WO2010080494A1

WO2010080494A1 - Methods for identifying degradation products in a polypeptide sample

Info

Publication number: WO2010080494A1
Application number: PCT/US2009/068462
Authority: WO
Inventors: Susan Park; Enona Gopinath
Original assignee: Tercica, Inc.
Priority date: 2008-12-19
Filing date: 2009-12-17
Publication date: 2010-07-15

Abstract

The present disclosure provides methods of detecting degradation products of one or more polypeptides in a formulation comprising the one or more polypeptides.

Description

METHODS FOR IDENTIFYING DEGRADATION PRODUCTS IN A POLYPEPTIDE SAMPLE

CROSS-REFERENCE [0001] This application claims the benefit of U.S. Provisional Patent Application No.

61/139,355, filed December 19, 2008, which application is incorporated herein by reference in its entirety.

BACKGROUND [0002] Monitoring the product purity profile for lot release and stability studies typically requires several orthogonal techniques to address different modes of degradation and, for small proteins, some type of chromatographic method is often the primary stability indicating assay.

In the case of a product with two active ingredients that are well-characterized proteins, the number of assays needed can be overwhelming for routine testing. [0003] There is a need in the art for methods of monitoring the purity profile of a polypeptide formulation.

Literature [0004] U.S. Patent No. 6,756,484; and Gellerfors et al. (1990) Acta Paediatr. Scand. Suppl.

370:93.

SUMMARY OF THE INVENTION

[0005] The present disclosure provides methods of detecting degradation products of one or more polypeptides in a sample comprising the one or more polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 presents an alignment of human growth hormone amino acid sequences.

DEFINITIONS

[0007] The terms "polypeptide" and "protein," used interchangeably herein, refer to a polymeric form (of any length) of amino acids, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term "polypeptide" includes synthetic polypeptides, semi-synthetic polypeptides, recombinant polypeptides, fusion proteins, and the like. The term "polypeptide" includes polypeptides modified with one or more non-amino acid moieties, where such moieties include, e.g., carbohydrates, amide groups, lipids, myristic acid, palmitic acid, phosphate groups, and the like.

[0008] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, 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, since the scope of the present invention will be limited only by the appended claims.

[0009] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0010] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0011] It must be noted that as used herein and in the appended claims, the singular forms "a,"

"an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a plurality of such polypeptides and reference to "the degradation product" includes reference to one or more degradation products and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0012] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

[0013] The present disclosure provides methods of detecting degradation products of one or more polypeptides in a sample comprising the one or more polypeptides. The present disclosure provides methods of detecting degradation products of insulin-like growth factor- 1 (IGF-I) and growth hormone (GH) in a sample comprising both IGF-I and GH.

[0014] A subject method involves applying a liquid sample comprising a polypeptide onto an ultra performance liquid chromatography (UPLC) column. The liquid sample can include one, two, three, four, five, or more different polypeptides, and will in some embodiments include two different polypeptides. In some embodiments, the one or more polypeptides in the liquid sample each has a molecular weight in the range of from about 5 kDa to about 100 kDa, or more than 100 kDa, e.g., from about 5 kDa to about 10 kDa, from about 10 kDa to about 25 kDa, from about 25 kDa to about 50 kDa, from about 50 kDa to about 100 kDa, or more than 100 kDa. In some embodiments, the polypeptide(s) each have a molecular weight in the range of from about 5 kDa to about 25 kDa.

[0015] A subject method provides for quantitative and/or qualitative determination of the presence of one or more degradation products of a polypeptide of interest in a sample. A "polypeptide of interest" will in some embodiments be a "native" polypeptide. A "polypeptide of interest" will in some embodiments be a naturally-occurring polypeptide. A "polypeptide of interest" will in some embodiments be referred to as a "parent" polypeptide, e.g., a desired polypeptide from which degradation product(s) arise. In some embodiments, a polypeptide of interest is a recombinant polypeptide. In some embodiments, a polypeptide of interest is a synthetic polypeptide. A polypeptide of interest can be a biologically active polypeptide that is included in a formulation intended for therapy, e.g., administration to an individual in need thereof.

[0016] Degradation products of a "parent" polypeptide of interest that can be identified using a subject method include, but are not limited to: a) enzymatic cleavage product(s) of a polypeptide; b) deamidated products of a polypeptide(s); c) modified products that contain a derivatized aspartic acid that is characterized by a cyclized imide; d) oxidized products; e) de- glycosylated products, e.g., where the parent polypeptide of interest is glycosylated; f) de- lipidated products, e.g., where the parent polypeptide of interest is lipidated; g) de- phosphorylated products, e.g., where the parent polypeptide of interest is phosphorylated; h) racemized polypeptides; i) mis-folded (e.g., folded into a non-native conformation) polypeptides; j) cyclized polypeptides; k) hydrolyzed polypeptides; 1) clipped products, etc. In some embodiments, the degradation product is missing from one to about 10 amino acids from the amino terminus and/or the carboxyl terminus, compared to the amino acid sequence of the parent polypeptide of interest.

