplifying the isolated population of phage clones by g up the population in a suitable bacterial host; (2) selecting antigen and a second n against which blocking and non-blocking activity, tively, is desired; (3) adsorbing the anti-antigen phage clones to immobilized antigen; (4) using an excess of the second protein to elute any red clones that recognize antigenbinding determinants which overlap or are shared with the binding determinants of the second protein; and (5) eluting the clones which remain adsorbed following step (4). ally, clones with the desired blocking/non-blocking properties can be further enriched by repeating the selection procedures described herein one or more times.

DNA encoding hybridoma-derived monoclonal antibodies or phage display Fv clones is readily isolated and sequenced using conventional ures (e.g. by using oligonucleotide primers designed to ically amplify the heavy and light chain coding regions of interest from hybridoma or phage DNA template). Once isolated, the DNA can be placed into sion vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin n, to obtain the sis of the desired monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of antibody-encoding DNA include Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones can be combined with known DNA sequences encoding heavy chain and/or light chain constant regions (e.g. the appropriate DNA sequences can be obtained from Kabat et al., supra) to form clones encoding full or partial length heavy and/or light chains. It will be appreciated that constant regions of any e can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species. An Fv clone derived from the variable domain DNA of one animal (such as human) species and then fused to constant region DNA of another animal species to form coding sequence(s) for “hybrid,” full length heavy chain and/or light chain is ed in the definition of “chimeric” and “hybrid” antibody as used herein. In n embodiments, an Fv clone derived from human variable DNA is fused to human constant region DNA to form coding sequence(s) for full- or partial-length human heavy and/or light chains.

DNA encoding anti-antigen antibody derived from a hybridoma can also be ed, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of homologous murine sequences derived from the hybridoma clone (e.g. as in the method of on et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). DNA encoding a hybridoma- or Fv clone-derived antibody or fragment can be further ed by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In this manner, “chimeric” or d” antibodies are prepared that have the binding icity of the Fv clone or hybridoma derived antibodies. (iv) zed and Human Antibodies Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.

Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 34-1536 ), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding ce from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR es and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called “best-fit” , the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 1 (1987)). r method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl.

Acad Sci. USA, 89:4285 (1992); Presta et al., J. l., 151:2623 (1993)).

It is further important that antibodies be humanized with ion of high affinity for the antigen and other favorable ical properties. To achieve this goal, according to one embodiment of the method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using threedimensional models of the al and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are ar to those d in the art.

Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits is of the likely role of the residues in the functioning of the candidate immunoglobulin ce, i.e., the analysis of residues that influence the ability of the candidate globulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody teristic, such as increased ty for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

Human antibodies in the formulations and compositions bed herein can be constructed by combining Fv clone variable domain sequence(s) ed from humanderived phage display libraries with known human constant domain sequence(s) as described above. Alternatively, human monoclonal antibodies can be made by the hybridoma method.

Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 l Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).

It is possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the gous deletion of the antibody chain joining region (JH) gene in chimeric and germ-line mutant mice s in complete inhibition of nous antibody production.

Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in ., 7:33 (1993); and Duchosal et al.

Nature 355:258 (1992).

Gene shuffling can also be used to derive human antibodies from non-human, e.g. rodent, antibodies, where the human antibody has similar affinities and specificities to the ng non-human antibody. According to this method, which is also called “epitope imprinting”, either the heavy or light chain variable region of a man antibody fragment obtained by phage display techniques as described herein is replaced with a repertoire of human V domain genes, creating a population of non-human chain/human chain scFv or Fab chimeras. Selection with antigen s in ion of a non-human chain/human chain chimeric scFv or Fab wherein the human chain restores the antigen binding site destroyed upon removal of the corresponding man chain in the primary phage display clone, i.e. the e governs nts) the choice of the human chain partner. When the process is repeated in order to replace the remaining non-human chain, a human antibody is obtained (see PCT WO 93/06213 published Apr. 1, 1993). Unlike traditional humanization of nonhuman antibodies by CDR grafting, this que provides completely human antibodies, which have no FR or CDR es of non-human origin. (v) Antibody Fragments dy nts may be generated by traditional means, such as enzymatic digestion, or by recombinant techniques. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors. For a review of n antibody fragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

Various techniques have been developed for the production of antibody fragments.

Traditionally, these fragments were derived via lytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by inant host cells. Fab, Fv and ScFv antibody fragments can all be sed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab’-SH fragments can be directly recovered from E. coli and ally coupled to form F(ab’)2 fragments (Carter et al., Bio/Technology 10:163-167 ). According to another approach, F(ab’) 2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab’) 2 fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Pat. No. ,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In n embodiments, an antibody is a single chain Fv fragment (scFv).

See WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. Fv and scFv are the only species with intact combining sites that are devoid of constant regions; thus, they may be suitable for reduced nonspecific binding during in vivo use. scFv fusion ns may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv. See Antibody ering, ed. Borrebaeck, supra. The antibody fragment may also be a “linear antibody”, e.g., as described in U.S. Pat. No. 5,641,870, for e. Such linear antibodies may be monospecific or bispecific. (vi) Multispecific Antibodies Multispecific antibodies have binding specificities for at least two different epitopes, where the epitopes are usually from different ns. While such molecules normally will only bind two ent epitopes (i.e. bispecific antibodies, BsAbs), antibodies with additional specificities such as trispecific antibodies are encompassed by this expression when used herein. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab’)2 bispecific antibodies).

Methods for making ific dies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities tein et al., Nature, 305:537-539 ). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas omas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the t le, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar ures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, sing at least part of the hinge, CH2, and CH3 regions. It is typical to have the first heavy-chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the globulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide nts in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

In one embodiment of this approach, the bispecific dies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid globulin heavy chain-light chain pair (providing a second binding icity) in the other arm. It was found that this tric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific le provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO96/27011, the interface n a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from inant cell e. One interface comprises at least a part of the CH 3 domain of an dy constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. ne or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted endproducts such as mers.

Bispecific antibodies include cross-linked or oconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any ient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact dies are proteolytically cleaved to generate F(ab’)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular ide formation. The Fab’ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’-TNB derivatives is then reconverted to the Fab’-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab’-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of H fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp.

Med., 175: 217-225 (1992) describe the tion of a fully humanized bispecific antibody F(ab’)2 molecule. Each Fab’ nt was separately secreted from E. coli and subjected to directed chemical ng in vitro to form the bispecific antibody.

Various techniques for making and ing bispecific antibody fragments directly from recombinant cell culture have also been described. For e, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper es from the Fos and Jun proteins were linked to the Fab’ portions of two ent antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.

The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444- 6448 (1993) has provided an alternative ism for making bispecific antibody fragments. The fragments comprise a chain le domain (VH) connected to a lightchain le domain (VL) by a linker which is too short to allow pairing between the two s on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody nts by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al, J. Immunol, 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147: 60 (1991). (vii) Single-Domain Antibodies In some embodiments, an antibody described herein is a single-domain antibody. A single-domain antibody is a single polypeptide chain comprising all or a portion of the heavy chain le domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1). In one embodiment, a single-domain antibody ts of all or a portion of the heavy chain variable domain of an antibody. (viii) Antibody Variants In some embodiments, amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid ce variants of the antibody may be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid alterations may be introduced in the t antibody amino acid sequence at the time that sequence is made. (ix) Antibody Derivatives The antibodies in the formulations and compositions of the ption can be r ed to contain additional nonproteinaceous moieties that are known in the art and readily available. In certain embodiments, the moieties suitable for derivatization of the antibody are water soluble polymers. Non-limiting es of water soluble polymers include, but are not limited to, hylene glycol (PEG), copolymers of ne glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, yethylated polyols (e.g., glycerol), polyvinyl l, and mixtures f. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular , and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not d to, the particular properties or functions of the antibody to be improved, r the dy derivative will be used in a therapy under defined conditions, etc. (x) Vectors, Host Cells, and Recombinant Methods Antibodies may also be produced using inant methods. For recombinant tion of an anti-antigen dy, nucleic acid encoding the antibody is isolated and inserted into a able vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody may be y isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally e, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. (a) Signal Sequence Component An antibody in the formulations and compositions described herein may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected preferably is one that is recognized and processed (e.g., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process a native antibody signal sequence, the signal sequence is substituted by a prokaryotic signal ce selected, for example, from the group of the alkaline atase, penicillinase, lpp, or heat-stable toxin II leaders. For yeast ion the native signal sequence may be substituted by, e.g., the yeast ase leader, a factor leader ding romyces and Kluyveromyces α-factor leaders), or acid phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available. (b) Origin of Replication Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this ce is one that enables the vector to replicate independently of the host chromosomal DNA, and es origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2µ, plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for g vectors in mammalian cells. Generally, the origin of ation component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter. (c) Selection Gene Component Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, in, methotrexate, or tetracycline, (b) ment auxotrophic deficiencies, or (c) supply critical nts not ble from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of a host cell.

Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug ance and thus survive the selection regimen. Examples of such dominant selection use the drugs in, mycophenolic acid and hygromycin.

Another example of le selectable markers for mammalian cells are those that enable the identification of cells competent to take up antibody-encoding c acid, such as DHFR, ine synthetase (GS), thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR gene are identified by culturing the transformants in a e medium ning methotrexate (Mtx), a competitive antagonist of DHFR. Under these conditions, the DHFR gene is amplified along with any other cotransformed nucleic acid. A Chinese hamster ovary (CHO) cell line deficient in endogenous DHFR activity (e.g., ATCC CRL-9096) may be used.

Alternatively, cells transformed with the GS gene are identified by culturing the transformants in a culture medium containing L-methionine sulfoximine (Msx), an inhibitor of GS. Under these conditions, the GS gene is amplified along with any other co-transformed nucleic acid. The GS selection/amplification system may be used in combination with the DHFR selection/amplification system described above. atively, host cells (particularly wild-type hosts that contain nous DHFR) transformed or co-transformed with DNA sequences encoding an antibody of interest, wild-type DHFR gene, and another selectable marker such as aminoglycoside 3’- phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). The trp1 gene es a selection marker for a mutant strain of yeast g the y to grow in tryptophan, for e, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are mented by known ds bearing the Leu2 gene.

In addition, vectors d from the 1.6 µm circular plasmid pKD1 can be used for transformation of Kluyveromyces yeasts. Alternatively, an expression system for large-scale tion of recombinant calf in was reported for K. lactis. Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copy expression s for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed. Fleer et al., chnology, 9:968-975 . (d) Promoter Component Expression and cloning vectors generally contain a promoter that is recognized by the host organism and is operably linked to nucleic acid encoding an antibody. Promoters suitable for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase er, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known ial promoters are suitable. ers for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding an antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where ription is initiated. Another ce found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3’ end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3’ end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.

Examples of suitable promoter sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehydephosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucosephosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehydephosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers also are advantageously used with yeast promoters.

Antibody ription from vectors in mammalian host cells can be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early er of the human cytomegalovirus is conveniently obtained as a HindIII E restriction nt. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature 297:598- 601 (1982) on expression of human β-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter. (e) Enhancer Element Component Transcription of a DNA encoding an antibody by higher eukaryotes is often increased by inserting an er ce into the vector. Many enhancer sequences are now known from mammalian genes n, elastase, albumin, α-fetoprotein, and insulin).

Typically, r, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the ation origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma er on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elements for tion of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5’ or 3’ to the antibody-encoding sequence, but is preferably located at a site 5’ from the promoter. (f) Transcription Termination Component Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also n sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5’ and, occasionally 3’, untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as enylated fragments in the untranslated portion of the mRNA encoding antibody. One useful transcription termination component is the bovine growth e polyadenylation region. See WO94/11026 and the expression vector sed therein. (g) ion and Transformation of Host Cells le host cells for g or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. le prokaryotes for this purpose e eubacteria, such as Gram-negative or Gram-positive sms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC ), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.

Full length antibody, antibody fusion proteins, and antibody fragments can be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, such as when the therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin) that by itself shows effectiveness in tumor cell destruction. Full length antibodies have greater half-life in circulation. tion in E. coli is faster and more cost efficient. For expression of antibody nts and polypeptides in bacteria, see, e.g., U.S. Pat. No. ,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), U.S. Pat. No. 5,840,523 (Simmons et al.), which describes translation initiation region (TIR) and signal sequences for optimizing sion and secretion. See also Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli. After expression, the antibody may be ed from the E. coli cell paste in a soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for ing antibody expressed e.g., in CHO cells.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding s. Saccharomyces cerevisiae, or common baker’s yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and s are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. amii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. tolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and ntous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger. For a review discussing the use of yeasts and filamentous fungi for the production of therapeutic proteins, see, e.g., oss, Nat. h. 22:1409-1414 .

Certain fungi and yeast s may be selected in which glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See, e.g., Li et al., Nat. Biotech. 24:210-215 (2006) (describing humanization of the ylation pathway in Pichia pastoris); and Gerngross et al., supra.

Suitable host cells for the expression of ylated antibody are also derived from ellular sms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as tera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the description, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed (Leninaceae), alfalfa (M. truncatula), and tobacco can also be utilized as hosts. See, e.g., U.S.

Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 ibing PLANTIBODIESTM technology for producing antibodies in transgenic plants).

Vertebrate cells may be used as hosts, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 , ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). Other useful mammalian host cell lines include e hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 6 (1980)); and myeloma cell lines such as NS0 and Sp2/0.

For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in lar Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 255-268.

Host cells are ormed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as riate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. (h) Culturing the Host Cells The host cells used to produce an antibody may be cultured in a variety of media.

Commercially available media such as Ham’s F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), 640 (Sigma), and Dulbecco’s Modified s Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, ium, and phosphate), s (such as , nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCINTM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.

The culture ions, such as temperature, pH, and the like, are those previously used with the host cell selected for sion, and will be apparent to the ordinarily skilled artisan. (xi) Purification of Antibody When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology :163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious inants.

The antibody composition ed from the cells can be purified using, for example, hydroxylapatite tography, hydrophobic interaction chromatography, gel electrophoresis, is, and affinity chromatography, with affinity chromatography being among one of the typically preferred purification steps. The suitability of protein A as an affinity ligand depends on the s and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. l. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as lled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter sing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABXTM resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for n cation such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, tography on silica, chromatography on heparin OSETM chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

In general, various methodologies for preparing dies for use in research, testing, and clinical are well-established in the art, consistent with the above-described methodologies and/or as deemed riate by one skilled in the art for a particular antibody of interest.

B. Selecting Biologically Active Antibodies Antibodies produced as described above may be subjected to one or more gical activity” assays to select an antibody with beneficial properties from a therapeutic ctive. The antibody may be screened for its ability to bind the antigen against which it was raised. For example, for an anti-DR5 antibody (e.g., drozitumab), the antigen binding properties of the antibody can be ted in an assay that detects the ability to bind to a death or 5 (DR5).

In another embodiment, the affinity of the antibody may be determined by saturation binding; ELISA; and/or ition assays (e.g. RIA’s), for example.

Also, the antibody may be subjected to other biological activity assays, e.g., in order to evaluate its effectiveness as a therapeutic. Such assays are known in the art and depend on the target antigen and intended use for the antibody.

To screen for antibodies which bind to a particular e on the antigen of interest, a routine cross-blocking assay such as that bed in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Alternatively, epitope g, e.g. as described in Champe et al., J. Biol. Chem. 270:1388-1394 (1995), can be performed to determine whether the antibody binds an epitope of interest.

C. Preparation of the Formulations Described herein are methods of preparing a liquid formulation comprising a protein and a compound which prevents ion of the protein in the liquid formulation.

The liquid formulation may be prepared by mixing the protein having the desired degree of purity with a compound which prevents oxidation of the protein in the liquid formulation. In certain embodiments, the protein to be formulated has not been subjected to prior lyophilization and the formulation of interest herein is an aqueous formulation. In some embodiments, the protein is a therapeutic protein. In n embodiments, the protein is an antibody. In r embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, or antibody fragment. In certain embodiments, the antibody is a full length antibody. In one ment, the antibody in the formulation is an antibody fragment, such as an F(ab’)2, in which case problems that may not occur for the full length antibody (such as ng of the antibody to Fab) may need to be addressed. The therapeutically effective amount of protein present in the ation is determined by taking into account the d dose volumes and mode(s) of administration, for example. From about 1 mg/mL to about 250 mg/mL, from about 10 mg/mL to about 250 mg/mL, from about 15 mg/mL to about 225 mg/mL, from about 20 mg/mL to about 200 mg/mL, from about 25 mg/mL to about 175 mg/mL, from about 25 mg/mL to about 150 mg/mL, from about 25 mg/mL to about 100 mg/mL, from about 30 mg/mL to about 100 mg/mL or from about 45 mg/mL to about 55 mg/mL is an exemplary protein concentration in the formulation. In some embodiments, the n described herein is susceptible to oxidation. In some embodiments, one or more of the amino acids ed from the group consisting of methionine, cysteine, ine, tryptophan, and tyrosine in the protein is susceptible to oxidation. In some embodiments, tryptophan in the protein is susceptible to oxidation. In some embodiments, methionine in the protein is susceptible to oxidation. In some embodiments, an antibody described herein is susceptible to oxidation in the Fab portion and/or the Fc portion of the antibody. In some ments, an antibody described herein is susceptible to oxidation at a tryptophan amino acid in the Fab portion of the antibody. In a r embodiment, the tryptophan amino acid susceptible to oxidation is in a CDR of the dy. In some embodiments, an antibody described herein is susceptible to oxidation at a nine amino acid in the Fc portion of the antibody.