[0017] As noted above, a liquid sample comprising one or more polypeptides of interest (and possibly one or more degradation products) is applied to a UPLC reverse-phase chromatography column. The one or more polypeptides of interest and the one or more degradation products (if present) are separated from one another. Peaks corresponding to the one or more polypeptides of interest and the one or more degradation products (if present) are generated. The separation of the one or more polypeptides of interest and the one or more degradation products (if present) is carried out in a single step, e.g., without the requirement for any other chromatographic or other separation steps. In addition to providing for identification and quantification of degradation products, a subject method provides for separation of the one or more polypeptides of interest from one another.

[0018] A non-limiting example of a suitable UPLC column is the Waters Aquity UPLC

BEH300 C18 1.7μm 2.1xl50mm (part 186003687) column. The detector is ultraviolet at 214 nm. See, e.g., Wales et al. (2008) Anal. Chem. 80:6815.

[0019] A UPLC column with particle sizes less than about 2 μm can be operated at a pressure of up to 1,000 Bar, and a flow rate of about 5 ml/minute. For example, the particle size can be from about 1.0 μm to about 1.5 μm, from about 1.5 μm to about 1.7 μm, or from about 1.7 μm to about 2.0 μm. The flow rate can be from about 0.2 milliliter/minute (ml/min) to about 0.5 ml/min, from about 0.5 ml/min to about 0.7 ml/min, from about 0.7 ml/min to about 0.8 ml/min, from about 0.8 ml/min to about 1.0 ml/min, from about 1 ml/min to about 2 ml/min, from about 2 ml/min to about 3 ml/min, or from about 3 ml/min to about 5 ml/min. A UPLC column can be operated at a pressure of from about 7000 pounds per square inch (psi) to about 7500 psi, from about 7500 psi to about 10,000 psi, from about 10,000 psi to about 11,000 psi, from about 11,000 psi to about 12,000 psi, from about 12,000 psi to about 13,000 psi, from about 13,000 psi to about 14,000 psi, or from about 14,000 psi to about 15,000 psi. A column can have a variety of dimensions, where the following are exemplary and are given as inner diameter x length: 2.1 mm (inner diameter) x 30 mm (length), 2.1 mm x 50 mm, 2.1 mm x 100 mm, 2.1 mm x 150 mm, etc. Exemplary conditions are: flow rate, 0.5 ml/min and 14,500 psi and particle size 1.7 μm. [0020] The column temperature during the separation process can be from about 2⁰C to about

6O⁰C, e.g., from about 2⁰C to about 5⁰C, from about 5 ⁰C to about 15⁰C, from about 15 ⁰C to about 25⁰C, from about 25⁰C to about 30 ⁰C, from about 3O⁰C to about 4O⁰C, from about 4O⁰C to about 50 ⁰C, or from about 50 ⁰C to about 6O⁰C. In one exemplary embodiment, the column temperature during the separation process is 52⁰C.

[0021] In some embodiment, the liquid sample that is applied to the UPLC column is at ambient temperature (e.g., about 22⁰C). Below are exemplary running (separating) conditions.

[0022] Mobile Phase A: 80% (25mM Sodium Phosphate pH 7.0) 20% Acetonitrile

[0023] Mobile Phase B: 100% Acetonitrile.

Table

[0024] The run time is from about 10 minutes to about 80 minutes, e.g., from about 10 minutes to about 15 minutes, from about 15 minutes to about 20 minutes, from about 20 minutes to about 25 minutes, from about 25 minutes to about 30 minutes, from about 30 minutes to about 40 minutes, from about 40 minutes to about 50 minutes, from about 50 minutes to about 60 minutes, from about 60 minutes to about 70 minutes, or from about 70 minutes to about 80 minutes. In some embodiments, the run time is about 60 minutes.

[0025] After the sample is run through the column, the area under the curve (AUC) of each peak is calculated. The AUC calculation can be performed manually, or can be calculated electronically, e.g., by a machine.

[0026] To quantitate the amount of degradation product(s) present in a liquid sample comprising a polypeptide of interest and one or more degradation products, the AUC of each peak is calculated. The AUC of a peak corresponding to a first degradation product (DPI), e.g., AUC_DPI is divided by the total AUC of all peaks (AUC_totai), and multiplied by 100. The percent of any other degradation product (e.g., a second degradation product (DP2), a third degradation product (DP3), a fourth degradation product (DP4), etc.) is calculated in a manner analogous to that given above for DPI. [0027] In some embodiments, a standard curve is generated, and the standard curve is used to calculate the percent by weight (e.g., percent of the total protein by weight) of a particular degradation product. The percent by weight of a first degradation product is calculated using data from the standard curve. The percent by weight of any other degradation product (e.g., a second degradation product (DP2), a third degradation product (DP3), a fourth degradation product (DP4), etc.) is calculated in a manner analogous to that for DPI. Polypeptides of interest

[0028] Exemplary polypeptides of interest include, e.g., an interferon (e.g., IFN-γ, IFN-α, IFN- β, IFN-ω; IFN-τ); an insulin (e.g., Novolin, Humulin, Humalog, Lantus, Ultralente, etc.); an erythropoietin (e.g., Procrit®, Eprex®, or Epogen® (epoetin-α); Aranesp® (darbepoietin-α); NeoRecormon®, Epogin® (epoetin- β); and the like); an antibody (e.g., a monoclonal antibody) (e.g., Rituxan® (rituximab); Remicade® (infliximab); Herceptin® (trastuzumab); Humira™ (adalimumab); Xolair® (omalizumab); Bexxar® (tositumomab); Raptiva™ (efalizumab); Erbitux™ (cetuximab); and the like), including an antigen-binding fragment of a monoclonal antibody; a blood factor (e.g., Activase® (alteplase) tissue plasminogen activator; NovoSeven® (recombinant human factor Vila); Factor Vila; Factor VIII (e.g., Kogenate®); Factor IX; β-globin; hemoglobin; and the like); a colony stimulating factor (e.g., Neupogen® (filgrastim; G-CSF); Neulasta (pegfilgrastim); granulocyte colony stimulating factor (G-CSF), granulocyte-monocyte colony stimulating factor, macrophage colony stimulating factor, megakaryocyte colony stimulating factor; and the like); a growth hormone (e.g., a somatotropin, e.g., Genotropin®, Nutropin®, Norditropin®, Saizen®, Serostim®, Humatrope®, etc.; a human growth hormone; and the like); an interleukin (e.g., IL-I; IL-2, including, e.g., Proleukin®; IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9; etc.); a growth factor (e.g., Regranex® (beclapermin; PDGF); Fiblast® (trafermin; bFGF); Stemgen® (ancestim; stem cell factor); keratinocyte growth factor; an acidic fibroblast growth factor, a stem cell factor, a basic fibroblast growth factor, a hepatocyte growth factor; and the like); a soluble receptor (e.g., a TNF-α-binding soluble receptor such as Enbrel® (etanercept); a soluble VEGF receptor; a soluble interleukin receptor; a soluble γ/δ T cell receptor; and the like); an enzyme (e.g., α-glucosidase; Cerazyme® (imiglucarase; β-glucocerebrosidase, Ceredase® (alglucerase; ); an enzyme activator (e.g., tissue plasminogen activator); a chemokine (e.g., IP-10; Mig; Groα/IL-8, RANTES; MIP-Ia; MlP-lβ; MCP-I; PF-4; and the like); an angiogenic agent (e.g., vascular endothelial growth factor (VEGF) ; an anti-angiogenic agent (e.g., a soluble VEGF receptor); a protein vaccine; a neuroactive peptide such as bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, warfarin, dynorphin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, a sleep peptide, etc.; other proteins such as a thrombolytic agent, an atrial natriuretic peptide, bone morphogenic protein, thrombopoietin, relaxin, glial fibrillary acidic protein, follicle stimulating hormone, a human alpha- 1 antitrypsin, a leukemia inhibitory factor, a transforming growth factor, a tissue factor, an insulin-like growth factor, a luteinizing hormone, a follicle stimulating hormone, a macrophage activating factor, tumor necrosis factor, a neutrophil chemotactic factor, a nerve growth factor, a tissue inhibitor of metalloproteinases; a vasoactive intestinal peptide, angiogenin, angiotropin, fibrin; hirudin; a leukemia inhibitory factor; an IL-I receptor antagonist (e.g., Kineret® (anakinra)); and the like.