The liquid formulations included in the description se a protein and a compound which prevents oxidation of the protein in the liquid formulation, wherein the compound is ed from the group consisting of oxy-tryptophan, 5-hydroxy indole, 7-hydroxy indole, and serotonin. In some embodiments, the compound in the formulation is at a concentration from about 0.3 mM to about 10 mM, or up to the highest concentration that the compound is soluble to in the formulation. In certain embodiments, the compound in the formulation is at a concentration from about 0.3 mM to about 9 mM, from about 0.3 mM to about 8 mM, from about 0.3 mM to about 7 mM, from about 0.3 mM to about 6 mM, from about 0.3 mM to about 5 mM, from about 0.3 mM to about 4 mM, from about 0.3 mM to about 3 mM, from about 0.3 mM to about 2 mM, from about 0.5 mM to about 2 mM, from about 0.6 mM to about 1.5 mM, or from about 0.8 mM to about 1.25 mM. In some embodiments, the compound in the formulation is about 1 mM. In some embodiments, the compound prevents oxidation of one or more amino acids in the protein. In some embodiments, the compound prevents ion of one or more amino acids in the protein selected from group consisting of tryptophan, methionine, tyrosine, histidine, and/or cysteine.

In some embodiments, the nd prevents oxidation of the protein by a reactive oxygen species (ROS). In a further embodiment, the reactive oxygen species is selected from the group consisting of a singlet oxygen, a superoxide (O2-), an l radical, a peroxyl radical, a hydrogen peroxide (H2O2), a dihydrogen trioxide (H2O3), a rioxy radical (HO3•), ozone (O3), a hydroxyl radical, and an alkyl de. In a further embodiment, the nd prevents oxidation of one or more amino acids in the Fab portion of an dy.

In another further embodiment, the compound prevents oxidation of one or more amino acids in the Fc portion of an antibody.

In some embodiments, the liquid formulation further comprises one or more excipients ed from the group consisting of a stabilizer, a buffer, a surfactant, and a tonicity agent. A liquid formulation of the description is prepared in a pH-buffered solution.

The buffer of this description has a pH in the range from about 4.5 to about 7.0. In certain embodiments the pH is in the range from pH 4.5 to 6.5, in the range from pH 4.5 to 6.0, in the range from pH 4.5 to 5.5, in the range from pH 4.5 to 5.0, in the range from pH 5.0 to 7.0, in the range from pH 5.5 to 7.0, in the range from pH 5.7 to 6.8, in the range from pH 5.8 to 6.5, in the range from pH 5.9 to 6.5, in the range from pH 6.0 to 6.5, or in the range from pH 6.2 to 6.5. In certain embodiments of the description, the liquid formulation has a pH of 6.2 or about 6.2. In certain ments of the description, the liquid formulation has a pH of 6.0 or about 6.0. Examples of buffers that will control the pH within this range include organic and inorganic acids and salts f. For example, acetate (e.g., histidine e, arginine acetate, sodium acetate), succinate (e.g., histidine ate, arginine succinate, sodium succinate), gluconate, phosphate, fumarate, oxalate, lactate, citrate, and combinations thereof.

The buffer concentration can be from about 1 mM to about 600 mM, depending, for example, on the buffer and the desired isotonicity of the formulation. In certain embodiments, the formulation comprises a histidine buffer (e.g., in the concentration from about 5 mM to 100 mM). Examples of histidine buffers include histidine chloride, histidine acetate, histidine phosphate, histidine e, histidine succinate, etc. In certain embodiments, the ation comprises ine and arginine (e.g., ine chloride-arginine chloride, histidine acetate- arginine acetate, histidine phosphate-arginine phosphate, histidine sulfate-arginine sulfate, ine succinate-arginine succinate, etc.). In n embodiments, the formulation comprises histidine in the concentration from about 5 mM to 100 mM and the arginine is in the concentration of 50 mM to 500 mM. In one embodiment, the formulation comprises histidine acetate (e.g., about 20 mM)-arginine acetate (e.g., about 150 mM). In certain embodiments, the formulation comprises histidine succinate (e.g., about 20 mM)-arginine succinate (e.g., about 150 mM). In certain embodiments, histidine in the formulation from about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 15 mM to about 35 mM, about 20 mM to about 35 mM, about 20 mM to about 30 mM or about 20 mM to about 25 mM. In further embodiments, the ne in the formulation is from about 50 mM to about 500 mM (e.g., about 100 mM, about 150 mM, or about 200 mM).

The liquid ation of the description can further comprise a saccharide, such as a disaccharide (e.g., trehalose or sucrose). A “saccharide” as used herein es the general composition (CH2O)n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, ng sugars, nonreducing sugars, etc.

Examples of saccharides herein include glucose, sucrose, trehalose, e, fructose, maltose, dextran, glycerin, erythritol, glycerol, arabitol, l, sorbitol, mannitol, mellibiose, tose, raffinose, mannotriose, stachyose, lactulose, maltulose, glucitol, maltitol, lactitol, iso-maltulose, etc.

A surfactant can optionally be added to the liquid formulation. Exemplary tants include nonionic surfactants such as polysorbates (e.g. polysorbates 20, 80, etc.) or poloxamers (e.g. poloxamer 188, etc.). The amount of surfactant added is such that it reduces aggregation of the formulated antibody and/or minimizes the formation of particulates in the formulation and/or reduces adsorption. For example, the surfactant may be present in the formulation in an amount from about 0.001% to about 0.5%, from about 0.005% to about 0.2%, from about 0.01% to about 0.1%, from about 0.02% to about 0.06%, or about 0.03% to about 0.05%. In certain embodiments, the tant is present in the formulation in an amount of 0.04% or about 0.04%. In certain embodiments, the surfactant is present in the formulation in an amount of 0.02% or about 0.02%. In one embodiment, the formulation does not comprise a surfactant.

In one embodiment, the formulation contains the above-identified agents (e.g., antibody, , saccharide, and/or surfactant) and is ially free of one or more preservatives, such as benzyl alcohol, phenol, m-cresol, chlorobutanol and benzethonium Cl.

In another embodiment, a preservative may be ed in the formulation, particularly where the formulation is a multidose formulation. The concentration of preservative may be in the range from about 0.1% to about 2%, preferably from about 0.5% to about 1%. One or more other pharmaceutically acceptable carriers, excipients or stabilizers such as those described in Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) may be included in the formulation ed that they do not adversely affect the desired characteristics of the formulation. Exemplary pharmaceutically acceptable excipients herein further include interstitial drug dispersion agents such as soluble l-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are bed in US Patent ation Nos. 2005/0260186 and 104968. In one ment, a sHASEGP is combined with one or more additional aminoglycanases such as chondroitinases.

The formulation may further comprise metal ion chelators. Metal ion chelators are well known by those of skill in the art and include, but are not necessarily limited to aminopolycarboxylates, EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycolbis (beta-aminoethyl ether)-N,N,N’,N’-tetraacetic acid), NTA (nitrilotriacetic acid), EDDS ene diamine disuccinate), PDTA (1,3-propylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), ADA (beta-alaninediacetic acid), MGCA (methylglycinediacetic acid), etc. Additionally, some embodiments herein comprise phosphonates/phosphonic acid chelators.

Tonicity agents are present to adjust or maintain the tonicity of liquid in a ition. When used with large, charged biomolecules such as proteins and antibodies, they may also serve as “stabilizers” because they can interact with the charged groups of the amino acid side chains, y lessening the potential for inter- and intra-molecular interactions. Tonicity agents can be present in any amount n 0.1% to 25% by weight, or more preferably between 1% to 5% by weight, taking into account the ve amounts of the other ingredients. red tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

The formulation herein may also n more than one protein or a small molecule drug as necessary for the particular indication being treated, preferably those with mentary activities that do not adversely affect the other protein. For example, where the antibody is anti-DR5 (e.g., drozitumab), it may be combined with another agent (e.g., a chemotherapeutic agent, and anti-neoplastic agent).

In some embodiments, the formulation is for in vivo administration. In some embodiments, the formulation is sterile. The formulation may be rendered sterile by filtration through sterile filtration membranes. The therapeutic formulations herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, l administration, inhalation or by ned release or extendedrelease means.

The liquid formulation of the description may be stable upon storage. In some ments, the protein in the liquid formulation is stable upon storage at about 0 to 5ºC for at least about 12 months, at least about 18 months, at least about 21 months, or at least about 24 months (or at least about 52 weeks). In some embodiments, the protein in the liquid formulation is stable upon storage at about -20ºC for at least about 12 months, at least about 18 months, at least about 21 months, or at least about 24 months (or at least about 52 weeks).

In some embodiments, the protein in the liquid formulation is stable during formulation manufacturing. In some embodiments, the protein in the liquid formulation is stable when stored in a metal alloy container (e.g., a stainless steel container). In some embodiments, the protein in the liquid formulation is stable when stored in a glass vial. In some embodiments, the protein in the liquid formulation is stable when stored in a plastic ner. In some embodiments, the physical stability, chemical stability, or biological activity of the n in the liquid formulation is evaluated or measured. Any methods known in the art may be used to te the stability and biological activity. In some embodiments, the stability is ed by oxidation of the protein in the liquid formulation after storage. ity can be tested by evaluating physical stability, al stability, and/or ical activity of the dy in the formulation around the time of formulation as well as following storage.