[0029] Specific examples of erythropoietin include, but are not limited to, human erythropoietin (see, e.g., Jacobs et al. (1985) Nature 313:806-810; and Lin et al. (1985) Proc Natl Acad Sci USA 82:7580-7584); erythropoietin polypeptides discussed in U.S. Patent Nos. 6,696,056 and 6,585,398; the amino acid sequences provided in GenBank Accession Nos. NP_00790 and CAA26095; Epoetin alfa (EPREX®; ERYPO®); Novel erythropoiesis stimulating protein (NESP) (a hyperglycosylated analog of recombinant human eryhropoietin (Epoetin) described in European patent application EP640619); human erythropoietin analog- human serum albumin fusion proteins described in International patent application WO9966054; erythropoietin mutants described in International patent application WO9938890; erythropoietin omega, which may be produced from an Apa I restriction fragment of the human erythropoietin gene described in U.S. Pat. No. 5,688,679; altered glycosylated human erythropoietin described in International patent application WO9911781; PEG conjugated erythropoietin analogs described in WO9805363 or U.S. Pat. No. 5,643,575. Specific examples of cell lines modified for expression of endogenous human erythropoietin are described in international patent applications WO9905268 and WO9412650.

[0030] Suitable insulin polypeptides include, but are not limited to, proinsulin, preproinsulin, and the insulin forms disclosed in U.S. Patent Nos. 4,992,417; 4,992,418; 5,474,978; 5,514,646; 5,504,188; 5,547,929; 5,650,486; 5,693,609; 5,700,662; 5,747,642; 5,922,675; 5,952,297; 6,034,054; and 6,211,144; and published PCT applications WO 00/121197; WO 09/010645; and WO 90/12814. Insulin analogs include, but are not limited to, superactive insulin analogs, monomeric insulins, and hepatospecific insulin analogs. Various forms of insulin include Humalog®; Humalog® Mix 50/50™; Humalog® Mix 75/25™; Humulin® 50/50; Humulin® 70/30; Humulin® L; Humulin® N; Humulin® R; Humulin® Ultralente; Lantus®; Lente® Iletin® II; Lente® Insulin; Lente® L; Novolin® 70/30; Novolin® L; Novolin® N; Novolin® R; NovoLog™; NPH Iletin® I; NPH-N; Pork NPH Iletin® II; Pork Regular Iletin® II; Regular (Concentrated) Iletin® II U-500; Regular Iletin® I; and Velosulin® BR Human (Buffered).

[0031] Insulin polypeptides include analogs of human insulin wherein position B28 is Asp,

Lys, Leu, VaI or Ala and position B29 is Lys or Pro; des(B28-B30) human insulin; des(B27) human insulin; des(B30) human insulin; an analog of human insulin in which position B28 is Asp and position B29 is Lys or Pro; an analog of human insulin in which position B28 is Lys, and position B29 is Lys or Pro; Asp^B28 human insulin; Lys^B28 Pro^B29 human insulin; B29-N^ε- myristoyl-des(B30) human insulin; B29-N^ε-palmitoyl-des(B30) human insulin; B29-N^ε- myristoyl human insulin; B29-N^ε-palmitoyl human insulin; B28-N^ε-myristoyl Lys^B28 Pro^B29 human insulin; B28-N^ε-palmitoyl Lys^B28 Pro^B29 human insulin; B30-N^ε-myristoyl -Thr^B29 Lys^B3° human insulin; B30-N^ε-palmitoyl- Thr^B29 Lys^B3° human insulin; B29-N^ε--(N-palmitoyl- γ-glutamyl)-des(B30) human insulin; B29-N^ε- (N-lithocholyl-γ-glutamyl)-des(B30) human insulin; B29-N^ε-(ω-carboxyheptadecanoyl)-des(B30) human insulin; and B29-N^ε-(ω- carboxyheptadecanoyl) human insulin.

[0032] The amino acid sequences of various insulin polypeptides are publicly available in, e.g., public databases such as GenBank, journal articles, patents and published patent applications, and the like. For example, the amino acid sequences of human insulin are found in GenBank under the following accession numbers: CAA00714; CAA00713; CAA00712; CAA01254; IHISA and IHISB; 1 HIQA and 1 HIQB; IHITA and IHITB; 1 HLSA and IHLSB; IVKTA and IVKTB.

[0033] Suitable colony stimulating factor polypeptides include, but are not limited to, granulocyte colony stimulating factor (G-CSF), such as NEUPOGEN® filgrastim and NEULASTA™ pegfilgrastim, granulocyte-monocyte colony stimulating factor (GM-CSF), such as LEUKINE® sargramostim, macrophage colony stimulating factor, megakaryocyte colony stimulating factor; IL-3; stem cell factor (SCF); and the like. The amino acid sequences of various colony stimulating factors are publicly available, e.g., in public databases such as GenBank; journal articles; patents and published patent applications; and the like. For example, amino acid sequences of IL-3 are disclosed in U.S. Pat. Nos. 4,877,729 and 4,959,455, and International Patent Publication No. WO 88/00598; amino acid sequences of human G-CSF are disclosed in U.S. Pat. No. 4,810,643; WO 91/02754 and WO 92/04455 disclose the amino acid sequence of fusion proteins comprising IL-3; WO 95/21197, WO 95/21254, and U.S. Patent No. 6,730,303 disclose fusion proteins capable of broad multi-functional hematopoietic properties; amino acid sequences of human G-CSF are found under GenBank Accession Nos. NP_757374, P09919, FQHUGL, and NP_000750; amino acid sequences of human GM-CSF are found under GenBank Accession Nos. NP_000749 and P04141; amino acid sequences of IL-3 are found under GenBank Accession Nos. AAH66272, AAH66273, and AAH66276; etc.

[0034] Suitable growth hormone polypeptides include, but are not limited to, somatotropin; a human growth hormone; any of the growth hormone variants disclosed in U.S. Patent Nos. 6,143,523, 6,136,563, 6,022,711, and 5,688,666; fusion proteins comprising a growth hormone, e.g., as disclosed in U.S. Patent No. 5,889,144; growth hormone fragments that retain growth hormone activity; a growth hormone receptor polypeptide agonist as disclosed in U.S. Patent No. 6,387,879; and the like. Growth hormones include alternative forms of known growth hormones, e.g., alternative forms of human growth hormone (hGH), including naturally-occurring derivatives, variants and metabolic products, biosynthetic hGH, and engineered variants of hGH produced by recombinant methods (see, e.g., U.S. Patent No. 6,348,444).

[0035] Suitable growth factor polypeptides include, but are not limited to, keratinocyte growth factor; an acidic fibroblast growth factor, a stem cell factor, a basic fibroblast growth factor, a hepatocyte growth factor, an insulin-like growth factor, etc.; active fragments of a growth factor; fusion proteins comprising a growth factor; and the like. The amino acid sequences of various growth factors are publicly available, e.g., in public databases such as GenBank; journal articles; patents and published patent applications; and the like. For example, amino acid sequences of bFGF are found under GenBank Accession Nos. AAB20640, AAA57275, A43498, and AAB20639; amino acid sequences of aFGF are found under GenBank Accession Nos. AAB29059, CAA46661, and 1605206A; amino acid sequences of stem cell factor are found under GenBank Accession Nos. AAH69733, AAH69783, and AAH69797; amino acid sequences of keratinocyte growth factor are found under GenBank Accession Nos. 035565, AAL05875, and P21781; amino acid sequences of hepatocye growth factor are found under GenBank Accession Nos. AAA64239, AAB20169, and CAA40802.

[0036] Suitable soluble receptor polypeptides include, but are not limited to, a TNF-α-binding soluble receptor; a soluble VEGF receptor; a soluble interleukin receptor; a soluble IL-I receptor; a soluble type II IL-I receptor; a soluble γ/δ T cell receptor; ligand-binding fragments of a soluble receptor; and the like. Suitable soluble receptors bind a ligand that, under normal physiological conditions, binds to and activates the corresponding membrane-bound or cell surface receptor. Thus, a suitable soluble receptor is one that functions as a receptor antagonist, by binding the ligand that would ordinarily bind the receptor in its native (e.g., membrane- bound) form. The amino acid sequences of various soluble receptors are publicly available, e.g., in public databases such as GenBank; journal articles; patents and published patent applications; and the like. For example, amino acid sequences of soluble VEGF receptors are found under GenBank Accession Nos. AAC50060 and NP_002010; soluble VEGF receptors are described in U.S. Patent Nos. 6,383,486, 6,375,929, and 6,100,071; soluble IL-4 receptors are described in U.S. Pat. No. 5,599,905; soluble IL-I receptors are described in U.S. Patent Publication No. 20040023869; etc.

[0037] Suitable chemokine polypeptides include, but are not limited to, IP-10; Mig; Groα/IL-8,

RANTES; MIP- lα; MIP- lβ; MCP-I; PF-4; and the like; as well as fusion proteins comprising a chemokine. The amino acid sequences of various chemokines are publicly available, e.g., in public databases such as GenBank; journal articles; patents and published patent applications; and the like. For example, amino acid sequences of IP-10 are disclosed in U.S. Patent Nos. 6,491,906, 5,935,567, 6,153,600, 5,728,377, and 5,994,292; amino acid sequences of Mig are disclosed in U.S. Patent No. 6,491,906, and Farber (1993) Biochemical and Biophysical Research Communications 192(l):223-230; amino acid sequences of RANTES are disclosed in U.S. Patent Nos. 6,709,649, 6,168,784, and 5,965,697; etc.