Physical and/or chemical stability can be evaluated qualitatively and/or tatively in a variety of different ways, including evaluation of aggregate formation (for example using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography or capillary zone electrophoresis; amino-terminal or carboxy-terminal ce analysis; mass spectrometric analysis; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS-C) is; evaluating biological activity or antigen binding function of the antibody; etc. ility may result in aggregation, deamidation (e.g. Asn deamidation), oxidation (e.g. Trp oxidation), isomerization (e.g. Asp isomeriation), clipping/hydrolysis/fragmentation (e.g. hinge region ntation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc. In some embodiments, the oxidation in a protein is determined using one or more of RPHPLC , LC/MS, or tryptic peptide mapping. In some embodiments, the oxidation in an antibody is determined as a percentage using one or more of RP-HPLC, LC/MS, or tryptic peptide mapping and the formula of: Oxidized Fab Peak Area % Fab Oxidation = 100× Fab Peak Area + Oxidized Fab Peak Area Oxidized Fc Peak Area % Fc Oxidation =100× Fc Peak Area + Oxidized Fc Peak Area The formulations to be used for in vivo administration should be sterile. This is readily accomplished by filtration through e filtration membranes, prior to, or following, preparation of the formulation.

Also bed herein are methods of making a liquid formulation or preventing oxidation of a protein in a liquid formulation comprising adding an amount of a compound that prevents oxidation of a protein to a liquid formulation, wherein the compound is selected from the group consisting of 5-hydroxy-tryptophan, 5-hydroxy indole, 7-hydroxy indole, and serotonin. The description herein also includes a method of preventing oxidation of a protein in a liquid formulation comprising adding an amount of a nd that prevents oxidation of the protein to the liquid formulation, wherein the compound is selected from the group consisting of 5-hydroxy-tryptophan, 5-hydroxy indole, 7-hydroxy indole, and serotonin. In certain ments, the liquid formulation comprises an antibody. The amount of the compound that prevents oxidation of the protein as described herein is from about 0.3 mM to about 10 mM or any of the amounts disclosed herein.

III. stration of Protein Formulations The liquid formulation is administered to a mammal in need of treatment with the protein (e.g., an antibody), preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, erobrospinal, subcutaneous, intra-articular, intrasynovial, hecal, oral, topical, or inhalation routes. In one embodiment, the liquid formulation is administered to the mammal by intravenous administration. For such purposes, the formulation may be injected using a syringe or via an IV line, for example. In one embodiment, the liquid formulation is administered to the mammal by subcutaneous administration.

The appropriate dosage (“therapeutically effective amount”) of the protein will , for example, on the condition to be treated, the ty and course of the condition, r the protein is administered for preventive or therapeutic purposes, us therapy, the patient’s clinical history and response to the protein, the type of protein used, and the discretion of the attending physician. The n is suitably administered to the patient at one time or over a series of treatments and may be administered to the patient at any time from diagnosis onwards. The protein may be administered as the sole treatment or in conjunction with other drugs or therapies useful in treating the condition in question. As used herein the term “treatment” refers to both therapeutic treatment and prophylactic or tative measures. Those in need of treatment include those y with the disorder as well as those in which the disorder is to be prevented. As used herein a “disorder” is any condition that would benefit from treatment including, but not limited to, chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.

In a pharmacological sense, in the context of the description, a “therapeutically effective amount” of a protein (e.g., an antibody) refers to an amount effective in the tion or treatment of a disorder for the treatment of which the antibody is ive. As a general ition, the therapeutically effective amount of the protein administered will be in the range of about 0.1 to about 50 mg/kg of patient body weight whether by one or more administrations, with the typical range of protein used being about 0.3 to about 20 mg/kg, preferably about 0.3 to about 15 mg/kg, administered daily, for example. However, other dosage regimens may be . For example, a n can be administered at a dose of about 100 or 400 mg every 1, 2, 3, or 4 weeks or is administered a dose of about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 15.0, or 20.0 mg/kg every 1, 2, 3, or 4 weeks. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. The progress of this therapy is easily monitored by conventional techniques.

IV. Methods of Screening for Compounds for the Prevention of Protein Oxidation Also described herein are s of screening a compound that prevents oxidation of a protein in a protein composition. In some embodiments, the method comprises selecting a nd that has lower oxidation potential and less photosensitivity as compared to L- tryptophan, and testing the effect of the selected compound on preventing oxidation of the protein. In some embodiments, the photosensitivity is measured based on the amount of H2O2 produced by the compound upon light exposure. For example, a liquid composition comprising the compound can be exposed to 250 W/m2 light for a certain amount of time and the ing H2O2 formation is quantified. A nd with less photosensitivity produces less H2O2 upon exposure to a certain amount of light than a compound that has a higher photosensitivity upon exposure to the same amount of light. In some embodiments, the compound that produces less than about 15%, less than about 20%, or less than about 25% of the amount of H2O2 is ed. H2O2 can be produced by oxidation of amino acid residues in a protein that are susceptible to oxidation. In some embodiments, the oxidation potential is measured by cyclic voltammetry.

In some embodiments, the selected compound is tested for the effect on preventing oxidation of the protein by reactive oxygen species generated by 2,2’-azobis(2- amidinopropane) dihydrochloride (AAPH), light, and/or a Fenton reagent. In any of the embodiments herein, a method described in the Examples (e.g., Examples 2 and 3) may be used for screening a compound that prevents oxidation of a n in a protein composition.

V. Articles of cture In another embodiment of the description, an article of manufacture is included comprising a container which holds the liquid formulation of the description and optionally es instructions for its use. Suitable containers include, for example, bottles, vials and syringes. The container may be formed from a variety of materials such as glass, metal alloy (such as ess steel) or plastic. An exemplary container is a 300 cc metal alloy container (e.g., for storing at -20ºC). An exemplary ner is a 3-20 cc single use glass vial.

Alternatively, for a multidose formulation, the container may be 3-100 cc glass vial. The container holds the formulation and the label on, or associated with, the ner may indicate directions for use. The article of cture may further include other materials ble from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The specification is considered to be sufficient to enable one skilled in the art to practice the ion. s modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

EXAMPLES The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for rative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be ed within the spirit and purview of this application and scope of the appended claims.

Example 1: The antioxidant L-Trp produces ROS that oxidize monoclonal antibodies in protein formulations.

Monoclonal antibodies have been shown to e ROS through the antibody catalyzed water oxidation pathway (ACWOP) wherein antibodies ially catalyze a reaction between water and singlet oxygen ting hydrogen peroxide (Wentworth et al., Science 293(5536):1806-11 (2001); Wentworth et al., Proc Natl Acad Sci U S A 97(20):10930-5 (2000)). In the ACWOP, a variety of ROS, ing superoxide anion, dihydrogen trioxide, ozone, and even hydrotrioxy radical are generated in the pathway toward tion of hydrogen peroxide (Zhu et al., Proc Natl Acad Sci U S A :2247-52 (2004)). It has been shown that surface exposed tryptophans in a monoclonal anti-DR5 antibody, drozitumab (CAS number 9126288), also referred to herein as mAb1, act as substrate (1O2 and O2-) generators that facilitate ACWOP even under mild light conditions in a time and concentration dependent manner (Sreedhara et al., Mol. Pharmaceutics (2013)). It was demonstrated that mAb1 was particularly susceptible to oxidation during e under pharmaceutically relevant conditions (Sreedhara et al., Mol. Pharmaceutics (2013)).

Oxidation was shown to be site ic and zed to Trp53 (W53) on the heavy chain CDR (Fab) as evaluated by tryptic peptide mapping. Additionally, a reverse-phase HPLC assay was used to measure the total oxidation in the HC Fab and Fc regions of mAb1 via a papain digestion, DTT reduction, and reverse-phase separation. Peaks from RP-HPLC were identified using LC/MS and showed a strong correlation with results of the tryptic e map, indicating that the RP method could be used as a surrogate for detection of W53 (i.e. % Fab) oxidation hara et al., Mol. Pharmaceutics (2013)). In the RP papain digest method, Fab and Fc ion peaks eluted before their respective main peaks, allowing the quantification of % Fab and % Fc oxidation in relation to their total peak areas. The study further showed that hydrogen peroxide could serve as a surrogate for a number of ROS, ing superoxide and singlet oxygen.