[0038] Suitable angiogenic polypeptides include, but are not limited to, VEGF polypeptides, including VEGF121, VEGF165, VEGF-C, VEGF-2, etc.; transforming growth factor-beta; basic fibroblast growth factor; glioma-derived growth factor; angiogenin; angiogenin-2; and the like. The amino acid sequences of various angiogenic agents are publicly available, e.g., in public databases such as GenBank; journal articles; patents and published patent applications; and the like. For example, amino acid sequences of VEGF polypeptides are disclosed in U.S. Patent Nos. 5,194,596, 5,332,671, 5,240,848, 6,475,796, 6,485,942, and 6,057,428; amino acid sequences of VEGF-2 polypeptides are disclosed in U.S. Patent Nos. 5,726,152 and 6,608,182; amino acid sequences of glioma-derived growth factors having angiogenic activity are disclosed in U.S. Patent Nos. 5,338,840 and 5,532,343; amino acid sequences of angiogenin are found under GenBank Accession Nos. AAA72611, AAA51678, AAA02369, AAL67710, AAL67711, AAL67712, AAL67713, and AAL67714; etc.

[0039] Suitable neuroactive polypeptides include, but are not limited to, nerve growth factor, bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, beta- endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone- releasing hormone, bombesin, dynorphin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagons, vasopressin, angiotensin II, thyrotropin- releasing hormone, vasoactive intestinal peptide, a sleep peptide, etc.

[0040] Other proteins of pharmacologic interest include, but are not limited to, a thrombolytic agent, an atrial natriuretic peptide, bone morphogenic protein, thrombopoietin, glial fibrillary acidic protein, follicle stimulating hormone, a human alpha- 1 antitrypsin, a leukemia inhibitory factor, a transforming growth factor, an insulin-like growth factor, a luteinizing hormone, a macrophage activating factor, tumor necrosis factor, a neutrophil chemotactic factor, a nerve growth factor a tissue inhibitor of metalloproteinases; a vasoactive intestinal peptide, angiotropin, fibrin; hirudin; a leukemia inhibitory factor; and the like. The amino acid sequences of various therapeutic proteins are publicly available, e.g., in public databases such as GenBank; journal articles; patents and published patent applications; and the like. For example, amino acid sequences of tissue plasminogen activator are found under GenBank Accession Nos. P00750, AAA01895, AAA01378, AAB06956, and CAA00642. IGF-I and hGH

[0041] In some embodiments, the liquid sample comprises insulin-like growth factor-1 (IGF-I) and growth hormone (GH), and may further include one or more degradation products of IGF- 1 and/or GH. In some embodiments, a subject method involves applying a liquid sample comprising IGF-I and GH onto a UPLC column. In some embodiments, the IGF-I is recombinant human IGF-I (rhIGF-1), and the GH is human GH (hGH). IGF-I and IGF-I degradation products

[0042] The complete amino acid sequence of human IGF-I is known. See, e.g., Rinderknecht and Humbel (J. Biol. Chem. 253 (8), 2769-2776 (1978)). See also, e.g., GenBank Accession No. CAA01954, providing the 71-amino acid sequence: mgpetlcgae lvdalqfvcg drgfyfnkpt gygsssrrap qtgmvdeccf rscdlrrlem ycaplkpaks a (SEQ ID NO:1). The IGF-I can also include the amino acid sequence gpetlcgae (SEQ ID NO:2) at the NH₂ terminus, e.g., where an exemplary IGF-I amino acid sequence is gpetlcgae mgpetlcgae lvdalqfvcg drgfyfnkpt gygsssrrap qtgmvdeccf rscdlrrlem ycaplkpaks a (SEQ ID NO:3). See also GB 2241703. See also, EP 123,228Bl. See also, e.g., GenBank Accession Nos. NP_000609, NPJ)Ol 104754, NPJX)1104755, and NPJ)Ol 14753. A native-sequence IGF-I can have the 70-amino acid sequence: gpetlcgae lvdalqfvcg drgfyfnkpt gygsssrrap qtgmvdeccf rscdlrrlem ycaplkpaks a (SEQ ID NO: 10).

[0043] An IGF-I can also be a variant of a native- sequence IGF-I. See, e.g., U.S. Patent no.