To determine if limited (i.e., controlled) light exposure can be used as an accelerated stress model to study protein oxidation, the same human monoclonal IgG1 antibody (mAb1) was used to screen and evaluate ial antioxidants. L-tryptophan (LTrp ), an antioxidant used in protein formulations, has been recently shown to be photosensitive (Igarashi et al., Anal Sci 23(8):943-8 (2007)) and to have the ability to produce H2O2 upon light exposure. The sensitivity of mAb1 to L-Trp under light stress was evaluated, with and without the addition of L-methionine (L-Met) as a potential antioxidant. mAb1 was expressed in Chinese Hamster Ovary (CHO) cells and purified by a series of chromatography methods including affinity purification by protein A chromatography and ion-exchange chromatography. mAb1 was prepared at 5 mg/mL in a formulation of 20mM histidine acetate, 250mM trehalose and 0.02% polysorbate 20 in a glass vial and with 1mM L-Trp and various concentrations of L-Met, ranging from 10 mM to 100 mM, and d to eight hours of light at 250 W/m2 in an Atlas SunTest CPS+ Xenon Test Instrument (Chicago, IL). l vials were wrapped in aluminum foil and treated rly. After light exposure, solutions were prepared for is by reverse-phase HPLC. For RP-HPLC, mAb1 solution from the stress study was prepared to 1.1 mg/mL in 0.1 M Tris, 4.4 mM EDTA, and 1.1 mM cysteine. 150 µL of 0.1 mg/mL papain was added to 1.35 mL of the mAb1 solution before incubation at 37°C for two hours. Following incubation, 900 µL of the solution was combined with 100 µL of 1 M dithiothreitol (DTT) and incubated for another thirty s at 37°C. Samples were then run on an Agilent, Inc. 1100/1200 HPLC system (Santa Clara, CA) equipped with UV detection at 280 nm in conjunction with a Varian, Inc. Pursuit 3 μm, 2 mm ID x 250 mm yl column (Palo Alto, CA). Mobile Phase A was 0.1% TFA in water. Mobile Phase B was 0.1% TFA in acetonitrile. The mobile phase gradient increased linearly from 34% B at 0 minutes to 43% B at 50.0 minutes, then to 95% B at 50.1 minutes.

The gradient remained at 95% B until 60.1 minutes, and then decreased linearly from 95% B to 34% B between 60.1 and 60.2 minutes. The gradient remained at 34% B until the end of the cycle at 80.2 minutes. The column ature was 65°C, total flow rate was 0.2 mL/min, and injection volume of each sample was 6 µL. Chromatograms were then integrated for quantification of oxidation.

Additionally, mAb1 was found to be stable in L-His based buffer at pH 6.0.

Analysis of the light exposure effects of L-Trp and L-Met on mAb1 Fab ion showed that the mAb1 reference material (no light exposure) and the foil control had about 2% Fab ion (Fig. 1A). Since the foil control and the reference material showed the same level of Fab oxidation, it was unlikely that heat alone is causing oxidation of the Fab. When mAb1 was exposed to light (“No Excipient” sample), the Fab oxidation doubled to 4%. With the on of 1mM L-Trp, the Fab oxidation increased to almost 9%, suggesting that free L-Trp was generating ROS under light exposure that may have ed in oxidation of W53 on the Fab. Further addition of 10, 25, 50, and 100mM L-Met to ation containing 1 mM L- Trp appeared to reduce Fab oxidation slightly, but even 100 molar excess of L-Met did not reduce Fab oxidation to the level of the foil control (Fig. 1A).

Oxidation in the Fc region of mAb1 has been shown to be predominantly of Met residues Met 254 and Met 430 (Sreedhara et al., Mol. Pharmaceutics (2013)). Analysis of the light exposure effects of L-Trp and L-Met on mAb1 Fc oxidation showed that the mAb1 reference al and foil control had about 8% Fc ion even before exposure to light (Fig. 1B). Exposure to light resulted in only a minor increase in Fc oxidation (“No Excipient”) for mAb1 in ation buffer. However, incubation with 1mM L-Trp resulted in over 20% oxidation at these Met sites in the Fc region as seen by the RP-HPLC assay.

Addition of various trations of L-Met (10, 25, 50 and 100 mM) to formulations containing 1mM L-Trp reduced the amount of Fc oxidation, although even 100mM L-Met did not reduce Fc oxidation to the level of the controls (Fig. 1B).

It was previously reported that L-Trp ed H2O2 via superoxide ion and in a sub-stoichiometric fashion while antibodies under similar conditions were producing catalytic amounts (Wentworth et al., e 293(5536):1806-11 (2001); McCormick et al., Journal of the American Chemical Society 100:312-313 (1978)). To test the susceptibility of free L-Trp under pharmaceutically relevant conditions, such as under both ICH and ambient light conditions, formulations comprising 0.32mM to 7.5mM of L-Trp (L-Trp was dissolved in sodium phosphate buffer at pH 7.1) were exposed for 3 hours at 250W/m2 UV light and about 150k lux visible light. Samples were taken and analyzed immediately via the Amplex assay to detect the amount of H2O2 generated under these ions. A large quantity of H2O2 was generated by free L-Trp upon light exposure in a tration dependent manner (Fig. 2).

This H2O2 generation was reduced greatly in the presence of 50mM sodium azide, a known quencher of singlet oxygen (Fig. 2). When L-Trp was incubated with a combination of 50mM NaN3 and 150 U superoxide dismutase (SOD) or SOD alone, significant amounts of H2O2 were still detected in the samples not containing NaN3. This indicated that, in on to singlet oxygen, superoxide ion was also generated upon photo-irradiation that was ted to H2O2 by SOD.

While confirming the photosensitivity of free L-Trp under ICH light conditions, the effect of ambient light that was lly seen in laboratories was studied. Measurements using a DLM1 digital light meter in various labs ted an average of 300 lux on a lab benchtop (with white fluorescent lighting), an average of 3000 lux in a laminar flow hood (with white fluorescent lighting) and about 10000 lux for a sill exposed to southeast sunlight. Under these conditions, L-Trp in formulation buffers containing 50mg/mL mAb1 produced hydrogen peroxide in the micromolar range as ed using the Amplex Ultra Red assay (Fig. 3A). Peroxide production increased with both luminosity (300, 3000, and 10000 lux) and time (1, 3, and 7 days). The protein samples were further analyzed using the mAb1 ic RP-HPLC assay and showed increased heavy chain Fab oxidation corresponding to oxidation in W53 with increased sity (Fig. 3B). At the same time, % Fc ion in mAb1 under these conditions increased from 5 to 40% between 300 and 10000 lux, respectively. These levels of light re and time were determined to be pharmaceutically relevant for drug substance handling under ambient light and temperature before fill/finish ions and potentially while inspecting drug product vials. These results supported that L-Trp is photosensitive and that it produces several reactive oxygen species, including singlet oxygen, superoxide and H2O2 that can be detrimental to mAb product quality and that care should be taken while handling and storing L-Trp containing buffers.

Example 2: Screening of candidate antioxidant compounds.

Tryptophan (Trp) is an electron rich amino acid that undergoes oxidative and electrophilic addition reactions in the presence of ROS such as hydroxyl radicals and singlet oxygen. Any potential antioxidant to t Trp oxidation in proteins should have similar if not superior reactivity towards these ROS. A series of compounds that were either based on the L-Trp structure or have been reported to have antioxidant properties were evaluated.

Compounds screened for antioxidant y in this study ed derivatives of tryptophan, indole, aromatic acids such as salicylic acid and anthranilic acid, and some vitamins. The chemical structures of the various compounds used were based on (A) Tryptophan derivatives (B) Kynurenine (C) Indole derivatives and (D) Aromatic acid tives: (A) Tryptophan Derivatives Name R X A L-Tryptophan COOH H H -Hydroxy-Tryptophan COOH OH H -Methoxy-Tryptophan COOH OCH3 H -Amino-Tryptophan COOH NH2 H -Fluoro-Tryptophan COOH F H N-Acetyl-Tryptophan COOH H CH3C(O) Tryptamine H H H Tryptophanamide CONH2 H H Serotonin H OH H Melatonin H OCH3 CH3C(O) (B) Kynurenine NH2 O (C) Indole Derivatives Name Y2 Y3 Y4 Y5 Y7 Indole H H H H H IndoleAcetic Acid H CH2COOH H H H 4-Hydroxy Indole H H OH H H -Hydroxy Indole H H H OH H oxy IndoleAcetic Acid H CH2COOH H OH H 7-Hydroxy Indole H H H H OH 7-Hydroxy IndoleCarboxylic Acid COOH H H H OH (D) ic Acid Derivatives HO O Name Z1 Z2 Salicylic Acid OH H -Hydroxy Salicylic Acid OH OH Anthranilic Acid NH2 H oxy Anthranilic Acid NH2 OH Candidate antioxidant compounds obtained from a photosensitivity screening assay.

While L-Trp may have been an effective antioxidant under certain circumstances, its photosensitivity may limit its utility during normal processing without special precautions.