6,506,874. For example, an IGF-I variant can be an IGF-I variant of the amino acid sequence set forth in SEQ ID NO: 10 in which: (a) an amino acid residue located at a single position selected from positions 4, 5, 7, 10, 14, 17, 23, 24, and 43 of the amino acid sequence set forth in SEQ ID NO: 10 is replaced with an alanine residue; or (b) amino acid residues at positions 1 and 70 of the amino acid sequence set forth in SEQ ID NO: 10 are replaced with a serine residue and a valine residue, respectively; or (c) amino acid residues at positions 1 and 70 of the amino acid sequence set forth in SEQ ID NO: 10 are replaced with a serine residue and a valine residue, respectively, and an amino acid residue located at a single position selected from the group consisting of positions 3, 4, 5, 7, 10, 14, 17, 23, 24, 25, and 43 of the amino acid sequence set forth in SEQ ID NO: 10 is replaced with an alanine residue.

[0044] In some embodiments, a polypeptide of interest is IGF-I, e.g., native- sequence, recombinant human IGF-I. Native-sequence IGF-I can be recombinantly produced and is available for clinical investigations {see, e.g., EP 123,228 and 128,733). The term "rhIGF-1" refers to recombinant human IGF-I. For example, rhIGF-1 can have a molecular weight of about 7.6 kDa.

[0045] IGF-I degradation products include, e.g., degradation products in which one, two, three, four, five, or more, amino acids are deleted from the amino terminus of native- sequence rhIGF-1. For example, an IGF-I degradation product can: 1) comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 2-71 of the amino acid sequence set forth in SEQ ID NO:1; and 2) lack the N- terminal methionine.

[0046] As another example, an IGF-I degradation product can: 1) comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 3-71 of the amino acid sequence set forth in SEQ ID NO:1; and 2) lack the N-terminal Met-Gly sequence (e.g., the Met-Gly sequence as shown in SEQ ID NO:1).

[0047] As another example, an IGF-I degradation product can: 1) comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 4-71 of the amino acid sequence set forth in SEQ ID NO:1; and 2) lack the N-terminal Met-Gly-Pro sequence (e.g., the Met-Gly-Pro sequence as shown in SEQ ID NO:1).

[0048] As another example, an IGF-I degradation product can: 1) comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 5-71 of the amino acid sequence set forth in SEQ ID NO:1; and 2) lack the N-terminal four amino acids as shown in SEQ ID NO:1 (e.g., lack the N-terminal Met-Gly- Pro-Glu sequence (SEQ ID NO:8) of the amino acid sequence set forth in SEQ ID NO:1).

[0049] As another example, an IGF-I degradation product can: 1) comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 6-71 of the amino acid sequence set forth in SEQ ID NO:1; and 2) lack the N-terminal five amino acids as shown in SEQ ID NO:1 (e.g., lack the N-terminal Met-Gly- Pro-Glu-Thr sequence (SEQ ID NO:9) of the amino acid sequence set forth in SEQ ID NO:1).

[0050] A another example, an IGF-I degradation product can: 1) comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 2-70 of the amino acid sequence set forth in SEQ ID NO: 10; and 2) lack the N-terminal glycine (e.g., the N-terminal GIy of the amino acid sequence set forth in SEQ ID NO: 10).

[0051] As another example, an IGF-I degradation product can: 1) comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 3-70 of the amino acid sequence set forth in SEQ ID NO: 10; and 2) lack the N-terminal GIy- Pro sequence (e.g., the N-terminal Gly-Pro sequence as shown in SEQ ID NO: 10).

[0052] As another example, an IGF-I degradation product can: 1) comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 4-70 of the amino acid sequence set forth in SEQ ID NO: 10; and 2) lack the N-terminal Gly-Pro-Glu sequence (e.g., the N-terminal Gly-Pro-Glu sequence as shown in SEQ ID NO: 10).

[0053] As another example, an IGF-I degradation product can: 1) comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 5-70 of the amino acid sequence set forth in SEQ ID NO: 10; and 2) lack the N-terminal four amino acids as shown in SEQ ID NO: 10 (e.g., lack the N-terminal Gly-Pro-Glu-Thr sequence (SEQ ID NO: 11) of the amino acid sequence set forth in SEQ ID NO:10). [0054] As another example, an IGF-I degradation product can: 1) comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 6-70 of the amino acid sequence set forth in SEQ ID NO: 10; and 2) lack the N-terminal five amino acids as shown in SEQ ID NO: 10 (e.g., lack the N-terminal GIy- Pro-Glu-Thr- Leu sequence (SEQ ID NO: 12) of the amino acid sequence set forth in SEQ ID NO: 10). Human GH and hGH degradation products

[0055] The amino acid sequence of hGH is known. See, e.g, Roskam and Rougeon ((1979)

Nucl. Acids. Res. 7:305); Martial et al. (1979) Science 205:602; GenBank Accession Nos. NP_000506 (SEQ ID NO:4), NP_072053 (SEQ ID NO:5), NP_072054 (SEQ ID NO:6), and NP_072055 (SEQ ID NO:7). In some embodiments, the polypeptide of interest is native- sequence hGH having a molecular weight of about 21 kDa. In some embodiments, the polypeptide of interest is native- sequence hGH comprising amino acids 27-217 of the amino acid sequence set forth in SEQ ID NO:4, where the hGH lacks amino acids 1-26 of the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the polypeptide of interest is native-sequence hGH that: 1) comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 27-217 of the amino acid sequence set forth in SEQ ID NO:4; and 2) lacks amino acids 1-26 of the amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, amino acid sequence identity to amino acids 1-26 of the amino acid sequence set forth in SEQ ID NO:4.