Hence the photosensitivity of the above molecules was investigated and the les were rated for their H2O2 generation lity with respect to L-Trp. As a ing tool, antioxidant candidates were exposed to light for four hours at 250 W/m2 and the resulting H2O2 formation was quantified by the Amplex Ultra Red assay. Specifically, antioxidants were ed to 1 mM in 20 mM histidine acetate buffer at pH 5.5. The 1 mM antioxidant solutions were aliquoted into glass vials (2 mL/glass vial) and exposed to four hours of light at 250 W/m2 in an Atlas SunTest CPS+ Xenon Test Instrument (Chicago, IL). Total UV dose was 90 watt-hours/square meter and total visible dose was 0.22 million lux hours over the 4- hour period. Control vials were wrapped in aluminum foil and d similarly. The amount of hydrogen peroxide generated after exposure to light was ed using the Amplex® Ultra Red Assay (Invitrogen, Carlsbad, CA) following the cturer’s recommended procedure. On addition of horseradish peroxidase (HRP), the dye reacted 1:1 stoichiometrically with H2O2, resulting in the tion of fluorescent oxidation product resorufin. In this study, fluorescence readings were obtained using a Spectra Max M2 Microplate Reader (Molecular s, Sunnyvale, CA) with excitation and emission set at 560 nm and 590 nm, tively. Final H2O2 concentrations were determined using a standard curve ranging from 0 μm to 20 μm.

Analysis of hydrogen peroxide (H2O2) generation by tryptophan derivatives upon light exposure showed that under r conditions of light (corresponding to 0.22 million lux hours over a 4-hour period) and buffer (20mM L-His-acetate, pH 5.5), 5-hydroxy-L- tryptophan (5-HT) produced about one tenth of the H2O2, while kynurenine produced about one fifth of the H2O2, when compared to L-Trp (Fig. 4A). Other tryptophan derivatives produced anywhere between 30% and 105% of the H2O2 produced by L-Trp. In comparison to L-Trp, Trolox (a water soluble Vitamin E derivative) produced 123 times more H2O2, and pyridoxine (Vitamin B6) produced 5 times more H2O2 (Table 1). Indole, which has a basic structure like L-Trp, behaved similarly to L-Trp, but indoleacetic acid ed twice as much H2O2 (Fig. 4B). The y derivatives of indole behaved like 5-HT in that they produced negligible amounts of H2O2 upon light exposure. Several biochemically relevant derivatives of L-Trp, namely tryptamine, serotonin and melatonin were also tested.

Tryptamine produced about half as much H2O2 as L-Trp (Table 1). stingly, serotonin roxytryptamine) behaved much like the 5-OH derivatives of indole and phan, producing very little H2O2 upon light exposure, while melatonin (N-acetyl methoxytryptamine) produced less than a third of the H2O2 produced by L-Trp (Table 1).

Table 1: Hydrogen Peroxide Production Ratio between Tested Compounds and L-Trp (H2O2 produced by Compound)/(H2O2 produced Compound by L-Trp) L-Trp 1 L-Trpamide 0.43 N-Acetyl-L-Trp 0.31 N-Acetyl-L-Trpamide 0.34 -Fluoro-L-Trp 0.71 -Hydroxy-L-Trp 0.09 -Methoxy-DL-Trp 1.05 -Amino-DL-Trp 0.29 L-Kynurenine 0.20 Trolox 122.75 Pyridoxine 5.16 Indole 0.95 IndoleAcetic Acid 2.40 4-Hydroxyindole 0.00 -Hydroxyindole -0.08 -HydroxyindoleAcetic 0.11 Acid 7-Hydroxyindole -0.03 7-HydroxyindoleCarboxylic 0.15 Acid Tryptamine 0.53 Serotonin (5- 0.03 Hydroxytryptamine) Melatonin (N-Acetyl 0.28 Methoxytryptamine) lic Acid 0.03 -Hydroxysalicylic Acid 0.84 Anthranilic Acid 2.50 oxyanthranilic Acid 0.44 In order to understand the ROS formed during photo-irradiation, several of the Trp derivatives in the presence of 50 mM NaN3, a known singlet oxygen quencher, were tested under light exposure as described above. All the nds tested showed a substantial decrease in the amount of hydrogen peroxide generated under these conditions, indicating that singlet oxygen was a major ROS d upon irradiation of Trp and its derivatives (Fig. 5).

Other aromatic compounds such as salicylic acid and derivatives were also tested based on their reported antioxidant properties (Baltazar et al., Curr Med Chem 18(21):3252- 64 (2011)). Salicylic acid produced very little H2O2 upon light exposure while its 5-OH derivative behaved like L-Trp (Table 1). On the other hand, anthranilic acid produced twice as much H2O2 as L-Trp but 5-OH-anthranilic acid produced half as much H2O2 compared to L-Trp (Table 1).

Candidate antioxidant nds ed from a CV screening assay.

Based on the results from the photosensitivity screening assay, compounds with aromatic ring substitutions appeared to impact the amount of hydrogen peroxide generated.

Since the goal was preferential oxidation of the excipient rather than the protein drug, excipients that had low oxidation potentials may have served as effective antioxidants. The oxidation/reduction characteristics of the compounds were investigated. Several compounds, including L-Trp and derivatives, were evaluated for the protection of Trp oxidation in proteins using cyclic voltammetry (CV) and rank ordered based on their oxidation potentials (Table 2). ically, the candidate antioxidants were dissolved in zed water and then added to a 0.2 M lithium perchlorate electrolyte solution. Solutions were characterized with an EG&G Princeton Applied Research Model 264A Polarograph/Voltammeter with a Model 616 RDE Glassy Carbon Electrode as working ode. Solutions were scanned from -0.10 V to +1.50 V at a scan rate of either 100 or 500 mV/sec. The analytical cell was purged for four minutes with nitrogen before scanning of each antioxidant solution. The input was a linear scan of the potential of a working electrode, and the output was measurement of the resulting current. As the ial was d (linearly increased or decreased), electrochemically active species in the CV cell underwent oxidation and reduction reactions at the surface of the working ode that resulted in a current which was continuously measured. Redox reactions were characterized by sharp increases or ses in current (peaks). The potential at which an oxidation reaction occurred was referred to as the anodic peak potential (or oxidation potential), and the potential at which a reduction occurred was referred to as the cathodic peak (or reduction) potential.

The oxidation potentials of the excipients in this study ranged from 0.410 to 1.080 V vs Ag/AgCl (Table 2). Under these conditions, L-Trp had an irreversible ion potential of 0.938 V vs Ag/AgCl. Nine compounds were found to have a lower oxidation potential than L-Trp, including all of the 5-OH compounds which had ion potentials between 0.535 and 0.600 V vs Ag/AgCl. Of all the compounds tested, 5-amino-DL- tryptophan had the lowest oxidation potential at 0.410 V, while the N-acetyl compounds (0.730-0.880 V), and 5-methoxy-DL-tryptophan (0.890 V) were also below L-Trp. Seven nds had higher oxidation potential than L-Trp (Table 2). These were indoleacetic acid, 5-fluoro-L-tryptophan, tryptamine, tophanamide, L-kynurenine, o-DL- phan, and salicylic acid. Salicylic acid had the highest oxidation potential in this study (1.080 V vs Ag/AgCl). All the tested compounds showed non-reversible CV indicating that once oxidized, the species did not tend to receive electrons and probably could not be involved in further electrochemical reactions.

Table 2: ion Potentials of Excipients Oxidation Potential (V vs Compound Ag/AgCl) -amino-DL-tryptophan 0.410 -hydroxyindoleacetic acid 0.535 -hydroxy-L-tryptophan 0.565 -hydroxyindole 0.580 Serotonin HCl (5-hydroxytryptamine 0.600 HCl) Melatonin (N-acetyl 0.730 methoxytryptamine) N-acetyl-L-tryptophan 0.875 N-acetyl-L-tryptophanamide 0.880 -methoxy-DL-tryptophan 0.890 L-tryptophan 0.938 Indoleacetic acid 0.948 -fluoro-L-tryptophan 0.965 Tryptamine HCl 1.010 L-tryptophanamide 1.015 L-kynurenine 1.040 -nitro-DL-tryptophan 1.055 Salicylic acid 1.080 Oxidation c peak) potentials were measured using cyclic voltammetry with a glassy carbon working electrode in 0.2 M lithium perchlorate.

A correlation was ined between oxidation potential and light-induced H2O2 tion for 16 compounds that had oxidation potentials above and below the oxidation potential of L-Trp, and H2O2 production levels above and below that of L-Trp (Fig. 6). Since indole and tryptophan behaved similarly in H2O2 production under light exposure, it was le that substitutions on the C3 position of the 5 membered ring did not affect this property. However, tryptamine with a –CH2CH2NH2 substitution and indoleacetic acid with a OH substitution at the C3 position produced two times less and two times more H2O2, respectively, than L-Trp. These data indicated that the C3 substitutions played a role in photo-activation and peroxide generation. The C3 tutions did not affect the oxidation potentials of the molecules, whereas indole per se had significantly lower oxidation potential than L-Trp under these experimental conditions. Substitutions at the C5 of the 6- ed aromatic ring behaved quite predictably. In general, nds with electron donating groups such as –NH2 and –OH had lower oxidation potentials than their parent compounds and also showed low levels of H2O2 production upon photo-activation (e.g. 5- amino-DL-tryptophan, 5-hydroxyindoleacetic acid, 5-hydroxy-L-tryptophan, 5- hydroxyindole, serotonin). Similarly, compounds with high oxidation potential produced more H2O2 hoxy-DL-tryptophan, L-Trp, indoleacetic acid, 5-fluoro-L-tryptophan) under these conditions. There were exceptions to this correlation; some nds had high oxidation potential but did not produce much H2O2 (e.g. salicylic acid and L-kynurenine) indicating that there were potentially other mechanisms that played an important role for these six membered aromatic compounds that may not have been observed with compounds containing the indole backbone of L-Trp. The area of interest was the quadrant which contained compounds with lower oxidation potential and lower H2O2 production upon light exposure than L-Trp (Fig. 6, dashed box). Compounds with these two qualities were considered as new candidate antioxidants because they could (1) oxidize faster than Trp on the protein and (2) produce very little H2O2 during long term storage and/or ambient processing during drug product tion and therefore could protect the protein from r oxidation under these conditions.

Example 3: Candidate antioxidant compounds d oxidation of monoclonal antibody formulations.

Compounds that, ed to L-Trp, produced less H2O2 upon light ent as well as those with lower oxidation potentials than L-Trp were chosen for evaluation of their possible antioxidant ties using AAPH, light, and Fenton reaction as oxidative stress models (Table 3). mAb1 was used as a model protein to evaluate the effectiveness of select candidate antioxidants to protect against Trp oxidation by the ent oxidation stress models. Each stress model produced ion through a different mechanism and therefore each was valuable in the assessment of biopharmaceutical stability. AAPH, or 2,2’-Azobis(2- Amidinopropane) Dihydrochloride, is used as a stress model to mimic alkyl peroxides potentially generated from formulation excipients such as degraded polysorbate.

Decomposition of AAPH generates alkyl, alkoxyl, and alkyl peroxyl ls that have been shown to e amino acid residues in proteins, including methionine, tyrosine, and tryptophan residues (Ji et al., J Pharm Sci 98(12):4485-500 ; Chao et al., Proc Natl Acad Sci U S A 94(7):2969-74 (1997)). Similarly, controlled light could be used as a stress model to mimic ambient light exposure that drugs may experience during processing and storage. Light-induced oxidation of rmaceuticals was shown to d through a singlet oxygen (1O2) and/or superoxide anion (O2-) ism (Sreedhara et al., Mol.

Pharmaceutics (2013)). The Fenton reaction is also commonly used as an oxidative stress model. This mixture of H2O2 and Fe ions generates oxidation through a metal catalyzed, hydroxyl radical mechanism (Prousek et al., Pure and Applied Chemistry 79(12):2325-2338 (2007)), and is used to model metal residue from stainless steel surfaces used in drug manufacturing and storage.

Table 3: ion Stress Models Stress Model Mechanism Purpose AAPH Alkyl peroxides, alkyl radical Mimic alkyl peroxides from catalyzed degraded polysorbate Light Singlet oxygen (1O2), superoxide Mimic t light exposure anion (O2-), during processing and storage H2O2 Fenton (H2O2 + Hydroxyl l, metal catalyzed Mimic metal residue from Fe) stainless steel surfaces Tryptophan (W53) oxidation on mAb1 was thoroughly characterized previously using a RP-HPLC and LC-MS method (Sreedhara et al., Mol. Pharmaceutics ).

Briefly, mAb1 was digested with papain to generate Heavy Chain (HC) Fab, HC Fc, and Light Chain nts. The fragments were reduced with DTT, and then separated and identified via Liquid Chromatography-Mass Spectrometry (LC-MS). Oxidized versions of the HC Fab and HC Fc were found to elute earlier than their native counterparts. Comparison of area integrated under the oxidized and native peaks was used to quantify HC Fab and Fc oxidation. In addition, LC-MS/MS peptide maps (by trypsin digestion and by Lys-C digestion) showed that oxidation of the HC Fab was primarily of a Trp residue, W53, while oxidation of the HC Fc was attributed predominantly to oxidation of two Met residues, M254 and M430. By using the papain digest RP-HPLC method in the present study it was possible to investigate Trp residue oxidation by quantifying HC Fab oxidation, and Met residue oxidation by quantifying HC Fc oxidation.

% Fab oxidation and % Fc oxidation were calculated as follows (note that each antibody molecule has two Fabs; therefore, the % Fab oxidation ed did not reflect the % oxidized intact antibody containing Fab oxidation): Oxidized Fab Peak Area % Fab Oxidation = 100× Fab Peak Area + Oxidized Fab Peak Area Oxidized Fc Peak Area % Fc Oxidation =100× Fc Peak Area + Oxidized Fc Peak Area For the mAb1 light stress study, mAb1 was prepared to 5 mg/mL in a formulation of 20 mM histidine acetate pH 6.0, 250 mM trehalose, and 0.02% Polysorbate 20.

Antioxidants were added at 1 mM (final concentration) from 10 mM stock ons prepared in 20 mM ine acetate pH 6.0, 250 mM trehalose, and 0.02% Polysorbate 20. The ion was L-Met which was added to a final concentration of 1, 10, 25, 50, and 100 mM from a stock on of 200 mM L-Met in the same buffer (i.e., 20 mM histidine acetate pH 6.0, 250 mM trehalose, and 0.02% rbate 20). Glass vials ning these formulations were exposed to 250 W/m2 light in an Atlas SunTest CPS+ Xenon Test Instrument (Chicago, IL) at ambient temperature. Control vials were wrapped in aluminum foil and d similarly. After light exposure, solutions were prepared for analysis by reverse-phase HPLC as described above.

For the mAb1 AAPH stress study, mAb1 was prepared to 4 mg/mL in a formulation of 20 mM histidine acetate pH 6.0, 250 mM trehalose, and 0.02% Polysorbate 20. idants were added at 1 mM (final concentration) from 10 mM stock solutions prepared in 20 mM histidine acetate pH 6.0, 250 mM trehalose, and 0.02% Polysorbate 20. 200 µL of mM AAPH was added to 2 mL of each mAb1 solution and then incubated at 40°C for 24 hours. After incubation, each on was buffer exchanged with formulation buffer (20 mM histidine acetate pH 6.0, 250 mM trehalose, and 0.02% Polysorbate 20) using a PD-10 column so that the final mAb1 tration was 2.3 mg/mL. After buffer exchange, each solution was prepared for analysis by reverse-phase HPLC as described above.

For the mAb1 Fenton stress study, mAb1 was prepared to 3 mg/mL in a formulation of 20 mM histidine hydrochloride pH 6.0. Antioxidants were added at a final concentration of 1 mM from 10 mM stock solutions prepared in 20 mM histidine hydrochloride. A final concentration of 0.2 mM FeCl3 and 10 ppm H2O2 were added to each mAb1 solution and then incubated at 40°C for 3 hours. After incubation, each reaction was quenched by addition of 100 mM L-Met (prepared from a stock solution of 200 mM L-Met in 20 mM histidine hydrochloride) and then ed for analysis by reverse-phase HPLC as described above.

It was determined that incubation of mAb1 with AAPH for 24 hours at 40°C resulted in 27% Fab (Trp residue) oxidation (Fig. 7A) and 47% Fc (Met residue) oxidation (Fig. 7B). Seven excipients that had been previously screened using light stress and cyclic voltammetry were incubated with mAb1 under the AAPH conditions to evaluate antioxidant capabilities. Six of the seven compounds were found to significantly reduce AAPH-induced Fab oxidation (Fig. 7A). All six of these compounds contained the indole backbone.

Moreover, all the hydroxy derivatives tested roxy-L-Trp, 5-hydroxyindole, 7- hydroxyindole, and serotonin) reduced Fab oxidation to close to l levels (about 2%).

Meanwhile, salicylic acid had almost no effect on Fab oxidation under AAPH stress. None of the excipients appeared to impact the level of AAPH-induced Fc oxidation (Fig. 7B).

For the light stress study, mAb1 was exposed to 16 hours of light at 250 W/m2 while testing the aforementioned seven excipients (Fig. 8). Exposure of mAb1 to light (“No ent”) increased Fab oxidation 3.5 times over the control level (“mAb1 Ref Mat”, Fig. 8A). It was usly shown that L-Trp could protect against Trp oxidation in the model protein Parathyroid Hormone (PTH) (Ji et al., J Pharm Sci 98(12):4485-500 (2009)).

However, this study found that addition of 1mM L-Trp to mAb1 sed the Fab ion over 11-fold, probably through the production of ROS such as singlet oxygen by lightexposed L-Trp (Fig. 2). Addition of the hydroxy compounds (5-hydroxy-L-Trp, 5- hydroxyindole, 7-hydroxyindole, and serotonin) protected against light-induced Fab oxidation, reducing Fab oxidation to near control levels (Fig. 8A). On the other hand, salicylic acid med similarly to tophanamide, increasing Fab oxidation 8-fold over the control level. Similar results were observed for Fc oxidation under light stress (Fig. 8B). Light exposure of mAb1 ed in a 40% increase in Fc oxidation over the control level, whereas addition of L-Trp increased Fc oxidation to 7 times the control level.