[0056] An alignment of hGH amino acid sequences is provided in Figure 1. The signal peptide

(amino acids 1-26) is shown in bold type. Amino acid numbering in the art generally refers to the mature protein (e.g., where amino acid 27 of SEQ ID NO:4 is referred to as amino acid 1), such that Met-40 of SEQ ID NO:4 is referred to as Met- 14; Met- 151 of SEQ ID NO:4 is referred to as Met- 125; Asp-156 of SEQ ID NO:4 is referred to as Asp-130; Asn-175 of SEQ ID NO:4 is referred to as Asn-149; and Asn-178 of SEQ ID NO:4 is referred to as Asn-152. Met- 14, Met- 125, Asp-130, Asn-148, and Asn-152 are bolded and underlined in Figure 1.

[0057] Degradation products of hGH include, e.g., NH₂-terminally cleaved hGH; deamidated hGH; sulfoxydated hGH; hGH comprising a cyclic amide (e.g., at Asp-130 as underlined and bolded in Figure 1; or an amino acid corresponding to Asp-156 of SEQ ID NO:4); dimerized hGH; polymerized hGH; and oxidized hGH species.

[0058] The predominant degradation reactions of hGH are 1) deamidation (e.g., of Asn-149 and/or Asn-152) by direct hydrolysis or via a cyclic succinimide intermediate to form various amounts of L-asp-hGH, L-iso-asp-hGH, D-asp-hGH, and D-iso-asp-hGH; and 2) oxidation of the methionine residues in positions 14 and 125 (e.g., at amino acids corresponding to Met-40 of SEQ ID NO:4 and Met-151, respectively, of the amino acid sequence set forth in SEQ ID NO:4; see, e.g., Teh et al. (1987) /. Biol. Chem. 262:6472). Deamidation especially takes place at the Asn in position 149 and to a minor extent in position 152 (e.g., at amino acids corresponding to Asn-175 and Asn-178, respectively, of the amino acid sequence set forth in SEQ ID NO:4; see, e.g., Gellerfors et al. (1990) Acta Paediatr. Scand. Suppl. 370:93).

[0059] In some embodiments, an hGH degradation product includes monodesamido-hGH

(e.g., where the hGH is deamidated at Asn-149, or an amino acid corresponding to Asn-175 of the amino acid sequence set forth in SEQ ID NO: 4). In some embodiments, an hGH degradation product includes didesamido-hGH (e.g., where the hGH is deamidated at Asn-149 and at Asn-152, or at amino acids corresponding to Asn-175 and Asn-178 of the amino acid sequence set forth in SEQ ID NO:4). In some embodiments, an hGH degradation product includes a cyclic amide (e.g., at Asp-130 as underlined and bolded in Figure 1; or at an amino acid corresponding to Asp-156 of SEQ ID NO:4).

[0060] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMSWhat is claimed is:

1. A method of identifying a degradation product of one or more polypeptides of interest in a sample comprising the one or more polypeptides of interest, the method comprising: a) applying the sample to an ultra performance liquid chromatography column; b) separating the one or more polypeptides of interest from one or more degradation product present in the sample, generating a series of peaks corresponding to the one or more polypeptides and the one or more degradation products; c) measuring the area under the curve for each peak; and d) calculating the percent by weight of the one or more degradation products present in the sample.

2. The method of claim 1, wherein the one or more polypeptides each has a molecular weight in the range of from about 5 kDa to about 30 kDa.

3. The method of claim 1, wherein the sample is a liquid sample comprising native- sequence recombinant human insulin-like growth factor- 1 (rhIGF-1) and human growth hormone (hGH).

4. The method of claim 3, wherein the one or more degradation products of rhIGF-1 lack from one to about 5 amino acids from the amino terminus, compared to the amino acid sequence of the rhIGF-1.

5. The method of claim 3, wherein the one or more degradation products of the hGH is an NH₂-terminally cleaved hGH degradation product, deamidated hGH, hGH comprising a cyclic imide at Asp-130, and an oxidized species of hGH.