Compared to the control (no excipient), L-Tryptophanamide and salicylic acid also resulted in more Fc oxidation. The hydroxy nds produced similar Fc ion as the no excipient control ially because they produce much fewer ROS than L-Trp under light re. The light screening and NaN3 study results in Example 2 showed a good correlation between the amount of H2O2 generated by an excipient and Fc Met oxidation of mAb1.

The Fenton reaction, using a mixture of H2O2 and Fe ions, generates oxidation through a metal catalyzed, hydroxyl l reaction (Prousek et al., Pure and Applied Chemistry 79(12):2325-2338 (2007)). This on generated Fab, i.e. tryptophan, ion in mAb1. The reaction was also carried out in the presence of select antioxidants that were useful against both AAPH and light induced oxidation as reported above. Data d to the antioxidant ties against Fenton mediated reaction were analyzed using the RP-HPLC assay as described above (Fig. 9). Under the conditions tested, the Fenton reaction produced about four times the oxidation in the Fab region of mAb1 over the control. Most of the antioxidants , except salicylic acid, showed similar hydroxyl radical quenching properties to L-Trp, which protected the Fab oxidation by about 25% with respect to the no ent case (Fig. 9A). In regards to protection against Fc oxidation, the tested excipients (other than salicylic acid) med slightly better than L-Trp (Fig. 9B).

Electron donating substitutions on the aromatic ring may facilitate formation of the indolyl radical cation and potentially faster reactivity with radicals. Hydroxyl groups ed to the aromatic rings are electron donors as the oxygen atom has a lone pair of electrons that can be involved in resonance structure leading to lower oxidation potentials and potentially more susceptibility to electrophilic attack. As seen in Table 2, the hydroxyl tutions led to substantially lower oxidation potentials indicating these compounds could make better antioxidants than L-Trp and/or indole. The hydroxyl substituted indole and Trp derivatives also produced the least amount of en peroxide upon light irradiation (Table 1). This could have been due to the low quantum efficiency of these molecules in transferring light energy to molecular , coupled with their high quenching constants as demonstrated for -hydroxy-L-tryptophan (Dad et al., J Photochem Photobiol B, 78(3):245-51 (2005)). As shown in Fig. 7 and Fig. 8, the hydroxyl substituted indole and tryptophan tives provided considerable protection against AAPH, light and Fenton induced ion to W53 in the Fab region of mAb1. r, none of these compounds provided substantial antioxidant protection to Met oxidation in the Fc region of mAb1 in the AAPH induced degradation. In contrast, the indole and tryptophan derivatives behaved as expected under light mediated oxidation. Molecules that produced higher amounts of peroxide upon photoactivation (e.g., L-Trp and Trp-amide) also produced higher Met oxidation in the Fc region of mAb1, while the –OH derivatives produced lower H2O2, and also the lowest amount of Met oxidation in the Fc region under photo-oxidation conditions. Methionine was readily oxidized to methionine sulfoxide by H2O2 and alkyl peroxides through a nucleophilic substitution reaction (Li et al., Biotechnology and Bioengineering 48:490-500 (1995)). Photooxidation of methionine to methionine sulfoxide occurs via singlet oxygen, though this reaction occurs via a different intermediate (Li et al., Biotechnology and Bioengineering 48:490-500 (1995)). AAPH degrades under thermal stress to give both alkyl des and alkoxyl radicals that have different reactivity towards Met and Trp respectively (Werber et al., J Pharm Sci 100(8):3307-15 ). Previous s have shown that L-Trp was able to prevent Trp oxidation in PTH induced by AAPH and that L-Trp did not prevent Met oxidation in PTH under the same conditions (Ji et al., J Pharm Sci 98(12):4485-500 (2009)). rly, L-Met was able to protect PTH against AAPH induced oxidation, but did not t Trp oxidation. These observations were in line with the reaction mechanisms wherein Met oxidation is predominantly via nucleophilic substitution reactions whereas Trp oxidation is mainly via free radical mechanisms.

A putative mechanism of photoactivation of L-Trp leading to singlet oxygen and tely to H2O2 and the formation and quenching of singlet oxygen by 5-HT is shown in Fig. 10.

Claims (36)

1. A liquid formulation comprising a protein and a compound which prevents oxidation of the n in the liquid formulation, wherein the compound is selected from the group consisting of N-acetyl-L-trytophan, N-acetyl-L-tryptophanamide, 5-amino-DL- tryptophan, and 5-hydroxyindoleacetic acid, or a pharmaceutically acceptable salt thereof.

2. The formulation of claim 1, which is a pharmaceutical formulation le for administration to a subject.

3. The formulation of claim 1 or 2 which is aqueous.

4. The formulation of any one of claims 1-3, wherein the compound in the formulation is from 0.3 mM to 5 mM.

5. The ation of any one of claims 1-4, wherein the nd in the formulation is from 0.3 mM to 1 mM.

6. The formulation of any one of claims 1-5, wherein the compound prevents oxidation of tryptophan, cysteine, histidine, tyrosine, and/or methionine in the protein.

7. The formulation of any one of claims 1-6, wherein the compound ts oxidation of the protein by a ve oxygen species.

8. The formulation of claim 7, wherein the reactive oxygen species is selected from the group consisting of singlet oxygen, a superoxide (O2-), hydrogen peroxide, a yl radical, and an alkyl peroxide.

9. The formulation of any one of claims 1-8, wherein the protein is susceptible to oxidation.

10. The ation of any one of claims 1-9, wherein tryptophan in the protein is susceptible to oxidation.

11. The formulation of any one of claims 1-10, wherein the protein is an dy.

12. The formulation of claim 11, wherein the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, or antibody nt.

13. The formulation of any one of claims 1-12, wherein the protein concentration in the formulation is 1 mg/mL to 250 mg/mL.

14. The formulation of any one of claims 1-13, which further comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, a surfactant, and a tonicity agent.

15. The formulation of any one of claims 3-14, wherein the formulation has a pH of 4.5 to 7.0.

16. The formulation of any one of claims 1-15, wherein the compound is N-acetyl-L- tryptophan.

17. A method of making a protein formulation comprising adding a compound that prevents oxidation of a protein to the protein ation, n the compound is selected from the group consisting of N-acetyl-L-tryptophan, N-acetyl-L- phanamide, o-DL-tryptophan, and 5-hydroxyindoleacetic acid, or a ceutically acceptable salt thereof.

18. A method of preventing oxidation of a protein in a protein formulation comprising adding a compound that prevents oxidation of the protein to the formulation, wherein the compound is selected from the group consisting of N-acetyl-L-tryptophan, N- -L-tryptophanamide, 5-amino-DL-tryptophan, 5-hydroxyindoleacetic acid and melatonin, or a pharmaceutically acceptable salt thereof.

19. The method of claim 17 or 18, wherein the protein formulation is a ceutical formulation suitable for administration to a subject.

20. The method of any one of claims 17-19, which is aqueous.

21. The method of any one of claims 17-20, wherein the compound in the formulation is from 0.3 mM to 5 mM.

22. The method of any one of claims 17-21, wherein the compound in the formulation is from 0.3 mM to 1 mM.

23. The method of any one of claims 17-22, wherein the compound prevents oxidation of tryptophan, cysteine, histidine, ne, and/or methionine in the protein.

24. The method of any one of claims 17-23, wherein the compound prevents oxidation of the protein by a reactive oxygen s.

25. The method of claim 24, wherein the reactive oxygen species is selected from the group consisting of singlet , a superoxide (O2-), hydrogen peroxide, a hydroxyl radical, and an alkyl peroxide.

26. The method of any one of claims 17-25, wherein the protein is susceptible to oxidation.

27. The method of any one of claims 17-26, wherein tryptophan in the n is susceptible to oxidation.

28. The method of any one of claims 17-27, wherein the protein is an antibody.

29. The method of claim 28, wherein the antibody is a onal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric dy, or antibody fragment.

30. The method of any one of claims 17-29, wherein the protein concentration in the formulation is 1 mg/mL to 250 mg/mL.

31. The method of any one of claims 17-30, wherein the formulation further comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, a surfactant, and a tonicity agent.

32. The method of any one of claims 20-31, wherein the formulation has a pH of 4.5 to 7.0.

33. The method of any one of claims 17-32, wherein the nd is N-acetyl-L- tryptophan.

34. A liquid formulation as claimed in any one of claims 1-16, substantially as herein described with reference to any example thereof, and to the accompanying drawings.

35. A method as claimed in any one of claims 17, and 19-33 of making a protein formulation, substantially as herein described with reference to any example thereof, and to the anying drawings.

36. A method as claimed in any one of claims 18 to 33 of preventing oxidation of a protein in a protein formulation, ntially as herein described with reference to any example thereof, and to the accompanying drawings. PCT/U